Aspartic Acid-based Ampholytic Amphiphiles: Synthesis

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Aspartic Acid-based Ampholytic Amphiphiles: Synthesis, Characterization, and pH-Dependent Properties at Air/Water and Oil/Water Interface Weiwei Cheng, Sampson Anankanbil, Bianca Perez, Jacob Nedergaard Pedersen, Guoqin Liu, and Zheng Guo J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05122 • Publication Date (Web): 05 Feb 2019 Downloaded from http://pubs.acs.org on February 8, 2019

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Journal of Agricultural and Food Chemistry

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Aspartic Acid-based Ampholytic Amphiphiles: Synthesis,

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Characterization, and pH-Dependent Properties at Air/Water and

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Oil/Water Interface

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Weiwei Cheng, †, ‡ Sampson Anankanbil, ‡ Bianca Pérez, ‡ Jacob Nedergaard Pedersen, ‡ Guoqin Liu,*,† and

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Zheng Guo*,‡

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†School

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‡Department

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8000 Aarhus C, Denmark

of Food Science and Engineering, South China University of Technology, Guangzhou 510640, China of Engineering, Faculty of Science and Technology, Aarhus University, Gustav Wieds vej 10,

10 11

*Corresponding

author: Tel: +45 87155528, [email protected]; [email protected] (Zheng Guo).

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ACS Paragon Plus Environment


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ABSTRACT: A facile and two-step strategy was employed to synthesize a series of novel aspartic

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acid-based ampholytic amphiphiles from sustainable and commercially viable substances as starting

3

materials. The molecular structures of the synthetic compounds were well identified by MS and

4

1H/13C

5

evaluated by the use of multiple techniques such as FTIR, DSC, Langmuir−Blodgett study, and

6

fluorescence microscopy imaging. Due to the coexistence of amino and carboxyl groups in the

7

synthetic compounds, the compounds presented varying charges (cationic, ampholytic, and anionic)

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depending on the pH of the medium compared to the dissociation constants (pKa). Compounds with

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cationic (pH 1.0) and anionic (pH 9.0) forms had significantly higher γ0.1 and CMC values than that

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with ampholytic forms (pH 7.0). sn-1-Lauroyl-sn-3-aspartic acid (compound 3) at neutral and alkaline

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conditions displayed comparable foaming properties including foaming, calcium-tolerance, and

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temperature-resistance ability with commercial sulfonate SDS, and thus might be a promising

13

alternative to SDS, applied in personal care products and detergent formula. sn-1-Palmtoyl-sn-3-

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aspartic acid (Compound 5a) with ampholytic structure was proved as the most excellent stabilizer

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for the preparation of oil-in-water emulsions compared with palmityl aspartic acid (compound 5b),

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commercial food ingredient DATEM, and glyceride monopalmitate at aqueous phase pH 7.0. Thus,

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it has promising use as a pH-dependence emulsifying agent in various fields.

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KEYWORDS: Aspartic acid, Monoglyceride, Ampholytic amphiphiles, pH-Dependent, Foam, Oil-

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in-water emulsion

analysis, and the physicochemical, pH-dependent foaming and emulsifying properties were

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2


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INTRODUCTION

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Amino acid-based amphiphiles (AAAs) are biocompatible and biodegradable surfactants and thus

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have been emerging as the green and sustainable alternatives to petrochemical-based amphiphiles

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which account for the large majority in the present market.1-3 They are characterized by the natural

5

amino acids (as polar head) attached to the fatty acids or the derivatives of oleochemical sources.4-6

6

According to the connection site of the hydrophobic tail and the charge of the amino acid side-chain,

7

AAAs are classified as anionic, cationic, or ampholytic.7 AAAs with different charge types are

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responsible for the various properties, for instance, cationic AAAs possess antimicrobial and

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hemolytic activities due to their electrostatic interactions with the negatively charged molecules of

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the bacterial membranes,8-10 and lysine- and arginine-based cationic amphiphiles have been well

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synthesized via the attachment of fatty acids by amide bonds;11-13 whereas anionic AAAs with low

12

toxicity and biodegradability are used in cosmetics and personal care formulations.14 and anionic

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glutamic acid-based amphiphiles have also been synthesized by aminoacylation reaction.15,

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However, the synthesis and property evaluation of ampholytic AAAs are seldom reported. Previously,

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Obata et al. synthesized a kind of glutamic acid-based ampholytic lipids which were used in

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liposomes to construct an efficient drug delivery system and showed comparable blood persistence

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to conventional phospholipid-based liposomes due to their pH-responsive properties.5 Furthermore,

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commercial betaine-based ampholytic amphiphiles have been applied as foam booster in personal

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care products due to their excellent foaming properties.17 Therefore, ampholytic AAAs characterized

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by the coexistence of amino and carboxyl groups are potentially needed to expand the molecular

21

library of ampholytic surfactants for the multi-faceted applications in cosmetic, food, detergent, and

22

pharmaceutical industries.

23

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To obtain the ampholytic AAAs, the acidic (Asp and Glu) and basic (Lys) amino acids with the 3



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two carboxyl and amino groups, respectively, one of which links with the hydrophobic chain, have

2

to be selected as the polar head donator. In the present work, we attempted to develop a facile method

3

for the synthesis of novel aspartic acid-based ampholytic amphiphiles; thus to introduce and explore

4

a new class of non-toxic surface active compounds which can be incorporated in various food

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nanoemulsion formulations. However, due to the higher reactivity of the amino group towards the

6

carboxyl group than that of the hydroxyl group, and the strong polarity of free aspartic acid, it is

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highly difficult to find a reaction system to obtain the target compounds with fewer side products as

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possible. Accordingly, we selected sustainable and commercially viable materials, Carboxybenzyl

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(Cbz)-N-aspartic anhydride (ZAA) as polar head donator, which not only protected the amino group

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from the formation of side products but also decreased the polarity of aspartic acid to easily find an

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appropriate reaction solvent. Most importantly, it is well known that organic anhydride is the

12

acylating reagent reacting with hydroxyl group based on a Sn2 reaction mechanism.18-20 The reaction

13

pathway between ZAA and 1-monoglycerides (MAGs, C8‒C18) for the synthesis of target

14

compounds are shown in Figure 1. MS and 1H/13C NMR were used to identify the molecular

15

structures of the synthetic compounds. The physicochemical, pH-dependence foaming, and

16

emulsifying properties were characterized by using multiple technologies including FTIR, DSC,

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Langmuir−Blodgett, and fluorescence microscopy imaging. Structure-property relationship of the

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synthetic compounds depending on medium pH (1.0, 5.0, 7.0, and 9.0) were investigated for their

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potentially specified application in various fields such as food, laundry, pharmaceutical, and cosmetic.

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MATERIALS AND METHODS

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Materials. Carboxybenzyl (Cbz)-N-aspartic anhydride (ZAA), Sodium dodecyl sulfate (SDS),

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Palladium on activated charcoal (10%, Pd/C), pyridine (anhydrous, >99%), 2-methyltetrahydrofuran

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(2-MTHF, anhydrous, ≥99%), Nile red, and all other chemicals or solvents used in this work were 4


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purchased from Sigma-Aldrich (St. Louis, USA). Diacetyltartaric acid esters of mono- and di-

2

glycerides (DATEM) was donated by Dupont Nutrition and Health (Brabrand, Denmark). Fish oil

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from cod liver was friendly provided by in Omega3, Denmark and manufactured in Norway.

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Novozym 435 (Candida Antarctica lipase B) was supplied by Novozymes A/S (Bagsvaerd,

5

Denmark). MAGs (C8‒C18) were prepared by the general enzymatic method in our lab.20 All used

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water with 18.2 mΩ was from a Milli-Q system (Millipore corp., MA).

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General synthesis process of aspartic acid-based amphiphiles. 1 mole equivalent of ZAA and

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1.5 mole equivalent of MAGs were weighed into 25-mL screw-top flasks with 5 mL acetonitrile

9

(ACN) as reaction solvent and then incubated in an oil bath at the temperature range of 50‒80 °C.

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Samples were taken out at a regular interval of 1 h and then evaporated by rotatory evaporation at

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reduced pressure. 5 mL of anhydrous ethanol and 10% (mol/mol) Pd/C were subsequently added into

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the reaction flask, followed by the dropwise slow addition of triethylsilane. After reacting at room

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temperature for 2 h, the mixture was suction filtered to remove the Pd/C and 20 μL aliquots were

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transferred into a 1 mL centrifuge tube, followed by diluting with 200 µL CHCl3/CH3OH (3:1, v/v).

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Subsequently, 1 µL of diluted samples were spotted on TLC plate (TLC silica gel 60, 5 × 10 cm,

16

Merk, Darmstadt, Germany) to monitor the reaction process. The plates were developed in

17

chloroform: methanol: acetic acid (18:4:1.5, v/v/v).

18

Purification of target compounds. The target compounds were purified by silica gel column

19

chromatography eluted with the same solvent system as TLC. In this work, the yields of the target

20

compounds were determined by Iatroscan Mark VI Thin Layer Chromatography with flame

21

ionization detector (TLC-FID, Iatroscan MK-6s, Japan).21 Briefly, a series of different concentrations

22

of the purified products (0.05‒5.0 mg/mL) were prepared for the external calibration curve. Exactly

23

1.0 μL of standard or diluted sample was spotted on silica gel-coated chromarods (Chromarod-S III, 5



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Japan) and developed by the developing solvent as shown above. The concentration of target

2

compound in the reaction mixture was obtained based on the respective calibration curve, and finally

3

their yields were calculated by the percentage of experimental value obtained above against the

4

theoretical value from stoichiometric calculation.

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Structural identification of target compounds. The molecular structures of synthetic compounds

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were identified by electrospray ionization quadrupole time-of-flight mass spectrometer (ESI-TOF-

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MS, Bruker Daltonic GmbH, Bremen, Germany) and 1H/13C NMR spectral analysis (Bruker Avance

8

III spectrometer, Bruker Biospin, Billerica, MA) at 400 MHz. The NMR spectra are presented in the

9

supporting information.

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2-Amino-succinic acid 4-(2-hydroxy-3-octanoyloxy-propyl) ester (compound 1). While solid; yield

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53%; 1H NMR (400 MHz, CDCl3: MeOD 3:1 v/v, 25 °C, TMS): δ = 4.21 (s), 4.16 (s), 3.91 (s), 2.88

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(s), 2.43 (s), 2.07 (s), 1.70 (s), 1.56 (d), 1.49‒1.29 (m), 0.96 (s). 13C NMR (400 MHz, CDCl3: MeOD

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3:1) δ = 180.03, 73.78, 69.00, 66.98, 53.12, 52.01, 38.03, 35.48, 32.75, 28.71, 26.43, 25.47, 17.74.

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HRMS: m/z calculated for C15H27NO7: 333.381; found: 334.1829 [M+H+].

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2-Amino-succinic acid 4-(-2-hydroxy-3-decanoyloxy-propyl) ester (compound 2). While solid;

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yield 59%; 1H NMR (400 MHz, CDCl3: MeOD 3:1, 25 °C, TMS): δ = 8.16 (s), 4.27 (d), 3.96 (s),

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3.88 (s), 3.07 (d), 2.82 (s), 2.43 (t), 2.04 (s), 1.70 (s), 1.35 (s), 1.15‒0.80 (m). 13C NMR (400 MHz,

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CDCl3: MeOD 3:1) δ = 182.77, 178.08, 73.93, 69.00, 68.58, 66.90, 53.04, 52.40, 52.19, 38.00, 35.70,

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33.11, 28.70, 27.01, 26.48, 17.80, 16.41. HRMS: m/z calculated for C17H31NO7: 361.435; found:

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362.1834 [M+H+].

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2-Amino-succinic acid 4-(-2-hydroxy-3-dodecanoyloxy-propyl) ester (compound 3). While solid;

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yield 71%; 1H NMR (400 MHz, CDCl3: MeOD 3:1, 25 °C, TMS): δ = 4.28 (d), 3.88 (s), 3.43 (s),

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3.11 (s), 2.80 (s), 2.43 (s), 2.19‒1.99 (m), 1.72 (s), 1.39 (d), 0.95 (d).13C NMR (400 MHz, CDCl3: 6


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MeOD 3:1) δ = 182.02, 178.09,175.48, 71.37, 69.93, 68.47, 53.01, 52.37, 52.16, 38.97, 37.92, 35.75,

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33.45, 33.00, 28.70, 26.50, 17.79. HRMS: m/z calculated for C19H35NO7: 389.489; found: 390.2140

3

[M+H+].

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2-Amino-succinic acid 4-(-2-hydroxy-3-tetradecanoyloxy-propyl) ester (compound 4). While solid;

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yield 63%; 1H NMR (400 MHz, CDCl3: MeOD 3:1, 25 °C, TMS): δ = 4.24 (m), 3.89 (s), 3.47 (d),

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3.12 (s), 2.89 (s), 2.42 (t), 2.05 (s), 1.70 (s), 1.36 (d), 0.96 (t).13C NMR (400 MHz, CDCl3: MeOD

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3:1) δ = 181.49, 178.38,178.04, 175.65, 73.71, 71.01, 69.98, 68.53, 66.91, 52.33, 52.12, 37.91, 35.77,

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33.49, 33.20, 28.73, 26.26, 17.81. HRMS: m/z calculated for C21H39NO7: 417.543; found: 418.2412

9

[M+H+].

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2-Amino-succinic acid 4-(-2-hydroxy-3-hexadecanoyloxy-propyl) ester (compound 5a). While

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solid; yield 50%; 1H NMR (400 MHz, CDCl3: MeOD 3:1, 25 °C, TMS): δ = 4.21 (s), 3.91 (s), 3.43

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(s), 3.04 (d), 2.43 (s), 2.43 (s), 2.10 (s), 1.69 (s), 1.34 (s), 0.95 (s).13C NMR (400 MHz, CDCl3: MeOD

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3:1) δ = 179.85, 178.43,73.75, 69.10, 66.98, 52.95, 52.73, 51.88, 37.95, 35.75, 33.17, 32.98, 28.71,

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26.51, 25.34, 17.73. HRMS: m/z calculated for C23H43NO7: 445.597; found: 446.2673 [M+H+].

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2-Amino-succinic acid 4-hexadecyl ester (compound 5b). White solid; yield 41%; 1H NMR (400

16

MHz, CDCl3: MeOD 3:1, 25 °C, TMS): δ = 4.20 (s), 3.94 (s), 3.42 (s), 2.42 (s), 2.08 (s), 1.70 (s),

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1.34 (s), 0.96 (s). 13C NMR (400 MHz, CDCl3: MeOD 3:1) δ = 176.10, 174.70, 70.02, 65.37, 63.25,

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49.00, 34.26, 32.02, 29.43, 24.98, 22.75, 14.00. HRMS: m/z calculated for C20H39NO4: 357.535;

19

found: 358.2930 [M+H+].

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2-Amino-succinic acid 4-(2-hydroxy-3-octadecanoyloxy-propyl) ester (compound 6). While solid;

21

yield 44%; 1H NMR (400 MHz, CDCl3: MeOD 3:1, 25 °C, TMS): δ = 4.22 (s), 4.01 (s), 3.88 (s), 3.16

22

(s), 2.93 (s), 2.41 (t), 2.06 (s), 1.88 (s), 1.67 (s), 1.51 (s), 1.33 (s), 0.96 (t).13C NMR (400 MHz, CDCl3:

23

MeOD 3:1) δ = 180.78, 177.20,174.01, 73.23, 52.66, 47.32, 35.13, 32.90, 31.51, 28.10, 25.89, 25.33, 7



1

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17.28, 14.84. HRMS: m/z calculated for C25H47NO7: 473.651; found: 474.2947 [M+H+].

2

pKa. The extent of proton dissociation for the synthetic compounds at a given pH can be estimated

3

by their apparent pKa values, which was determined by potentiometric pH titration as described by

4

Mezei et al22 with minor modifications. Briefly, 25 mL of 0.5 mg/mL compound solutions in

5

duplicate were titrated stepwise by 0.1 M HCl and NaOH solution, respectively, at room temperature.

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The pH value was measured with a WTW Inolab pH-meter (Weilheim, Germany) and then plotted

7

against the volumes of HCl and NaOH addition. pKa refers to the pH value of the corresponding

8

adjacent semi equivalence.

9

Packing behavior. The molecular packing behaviors of the synthetic amphiphiles were analyzed

10

by temperature-ramp fourier transform infrared spectroscopy (FTIR) based on the previous method

11

developed in our lab.23 In brief, about 5 mg of samples dried under vacuum overnight were pressed

12

onto a temperature-controlled ZnSe crystal of ATR accessory (Pike Technologies, Madison, WI) in

13

a horizontal Bruker FTIR spectrometer (Ettlingen, Germany). Spectra in the range of 4000‒400 cm-1

14

were acquired at intervals of 2 °C from 40 to 100 °C and collected with an unpolarized electron beam

15

at a 4 cm-1 resolution and 8 scans per sample. The FTIR spectra were analyzed using Origin 9.3

16

software (OriginLab Corp. Northampton, MA).

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Melting point (Tm). The melting points of the synthetic compounds were determined by using

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differential scanning calorimetry (DSC) on a Pyris 6 system (Perkin-Elmer Cetus, Norwalk, CT).

19

8‒12 mg of each compound was weighed into an aluminum pan and hermetically sealed by using a

20

crimping device. Subsequently, the measurement was carried out under 20 mL/min of nitrogen gas

21

flow scanned from -20 °C to 120 °C at a heating rate of 5 °C/min with an empty pan as the reference.

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The melting point (Tm) was obtained from the fitting of thermograms by using MicroCal Origin 9.3

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software (Microcal Software Inc., Northampton, USA). 8


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Foaming ability and foam stability. Foaming ability and foam stability were determined by the

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methods of Nandi et al.24 and Altenbach et al.25 with some modifications. Briefly, approximately 20

3

mg aliquot of each compound was weighed into 50 mL graduated centrifuge tubes containing 20 mL

4

of water at the final concentration of 1.0 mg/mL. Then the solution was manually beaten by vigorous

5

shaking the tube for 1 min. The volume of formed foam was recorded after standing for 30 s and 5

6

min, which were referred to as foaming ability and foam stability, respectively. Additionally, effects

7

of pH (1, 5, 7, and 9), temperature (25, 30, 35, and 40 °C), and Ca2+ addition (100, 200, 300, and 400

8

ppm) in water medium on the foaming ability and foam stability of synthetic compound were also

9

investigated in the present study. All measurements were performed in duplicate.

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Surface tension (γ). The determination of γ value in aqueous solution was conducted at room

11

temperature by using Langmuir‒Blodgett Microthrough X (Kibron Inc., Helsinki, Finland) equipped

12

with a surface pressure sensor and a platinum rod probe. The sample solutions were prepared at a

13

final concentration of 0.1 mM in buffers (pH=1.0, 5.0, 7.0, and 9.0) or water with different

14

concentrations of Ca2+ (100‒400 ppm). All freshly prepared solutions were gradually transferred into

15

the 96-multiwell plate until liquid barely overflowed in every well. Before measurement, the surface

16

pressure was first cleared zero while the probe was in the air. Subsequently, the probe was lowered

17

just to touch the surface of the liquid at room temperature. The γ value was collected by using

18

FilmWare version 3.62 software. All measurements were performed in triplicate.

19

Critical micelle concentration (CMC). The CMC values of the compounds were detected at room

20

temperature by γ measurement of various concentrations (0.01‒5.0 mg/mL) of aqueous compound

21

solutions in water or buffer (pH=1.0, 5.0, 7.0, and 9.0). The γ values were determined by the method

22

as depicted above and eventually plotted against the concentrations of compound solutions, where

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the concentration of the first break point was commonly referred to as the CMC value of 9



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corresponding compound.

2

Preparation of oil-in-water emulsion. Oil-in-water emulsions were formulated to evaluate the

3

oil/water interfacial properties of the synthetized compounds with commercial DATEM as a control

4

as described in the previous reports from our lab.26, 27 In brief, the aqueous phase containing 2.0%

5

(w/w) of synthetic compounds were first prepared with a pH adjustment to 1.0, 5.0, 7.0, and 9.0,

6

respectively by using 0.1 M NaOH or HCl solution. Subsequently, 2 mL of fish oil was mixed with

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8 mL of aqueous phase prepared above, followed by magnetic stirring for 15 min. The biphasic system

8

was sonicated on ice at 70% power for 3 min to form O/W emulsions.

9

Particle size and zeta potential. The measurements of oil droplet size and zeta potential in freshly

10

prepared emulsions were conducted by photon correlation spectroscopy using a Zetasizer Nano ZS

11

instrument (Malvern Instruments Ltd., Worcestershire, UK) at room temperature. The emulsions

12

stored at 5 °C for 0‒30 days were diluted one hundred times in the corresponding pH buffer (pH=1.0,

13

5.0, 7.0, and 9.0) prior to analysis. Due to the rather short timespan between dilution and analysis, the

14

destabilization of the emulsions was negligible. The size distribution of oil droplets was determined

15

by dynamic light scattering (DLS) using non-invasive backscatter optics and expressed as the

16

intensity-weighted harmonic mean size (Dz). All measurements were performed in triplicate.

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Creaming index (CI). The freshly prepared emulsions were firstly transferred into a transparent

18

15 mL screw-top glass tubes (21 × 70 mm) and stored at 5 °C. The creaming stability in each emulsion

19

was monitored every 10 days until 30 days and characterized by the CI value based on the following

20

equation28: CI (%) = 100 × Hc/HE, where HC refers to the height of upper cream layer and He refers

21

to the total height of each emulsion.

22

Fluorescence microscopy imaging of emulsion droplets. Droplet distribution of freshly prepared

23

emulsions was visualized by confocal laser scanning microscope (CLSM) using a Zeiss LSM 700 10


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Meta confocal microscope (Carl Zeiss, Oberkochen, Germany) with the excitation and emission

2

wavelength at 488 and 518 nm, respectively. The location of the amphiphilic compound in O/W

3

emulsion system was visualized by using a Nikon structured illumination microscopy (N-SIM)

4

system with 100×/1.49 NA oil-immersion objective (Tokyo, Japan). 2 µL 1 mg/mL Nile red/ C11-

5

BODIPY (581/591) in acetone was vigorously mixed by vortexing with 20 µL emulsion, which was

6

thereafter dropped onto a concave microscope slide. After covered with glycerol‐coated cover slips,

7

the CLSM and N-SIM images were reconstructed by ZEN and Nis Elements software, respectively.

8

RESULTS AND DISCUSSION

9

Synthesis of Aspartic Acid-based Ampholytic Amphiphiles. To obtain the target ampholytic

10

amphiphiles based on Asp, we first took natural Asp as the starting material and used Novozym 435

11

as biocatalyst in ACN. However, due to the specifity of lipase and biphasic reaction system, the target

12

compounds were not successfully synthesized by the enzymatic method. It is well-known that Asp is

13

a highly polar compound and cannot be dissolved in most solvents. Thus, it is difficult to find a

14

suitable cosolvent to dissolve both Asp and MAGs. We tried to apply the low melting point of MAGs

15

(39‒81 °C for C8‒C18) in water medium and perform the reaction at temperatures higher than that

16

of the melting point of corresponding MAG, which has been successfully carried out in another

17

publishing reaction between free lysine and low melting point of anhydride. However, MAG is

18

amphiphilic containing a hydrophilic hydroxyl group and a hydrophobic carbon chain, leading to

19

bubble formation during reaction with magnetic stirring in water. Moreover, MAG heated in water is

20

readily hydrolyzed. Most importantly, the amino group has a higher reactivity towards the carboxylic

21

group compared to the hydroxyl group, which means that the amidation reaction between Asps takes

22

place preferentially (less reversible) and thus leads to a greatly difficult esterification reaction due to

23

a high reversibility of esterification. That is why we could not always get the desired compounds 11



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linking Asp with MAG using chemical methods with scandium(III) triflate29 or methanesulfonic

2

acid30 as catalysts. Therefore, it is imperative to first protect the amino group of Asp to prevent the

3

amidation reaction.

4

Based on our previous work in our lab, anhydrides can be used as excellent electrophiles which

5

can react with a series of nucleophiles such as MAGs, polysaccharides, sugar alcohols, and phenolic

6

acid.20, 23, 26, 31, 32 Accordingly, we were inspired to synthesize the aspartic acid-based amphiphiles

7

using N-protected aspartic anhydride as the acyl donor and MAGs/hexadecanol as nucleophiles in

8

the absence of catalyst (Figure 1). Taking into account the solubility of reactants and less toxicity, the

9

reactions were carried out in ACN at 50‒80 °C depending on the different carbon chain of MAG (50

10

°C for compounds 1‒3, 70 °C for compound 4, and 80 °C for compounds 5a, 5b, and 6). The Cbz

11

deprotection process was conducted based on a previous report.33 From Table 1, the isolation yields

12

of the target compounds ranged from 44% to 71% with an increasing trend from 8 to 12 carbon chain

13

length MAGs and a subsequently decreasing trend from 14 to 18 carbon chain length MAGs. This

14

changing trend might be attributed to the poor solubility of MAGs with long fatty acid side chains in

15

CAN, which was consistent with previous findings.20 The molecular structures of the synthetic

16

amphiphilic compounds were identified by MS, and 1H/13C NMR. The spectra are presented in the

17

Supporting Information.

18

Physicochemical Properties of Aspartic Acid-based Ampholytic Amphiphiles. As far as we

19

know, the array of ampholytic aminolipids based on Asp with varying hydrophobic carbon chains in

20

length from C8 to C18 were synthesized for the first time in the present work. Therefore, their

21

physicochemical properties, such as melting transition, molecular packing behavior, pKa, etc., are of

22

fundamental interest in the development of application in various fields. As shown in Figure 1, all

23

synthetic compounds contain the common polar head with an amino group and a carboxyl group, and 12


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the molecular size and volume increase as the carbon number of hydrophobic tail increases. The

2

synchronous presence of amino and carboxyl groups as well as the long aliphatic chain endow the

3

compounds with pH-regulatable amphiphilic properties. It was assumed that different carbon chain

4

length might lead to the differing properties of synthetic compounds, which could be assigned to

5

various applications. Furthermore, the protonation state of common polar head depending on the

6

medium pH was also a key factor to be considered when the surface and emulsion properties of the

7

synthetic compounds were analyzed.

8

DSC Characterization. To understand the thermal transitions of the synthetic amphiphilic

9

compounds, temperature scans from -20 to 120 °C were performed by DSC and Tm values are

10

presented in Table 1. As expected, the Tm value increased from 47 to 92 °C with a general order of

11

compound 1‒6, except for compound 5b. This suggested that the increase of the hydrophobic chain

12

length from C8 to C18 led to the corresponding increase of melting temperatures due to the enhancing

13

van der Waals interactions, which was in agreement with the previously published reports.31,

14

Additionally, the Tm values of the synthesized compounds were remarkably higher than the

15

corresponding MAGs. For example, compound 5a presented a higher Tm value (89 °C) than that of

16

glycerol monopalmitate (GMP, 77 °C), suggesting that the introduction of Asp resulted in the rise of

17

Tm. This can be explained by the stronger hydrogen bond interactions of the compound compared

18

with MAGs due to the presence of amino and carboxyl groups. In the case of compounds 5a and 5b

19

with the same hydrophobic tail, the Tm of compound 5a was higher than that of compound 5b (74 °C),

20

which could be attributed to the varying of hydrogen bond interactions resulting from the hydroxyl

21

bond, leading to the difference in Tm between the two compounds. Accordingly, based on the DSC

22

data, the resulting ampholytic aminolipids display better structural organization and thus superior

23

packing behavior compared to the corresponding MAGs. 13


34


Page 14 of 38

1

FTIR Characterization. To further understand the packing behavior and intermolecular

2

interactions of the synthetic amphiphiles, FTIR spectroscopy studies were performed.35 Based on the

3

previous reports,31, 34, 36 the CH2 bending bands (~725‒720 cm-1) and CH2 symmetric stretching bands

4

(~2920 and 2850 cm-1) can provide valuable information about lateral organization of alkyl chain of

5

compounds, and thus were selected as the regions of interest in the FTIR spectrum. As shown in Table

6

1, all compounds behaved in both peaks at ~2920 and ~2850 cm-1, and no significant frequency shift

7

was observed at room temperature in those two regions. The packing mode of the alkyl chains was

8

identified by the peak number in the region of ~725‒720 cm-1, where single and double peak refer to

9

the hexagonal and orthorhombic packing, respectively. From Table 1, compounds 1‒3 and 5b

10

displayed hexagonal packing, while compounds 4, 5a, and 6 showed orthorhombic packing, which is

11

the more densely packing mode due to all-trans conformation of the alkyl chains organized in a highly

12

dense rectangular crystalline lattice. Therefore, long alkyl chains (≥14) promoted tighter packing,

13

accounting for the relatively higher Tm value of compounds 4, 5a, and 6 than the rest of compounds

14

(see DSC data).

15

pKa. As depicted above, the common polar head of the synthetic compounds contains an amino

16

group and a carboxyl group (Figure 1), suggesting that they all have similar proton activity and can

17

display alkaline-acid equilibrium in aqueous solutions. The dissociation states of the synthetic

18

compounds in aqueous solutions are presented in Figure 2. The compounds bear different charges

19

(positive, ampholytic, or negative) depending on the pH of medium. It has been well known that the

20

charge type of ionic amphiphiles is responsible for some of their properties, such as self-assembly,22

21

calcium-resistance,37 foam stability,38 etc.. When the pH of the medium lies between pKa1 and pKa2,

22

the synthetic compounds belong to ampholytic amphiphiles bearing positively and negatively mixed

23

charged moieties (Figure 2). The determined pKa1 and pKa2 values are shown in Table 1. The slight 14


Page 15 of 38


1

decrease of pKa1 (from 1.75 to 1.58) and pKa2 (from 8.90 to 8.49) values with the increase of

2

hydrophobic chain length was observed. Furthermore, taking into account that the pKa of the α-

3

carboxyl and α-amino groups of Asp are 1.88 and 9.60, respectively, we could state that the

4

introduction of the hydrophobic groups in Asp decreased the apparent pKa, which could be an

5

inductive effect due to aggregation behavior.22

6

pH-Dependent Interfacial Properties at Air/Water Interface. To gain more information for the

7

specific applications of the synthetic amphiphiles in various fields, their surface-active properties at

8

air/water interface were investigated by checking foaming, CMC, and γ (Table 2). Compound 3

9

exhibited excellent foaming ability (38.4 mL) which amounted to 91% of commercial sulfonate

10

detergent SDS (42.0 mL), indicating that it might be applied as a promising alternative to SDS with

11

unexpected side effects.24 The foaming ability decreased for the compounds with longer (>12) and

12

shorter (

Aspartic Acid-based Ampholytic Amphiphiles: Synthesis

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