Amino acid-derivatized slide-ring gels: Chemical crosslinking of polyrotaxane conjugates with different amino acid pendant groups

Accepted Manuscript Amino acid-derivatized slide-ring gels: chemical crosslinking of polyrotaxane conjugates with different amino acid pendant groups Jun Araki, Naoki Sainou PII:

S0032-3861(15)30139-7

DOI:

10.1016/j.polymer.2015.07.060

Reference:

JPOL 18020

To appear in:

Polymer

Received Date: 9 June 2015 Revised Date:

27 July 2015

Accepted Date: 31 July 2015

Please cite this article as: Araki J, Sainou N, Amino acid-derivatized slide-ring gels: chemical crosslinking of polyrotaxane conjugates with different amino acid pendant groups, Polymer (2015), doi: 10.1016/j.polymer.2015.07.060. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Amino acid-derivatized slide-ring gels: chemical crosslinking of polyrotaxane conjugates with

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different amino acid pendant groups

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Jun Araki* and Naoki Sainou†

*Faculty of Textile Science and Technology, Shinshu University, Tokida 3-15-1, Ueda, Nagano prefecture, 386-8567, Japan, and Division of Biological and Medical Fibers, Institute for Fiber Engineering (IFES), Interdisciplinary Cluster for Cutting Edge Research (ICCER), Shinshu

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University, Tokida 3-15-1, Ueda, Nagano prefecture, 386-8567, Japan. Tel. & FAX: +81-26821-5587, E-mail: [email protected]

Graduate School of Science and Technology, Shinshu University, Tokida 3-15-1, Ueda 386-

8567, Japan.

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†

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Running Title: Amino Acid-Derivatized Slide-ring Gels

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ABSTRACT

Two different amino acid-polyrotaxane (AA-PR) conjugates were synthesized and examined

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using two types of amino acids, i.e., alanine (Ala) and phenylalanine (Phe), and a PR containing poly(ethylene glycol) and α-cyclodextrin (CD) axes via the activation of the carboxyl groups in the AAs using 1,1′-carbonyldiimidazole and subsequent esterification with the hydroxyl groups

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in the CDs. The degree of substitution (DS) of the obtained conjugates was determined using 1H NMR spectroscopy, titration after deprotection and ultraviolet absorbance analyses (in the case

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of the Phe-PR conjugates). The DS values obtained using these were in good agreement, indicating that the DS could be controlled by varying the reaction temperature, reaction time, and [AA]/[−OH] stoichiometric ratio, the last of which provided the widest DS control. The BocPhe-PR conjugate exhibited solubility behavior dependent on the solvent and temperature, i.e.,

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an upper critical solution temperature in methanol and a lower critical solution temperature in ethanol. Various AA-PR slide-ring gels were prepared by crosslinking the deprotected cationic conjugates using hexamethylene diisocyanate. The degree of swelling was highest at pH = 4.64

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and much lower at higher and lower pH. The low swelling of the gels at higher and lower pH is attributed to deprotonation of the primary amino groups in the gels and hydrolytic dissociation of

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the ester linkages between the AAs and PRs, respectively.

Keywords: Polyrotaxanes; Amino acids; Degree of substitutions

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1. INTRODUCTION The incredible usefulness of polyrotaxane (PR), i.e., supramolecules consisting of many ring molecules and linear penetrating axes, and their establishment as a novel category of

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compounds within the field of materials chemistry has been well recognized [1–5]. Since the pioneering discovery of the PR consisting of a poly(ethylene glycol) (PEG) axis and cyclodextrin (CD) rings by Harada and coworkers [6–8], it has been the focus of numerous studies, and the

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syntheses of many analogs of this compound have been reported. More recently, the discovery of novel “slide-ring materials,” i.e., supramolecular materials possessing mobile crosslinks, has

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stimulated research targeting the development of additional cutting-edge functional materials [1– 4].

Greater understanding of the properties of PRs has further accelerated this trend. For instance, the poor solubility of PRs in water and general organic solvents was recently overcome

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with the discovery of solvent systems possessing hydrogen-bond-breaking abilities [9–11], and the synthesis of various PR derivatives [12,13] has enhanced the utility of PRs in materials chemistry. Such PR derivatives have nonionic [12], ionic [13], polymeric [14–16], and liquid

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crystalline [17,18] functional groups. In addition to their outstanding properties attributable to the free movement of ring moieties and/or crosslinks [1–4], these functionalized PRs have

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additional interesting properties that have extended their use to all aspects of materials science. The authors’ research group has been involved in the synthesis of amino acid (AA)-PR

conjugates, i.e., PR derivatives with pendant AA moieties [19,20]. Similar conjugates with functional dipeptides were previously synthesized and used for the digested uptake of another model dipeptide [21]. To develop a more versatile method, we prepared the conjugates of PR and glycine (Gly) via an esterification between carboxyl groups in N-protected Gly and the hydroxyl

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groups in PR, followed by subsequent deprotection. While the Boc groups could be readily removed via conventional treatment using neat trifluoroacetic acid (TFA) [19], the removal of Z groups in the conjugates could not be achieved using common methods, including hydrogenation

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and hydrobromic acid/acetic acid treatment; it can be achieved only with a TFA/thioanisole system [20]. The variation of the reaction temperature, reaction time, and [AA]/[−OH]

5%–60% compared with the PR hydroxyl content [20].

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stoichiometric ratio enabled control of the degree of substitution (DS) of AA over the range of

Results of comprehensive studies of various AA-polymer conjugates [22,23] suggested

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that the properties of these types of conjugates are quite dependent on the type and number of grafted AA moieties. Therefore, a more detailed investigation of changes in the DS for different AAs is of significant interest. Such a study is also required to gain further knowledge of the properties of conjugates prepared using various functional AAs and peptides in order to evaluate

protected

AAs,

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their potential for practical applications. In the present study, conjugation with two different namely

N-α-(t-butoxycarbonyl)-L-alanine

(Boc-Ala)

and

N-α-(t-

butoxycarbonyl)-L-phenylalanine (Boc-Phe), was examined. Calculation of the DS values was

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performed using 1H NMR spectroscopic data, colloidal titration after deprotection, and UV-vis absorption analysis (for the conjugates with Phe as a UV-absorbing pendant AA). Both the

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protected and deprotected conjugates were investigated for solubility using a wide variety of solvents. The Boc-Phe-PR conjugates exhibited intriguing solution behavior dependent on the solvent type and temperature, including an upper critical solution temperature (UCST) in methanol and a lower critical solution temperature (LCST) in ethanol. Novel AA-PR slide-ring gels with freely mobile crosslinks bearing pendant AA moieties were also prepared via chemical crosslinking of the deprotected conjugates. Slide-ring gels, which are chemically crosslinked PR

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gels, have been reported to show novel properties including significant swelling and stretching behavior because of the free movement of the crosslinks of the CD rings [24]. The present AAPR slide-ring gels were therefore expected to exhibit such improved properties in addition to the

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functionality attributed to the AA-polymer conjugates [22,23]. Thus, the swelling behavior of the obtained AA-PR slide-ring gels was examined under various pH environments.

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2. EXPERIMENTAL SECTION 2.1. Materials.

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Using previously reported methods [25,26], the PR used in this study was prepared from PEG with a molecular weight of 35000 and α-CD. The values for the weight-average molecular weight and polydispersity index, determined using gel-permeation chromatography (GPC) under conditions described in previous studies [19,20], are summarized in Table S1 in the Supporting

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Information. The average value of the hydroxyl content (HC) values for the PR, which were determined from the 1H NMR analysis of the PR hydrolyzed using 20% D2SO4, was 13.89 ± 0.05 and was considered to be nearly constant (see Supporting Information for details). 1,1′-

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carbonyldiimidazole (CDI) was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). The amino acids Boc- L-Ala and Boc- L-Phe were purchased from Peptide Institute Inc.

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(Osaka, Japan). All other chemicals were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). All chemicals were of reagent grade and used without any special purification. Boc-Phe was prepared from Boc-L-Phe (or Boc-D,L-Phe) and di-t-butyl dicarbonate ((Boc)2O) according to the reported procedure [27]. Dimethyl sulfoxide was dehydrated overnight by stirring with calcium hydride followed by distillation under reduced pressure.

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Scheme 1. Synthesis and deprotection schemes for the AA-PR conjugates.

2.2. Measurements.

The 1H NMR spectra at 400 MHz were recorded in deuterated dimethyl sulfoxide (DMSO-d6) or 4% NaOD/D2O using a Bruker AVANCE spectrometer (Bruker BioSpin K.K., Yokohama,

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Japan) at room temperature. The chemical shifts were referenced to tetramethylsilane (δ = 0) in the case of DMSO-d6 and residual protons in the deuterated solvents (δ = 4.70) in the case of

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NaOD/D2O. The latter solvent system was used to determine the DS values for the Boc-Gly-PRs and Z-Gly-PRs after complete hydrolysis of the ester linkages between the PR and the protected

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glycines in NaOD/D2O [19]. The DS values for the Boc-AA-PRs were calculated from the ratios of ABoc to AH1, where ABoc and AH1 are the integrated areas of the proton signals from the Boc groups (near 1.39 ppm) and the anomeric protons in CD (near 4.8 ppm), respectively. The DS values calculated using the 1H NMR data are denoted by DSNMR. GPC analyses of all the conjugates and colloidal titration for determining the primary amino group content in the deprotected samples were performed in a manner similar to those reported previously [19,20].

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Attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectra of the freeze-dried conjugates were recorded using a Shimadzu IRPrestage-21 spectrometer (Shimadzu Corporation, Kyoto, Japan) equipped with a diamond ATR accessory (SensIR Technologies DurasampleIR II,

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Smiths Detection, UK) in air at a 4 cm−1 resolution with 32 scans. The qualitative solubilities of the conjugates in various solvents (water, methanol, ethanol, acetone, DMSO, dimethyl acetamide (DMAc), dimethylformamide (DMF), tetrahydrofuran (THF), toluene, and

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dichloromethane) were determined through visual observation of the mixtures (5 mg/2 mL) at

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room temperature.

2.3. Synthesis of the AA-PR conjugates from PR and Boc-protected amino acids. Syntheses of the various AA-PR conjugates was performed according to previously reported procedures (Scheme 1) [19,20]. A Typical example using PR and Boc-Ala is briefly

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described as follows. CDI (6.55 mmol, 1.06 g) was added to a solution of Boc-Ala (1.24 g, 6.55 mmol) in DMAc (20 mL), followed by stirring for 2 h at room temperature. The mixture was then added to a solution of PR (500 mg, 6.55 mmol of −OH) in DMAc (20 mL) containing 6

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wt% lithium chloride, and the resultant solution was further stirred for 24 h at 60 °C under an argon atmosphere. Subsequently, the conjugate was precipitated by dropwise addition of the

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mixture into vigorously stirred 2-propanol (400 mL). The precipitated solid was collected and washed via centrifugation with 2-propanol and then vacuum dried to yield a white solid. Other conjugates were also prepared using Boc-Ala or Boc-Phe under different conditions, which are summarized in Table 1 (note that the precipitation solvent used during the purification stage was dependent on the type of conjugate). The obtained conjugates were designated using the name of the amino acid and the employed conditions; e.g., the conjugate prepared from Boc-Ala at [Boc-

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Ala]/[−OH] = 2:1 for a reaction time of 48 h and that prepared from Boc-Phe at [BocPhe]/[−OH] = 1:1 at 40 °C were designated as Boc-Ala200-PR35k_48h and Boc-Phe100-

time of 24 h and a reaction temperature of 60°C were employed.

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PR35k_40C, respectively. For the conjugates without specified reaction conditions, a reaction

Table 1. Reaction conditions for preparation of the Boc-AA-PR conjugates. Types of

PR a

AA

Boc-Ala50-PR35k

1

Ala

Boc-Ala100-PR35k

1

Ala

Boc-Ala200-PR35k

2

Ala

Boc-Ala300-PR35k

1

Ala

Boc-Ala100-PR35k_40C

2

Ala

Boc-Ala100-PR35k_80C

2

Ala

Boc-Ala100-PR35k_12h

3

Ala

Reaction

Precipitation

temperature, °C

time, h

solvents

60

24

Isopropanol

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0.5

Reaction

1.0

60

24

Isopropanol

2.0

60

24

5% NaHCO3aq.

3.0

60

24

5% NaHCO3aq.

1.0

40

24

Isopropanol

1.0

80

24

5% NaHCO3aq.

1.0

60

12

Isopropanol

3

Ala

1.0

60

48

5% NaHCO3aq.

1

Phe

0.5

60

24

Isopropanol

1

Phe

1.0

60

24

Isopropanol

1

Phe

2.0

60

24

80% MeOH/H2O

1

Phe

3.0

60

24

80% MeOH/H2O

2

Phe

1.0

40

24

70% MeOH/H2O

Boc-Phe100-PR35k_80C

2

Phe

1.0

80

24

70% MeOH/H2O

Boc-Phe100-PR35k_12h

3

Phe

1.0

60

12

70% MeOH/H2O

Boc-Phe100-PR35k_48h

3

Phe

1.0

60

48

70% MeOH/H2O

Boc-D,L-Phe100-PR35k

3

D,L-Phe

1.0

60

24

70% MeOH/H2O

Boc-Phe200-PR35k_1W

3

Phe

2.0

60

168

80% MeOH/H2O

Boc-Phe300-PR35k_80C

3

Phe

3.0

80

24

80% MeOH/H2O

Boc-Phe50-PR35k Boc-Phe100-PR35k Boc-Phe200-PR35k Boc-Phe300-PR35k

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Boc-Phe100-PR35k_40C

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Boc-Ala100-PR35k_48h

[AA]/[−OH]

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Starting

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Samples

a

Batch number of the starting PR (see Table S1 in the Supporting Information for details).

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2.4. Deprotection of the Boc groups. Deprotection was also performed according to previously reported procedures [19,20]; in brief, the conjugates were dissolved in TFA at 0.1 g/mL and stirred for 3 h at room temperature,

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followed by precipitation in diethyl ether (10 times the volume of the TFA solution). The precipitates were washed repeatedly with diethyl ether via centrifugation and then air dried to

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yield white solids.

2.5. Determination of the DS via titration.

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The DS values of the deprotected conjugates were determined using colloidal titration according to previously reported procedures [19,20]; in brief, weighed samples (the sample weights were chosen such that each sample contained 20–30 µmol of amino groups) were dissolved in deionized water (50 mL), and was titrated using a 2.5 mM potassium poly(vinyl

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sulfate) (PVSK) titrant and a few drops of 0.1 w/v% toluidine blue solution as an indicator. The DS values determined on the basis of the titration data, DStit, were calculated using the following equations: ∙∙

and

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 (mmol⁄g) = 

∙

,

!×" ×)

(2)

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 (%) = ×(

(1)



where AAC is the amino acid content (in mmol/g), V is the volume of the PVSK solution (in mL) used for titration of the primary amino groups in the deprotected conjugates, f and c are a factor (i.e., the ratio of the actual concentration to a prescribed concentration) and the molar concentration (in mol/L) of the used PVSK solution, respectively, and M is the molecular weight of the amino acid moiety (186 for Boc-Ala- and 262 for Boc-Phe-).

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2.6. Determination of the DS of the Phe-PR conjugates using UV absorbance measurements. The DS values for the conjugates containing Phe groups were also determined using UVvisible spectroscopy. Phe-PR or Boc-Phe-PR was stirred in a 6 wt% aqueous NaOH solution for

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24 h to afford a transparent solution of a mixture of Phe (or Boc-Phe) and PR via alkaline hydrolysis of the PR-Phe ester linkage. The UV-vis transmittance spectra of the obtained solutions were recorded using a HITACHI U-2000A spectrophotometer at wavelengths in the

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range of 210–400 nm using a 1 × 1 × 4 cm quartz cell. The molar extinction coefficient of Phe at 257 nm, ε, was determined according to the Lambert–Beer law using a separate set of spectra

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obtained for aqueous NaOH solutions containing different concentrations of Phe. The DS values based on the UV data, DSUV, were determined using the following equations: #$%& (mol) =

'()

∙ ,,

(3)

-./0 (1) = #./0 ∙ 2,

(4)

-$3 (g) = -$%&

$3

9:;

78 (%) = 

 ∙

,

− -$%& + -6 , and

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*∙+

(5)

(6)

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where A257 is the absorbance of the sample solution at 257 nm, V is the volume of the prepared solution, NPhe is the molar number of the grafted Phe, WPhe and M are the weight and molecular

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weight values for the amino acid moiety (M corresponds to 262 for Boc-Phe- and 248 for Phe-), and WPhe-PR, WPR, and WH are the weights of the conjugates, the starting PR, and the dissociated protons, respectively.

2.7. Preparation of the AA-PR slide-ring gels. The crosslinking reaction of the AA-PRs using HMDI to produce the corresponding slide-ring gels is shown in Scheme 2. Fifty milligrams of each of the various deprotected conjugates were

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dissolved in anhydrous DMSO. After the addition of hexamethylene diisocyanate (HMDI), each aliquot was rapidly stirred and poured into a Teflon® beaker (2 mL) or a polyvinyl chloride tube (Elicon tube, Imamura Co., Ltd., Tokyo; inner diameter: 5 mm), followed by sealing and gelation

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at a constant temperature. After removal from the mold, to remove any impurities, the obtained gels were sequentially soaked in DMSO and deionized water for 2 days each. The content of the primary amino groups in each of the conjugates, the volumes of dehydrated DMSO, the

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stoichiometric ratios of [HMDI]/[−NH2], and the gelation temperatures for each Phe-PR gel

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preparation are summarized in Table 2.

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Scheme 2. Crosslinking of the AA-PRs with HMDI to produce slide-ring gels.

mmol/g

A

Phe300-PR35k

35.7

2.16

2.0

250

60

25

7.6

B

Phe300-PR35k

35.7

2.16

2.0

500

60

65

16.4

C

Phe300-PR35k

35.7

2.16

1.0

250

60

65

3.8

D

Phe300-PR35k

35.7

2.16

1.0

250

60

25

11.9

Table 2. Crosslinking conditions for preparation of the Phe-PR slide-ring gels. Entry

Starting Phe-PR

DStit,

[−NH2],

[HDMI]

Volume of

Gelation

Gelation

Swelling

%

/[−NH2]

DMSO, µL

time, min

temperature, °C

ratio

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Phe200-PR35k

26.3

1.87

0.61

250

60

25

21.9

F

Phe200-PR35k

26.3

1.87

0.61

250

600

25

15.3

G

Phe300-PR35k_80C

37.9

2.21

0.66

250

60

25

7.5

H

Phe300-PR35k_80C

37.9

2.21

0.5

250

60

25

10.8

I

Phe300-PR35k_80C

37.9

2.21

0.34

250

60

25

13.3

2.8.

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E

Determining the swelling ratios of the AA-PR slide-ring gels.

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The swelling ratios Q of the Phe-PR gels were determined as follows. First, the gels A–I in Table 2 were soaked in deionized water for more than 24 h to allow for equilibrated swelling.

according to the following equation: <=

= >

,

(7)

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The fully swollen gels were then dried at 65 °C for 48 h, and the Q values were calculated

where Ws and Wd are the weight of the swollen and dried gels, respectively.

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The gels F and H were further subjected to swelling with various electrolyte solutions; aliquots of the split gel F were immersed separately in sufficient quantities of aqueous NaCl solutions with different molar concentrations (0.01, 0.1, and 0.5 M) for 24 h, with two solution

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exchanges. The Ws values for each aliquot were determined, and then, each swollen gel was placed in deionized water for 24 h with two water exchanges to remove the NaCl. After drying at

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65 °C for 48 h, the Wd values for each sample were determined. The Ws and Wd values were then used to determine the Q values for the aliquots exposed to the different NaCl solution according to equation 7. The gel H was similarly split into pieces that were further immersed in three types of aqueous buffers with an identical ionic strength of 0.1: a 0.1 M acetate buffer (pH = 3.68), a 0.1 M ammonium buffer (pH = 8.54), and a 0.1 M sodium carbonate buffer (pH = 9.99). After determining the Ws value in each buffer, the gels were desalted and dried as described above to

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determine the Wd values. The Q values for these samples were also calculated. Preparation of the different buffers is summarized in the Supporting Information.

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2.9. Determination of the pKa values of the primary amino groups in the deprotected conjugates via titration.

Boc-Phe200-1W (50 mg) was dissolved in deionized water (10 mL) and then titrated with

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a 0.01 M NaOH solution and monitored using a pH/conductivity meter (Seven Go Duo-SG78, METTLER TOLEDO, OH, USA). As previously reported [27], the pH value at the point when

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half of the primary amino groups were consumed was used as the pKa value. Typical titration curves for the pH and conductance of Phe200-PR_1W are presented in the Supporting Information.

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2.10. Evaluation of conjugate degradation at various pH.

One of the conjugates, Boc-Phe300-PR35k-80C_03, was dispersed in the three buffer solutions used in the swelling experiment (described above) at 100 mg/10 mL, and then, the solutions were

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allowed to stand for 24 h at room temperature. After removal of the solution by vacuum drying (salts remained), the samples were subjected to GPC analysis as described in the Measurements

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section. The degree of degradation (DD) of each of the conjugates was calculated according to the following equation:  (%) =

?'?" ×

?'?" @A?B

,

(8)

where A8-10 and A12-13 are the peak areas for the peaks appearing with retention times of 8–10 min and 12–13 min, respectively, in the GPC chromatograms.

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2.11. Determination of the cloud point temperature using transmittance measurements. Each of the protected conjugates was dispersed in methanol or ethanol and transferred to a 1 × 1 × 4 cm quartz cell, which was maintained in a water bath at the desired temperature for 4

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min. The cell was rapidly wiped and the transmittance spectrum at 500 nm was obtained using a UV–vis spectrophotometer (HITACHI U-2000A). The conjugate/solvent ratio was selected to afford an appropriate range of transmittance values over the employed temperature range: 1

3. RESULTS AND DISUCSSION

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and 3.7 mg/mL for Boc-Phe300-PR35k-80C_02/ethanol.

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mg/mL for Boc-Phe300-PR35k-80C_02/methanol and Boc-Phe300-PR35k-80C_03/methanol,

3.1. Synthesis of AA-PR conjugates using Ala and Phe.

Two types of AA-PR conjugates were prepared using Ala and Phe as pendant AAs. GPC

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analyses of all of the conjugates indicated unimodal chromatograms with earlier elution times than that of the starting PR and corresponding to weight average molecular weights (Mw) higher than that of the starting PR (Table 3). The chromatograms of all the conjugates containing Phe

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also had UV-detected peaks of higher molecular weight similar to those observed using a refractive index (RI) detector. Figure 1(a) shows the 1H NMR spectrum of the Boc-Ala200-

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PR35k, indicating the signals attributable to the grafted Boc-Ala (CH3 of the Boc groups at δ = 1.3) in addition to those for the PR [25,26]. Similar results were observed for all the other conjugates examined. These results clearly indicated both successful grafting of the selected AAs on the PR without PR decomposition and efficient removal of unreacted reagents and byproducts via precipitation using the poor solvents listed in Table 1.

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Figure 1. 1H NMR spectra of (a) Boc-Ala200-PR35k in DMSO-d6, (b) Phe100-PR35k_80C in

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DMSO-d6, (c) Boc-Phe100-PR35k_12h in 11% NaOD/D2O, and (d) Boc-Phe100-PR35k in 20% D2SO4/D2O. Asterisks indicate signals for residual protons in the deuterated solvents. Signals around 2.4 and 2.8 ppm in Figure 1(d) are those from residual dimethylacetamide and its acid

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hydrolysis products, namely dimethyamine, respectively.

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Table 3. Weight average molecular weights Mw and polydispersity indices Mw/Mn of various AA-PRs determined by GPC measurements. Mw, × 105

Mw/Mn

Boc-Ala50-PR35k

1.14

1.6

Boc-Ala100-PR35k

1.28

1.3

Boc-Ala200-PR35k

1.25

1.6

Boc-Ala300-PR35k

1.58

1.4

Sample

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1.7

Boc-Ala100-PR35k_80C

1.12

1.7

Boc-Ala100-PR35k_12h

1.11

1.6

Boc-Ala100-PR35k_48h

1.24

1.7

Boc-Phe50-PR35k

1.39

1.7

Boc-Phe100-PR35k

1.60

1.5

Boc-Phe200-PR35k

1.32

1.6

Boc-Phe300-PR35k

1.62

1.6

Boc-Phe100-PR35k_40C

1.01

1.8

Boc-Phe100-PR35k_80C

1.12

1.5

Boc-Phe100-PR35k_12h

1.18

1.8

Boc-Phe100-PR35k_48h

1.24

1.7

Boc-D,L-Phe100-PR35k

1.21

1.7

Boc-Phe300-PR35k_80C

1.98

1.6

3.2. Deprotection of the conjugates.

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1.00

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Boc-Ala100-PR35k_40C

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Previously, deprotection of the conjugates was discovered to be achieved by treating them with neat TFA [19,20]. This method was therefore used in the present study. Proton NMR spectra of

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the obtained deprotected conjugates, i.e., Phe100-PR35k_80C (Figure 1(b)) showed no signals attributable to Boc groups at δ = 1.3 ppm, indicating complete deprotection.

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Figure 2 presents FT-IR spectra of the starting PR, Boc-Phe100-PR35k_80C, and Phe100-PR35k_80C. The absorption at 1743 cm−1, which is absent in the former spectrum but present in the latter two, is attributed to the C=O stretching of the ester linkages between PR and the AAs. The presence of an identical absorption band in the spectrum of Phe100-PR35k_80C (Figure 2(c)) supports the conclusion that cleavage of the ester linkages did not occur during TFA treatment. Absorptions at 1684 and 1672 cm−1 in Figures 2(b) and 2(c) are attributed to stretching vibrations of C=O groups in Boc groups and those in trifluoroacetate ions, respectively

16

ACCEPTED MANUSCRIPT

[19]. In addition, three sharp absorptions can also be seen at 721, 799, and 839 cm−1. These bands are attributed to C–F stretching in the trifluoroacetic acid salts of the primary amines, indicating the formation of TFA·NH2 groups after successful deprotection. These observations

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clearly indicate a successful conjugation of AAs and PRs, as well as subsequent deprotections of

EP

TE D

M AN U

SC

Boc groups without loss of AA moieties.

AC C

Figure 2. FT-IR spectra of (a) unmodified PR, (b) Boc-Phe100-PR35k_80C, and (c) Phe100PR35k_80C.

3.3. Changes in the DS values with the reaction conditions. Determination of the DS values for the prepared conjugates was first accomplished using 1H NMR spectroscopy. NMR analyses for DS determination were performed in 11 wt % NaOD/D2O or 20% D2SO4/D2O solutions. The former was used to hydrolyze the ester linkages

17

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between the PR and AA, and the latter was used to hydrolyze the conjugates into a mixture of PEG, AA, and glucose residues (from CD). Although both treatments were effective for clarifying the obscured spectra of the conjugates, the NaOD/D2O method occasionally resulted in

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spectra wherein recognizing the H-1 signal near 4.8 ppm was difficult because of overlapping with shifted signals of residual protons in the NaOD. Specifically, this signal is known to shift toward the lower field with an increase in NaOD concentration, while the signals attributed to

SC

carbohydrate structures shift toward higher field in more concentrated NaOD [28]. Thus, the H-1 proton signal at 4.6 ppm in D2O [29] and the residual proton signal for D2O at 4.4 ppm [29] may

M AN U

approach one another and ultimately overlap to become indiscernible in some cases, viz., with an increase in the NaOD concentration. In these cases, the use of D2SO4 hydrolysis was effective because the residual proton signal was shifted to a far higher value at ca. 7.0 ppm. Figures 1(c) and 1(d) show the 1H NMR spectra of Phe100-PR35k_12h in NaOD and Boc-Phe100-PR35k in

TE D

20% D2SO4, respectively. In the latter, signals attributed to the Boc groups are absent because of the release of the Boc protective groups as isobutylene under acidic condition. The values for DSNMR were calculated from the ratios of the areas of the H-1 proton signals (δ = 4.9 ppm in

spectra).

EP

Figure 1(c) and 4.6–5.2 ppm in Figure 1(d)) to those of the aromatic protons (δ = 7.3 ppm in both

AC C

Figure 3 plots changes in the DSNMR values as a function of different reaction condition

parameters, i.e., the stoichiometric ratio of the AA to PR hydroxyls, the reaction temperature, and the reaction time. When one parameter was varied, the other two were fixed; i.e., when the stoichiometric ratio was varied, the reaction temperature and time were fixed at 60 °C and 24 h, respectively. The DSNMR values increased with an increase in each of the parameters (see Table 4). Under the conditions examined in the present study, the widest control of the DSNMR value

18

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was achieved by varying the [AA]/[−OH] stoichiometric ratio. Figures 3(a)–(c) also clearly indicate good agreements of the values for the protected conjugates and those for the deprotected conjugates, and therefore, it can be concluded that no AA detachment occurred during the

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employed deprotection treatment. Also, the figures demonstrate that the DSNMR values for the Ala and Phe conjugates are similar. To gain more insight, the DSNMR values of the protected and deprotected conjugates were compared, as shown in Figure 4; this result clearly indicates that the

TE D

M AN U

differences were observed at lower DSNMR values.

SC

DSNMR values before and after deprotection were also fairly comparable, although slight

Figure 3. Changes in the DSNMR values as a function of the (a) stoichiometric ratio of

EP

[AA]/[−OH], (b) reaction temperature, and (c) reaction time. Red circles: Boc-Ala-PR; blue

AC C

circles: Ala-PR; red triangles: Boc-Phe-PR; blue triangles: Phe-PR.

19

SC

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ACCEPTED MANUSCRIPT

Ala-PR; open circles Boc-Phe-PR.

M AN U

Figure 4. Comparison of the DSNMR values before and after deprotection. Filled circles: Boc-

Table 4. DS values for various AA-PR conjugates determined using 1H NMR and UV–vis

TE D

absorption spectroscopy and titration. DSNMR, % 3.8

Boc-Ala100-PR35k

11.8

Boc-Ala200-PR35k

33.6

Boc-Ala300-PR35k

a

37.5 5.6

8.5

Boc-Phe100-PR35k

14.0

16.2

Boc-Phe200-PR35k

34.2

30.3

Boc-Phe300-PR35k

37.5

39.2

Ala50-PR35k

7.5

3.3

Ala100-PR35k

14.0

10.7

Ala200-PR35k

33.2

25.5

Ala300-PR35k

41.1

32.1

AC C

Boc-Phe50-PR35k

DStit, %

EP

Boc-Ala50-PR35k

DSUV, %

20

6.5

8.6

3.9

Phe100-PR35k

15.8

17.4

12.7

Phe200-PR35k

30.7

28.1

20.3

Phe300-PR35k

41.8

44.8

35.7

Boc-Ala100-PR35k_40C

5.4

Boc-Ala100-PR35k_80C

15.0

Boc-Phe100-PR35k_40C

4.2

4.1

Boc-Phe100-PR35k_80C

21.4

32.7

Ala100-PR35k_40C

6.8

3.1

Ala100-PR35k_80C

14.5

10.0

Phe100-PR35k_40C

3.8

5.9

Phe100-PR35k_80C

20.3

31.3

Boc-Ala100-PR35k_12h

12.3

Boc-Ala100-PR35k_48h

17.2

Boc-Phe100-PR35k_12h

9.0

11.5

Boc-Phe100-PR35k_48h

20.5

19.8

Ala100-PR35k_12h

12.0

Ala100-PR35k_48h

20.0

Phe100-PR35k_12h

8.0

Phe100-PR35k_48h

16.0

M AN U

TE D

13.2

7.5

6.0

23.8

11.2

15.3

37.9

AC C

Boc-Phe300-PR35k_80C

10.8

7.9

EP

Boc-D,L-Phe100-PR35k

2.8

SC

Phe50-PR35k

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ACCEPTED MANUSCRIPT

3.4. Determining the DS values using different methods. In addition to NMR spectroscopy, the DS values of the prepared conjugates were also determined using two other methods, i.e., colloidal titration and UV–vis spectroscopy, although the latter was only applicable to the PR-Phe conjugates with UV-detectable pendant AAs. The results are summarized in Figure 5. Although slight deviations of the values were observed for the conjugates prepared at [AA]/[OH] = 2, all the DS values obtained using the three methods

21

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were in satisfactory agreement, confirming the usability of all these methods. At a glance, Figure 5 seems to demonstrate slightly lower values of DStit than DSNMR or DSUV. Two possibilities can be speculated for this observation; the one is a loss of AAs during deprotection, although the

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possibility is quite implausible as shown in our previous study [19]. The other is related to the nature of colloidal titration measurements; since the measurements is based on polyion complexation between the cationic AA-PR conjugates and anionic titrant, i.e. PVSK, some

SC

amino groups might be enclosed within insoluble aggregations of PVSK and AA-PR conjugates, yielding untitrated amino groups leading to lower amino group contents. Even if the latter may

AC C

EP

TE D

can be considered to be minor.

M AN U

be plausible in our present situation, we consider that the difference produced with this reason

Figure 5. DS value as a function of the [AA]/[−OH] molar ratio for Boc-Phe-PR and Phe-PR calculated using 1H NMR, UV absorbance, and colloidal titration data. Filled circles: DSNMR of Boc-Phe-PR; open circles: DSUV of Boc-Phe-PR; filled squares: DSNMR of Phe-PR; open squares: DSUV of Phe-PR; and crosses: DStit of Phe-PR.

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3.5. Solvent solubility of the conjugates. In contrast to the poor solubility of the starting PR in general solvents [6–11], the synthesized conjugates exhibited a wide variety of solvent solubilities, as previously observed

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for PR conjugates [19,20]. As examples, the solubilities of the Boc-Phe-PR conjugates are summarized in Table 5 (for the solubilities of Boc-Ala-PR, Ala-PR and Phe-PR, see the Supporting Information). The DS values described together in Table 5 are that of DSNMR values,

SC

because the other two, i.e. DStit and DSUV, can characterize only limited types of the conjugates (namely, the deprotected ones and those containing aromatic rings, respectively). All the

M AN U

protected conjugates were insoluble in water and soluble in various organic solvents, while most of the deprotected conjugates indicated good water solubility and restricted solubility in organic solvents (see Supporting Information). Good solvents for the protected conjugates tended to increase with an increase in DS. These tendencies are quite similar to those in our previous

TE D

results [19,20]. The good water solubility of the deprotected conjugates is undoubtedly due to the presence of the primary amino groups that were generated following deprotection. The BocPhe300-PR35k conjugate with a DSNMR value of 37.5% exhibited good solubility in the widest

EP

range of solvents; it was soluble in all the organic solvents examined. Therefore, it can be concluded that the solubility of the conjugates could be widely controlled by changing the type

AC C

of grafted AA and the DS value and through the presence/absence of Boc protecting groups.

Table 5. DSNMR values for various Boc-Ala-PRs and their solubilities in water and general organic solvents.ab Samples

DSNMR,

H2O

MeOH

EtOH

Acetone

DMSO

DMAc

DMF

THF

DCM

Toluene

i

i

i

i

s

s

s

i

i

i

% Boc-Ala50-PR35k

3.8

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11.8

i

s

i

i

s

s

s

i

i

i

Boc-Ala200-PR35k

33.6

i

s

s

s

s

s

s

s

i

i

Boc-Ala100-PR35k_40C

5.4

i

i

i

i

i

i

i

i

i

i

Boc-Ala100-PR35k_80C

15.0

i

i

i

i

s

s

i

i

i

i

Boc-Ala100-PR35k_12h

12.3

i

i

i

i

s

s

s

i

i

i

Boc-Ala100-PR35k_48h

17.2

i

s

s

i

s

s

a

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Boc-Ala100-PR35k

s

i

i

i

MeOH: methanol: EtOH: ethanol; DMSO: dimethyl sulfoxide; DMAc: dimethyl acetamide;

DMF: dimethylformamide; THF: tetrahydrofuran; DCM: dichloromethane. s: soluble: i: insoluble.

SC

b

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3.6. Temperature- and solvent-dependent solution properties of the Boc-Phe-PR conjugates. To gain further insight into the solubility behavior of the conjugates, Boc-Phe300-PR35k_80C was further investigated. The conjugate was dissolved in methanol or ethanol and subjected to solubility examination at temperatures in the range of 0–65 °C. The results are summarized in

TE D

Figures 6 and 7, which reveal intriguing temperature- and solvent-dependent behavior. The solution of Boc-Phe300-PR35k_80C_03 in methanol exhibited typical UCST, above which the conjugate was soluble, while a solution of the same conjugate in ethanol exhibited LCST, only

EP

below which the conjugate formed a transparent solution. Another curiosity was the difference in the LCSTs for ethanolic solutions of different batches of the conjugate with similar DS values;

AC C

the LCSTs were quite different at 0 °C for Boc-Phe300-PR35k_80C_02 with DSUV = 71.5% and at 50 °C for Boc-Phe300-PR35k_80C_03 with DSUV = 70.5%. Although both the batches were synthesized using the same procedure, had comparable DS values, and were similarly soluble at lower temperatures, they exhibited very different LCSTs. Some polymeric derivatives possessing pendant AA moieties have been reported to show quite complicated temperature-dependent solution phenomena; e.g., Endo et al. reported various

24

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types of thermo-responsive polymer-AA conjugates exhibiting both UCSTs and LCSTs, and others possessing dual LCSTs [22]. However, to the best of our knowledge, no study has reported of polymer derivatives exhibiting different thermo-responses in different solvents.

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Although the details of this phenomenon are not clarified yet, the present results may be promising for the preparation of functional materials exhibiting complicated responses with

TE D

M AN U

SC

changes in their environments.

Figure 6. Transmittance of Boc-Phe300-PR35k_80°C_03 in ethanol (open squares), Boc-

EP

Phe300-PR35k_80°C_02 in methanol (filled circles), and Boc-Phe300-PR35k_80°C_03 in

AC C

methanol (filled squares).

25

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ACCEPTED MANUSCRIPT

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3.7. Preparation of AA-PR slide-ring gels.

SC

Figure 7. Appearances of Boc-Phe300-PR35k_80°C_03 in methanol (a) at 40°C and (b) 2°C.

The synthesized and deprotected AA-PR conjugates, e.g., Phe-PR, were further subjected to gelation in dehydrated DMSO using HMDI as a crosslinker, affording AA-PR slide-ring gels. Figure 8 shows the appearance of the Phe-PR slide-ring gel swollen with DMSO and water. Both

TE D

gels in Figure 8 were prepared using the same mold; thus, their sizes were identical before swelling (although the gel in Figure 8(b) was cut in half). The figure indicates that greater

AC C

EP

swelling of the gel occurred in DMSO than in water.

Figure 8. Appearance of gel D swollen with (a) DMSO and (b) water.

Table 6 summarizes the swelling ratio values Q for various Phe-PR slide-ring gels prepared from different Phe-PRs under different gelation conditions. Here the values of DStit, not those of DSNMR or DSUV, are listed, since DStit most directly reflects values of primary amino group

26

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contents, which relates to stoichiometric ratio at cross-linking with HMDI. Comparison of gels A and D and C and D suggests that higher Q values were obtained when greater quantities of HDMI or lower gelation temperatures were used, likely due to the formation of a lower

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crosslinking density under these conditions. A comparison of gels E and F also suggests a lower Q for the latter because of an increased crosslinking density as the result of the longer crosslinking time. These results imply incomplete crosslinking at a gelation time of 60 min. For

SC

gels G, H, and I, a decrease in the [HMDI]/[−NH2] value clearly induced a decrease in Q, which can also be explained by an increase in the crosslinking density. The apparent behavior of gels

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A–C is somewhat inexplicable; the Q for gel B was higher than that for gels A and C, even though the gelation temperature and [HMDI]/[−NH2] value for gel B were higher than that for gel A and gel C, respectively. These results can be speculated to be attributable to the higher pregel concentration of B compared with that of A and C. The gel B was prepared using a

TE D

greater quantity of anhydrous DMSO than was used for A and C, resulting in a more dilute pregel solution and therefore likely a lower crosslinking density for B. All these tendencies are

EP

similar to those observed for typical ionic hydrogels [30,31].

Table 6. Crosslinking conditions for the Phe-PR slide-ring gels and their swelling ratios Q. Starting Phe-PR

DStit,

[−NH2],

%

mmol/g

AC C

Entry

[HDMI]/[−NH2]

Volume of

Gelation

Gelation

DMSO, µL a

time, min

temperature, °C

Qb

A

Phe300-PR35k

35.7

2.16

2.0

250

60

25

7.6

B

Phe300-PR35k

35.7

2.16

2.0

500

60

65

16.4

C

Phe300-PR35k

35.7

2.16

1.0

250

60

65

3.8

D

Phe300-PR35k

35.7

2.16

1.0

250

60

25

11.9

E

Phe200-PR35k

26.3

1.87

0.61

250

60

25

21.9

F

Phe200-PR35k

26.3

1.87

0.61

250

600

25

15.3

G

Phe300-PR35k_80C

37.9

2.21

0.61

250

60

25

7.5

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ACCEPTED MANUSCRIPT

H

Phe300-PR35k_80C

37.9

2.21

0.5

250

60

25

10.8

I

Phe300-PR35k_80C

37.9

2.21

0.34

250

60

25

13.3

Volume of anhydrous DMSO used to prepare each pregel solution.

b

In deionized water.

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a

The Q values summarized in Table 6 appear to be relatively lower than previously reported values for slide-ring gels, which exceeded several hundreds [13,24]. Numerous factors may be

SC

responsible for this phenomenon, including the consumption of the primary amino groups of

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Phe-AA during crosslinking. The used crosslinker HDMI reacts with primary amino groups to form urea linkages, leading to a decrease in the amino group content. Therefore, the formation of the Phe-PR slide-ring gels using HDMI as the crosslinker results in a lower degree of swelling than has been reported for other ionic slide-ring gels [13] (note that the first reported slide-ring gels [24] were also ionic gels because cyanuric chloride was employed as the crosslinker, and

TE D

cyanuric acid moieties were generated after crosslinking via hydrolysis under alkaline conditions [32]). The swelling of slide-ring gels prepared from nonionic PRs and a nonionic crosslinker (divinyl sulfone) was <10 [33]. In the present study, when [HDMI]/[−NH2] = 0.5, complete

EP

consumption of the primary amino groups should theoretically occur. However, the present

AC C

crosslinking reaction is not complete because some of the isocyanate groups in HDMI do not react with the amino groups, which therefore remain intact. The unreacted isocyanate groups are also converted to primary amino groups via hydrolysis. Thus, the amino group content decreases only when two amino groups are connected via HDMI. The presence of the hydrophobic aromatic moieties in Phe-AA possibly contributes to the lower Q values of the present slide-ring gels. These hydrophobic groups may associate in an aqueous environment, leading to shrinkage

28

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of the gels. For further detailed interpretation of the Q values, precise determination of the crosslinking points and the free amino group content will be required.

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3.8. Change in the swelling ratio with ionic strength.

Table 7 indicates the change in the swelling ratio Q for gel E with an increase in the ionic strength of the system. An increase in ionic strength from 0 to 0.5 induced shrinkage of the gel,

SC

and the Q value decreased from 14.4 to 1.7. This result clearly indicates the ionic nature of the gel. A remarkably steep decrease in Q was also observed when the ionic strength was increased

M AN U

from 0.01 and 0.1. Similar behavior has been observed for sulfoethylated slide-ring gels [13] and slide-ring gels with pendant quaternary ammonium groups (Takamizawa, Y.; Araki, J., unpublished data).

0

Q

14.4

3.9.

EP

Ionic strength

TE D

Table 7. Swelling ratios of gel E at different ionic strengths (pH 5.17–5.40). 0.01

0.1

0.5

11.5

3.68

1.70

Change in the swelling ratio with pH.

The swelling ratio of the AA-PR gels was

AC C

also expected to change as the pH of the surrounding medium changed because the pKa value for the amino groups in the pendant AA moieties was found to be 6.3, above and below which the Q value changed dramatically. Table 8 lists the Q values for gel H at various pH (3.86, 4.61, 8.85, and 10.2), which were conditioned by the use of various aqueous buffers with an identical ionic strength of 0.1. The highest Q value of ca. 12 was obtained at pH = 4.61, while the lowest Q values were all near 2 and were observed at all the other pH values. Because this Phe-PR slide-

29

ACCEPTED MANUSCRIPT

ring gel is a cationic gel with pendant amino groups that have a pKa = 6.3, the Q values were expected to be higher and lower below and above the pKa value, respectively. Most Q values

which the Q value was unexpectedly low.

Table 8. Q values for gel H at different pH (ionic strength = 0.1). 4.61

Q

2.10

12.1

8.54

SC

3.86

2.50

10.2

2.10

M AN U

pH

RI PT

included in Table 8 reasonably follow this prediction, with only one exception at pH = 3.86, for

The low Q value at pH = 3.86 implied the undesirable hydrolysis of the ester linkages between Phe and PR under relatively acidic conditions. Therefore, the hydrolytic degradation behavior of one of the Phe-PR compounds (Boc-Phe300-PR35k_80C_03) was investigated at several pH

TE D

values using GPC. The Phe-PR was immersed in various buffers at room temperature for 24 h and then analyzed via GPC, and the results are shown in Figure 9. A large peak at 12.1 min can be clearly seen for the pH = 3.86 solution ((Figure 9(a)). This peak corresponds to a substance

EP

with a molecular weight of 460, viz., cleaved Phe. The chromatograph of the sample treated at pH = 4.61 (Figure 9(b)) also exhibited a peak at 11.2 min, which corresponds to a substance with

AC C

a molecular weight of ca. 2010. The result should be carefully examined; its molecular weight value, which corresponds to AA-CD adducts, indicates that the dominant reason for generating this peak is not a cleavage between CDs and AAs but some dissociation (unthreading) of AA-CD adducts from the conjugates. Absence of a peak corresponding to free Phe (12.1 min, as stated above) also supports the hypothesis. The dissociation of the AA-CD adducts might occur during the treatment at pH = 4.61, since the used conjugates was thoroughly purified prior to treatment

30

ACCEPTED MANUSCRIPT

at pH = 4.61, whereas no purification was performed between the treatment at the pH and subsequent GPC analysis. Although a reasonable explanation for this undesired dissociation of AA-CDs is still difficult, a previous observation have reported some unexpected decomposition

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during storage, yielding a similar dissociation of CDs [26]. Nonetheless, the results also indicate that the remaining conjugates still have sufficient AA moieties, due to an absence of AA-PR cleavage; therefore, we concluded that the conjugates treated at pH = 4.61 indicate the DD value

SC

of 0%. For the samples immersed in other buffers (Figure 9(c)–(e)), almost no elution of low molecular weight substances was observed. In addition, in all the chromatograms, no drastic

M AN U

changes in the elution of high molecular weight fractions, particularly at the peak at 8.62 min (molecular weight of 1.98 × 105), were observed, although very slight decreases in the peak intensity were detected for the for pH = 3.86 and 4.61 solutions.

Table 9 lists the degree of degradation (DD) values for Boc-Phe300-PR35k_80C_03 after

TE D

treatment at various pH, calculated using equation 8. As suggested by the chromatograms, the highest and a moderate degree of degradation were observed for the Phe-PR recovered from the pH 3.86 and 4.61 buffers, respectively. Calculating from molecular weight values of the Boc-

EP

Phe300-PR35k_80C and its parent PR, namely 1.98 × 105 and 9.30 × 104 respectively, decomposition of 56.6% of Phe moiety corresponds to a decrease in molecular weight to 1.39 ×

AC C

105, giving a difference in less than 10 seconds in the present GPC system; therefore, the shift of the peak corresponding to the conjugates was almost indiscernible, as stated above. However, a lower to nearly negligible degree of degradation was observed for the samples recovered from higher pH solutions. These results also support the data presented in Table 8. The lower Q value at pH = 3.86 can be attributed to hydrolytic loss of the grafted Phe groups, which may contribute to decrease in Q in two different ways; the one is an ionic interaction between bare, slightly

31

ACCEPTED MANUSCRIPT

anionic PRs and intact, cationic AA-PRs, and the other is a shrinkage of the network by severe associations of partial, water-insoluble PR-like moieties without AAs, formed between crosslinked points. On the other hands, the low Q values decreased at higher pH due to

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deprotonation of the primary amino groups in the Phe-PR slide-ring gel. Although some PheCDs were dissociated from the slide-ring gel at pH = 4.61, the repulsive effect of the remaining protonated amino groups overcame the effect of the decrease in the primary amino group

SC

content.

temperature. pH

3.86 4.61 8.54 10.2

DD, % 56.5 0 a

0

0

M AN U

Table 9. DD values for Boc-Phe300-PR35k_80C after treatment at various pH for 24 h at room

TE D

a Since the corresponding chromatogram (Figure 9(b)) indicates a considerable peak at 11.2 min, it corresponds to a slight dissociation of CDs grafted with AAs, and do not suggest the

AC C

EP

cleavage between CDs and AAs (see the text in detail).

32

AC C

EP

TE D

M AN U

SC

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ACCEPTED MANUSCRIPT

Figure 9. Chromatograms of Boc-Phe300-PR35k_80°C after treatment for 24 h at room temperature at (a) pH 3.86, (b) pH 4.61, (c) pH 8.54, (d) pH 10.2, and (e) untreated.

4. CONCLUSION

33

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Two new types of AA-PR conjugates, i.e., Boc-Ala-PR and Boc-Phe-PR, with DS values in the range of 6%−60% were obtained by varying the reaction conditions, including the reaction time and temperature and the [AA]/[−OH] stoichiometric ratio of the PRs. Deprotection was

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readily performed using neat TFA treatment. The DS values obtained using different methods, including 1H NMR spectroscopy, titration, and UV absorption analysis (only in the case of the Phe conjugates), were in good agreement. The conjugates exhibited good solubility in a wide

SC

variety of solvents, and some Phe-PR conjugates showed curious temperature- and solventdependent solubilities; i.e., UCSTs and LCSTs were observed in methanol and ethanol,

M AN U

respectively. Furthermore, the swelling behavior of the AA-PR slide-ring gels prepared via crosslinking of the conjugates was dependent on the ionic strength and pH of the system. The swelling maximum of the gels was reached at pH = 4.61, above and below which shrinkage due to deprotonation of the primary amino groups and hydrolytic dissociation of the pendant AAs

TE D

were observed, respectively. The present results, along with the observations made in previous studies [19,20], provide fundamental knowledge regarding the synthesis of various AA-PR conjugates and also reveal unique behaviors of these materials, such as switching of the UCST

EP

and LCST as a function of the solvent. Further investigation of the hydrolytic degradation at low pH is needed to further confirm the good swelling properties of AA-PR gels under various

AC C

conditions.

Supplementary data.

Supplemaentary data includes (i) Determination of the numbers of

included CDs (N), the inclusion ratio (R), and the hydroxyl content (HC) using 1H NMR spectroscopy, (ii) Mw, Mw/Mn, N, R, and HC values for the starting PRs, (iii) recipes for the aqueous buffer solutions used, (iv) conductimetric and pH titration curves for Dep-Boc-

34

ACCEPTED MANUSCRIPT

Phe200-PR_1W, and (v) DSNMR and solubilities in water and organic solvents for the Boc-Phe-

AUTHOR INFORMATION

ACKNOWLEDGMENTS

SC

*E-mail: [email protected] (J. A.).

M AN U

Corresponding Author.

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PRs, Ala-PRs, and Phe-PRs. These data are available online free of charge.

This research was partially supported by Program for Dissemination of Tenure-Track System funded by the Ministry of Education and Science, Japan. The authors are grateful to Professor Kousaku Ohkawa (Shinshu University) for the use of the UV–Vis spectrometer and Professor

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Mutsumi Kimura (Shinshu University) for the use of the ATR-FTIR spectrometer. The authors also thank Prof. Kohzo Ito and Dr. Kouichi Mayumi of the University of Tokyo for their

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REFERENCES

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allowance of the use of the master thesis of their graduate.

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[5] Loethen, S.; Kim, J.-M.; Thompson, D. H. Polym. Rev. 2007, 47, 383–418. [6] Harada, A.; Li, J.; Kamachi, M. Nature 1992, 356, 325–327.

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[9] Araki, J.; Ito, K. J. Polym. Sci. A Polym. Chem. 2006, 44, 532–538.

[10] Samitsu, S.; Araki, J.; Kataoka, K.; Ito, K. J. Polym. Sci. B Polym. Phys. 2006, 44, 1985–

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[11] Araki, J.; Kataoka, T.; Ito, K. J. Appl. Polym. Sci. 2007, 105, 2265–2270. [12] Araki, J.; Ito, K. J. Polym. Sci. A Polym. Chem. 2006, 44, 6312–6323.

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[13] Araki, J. J. Polym. Sci. A Polym. Chem. 2011, 49, 2199–2209. [14] Araki, J.; Kataoka, T.; Ito, K. Soft Matter 2008, 4, 245–249.

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[16] Araki, J.; Sato, H.; Takagi, Y.; Ohta, H. Mol. Cryst. Liq. Cryst. 2014, 592, 99–105. [17] Kidowaki, M.; Nakajima, T.; Araki, J.; Inomata, A.; Ishibashi, H.; Ito, K. Macromolecules 2007, 40, 6859–6862.

[18] Inomata, A.; Kidowaki, M.; Sakai, Y.; Yokoyama, H.; Ito, K. Soft Matter 2011, 7, 922–928. [19] Araki, J.; Kagaya, K.; Ohkawa, K. Biomacromolecules 2009, 10, 1947–1954.

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[20] Araki, J.; Kagaya, K. Polym. J. 2013, 45, 1081–1086. [21] Yui, N.; Ooya, T.; Kawashima, T.; Saito, Y.; Tamai, I.; Sai, Y.; Tsuji, A. Bioconjugate

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Chem. 2002, 13, 582–587 [22] Mori, H.; Endo, T. Macromol. Rapid Commun. 2012, 33, 1090−1107.

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[25] Araki, J.; Zhao, C.; Ito, K. Macromolecules 2005, 38, 7524–7527.

[26] Araki, J. J. Polym. Sci. A Polym. Chem. 2010, 48, 5258–5264 (Erratum. J. Polym. Sci. A Polym. Chem. 2011, 49, 1298).

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[27] Li, J.; Revol, J-F.; Marchessault, H. R.; J. Colloid. Interface Sci., 1996, 183, 365–373. [28] Isogai, A. Cellulose 1997, 4, 99–107

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1990, 29, 554–560.

[31] Anbergen, U.; Oppermann, W. Polymer 1990, 31, 1854–1858. [32] Titration of the freeze-dried slide-ring gel (prepared by crosslinking with cyanuric chloride according to the method in ref. 24) using an aqueous 0.01 M NaOH solution yielded a value of 6.85 × 10-4 mol/g dry gel, as a weak acid group content. Details are described in: Kasahara, R. Master thesis, the university of Tokyo, 2005 (in Japanese).

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[33] Fleury, G.; Schlatter, G.; Brochon, C.; Hadziioannou, G. Polymer 2005, 46, 8494–8501.

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Table of Contents graphic

Highlights

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Amino acid-polyrotaxane (AA-PR) conjugates were prepared using Ala and Phe. DS values obtained by NMR, titration and UV analysis were in satisfactory agreements.

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One of the conjugate indicated UCST in methanol and LCST in ethanol.

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AA-PR slide-ring gels indicated the largest swelling at pH = 4.61. Lower and Higher pH caused cleavage of AA and gel shrinkage by deprotonation.

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Supporting Information for “Amino acid-derivatized slide-ring gels: chemical crosslinking of polyrotaxane conjugates with different amino acid pendant groups”

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Jun Araki* (Corresponding author) Faculty of Textile Science and Technology, Shinshu University, Tokida 3-15-1, Ueda 386-8567, Japan, and Division of Biological and Medical Fibers, Institute for Fiber Engineering (IFES), Interdisciplinary Cluster for Cutting Edge Research (ICCER), Shinshu University, Tokida 3-151, Ueda, Nagano prefecture, 386-8567, Japan.

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Naoki Sainou Graduate School of Science and Technology, Shinshu University, Tokida 3-15-1, Ueda 3868567, Japan.

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.  .  

(1)

.  .  .  . 

 % 
⁄325

'' ( &

(3)

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 mmol/g 

(2) % & %'

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Determination of numbers of included CDs N, inclusion ratio R and hydroxyl content HC by 1H NMR. About 10 mg of the conjugate was mixed with 20% D2SO4/D2O, followed by heating at 90 °C for 45–90 min (until a transparent solution was obtained). With this treatment, the conjugate was decomposed into a mixture of glucose, dissociated AAs, PEG35000 and cleaved adamantine (see the following Scheme 1). The mixture was directly subjected to 1H NMR measurement, using a drop of acetic acid as an internal standard (δ = 2.04 ppm). Values of N, R and HC were calculated according to the following equations;

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where An is an integrated area of the signals around n ppm (for example, A3.3–4.1 means an integrated areas of the signals observed between 3.3–4.1 ppm).

Hydrolysis of AA-PR derivatives in 20% D2SO4/D2O

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Scheme S1.

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Table S1. Weight average molecular weight Mw, polydispersity index Mw/Mn, numbers of included CDs N, inclusion ratio R and hydroxyl content HC of the starting PRs. Mw/Mn a Nb R, % b HC, mmol/g b Starting PR Mw a 5 1 1.7 88.3 27.2 13.89 1.07 × 10 4 2 1.7 87.8 27.0 13.87 9.90 × 10 4 3 1.8 87.4 26.9 13.85 9.30 × 10 4 1.5 90.1 27.7 13.96 1.23 × 105 a Determined by GPC. b Determined by 1H NMR.

Preparation of aqueous buffer solutions. Five types of aqueous buffers with different pH values and an identical ionic strength of 0.1 were prepared as in the following table.

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Table S2. Recipes of various aqueous buffers with different pH values and identical ionic strength of 0.1 pH Types and cencentrations of Recipes values buffers 3.68 0.02 M citric buffer Mixing of 100 mL of 1 M NaOH, 100 mL of citric acid solutions and 800 mL of water, followed by dilution with water to five volumes 4.61 0.1 M acetate buffer Dissolution of 41.0 g of CH3COONa and 30.0 g of CH3COOH in water to yield 1 L solution, followed by dilution with water to five volumes 8.54 0.1 M ammonium buffer Mixing of 90 mL of 0.5 M aqueous ammonia and 10 (0.02 M NH3/0.18 M NH4Cl) mL of 0.5 M of aqueous NH4Cl solution 9.99 0.1 M sodium carbonate Mixing of 100 mL of 1 M aqueous NaHCO3, 100 mL buffer of 1 M aqueous Na2CO3 and 800 mL of water 10.20 0.025 M sodium carbonate Dilution of the above 0.1 M sodium carbonate buffer buffer with water to four volumes

Figure S1. Typical conductimetric and pH titration curves of Dep-Boc-Phe200-PR_1W.

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DSNMR of various Boc-Phe-PRs and their solubility in water and general organic

Samples Boc-Phe50-PR35k Boc-Phe100-PR35k Boc-Phe200-PR35k Boc-Phe300-PR35k Boc-Phe100-PR35k_40C Boc-Phe100-PR35k_80C Boc-Phe100-PR35k_12h Boc-Phe100-PR35k_48h a

DSNMR, % 5.6 14.0 34.2 37.5 4.2 21.4 9.0 20.5

H2O

MeOH

EtOH

Acetone

DMSO

DMAc

DMF

THF

DCM

Toluene

i i i i i i i i

i s s s i s i i

i i s s i i i i

i i s s i i i i

s s s s s s s s

s s s s i s s s

s s s s i s s s

i s s s i i i s

i i s s i i i i

i i i s i i i i

s; soluble, i; insoluble.

Ala50-PR35k Ala100-PR35k Ala200-PR35k Ala300-PR35k Ala100-PR35k_40C Ala100-PR35k_80C Ala100-PR35k_12h Ala100-PR35k_48h a

DSNMR, % 7.5 14.0 33.2 41.1 6.8 14.5 12.0 14.5

H2O

MeOH

EtOH

Acetone

DMSO

DMAc

DMF

THF

DCM

Toluene

s s s s i s s s

s s s s i i s s

i i s s i i i s

i i i i i i i i

s s s s s s s s

s s s s s s s s

s s s s s s s s

i i i i i i i i

i i i i i i i i

i i i i i i i i

s; soluble, i; insoluble.

Phe50-PR35k Phe100-PR35k Phe200-PR35k Phe300-PR35k Phe100-PR35k_40C Phe100-PR35k_80C Phe100-PR35k_12h Phe100-PR35k_48h a

DSNMR, % 6.5 15.8 30.7 41.8 3.8 20.3 8.0 16.0

H2O s s s s i s s s

MeOH

EtOH

Acetone

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Samples

DSNMR of various Phe-PRs and their solubility in water and general organic

s s s s i s s s

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Table S5. solvents.a

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Samples

DSNMR of various Ala-PRs and their solubility in water and general organic

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Table S4. solvents.a

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Table S3. solvents.a

i s s s i s i s

i i i s i i i i

DMSO

DMAc

DMF

THF

DCM

Toluene

s s s s s s s s

s s s s s s s s

s s s s s s s s

i i i s i i i i

i i i i i i i i

i i i i i i i i

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s; soluble, i; insoluble.

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