Synthesis, self-assembly, bacterial and fungal toxicity, and preliminary biodegradation studies of a series of l-phenylalanine-derived surface-active ionic liquids

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Synthesis, self-assembly, bacterial and fungal

toxicity, and preliminary biodegradation studies of

a series of ^L-phenylalanine-derived surface-active

ionic liquids †

Cite this: DOI: 10.1039/c9gc00030e

a

Illia V. Kapitanov, ‡^aAndrew Jordan, ‡^bYevgen Karpichev,

Marcel Spulak, ^cLourdes Perez, ^dAndrew Kellett, ^bKlaus Kümmerer ^eand

a

Nicholas Gathergood

*

We report for the ﬁrst time a comprehensive study on the synthesis (supported by green chemistry

metrics), aggregation properties, bacterial/fungal toxicities and preliminary data on biodegradation of a

series of 24 ^L-phenylalanine derived surface-active ionic liquids (SAILs). The various cationic headgroups

included pyridinium, imidazolium, and cholinium groups and enabled a comprehensive analysis of the

eﬀect of the alkyl ester chain (from C₂to C₁₆) on the synthesis, toxicity, biodegradability, and surfactant

properties of the novel SAILs. The evaluation of the SAILs revealed that a wide variety of properties were

strictly dependent on the side chain length, including their bacterial and fungal toxicities (from low toxi-

city to high toxicity), and aggregation properties. Addition of the ^L-phenylalanine moiety which connects

the lipophilic side chain to the cationic head group results in the phenyl group essentially contributing to

the self-assembling properties. The interplay of dispersion interactions of the phenyl ring and the side

chain hydrophobicity allows us to rank the novel SAILs (thus identifying the remarkable ones) as compared

to other surfactants. The CMC values for the SAILs reported in this study are signiﬁcantly (up to 10 times)

lower than those reported for conventional surfactants with the same length of the side chain. Adsorption

and micellization are among the factors aﬀecting the toxicity of the studied SAILs. Preliminary bio-

degradation studies have shown that no clear trend was observed when comparing the closed bottle test

results of the SAIL C₂and C₁₀derivatives. Medium chain length (C₆to C₈) pyridinium SAILs have been

recommended as the most prospective green alternatives for conventional cationic surfactants. These

ﬁndings can contribute to designing new eﬃcient amphiphiles with optimized antimicrobial activities and

to employ them as potential environmentally benign mineralisable surfactants.

Received 4th January 2019,

Accepted 25th February 2019

DOI: 10.1039/c9gc00030e

rsc.li/greenchem

Over the past few decades, the principles of green chemistry green chemistry applications^4–6due to their specific properties

have been proposed to develop a new approach for the design as solvents,⁷potential for high recyclability,⁸low toxicity,^9–11

and implementation of chemical products and processes.^1–3and synthesis from renewable resources.^12,13

Ionic liquids (ILs) are increasingly seen as an integral part of

The overlap of surfactant and IL chemistry has been clearly

observed from the very beginning of the current upsurge in IL

research and development.⁴Indeed, diluted solutions of ILs con-

taining ions with long chains can exhibit self-aggregation similar

to that of conventional surfactants. The unique properties of

these surface-active ionic liquids (SAILs), in particular, the abnor-

mally low melting point and/or low Kraﬀt temperature, provide

an obvious advantage to the IL-based surfactants and foster

growing interest in synthesizing and studying the self-organiz-

ation of SAILs.^14–18This resulted in an increase in the study of

miscellaneous applications of SAILs, including biomass extrac-

tion¹⁹and micellar catalysis of various organic reactions.^20–22

Since the implementation of the EU regulation on deter-

gents,²³investigations into the ultimate biodegradability of

^aDepartment of Chemistry and Biotechnology, Tallinn University of Technology,

Academia tee 15, 12618 Tallinn, Estonia. E-mail: nicholas.gathergood@taltech.ee;

Fax: +372 62 311-6830; Tel: +372 63 570-8636

^bSchool of Chemical Sciences, Dublin City University, Glasnevin, Dublin 9, Ireland

^cDepartment of Organic and Bioorganic Chemistry, Faculty of Pharmacy,

Heyrovského 1203, Hradec Kralove, CZ500 05, Czech Republic

^dDepartment of Surfactants and Nanobiotechnology, IQAC-CSIC, Jordi Girona 18-26,

ES-08034 Barcelona, Spain

^eInstitute of Sustainable and Environmental Chemistry, Leuphana University of

Lüneburg, Scharnhorststraße 1, D-21335 Lüneburg, Germany

†Electronic supplementary information (ESI) available. See DOI: 10.1039/

c9gc00030e

‡These authors contributed equally to the publication.

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surfactants have been a requirement that has driven a new adsorption, CMC value, transport across the membrane, and

wave of research on the preparation of biodegradable solubility in the aqueous media), complicated by the problem

SAILs.^24–26These strategies employed to promote bio- that some of these parameters may show opposite trends.^41,46

degradation are well documented, including many cases utilis- The antimicrobial activity of cationic amino acid derived SAILs

ing hydrolysable ester bonds and cleavable amide links.²⁷The depends on their structures and size (specifically, the amino

legislation intends that for a surfactant that does not ulti- acid residue and the chain length are key parameters), the

mately undergo biodegradability, the derogation may be molecule hydrophilic/lipophilic balance, and the cationic

applied for depending on the end use application, and the cal- charge density.³⁰

culated risk to the environment and health by the expected

Recently, we reported a ‘benign by design’⁴⁷approach for

use of a surfactant. Depending on the concentration in an the elaboration of the ^L-phenylalanine ethyl ester platform for

aquatic environment, surfactants (cationic quaternary designing completely mineralizable ILs;^48,49see Fig. 1 (see also

ammonium salts, in essence²⁸) can be toxic for living (micro) Fig. 3 in ref. 49 for IL cation variation). The biodegradation

organisms.²⁹The adsorption of surfactants may cause the dis- was investigated with a modified closed bottle test based on

ruption of the cellular membranes and result in acute or OECD guideline 301D and the liquid chromatography method

chronic eﬀects on sensitive organisms through diﬀerent non- combined with high-resolution mass spectrometry analysis to

specific modes of action. The hydrophobic/hydrophilic identify the chemical structures of products resulting from

balance of the molecule and the cationic charge density aﬀect incomplete biodegradation.

the antimicrobial activity.³⁰

In the present work, we undertook the preparation of green

Therefore, an ecotoxicological study of surfactants is essen- potentially degradable SAILs by structural modification of the

tial to establish safe concentrations for the environment and recently reported mineralizable ILs (see Fig. 1). We report for

compare with predicted or measured environmental concen- the first time a comprehensive study on the synthesis (sup-

trations.³¹Hence, gathering of additional ecotoxicity data is ported by green chemistry metrics), aggregation properties,

essential to ascertain the balance of toxicity vs. eﬃciency.³²

bacterial/fungal toxicity and preliminary data on bio-

An imbalance in the IL research field between the assess- degradation of ^L-phenylalanine based SAILs. The various cat-

ment of the environmental impacts of ILs and the impact of ionic headgroups included pyridinium, imidazolium, and cho-

manufacturing ILs has recently been pointed out.³³Some linium and enabled a comprehensive analysis of the eﬀects of

confusion has arisen from the estimation of the ready bio- the alkyl ester chain on the synthesis, toxicity, biodegradabil-

degradability test as an indicator of ultimate degradation, ity, and surfactant properties of the newly synthesized

whereas the life cycle assessment is often considered as a SAILs. For a detailed associated biodegradation study see

“gold standard method” for determining whether a method or Kümmerer et al.⁵⁰

technology is green. Therefore, when designing new green ILs,

undertaking a comprehensive yet feasible green chemistry

assessment still remains a major challenge.³⁴

Results and discussion

ILs based on amino acids can be considered as one of the

green alternatives.¹²The synthesis, physicochemical properties The main objective of this study is to determine if green sur-

and industrial applications of amino acid surfactants demon- factants can be identified by evaluating their synthesis,

strate wide commercial opportunities for this ever-growing microbial toxicity and aggregation properties. A preliminary

class of surfactants.^35,36Special attention should be paid to biodegradation study is also included in the assessment. The

the plausible synergetic eﬀect of a combination of ILs contain- Results and discussion section is therefore split into the afore-

ing natural biologically active molecules, as compared with mentioned subsections.

conventional ILs.³⁷Development of green ionic liquids

demands toxicological studies to facilitate

favourable of SAILs,²⁸which is assumed to increase with the extension of

As increased microbial activity is linked to the lipophilicity

a

outcome.³⁸The toxicity of cationic surfactants towards (micro) the side chain, a comprehensive study is required in which the

organisms is generally considered to be primarily related to analysis of the extent of surface activity of the SAILs

the sorption of a surfactant molecule to biological mem- is accompanied by their toxicity (bacterial and fungal) and bio-

branes, which is driven by nonspecific hydrophobic inter- degradability evaluation. This enables us to establish,

actions.^39,40Since the free energy of adsorption of cationic sur- which compounds in the series of the studied cationic

factants (including SAILs) largely increases with the hydro- SAILs demonstrate remarkable surfactant properties,

phobic chain length,⁴¹the migration rate of the surfactant to while are also considered as not exhibiting high toxicity.

the cell wall interface with the lengthening of the alkyl chain The result is that we can identify the greener surfactant

also increases. The longer the alkyl chain, the easier it is for chemicals.

the molecule to insert through the polar head-group region of

Our hypothesis as shown in Fig. 2 is that the cleavage of the

the membrane bilayer inducing membrane damage and cell amide bond vs. ester bond in the IL molecule (Fig. 1) will even-

death.^42–45At the same time, the maximum biological eﬀect at tually lead to mineralizable transformation products (TPs).

a specific chain length is a result of the combination of This has previously been shown as the breakdown pathway of

various physicochemical parameters (e.g. hydrophobicity, pyridinium ethyl ester derivatives.^48,49

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Fig. 1 Structures of L-phenylalanine based ILs studied.

Fig. 2 Proposed ﬁrst step of biodegradation of L-Phe derived ILs with pyridinium cations resulting in biodegradable TPs (for R = C₂see ref. 48 and 49).

Synthesis

The yield, conversion and selectivity for all the steps of Phe

IL synthesis were high (>89%), and according to Clark et al.⁵³

they can be marked with a green flag (see ESI†). The atom

economy (AE), reaction mass eﬃciency (RME) and optimum

eﬃciency (OE) in the cases of ester 1–8 and salts 17–40 were

no less than 91.5% (AE), 83.3% (RME) and 87.5% (OE),

respectively. For the synthesis of alkylation agents 9–16, the

parameters AE, RME and OE were lower than those in the case

of ester and salt synthesis due to the formation of stoichio-

metric quantities of HBr (AE: 79.5–86.3%; RME: 64.0–75.9; OE:

80.5–88.6%). Analysis of the process mass intensity (PMI) para-

meters has demonstrated that a major contribution to the PMI

total parameter is from the PMI workup solvents. Thus,

workup solvent use has been identified as the problem of the

synthetic procedure which should be tackled first in future

studies.

L-Phenylalanine derived surfactants were synthesised in three

stages using methods similar to those reported earlier;^48,49,51

see Scheme 1, Experimental section, and ESI.†

The first step involved acid catalysed esterification of ^L-Phe

with a fatty alcohol using p-toluene sulfonic acid (PTSA) to give

esters (1–8).⁵²Next was alkylation of the esters using bromo-

acetyl bromide to form alkylating reagents (9−16). Last was the

alkylation of pyridine, dimethylethanolamine (DMEA) or

1-methylimidazole to furnish the respective ionic liquid surfac-

tants (17–40). The molecular structures of the products and

their synthesis are shown in Scheme 1. All SAILs (17–40) were

white solids with melting points in the range 56–120 °C except

for 26 which was a liquid at room temperature. It is noted that

1

the isolation of solid SAILs in high purity (as confirmed by H

NMR) is easier and more convenient compared to that of high

viscosity ILs. Detailed synthesis and characterisation of the

products are included in the Experimental part and the ESI.†

Concerning solvents which have been used for reactions

and workup, the most problematic were DCM, petroleum ether

and diethyl ether (red flag), followed by more acceptable,

toluene and acetonitrile (amber flag), and finally non-proble-

matic – water (green flag). After synthesis and workup the pro-

Green chemistry metrics

Green chemistry metrics are an important benchmark with blematic solvents were recovered with yields in the range

which a synthetic route to a target could be analysed. A green 80–85%. Due to the low boiling point temperature of DCM,

chemistry metric approach described by Clark et al.⁵³has been petroleum ether and diethyl ether, recovery does not have a

used for calculation of the parameters shown in Table 1 high energy demand. The substitution of problematic solvents

(^L-phenylalanine esters and pyridinium SAILs) and Table S2† with greener alternatives (e.g. replacing DCM with ethyl acetate

(alkylating agents; imidazolium and cholinium SAILs).

for the extraction of esters 1–8 and alkylation agents 9–16) had

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Scheme 1 A synthetic route for the ILs (17–40) via esters (1–8) and alkylating reagents (9–16).

a significant negative eﬀect. Previously high product yields from C₂to C₁₆(91.5 to 95.6 and 79.5 to 86.3, respectively). This

decreased to low–moderate (35–60%) for esters and moderate is due to the increased MW of the product as the sidechain

(50–80%) for alkylation agents due to the diﬃculties in separ- increases in length (from C₂to C₁₆); however the same by-

ating the water–ethyl acetate phases during the workup step. product is formed (water or HBr). In this evaluation we do not

Formation of a stable emulsion hindered product isolation and consider (based solely on atom economy) that the synthesis of

loss of the product into the aqueous phase. Our final optimised the C₁₆derivative is greener than that of C₂, but that they are

synthetic procedure used less favorable (from a green chemistry comparable. Analysis of the PMI data for all compounds prepared

point of view) solvents, but incorporation of a solvent recovery shows three distinct groups (Table 1 and Table S2†). In the last

step led to a positive green chemistry metrics result overall, step, the PMI contributions from the reaction and workup com-

including high yields. In the reactions we also did not use criti- ponents are approximately equal. Future eﬀorts to reduce solvent

cal elements (with only 5–50 year supply remaining). Energy use should investigate both reaction solvent and work-up solvent

parameters were marked with a green flag (e.g. reaction run requirements. Considering the PMI data for the amino ester syn-

between 0 to 70 °C for the synthesis of alkylation agents 9–16 thesis, the work-up solvent value is the dominant contributor;

and salts 17–40) or an amber flag (e.g. reaction run between −20 therefore the work-up should be targeted for reducing solvent

to 0 °C or 70 to 140 °C for the synthesis of esters 1–8).

use. Synthesis of the acyl bromides also has a larger PMI contri-

Improvements in the green chemistry metrics compared to bution due to work-up solvents albeit not as overriding as that

our previous study of ethyl ester derivatives are as follows. The observed for the aminoester synthesis (Table S2).†

yields are consistently excellent for 1–8 (97–99%) compared to

Microbial toxicity

43–99% for ethyl derivatives. For the acyl halides the range of

yields improved from 54–89% to 93–98% (see Table S2†). Also, Antimicrobial activity denotes a desirable biological activity

for the Phe ILs the yield improved from 13–98% to 91–98% while toxicity denotes a negative behaviour. The following

(e.g. pyridinium IL 22, 98% yield, see Table 1). The atom results report our eﬀorts to identify low/moderate microbial

economy for the last step is 100% as the halogen atom is incor- toxicity surfactants in our SAIL series (17–40). We also

porated into the product as a bromide anion. Atom economy acknowledge that nowadays, given the huge problem with

for the synthesis of amino esters and acyl halides increases resistant bacteria, it is of great interest to prepare new anti-

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Table 1 Green chemistry metrics calculated for pyridinium SAILs (17–24) and their precursor L-phenylalanine esters (1–8)

PMI

Compounds

(chain

length)

reactants, PMI

reagents, reaction PMI

total reaction catalyst solvents workup chemical solvents

PMI

workup

PMI

workup

PMI PMI

Yield Conversion Selectivity AE

RME OE

1 (C2)

2 (C4)

3 (C6)

4 (C8)

5 (C10)

6 (C12)

7 (C14)

8 (C16)

97.0

98.0

99.0

97.1

98.9

97.9

100.0

97.0

98.0

99.0

97.1

98.9

97.9

91.5 88.9

97.2 40.0 4.9

2.4

2.2

2.1

2.0

1.9

1.8

2.5

6.7

5.9

5.3

4.8

4.4

4.0

3.8

35.1

38.1

33.4

30.1

27.0

25.2

22.9

21.4

1.2

1.1

1.0

0.9

0.8

0.7

0.6

33.8

36.9

32.4

29.2

26.2

24.5

22.2

20.8

92.5 84.5

93.3 85.0

93.9 84.5

94.4 85.1

94.9 83.3

95.3 84.7

95.6 83.7

91.4 47.0 8.9

91.1 41.4 8.0

90.0 37.4 7.3

90.1 33.7 6.7

87.8 31.6 6.3

88.9 28.7 5.8

87.5 27.0 5.6

17 (C2)

18 (C4)

19 (C6)

20 (C8)

21 (C10)

22 (C12)

23 (C14)

24 (C16)

94.1

93.0

97.0

95.9

98.0

96.0

97.0

100.0

94.1

93.0

97.0

95.9

98.0

96.0

97.0

100.0 94.1

100.0 93.0

100.0 97.0

100.0 95.9

100.0 98.0

100.0 95.9

100.0 97.0

94.1 10.7 4.9

93.0 10.2 4.7

1.1

1.0

3.9

3.6

3.3

3.1

2.9

4.0

3.9

3.7

5.8

5.5

4.9

4.7

4.4

5.5

5.3

5.0

0.0

5.8

5.5

4.9

4.7

4.4

5.5

5.3

5.0

97.0

95.9

9.2 4.3

8.8 4.2

8.4 4.0

98.0 10.5 5.1

95.9 10.3 5.0

97.0

9.7 4.7

microbial and antifungal surfactants. As such, one can con- bacterial toxicity observed.^54,55Nevertheless, further increasing

sider compounds shown to have high microbial toxicity as of the side chain length does not result in the lower MIC

potential drug discovery ‘hit compounds’ from an anti- values (i.e. at higher toxicity). Such a levelling-oﬀ was observed

microbial activity perspective. The priority of this work, for the derivatives from decyl compounds (21, 29 and 37) to

however, is the development of greener (inc. low microbial tox- tetradecyl compounds (23, 31 and 39) against Gram-positive

icity) surfactants.

bacterial strains up to dodecyl compounds (22, 30 and 38)

against the Gram-negative bacterial strains scanned. The mode

of action of quaternary ammonium compounds (QAC) against

Bacterial toxicity

The in vitro antibacterial eﬀects of the linear alkyl ILs (17–40) bacterial cells is thought⁵⁶to involve a general perturbation of

and corresponding Phe esters (1–8) were investigated. The lipid bilayer membranes which are found to constitute the bac-

results collected in Table 2 and Table S3† demonstrate a broad terial cytoplasmic membrane and the outer-membrane of

spectrum of bacterial toxicities for the compounds screened. Gram-negative bacteria. Such action leads to a generalized and

Phe esters (1–8) do not demonstrate toxicity with respect to progressive leakage of cytoplasmic materials to the environ-

Gram-negative bacteria. This behaviour is probably due to the ment. The presence of the outer-membrane makes some

cationic charge of these molecules, although these compounds Gram-negative species of bacteria relatively insensitive to the

are pH/sensitive, and the cationic charge depends on the pK_abiocides. This might be related to a failure of the compounds

of the molecule and the pH of the medium. For the SAILs toxic to Gram-positive strains to penetrate the outer membrane

(17–40), a clear trend of increasing toxicity with alkyl chain of the Gram-negative strains and to access the cytoplasmic

elongation is observed up to a certain chain length, irrespec- membrane. It is believed for n-alkyl SAILs^57,58that in the case

tive of the cation (pyridinium, imidazolium, or cholinium). of Gram-positive bacteria and yeast, such activity maximizes

The Gram-positive strains generally demonstrate higher sensi- with n-alkyl chain lengths of n = 12–14, while for Gram-nega-

tivity (lower MIC₉₅values) to the chain length than the Gram- tive bacteria, optimal activity is achieved for compounds with

positive strains; i.e. compare E. coli vs. S. aureus as shown in chain lengths of n = 14–16; further side chain extension is con-

Fig. 3. Decyl chain length SAILs (compounds 21, 29 and 37) sidered to make compounds with n ≥ 18 virtually inactive.

appear to be in an optimum region to demonstrate the There has been a mention of some examples⁵⁸of imidazolium

microbial toxicity where the lipophilicity, solubility and thus ILs when Gram-positive strains were both the most and the

bioavailability are at a maximum, hence the highest levels of least resistant strains, but the toxicity increased with the alkyl

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Table 2 Bacterial toxicity results obtained for the SAILs (17–24)

MIC₉₅, µM

Gram positive

Gram negative

IL

Side chain length

Time

SA

MRSA

SE

EF

EC

KP

KP-E

PA

17

18

19

20

21

22

23

24

C2^48,49

C4

24 h

48 h

24 h

48 h

24 h

48 h

24 h

48 h

24 h

48 h

24 h

48 h

24 h

48 h

24 h

48 h

2000

>2000

500

1000

62.5

7.81

125

1.95

15.62

3.9

>2000

2000

125

31.25

250

>2000

500

>2000

2000

>2000

500

31.25

250

>2000

500

>2000

2000

>500

3.9

62.5

>500

>2000

2000

>500

3.9

62.5

>500

>2000

2000

>500

15.62

62.5

500

C6

31.25

250

1.95

62.5

7.81

250

500

C8

>500

3.9

62.5

C10

C12

C14

C16

1.95

3.9

1.95

3.9

62.5

1.95

31.25

7.81

62.5

1.95

62.5

7.81

250

>500

31.25

SA: Staphylococcus aureus subsp. aureus (ATCC 29213), MRSA: Staphylococcus aureus subsp. aureus methicillin resistant (ATCC 43300), SE:

Staphylococcus epidermidis (HK6966, 112-2016, dialysis tube, University Hospital Hradec Králové, Institute of Clinical Microbiology), EF:

Enterococcus faecalis (ATCC 29212), EC: Escherichia coli (ATCC 25922), KP: Klebsiella pneumoniae (HK 11750, 64-2016, urine catheter tube,

University Hospital Hradec Králové, Institute of Clinical Microbiology), KP-E: Klebsiella pneumoniae (ESBL positive, HK 14368, University Hospital

Hradec Králové, Institute of Clinical Microbiology), PA: Pseudomonas aeruginosa (ATCC 27853).

chain length. In the present work, we report the derivatives aﬀecting the IL interaction with the cell surface or preferable

with the longest chain (cetyl 24, 32, and 40 for Gram-positive self-aggregation of these salts as compared to their interaction

strains, and tetradecyl 23, 31, and 39 in addition to them for with the lipid membrane. The strong correlation of IL toxicity

Gram-negative strains) to exhibit even lower toxicity. This towards both bacteria and aquatic organisms with their lipo-

phenomenon called a “cut-oﬀ eﬀect” has been reported for philicity has been widely discussed elsewhere to be an impor-

diﬀerent cationic SAILs, e.g. 1-alkyl-⁴³and 1-alkoxyl-3-methyl- tant factor determining the environmental application of

imidazoliun⁴⁰salts or 1-alkyl-3-hydroxypyridinium salts,⁵⁹ILs.^61,62The antimicrobial eﬃcacy of SAILs can be reported

towards miscellaneous bacterial strains. According to the relative to their critical micelle concentration (CMC) as one of

studies of Ruokonen et al.⁶⁰on the liposome–IL interactions, the reference concentrations⁶³reflecting the interplay of self-

the toxic eﬀect of the shortest chain-length ILs which do not aggregation with the cut-oﬀ eﬀect. As can be seen from Fig. 3,

rupture the cell membrane consists predominantly in aﬀecting the CMC values (see the discussion below) interfere with the

cell metabolism at the toxic concentration. The biodegradable ascending arm of the curve for some of the strains studied

ethyl derivatives 17, 25 and 33 reported earlier,^48,49along with supporting the predominant role of the micellization of the

ethyl esters with the ethyl to hexyl chain length compounds SAILs as a factor decreasing the toxicity of these long-chain

(1–3), fall in this category. Most of the SAILs reported in this salts at concentrations above the CMC.

work bearing medium and long lengths of the n-alkyl ester

with n = 4 to 14 (n = 12 in the case of Gram-negative bacterial

Fungal toxicity

strains) show a dependence on gradually increasing toxicity The linear alkyl ILs (17–40) along with the esters (1–8) have

caused by a partial cell membrane rupturing eﬀect. This ten- also been screened for their fungal toxicity. The results

dency is followed by levelling-oﬀ the toxicity for the longer obtained for pyridinium SAILs (17–24) are shown in Table 3

chain salts to reach the highest toxic eﬀect (lowest MIC) in the with representative strains included in Fig. 3a. The fungal tox-

series. The “cut-oﬀ” eﬀect turns to the reverse dependence of icity results for phenylalanine esters (1–8) and imidazolium

toxicity on the hydrophobicity for the longest chain length ILs, and cholinium SAILs (25–40) are provided in Table S4† and

n = 14 (Gram-negative bacteria) or n = 16 (Gram-positive bac- Fig. 3b and c. Overall, it can be shown that as the alkyl chain

teria): esters 7 and 8 and cetyl chain length SAILs 24, 32, and length increases so does the antifungal activity. The fungal tox-

40. This may be connected either to the high steric eﬀect icity for the octyl derivatives is generally moderate,

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Fig. 3 Bacterial and fungal toxicities (MIC, μM) as a function of the side chain length of the Phe derived SAILs. (a) Pyridinium (17–24), (b) imidazo-

lium (25–32), and (c) cholinium (33–40) head groups towards selected Gram-positive (S. aureus, SA) and Gram-negative (E. coli, EC) bacteria and

selected fungi (C. albicans CA2; A. corymbifera, AC) strains.

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Table 3 Fungal toxicity results obtained for the pyridinium SAILs (17–24)

MIC₉₅, µM

IL

Side chain length Time (h) CA1

CA2

CP

CK1

CK2

CT

CG

CL

TA

AF

AC

TM^a

17 C2^48,49

18 C4

24 h

48 h

24 h

48 h

24 h

48 h

24 h

48 h

24 h

48 h

24 h

48 h

24 h

48 h

24 h

48 h

>2000 >2000 >2000 >2000 >2000 >2000 >2000 >2000 >2000 >2000 >2000 >2000

>2000 >2000 2000

2000

>2000 2000

>2000 >2000 2000

>2000 >2000 >2000

2000

1000

125

>2000 >2000 >2000 >2000 >2000 >2000 >2000 >2000 >2000 >2000 >2000

19 C6

2000

125

500

1000

250

2000

31.25

125

1000

62.5

250

500

250

125

62.5

3.9

2000

250

2000

>2000 1000

500

20 C8

31.25

62.5

15.62

31.25

7.81

1.95

7.81

31.25

62.5

7.81

31.25

7.81

1.95

3.9

31.25

62.5

7.81

31.25

7.81

1.95

3.9

1000

2000

31.25

15.62

7.81

500

2000

62.5

500

250

21 C10

22 C12

23 C14

24 C16

15.62

3.9

1.95

3.9

15.62

31.25

3.9

15.62

7.81

31.25

15.62

1.95

15.62

62.5

7.81

0.98

1.95

7.81

62.5

7.81

3.9

>500

3.9

7.81

3.9

7.81

15.62

31.25

62.5

15.62

7.81

15.62

3.9

31.25

15.62

7.81

15.62

7.81

15.62

31.25

^aEvaluation after 72 h and 120 h, respectively. CA1: Candida albicans (ATCC 24433), CA2: Candida albicans (ATCC 90028), CP: Candida parapsilosis

(ATCC 22019), CK1: Candida krusei (ATCC 6258), CK2: Candida krusei (E28, University Hospital Hradec Králové, Institute of Clinical

Microbiology), CT: Candida tropicalis (ATCC 750), CG: Candida glabrata (ATCC 90030), CL: Candida lusitaniae (2446/I, University Hospital Hradec

Králové, Institute of Clinical Microbiology), TA: Trichosporon asahii (1188, University Hospital Hradec Králové, Institute of Clinical Microbiology),

AF: Aspergillus fumigatus (ATCC 204305), AC: Absidia corymbifera (CCM 8077), TM: Trichophyton mentagrophytes (445, University Hospital Hradec

Králové, Institute of Clinical Microbiology).

250–1000 µM for imidazolium and cholinium derivatives (28 fungicidal eﬀects of cationic amphiphilic compounds as was

and 36), whereas the pyridinium derivative (20) is slightly supposed after studying Candida albicans.⁶⁵Indeed, as can be

more toxic, with MIC values ranging from 125 to 1000 µM. seen from Fig. 3, the CMC values are far above the MIC values

When the alkyl chain lengths were extended to n = 10, an and if the aggregation can be considered among the factors

order of magnitude increase in the toxicity was observed for all aﬀecting antifungal toxicity, the submicellar (premicellar)

three decyl derivatives 21, 29, and 37 ranging from 15.62 to aggregates may play a role in the interaction with the fungal

250 µM. The pyridinium C₁₀SAIL (21) is more toxic than its membrane. For conventional cationic surfactants it was

imidazolium (29) and cholinium (37) counterparts with MIC suggested that the mechanism of the antifungal action of

values reaching as low as 3.9 µM for the Aspergillus fumigatus micelle-forming cationic detergent does not involve fungal cell

(AF) fungus (Table 3 and Fig. 3). As the alkyl chain is extended lysis but rather the change of the cell surface charge from

to the dodecyl level, a broad spectrum fungal toxicity is negative to positive. Mammalian cells are more fragile regard-

observed for all three compounds 22, 30, and 38 with MIC ing CTAB induced cell lysis, being disrupted by cationic

values in the region of 3.9–125 µM. Further extension of the amphiphiles over a range of micromolar concentrations.⁶⁶The

chain length results in the “cut-oﬀ” eﬀect. Some fungal strains fungal toxicity data can be considered as more predictive, as

(in particular, yeasts Candida albicans and Candida lusitaniae) compared to the bacterial toxicity screening data to estimate

demonstrate better resistance towards cetyl derivatives 24, 32, potential biodegradability.

and 40 whereas other SAILs follow the levelling-oﬀ tendency of

showing even slightly lower MIC values (e.g. Trichosporon

Biodegradation studies

asahii). The mechanism of fungal toxicity for linear alkyl qua- Along with toxicity data, biodegradation studies are an integral

ternary ammonium compounds has been investigated in the part of the green chemistry assessment of a chemical. In 2013,

literature⁶⁴and is considered to be similar to that discussed Gathergood and Connon published a combined microbial tox-

for bacteria. QACs can insert into the plasma membrane of a icity, biodegradation, green chemistry metrics and catalyst per-

fungus and cause disruption of the membrane and eventually formance study of a series of ILs.^67,68One of the findings from

lysis as well as inhibiting spore growth.⁶⁴Another hypothesis this work was that the studied ILs containing amide group(s)

was connected with an assumption that interfacial micelle-like exhibited very low biodegradation in the CO₂headspace test.

aggregates are formed at the cell surface as a step in the This was in remarkable contrast to ILs containing an ester

binding process. Even submicellar complexes may initiate the functional group, where facile breakdown to the carboxylic

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suitable model for the long chain examples, while high

microbial toxicity had not been established in both our bac-

terial and fungal toxicity assessment. Therefore, we would not

expect toxicity complications during the CBT with these C₁₀

compounds. The C₁₀SAILs could primarily breakdown accord-

ing to two likely pathways: amide hydrolysis and ester hydro-

lysis. An illustration of these potential breakdown sites is pro-

posed for the pyridinium IL (21) (Fig. 5), and the bio-

degradation data from CBT are shown in Table 4.

As can be seen from Table 4, none of the longer chain (as

compared to the ethyl ester) SAILs passed the CBT. There are

three possible reasons for the reduction in biodegradability

observed for the C₁₀chain length SAIL (21, 29, and 37) when

compared to the short chain C₂pyridinium IL (17) previously

reported to be mineralisable. The SAIL (21, 29, and 37) could

undergo slow hydrolysis (compared to C₂) to form n-decanol

and pyridinium ^L-phenylalanine carboxylic acid transform-

ation products. The latter may be an unsuitable substrate for

further amide cleavage and therefore be persistent (see Fig. 5,

right). Another reason could be that decyl chain length SAILs

undergo amide hydrolysis first (see Fig. 5, left). The second

option follows the mechanism confirmed for the ethyl deriva-

tive (21) to be the favourable route for mineralisation.^48,49

Therefore, upon elongation of the alkyl chains from C₂to

C₁₀, a dramatically reduced biodegradability was observed for

the SAILs with all three headgroups (21, 29 and 37).

Biodegradation of 36% was observed for the pyridinium C₁₀IL

(21), 2% less than the biodegradation attributed to total ester

mineralisation (assuming that the acid remains persistent in

Fig. 4 Structural features that promote IL biodegradation.⁹(Courtesy of

the Royal Society of Chemistry.)

acid and conversion of the alcohol to CO₂have been widely

reported.^9,26Fig. 4 shows examples of ILs divided into three

categories with respect to their abilities to pass the biodegrad-

ability tests, according to the review of Jordan and

Gathergood.⁹This demonstrates the importance of the struc-

tural features to promote or impede the biodegradation of ILs

and highlights, using corresponding traﬃc light colours, the

goal of attaining a green rating. It is a major challenge, as even

in the case of ester containing ILs, the carboxylic acid trans-

formation product has been shown to persist after a 28 day

CBT.⁶⁹Therefore, the development of mineralisable ILs

remains a demanding task.

Our study published previously⁴⁹provided an insight into

the biodegradability of the ethyl ester of cationic phenyl-

alanine-based ILs. The pyridinium IL derivative 7 was found to

be the preferred green IL of the studied series (see Fig. 1),

based on synthesis, toxicity and biodegradation consider-

ations. The longer alkyl chain ILs studied in the present work

demonstrate that the toxicity level increases with the increase

in the alkyl chain length up to C₁₀–C₁₂– the chain length for

which the “cut-oﬀ” eﬀect appears. Initially, we screened the

biodegradation of C₁₀ILs with pyridinium (21), imidazolium

(29) and cholinium (37) headgroups. These SAILs were chosen

as they contain a side chain length C₁₀, in the middle of our

series C₂to C₁₆. We would expect these compounds to be a

Table 4 Biodegradability and carbon distribution of selected ILs;

colour coded according to the traﬃc light classiﬁcation⁶⁷(green ≥60%,

amber ≥20% and <60%, and red <20%)

IL

Chain length

Head group

Biodegradation %

(ref. 49)

17

21

25

29

33

37

C2

C10

C2

C10

C2

C10

Pyridinium

Imidazolium

Cholinium

(ref. 48 and 49)

Fig. 5 Potential sites for the ﬁrst stage of pyridinium SAIL (21) breakdown.

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that case); a value of 44% was observed for the imidazolium

SAIL (29), 4% above the theoretical degradation according to

total ester degradation; and, finally, the cholinium SAIL (37)

displayed degradation levels 13% less than total ester degra-

dation. These data cannot confirm whether the amide bond of

the C₁₀SAILs underwent hydrolysis. Reduced levels of biode-

gradability may be attributed to the level of the broad spec-

trum antimicrobial activity of the parent SAILs discussed

above (1.96 µM for S. aureus, 3.9 µM for E. coli, 15.62 µM for

C. albicanis CA2, and 31.25 µM for A. fumigatus, Tables 2 and

3, Tables S3 and S4†). According to Boethling’s rules of thumb

an extension in the alkyl chain length should increase biode-

gradability.⁷⁰However, as the toxicity levels increased with the

alkyl chain length, the potential increase in biodegradability is

oﬀset by the inability of the bacteria to degrade the IL. Indeed,

Boethling states that in many cases structural changes made

to reduce the toxicity of small molecules can reduce biodegrad-

ability.⁷⁰Although the longer chain pyridinium derivative (21)

undergoes a remarkably reduced level of biodegradation com-

pared to the C₂compound (17), conclusions about their bio-

degradation pathways and ultimate biodegradability cannot be

drawn without a full metabolite analysis and test time exten-

sions. A detailed study of the biodegradability of the series of

Phe-derived SAILs is presented in a separate publication of

Kümmerer et al.⁵⁰

Fig. 6 Surface tension as a function of the total surfactant concen-

tration for the ^L-phenylalanine derived pyridinium SAILs (17–24); water,

25° C.

As expected, a progressive CMC diminution when the alkyl

chain length increased was observed for the three SAIL

families (Table 5). This behaviour is common in all types of

surfactants and it is attributed to the higher hydrophobic

content of the molecule. The log CMC value decreases linearly

with the chain length extension, following the empirical

Klevens equation:⁷⁴log CMC = A − Bn, where A and B are con-

stants for a particular homologous series at a constant tem-

perature. The parameter A is related to the contribution of the

polar head to micelle formation, and the parameter B indicates

the average contribution to the micelle formation of each

additional methylene group in the hydrophobic chain. The

Klevens dependency for the SAILs studied in this work is pre-

sented in Fig. 7 separately for the salts with pyridinium (a),

imidazolium (b), and cholinium (c) head groups, along with

selected data on the structurally similar salts reported in the

literature. All three series exhibit about the same value of the

slope close to −0.30; see eqn (1)–(3).

Aggregation behaviour in aqueous solution

The aggregation properties, as well as the adsorption behav-

iour of the linear alkyl ^L-phenylalanine IL surfactants 17–40,

have been studied by means of tensiometry and conductivity.

Plots of the surface tension of aqueous solutions of SAILs

versus the log of their bulk concentration are shown in Fig. 6.

The surface tension curves are characteristic of surfactants. At

low concentrations, high surface tension values were obtained.

On increasing the SAIL concentration, the surface tension

values decreased, due to the adsorption of the SAIL molecules

at the air/liquid interface, until a plateau attributed to the for-

mation of micelles is observed. Subsequently, the surface

tension values do not change with the concentration.⁴⁵

For the short and medium-chain homologues in each

series, a minimum of surface tension appears before attaining

a plateau. The appearance of a minimum in the surface

tension isotherm is usually attributed to impurities (which

generally can be reduced or eliminated by purification).⁷¹The

log CMC_Py¼ ꢀ0:297n ꢀ 0:157

log CMC_Im¼ ꢀ0:305n þ 0:062

log CMC_Chol¼ ꢀ0:296n ꢀ 0:178

ð1Þ

ð2Þ

ð3Þ

The slopes ca. −0.30 are similar to those for either conven-

repeated purification of the studied Phe derived SAILs did not tional ionic surfactants (alkyl pyridinium,⁷⁵[C_nmim],⁷⁶or alkyl

eliminate the minimum in the tensiometry plot, which may cholinium⁷⁷salts), or ester-functionalized⁷³and amide-func-

support another origin of this phenomenon. Similar obser- tionalized⁷⁸SAILs. They all form a series of nearly parallel

vations have been found in the literature for short or medium plots, as shown in Fig. 7. It is worth noting that this regularity

chain 1-alkyl-3-methylimidazolium salts ([C_nMIM]),⁷²1-alkyl- has been reported for various conventional ionic surfactants⁷¹

pyridinium,⁴⁴and amide-functionalized pyridinium and imi- and SAILs.^45,79Therefore, an extension of the side chain on

dazolium salts.^15,73This behavior has been ascribed to the for- one more CH₂group aﬀects similarly the aggregation pro-

mation of the surface micelles prior to bulk aggregation. It perties

in

all

these

classes

of

surfactants.

leads to a minimum in the area per molecule and a maximum Diethylmethylammonium iodides of Phe esters⁸⁰with side

in the adsorbed film thickness, as well as the formation of a chains from C₁₀to C₁₆exhibited a slope of the Klevens

surface monolayer at concentrations higher than the CMC.

equation of ca. 0.4, which is steeper than that in eqn (1)–(3).

Green Chem.

This journal is © The Royal Society of Chemistry 2019

Table 5 Aggregation properties of the surface-active ILs (17–40, 19Ala, 27Ala and 35Ala)

γ_CMC

π_CMC

Γ_max

A_min

IL

Chain length CMC^a(mM)

β

ꢀΔG^°_micðkJ mol^ꢀ1

Þ

(mN m⁻¹

)

(mN m⁻¹

)

pC₂₀CMC/C₂₀(mmol m⁻²

)

(nm²) ꢀΔG_a^°_dsðkJ mol^ꢀ1

Þ

ꢀΔG^°_ad=A_minðmJ m^ꢀ2

Þ

17

18

19

19Ala

20

21

22

23

24

2

4

6

187 (ST)

57 (ST); 89 (C)

—

42.86

32.90

29.27

34.95

29.16

30.70

33.90

38.07

41.98

29.14

39.1

42.73

37.05

42.84

41.3

38.10

33.93

30.02

1.02

2.38 13.4

3.06 12.1

1.74

3.42

3.93

4.39

4.76

5.21

2.0

2.14

1.49

1.87

2.03

2.76

2.47

2.34

2.39

2.89

0.77

1.11

0.89

0.82

0.60

0.67

0.71

0.69

0.58

25.06

36.89

38.03

29.65

36.66

40.42

43.52

45.43

46.56

34.00

35.30

51.53

40.53

81.34

80.02

81.91

88.90

114.60

0.55 24.70

0.52 24.10

0.52 29.15

0.58 36.55

0.56 40.40

0.58 45.50

0.58 49.50

11 (ST); 19.9 (C)

107 (ST); 92 (C)

2.25 (ST); 4.90 (C)

0.65 (ST); 1.60 (C)

0.19 (ST); 0.50 (C)

0.056 (ST); 0.18 (C)

5.9

5.5

4.7

3.2

2.0

8

10

12

14

16

0.0125 (ST); 0.037 (C) 0.48 52.15

26

27

27Ala

28

29

30

31

32

4

6

8

10

12

14

16

9.0 (ST); 7.90 (C)

23 (ST); 2.50 (C)

122 (ST); 122 (C)

3.0 (ST); 5.50 (C)

1.2 (ST); 1.30 (C)

0.20 (ST); 0.44 (C)

0.05 (ST); 0.18 (C)

0.016 (ST); 0.046 (C)

0.47 23.94

0.51 28.79

0.52 23.05

0.59 36.43

0.51 39.81

0.55 44.97

0.55 48.66

0.47 50.90

33.85

29.98

38.60

29.30

34.30

36.86

38.75

40.50

38.15

42.02

33.40

42.70

37.70

35.14

33.25

31.50

2.50 28.6

3.09 22.0

1.24

1.61

1.18

2.24

2.53

2.83

2.75

3.26

1.34

1.03

1.41

0.74

0.66

0.59

0.60

0.51

40.29

39.96

28.84

40.81

38.94

40.94

45.40

45.66

30.19

44.41

43.06

71.70

78.90

96.03

105.02

128.78

1.68

6.3

3.85 21.4

3.70

4.20

4.94

5.19

4.8

4.1

4.8

3.1

34

35

35Ala

36

37

38

39

40

4

6

8

10

12

14

16

80 (ST); 72 (C)

18 (ST); 23 (C)

0.47 24.26

0.52 29.31

0.58 24.12

0.59 36.16

0.56 41.01

0.57 46.06

0.50 47.37

0.56 52.97

33.85

29.98

38.60

29.30

34.30

36.86

38.75

40.50

38.15

42.02

33.4

42.7

37.7

35.14

33.25

31.50

2.41 22.9

3.06 23.1

1.39

1.57

1.95

2.70

2.36

2.09

2.53

2.41

1.19

1.06

0.85

0.62

0.70

0.79

0.66

0.69

37.95

40.12

28.77

37.54

39.08

43.62

45.03

48.58

33.16

43.13

36.46

81.56

72.47

71.53

94.05

97.00

124 (ST); 117 (C)

4.0 (ST);5.70 (C)

1.30(ST); 1.40(C)

0.28 (ST); 0.40 (C)

0.08 (ST); 0.16 (C)

0.024 (ST); 0.061 (C);

1.51

4.1

3.54 10.5

3.63

4.23

4.77

5.32

5.1

3.5

5.0

^aObtained from conductivity (C) and surface tension (ST) measurements.

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cates that the polarity of these three structural groups is

similar and consequently their derivatives have the same

hydrophobic/hydrophilic balance.

When compared with cholinium, imidazolinium and

pyridinium ILs without phenylalanine in the polar head, these

SAILs have much lower CMC values. Quantitatively, the CMC

values for ILs (17–24) are similar to the CMC values for alkyl-

pyridinium bromides⁷⁵(conventional cationic surfactants with

the pyridinium headgroup) with the side chain longer by six

CH₂groups (C_n+6) and comparable to pyridinium ILs functiona-

lized with an ester or amide moiety,⁷³longer by four CH₂

groups (C_n+4). This tendency can also be seen in the case of imi-

dazolium SAILs (26–32) as compared to [C_nmim] salts (C_n+6) or

imidazolium ILs with the ester moiety⁷³(C_n+4), and for choli-

nium SAILs (34–40) as compared to n-alkyl cholinium bro-

mides.^77,89These results show how the introduction of the aro-

matic moieties into the IL molecule aﬀects the micellization of

SAILs.

Structural changes of the headgroup with non-aromatic

moieties do not aﬀect significantly the micellization of SAILs;

nicotine-based SAILs containing N-methyl pyrrolidine as the sub-

stituent⁸⁴show almost the same CMC values as the ester-contain-

ing pyridinium ILs,⁷³Fig. 7a. The plot for a series of imidazolium

amino acid-based ILs, histidine surfactants,⁸⁵is intermediate

between those of conventional alkyl imidazolium salts and ester-

containing imidazolium ILs,^44,73and the CMCs for a novel series

of cleavable surfactants, alkyloxycarbonyloxyethyl [mim] ILs,⁴¹

eventually coincide with the CMCs of those bearing an ester or

amide reported by the same group earlier;⁷³see Fig. 7b. It

has been recently demonstrated⁹⁰that the ILs with ether

group in the side chain have higher polarity relative to those

with alkyl side-chains, which was ascribed to intramolecular

hydrogen bonding involving the oxygen atom(s) and the relatively

acidic imidazolium hydrogens. Nevertheless, within the series

we have found in the literature, there is no evident influence of

the alkoxy side-chains vs. alkyl side-chains on the aggregation.

An increase of the hydrophobicity of the head group by an

extension of the chain at position 3 of the imidazolium ring

(replacing Me with Bu)⁸⁷also had a minor eﬀect on the CMC

values with regard to the imidazolium ILs with the ester

group^44,73(see Fig. 8b). This is in spite of the fact that this modi-

fication facilitates vesicle formation. A series of cholinium car-

boxylates (using the cholinium ion as a cation and an anion with

variable chain lengths)⁸⁹demonstrates that the Klevens plot just

slightly shifted rightwards compared to that of the same chain-

length n-alkylcholinium bromides with about double CMC

values, Fig. 7c.

Fig. 7 Eﬀect of the side alkyl chain on the CMC of Phe-based cationic

surface active ILs ( ) with pyridinium 17–24 (a), imidazolium 26–32 (b),

and cholinium 34–40 (c) headgroups; water, 25 °C. Data on CMC values

for the series of phenylalanine alkyl ester hydrochlorides⁸¹and the

diﬀerent surfactants with tetraalkylammonium,⁸²pyridinium,^73,75,83,84

imidazolium,^{41,44,85–88}and cholinium^77,89head groups are taken from

the literature (see Table S1†).

Therefore, we suggest that a significant factor aﬀecting the

aggregation properties of the SAILs (17–40) is the presence of

the phenyl group of Phe capable of inducing micellization. It

is noteworthy that a series of cationic surfactants, hydrochlo-

rides of Phe esters, studied by Joondan,⁸¹have similar CMC

values to the SAIL reported in this work while the CMC values

On comparing cholinium, pyridinium and imidazolinium of ^L-alanine (19Ala, 27Ala, and 35Ala) ILs with the same chain

polar heads, it can be observed that very similar CMC values length (C₆), see Fig. 7 and Table 5, are one order of magnitude

were obtained for SAILs with the same alkyl chain. This indi- higher than that of phenylalanine. These results evidently

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The eﬀective range for the surface active ILs examined was

determined to be not very diﬀerent from those of the long

chain cationic surfactants and displayed a larger eﬀective

range than those of traditional n-alkyl surfactants.

The γ_CMCvalues for the studied ILs are between 30 to

40 mN m⁻¹and are dependent on the chain length. As plotted

in Fig. 8, the γ_CMCslightly decreases monotonously with the

reducing chain length from C₁₆to C₁₀and reaches a minimum

for the medium-chain ILs (C₆and C₈). The γ_CMCincreases

again in the case of the shortest chain ILs, C₄and C₂. This

shape of the plot may be ascribed to the increased viscosity of

the solution of the short chain ILs at concentrations above the

aggregation concentration needed for the correct determi-

nation of the CMC. Although the short chain ILs are known to

form aggregates other than micelles, they are usually con-

sidered to behave like short-chain cationic surfactants.⁹⁴The

C₂and C₄Phe ILs fall on the Klevens plot for the whole series

shown in Fig. 8, which indirectly supports the fact that all ILs

in the series undergo the same type of aggregation.

Fig. 8 Eﬀect of the side chain lengths of the SAILs (17–40) on the

values of the surface tension observed at the CMC (γ_CMC).

The ability of a surfactant to reduce the surface tension by

20 mN m⁻¹(pC₂₀) was found as:

demonstrate the pronounced role of the phenyl ring in micelli-

zation of the amino acid-based SAILs.

The literature data indicate that the CMC is significantly

influenced by the location of the phenyl ring within the hydro-

phobic tail segment in the trimethylammonium surfactants.⁹¹

The closer the phenyl moiety is located to the head group, the

lower the CMC. The IL series of benzyl(2-acylaminoethyl)di-

methylammonium chlorides,⁸²bearing both a benzyl moiety

in the headgroup and an amide group close to the head group,

have low CMCs. However, these are still approximately an

order of magnitude higher than the CMC values for ILs

reported in the present work; see Fig. 7a. A similar observation

has been reported for nonionic surfactants, poly(oxyethylene)

glycol alkyl ethers, showing that moving the phenyl group

from the terminal region of the hydrophobic chain to the

neighboring hydrophilic headgroup, leads to the decreased

CMC.⁹²In the case of N-arylimidazolium ILs, the authors⁸⁶

observed lower CMC values than those of [C_nmim]Br,

suggesting that the incorporation of the 2,4,6-trimethylphenyl

group results in weakening of the electrostatic repulsion

between the head groups, and promotes micelle formation.

Kunitake and co-authors⁹³have highlighted that the

inclusion of aromatic (“mesophase-inducing”) segments,

within the hydrophobic tail region of a single-tailed surfactant,

can significantly enhance the interactions between the tail seg-

ments. This causes such single-tailed surfactants to form ves-

icular aggregates, which are otherwise seen only in two-tailed

or gemini surfactants.

pC₂₀¼ ꢀlog C₂₀

;

ð5Þ

where C₂₀is the molar concentration required to reduce the

surface tension by 20 mN m^{−1 71}

.

The larger the pC₂₀the more eﬃciently the surfactant is

adsorbed at the interface and the more eﬃciently it reduces

the surface tension. The pC₂₀values for SAILs (17–40) increase

gradually with the elongation of the chain length, irrespective

of the head group structure (pyridinium, imidazolium, or cho-

linium), from ca. 2.5 for C₄derivatives to ca. 5.2 for C₁₆ILs.

The C₆Ala derivative SAILs show much lower adsorption

eﬃciency as compared to Phe derivatives, and the values are in

the range from 1.51 (35Ala) to 1.68 (27Ala) and then 1.74

(19Ala). It corresponds to the pC₂₀values of shorter chain

length Phe SAILs, between those for C₂and C₄. The obtained

pC₂₀values also indicate that the adsorption eﬃciency of these

SAILs is superior to that shown by ILs without Phe in their

chemical structure.^41,45,75,89

The average area per molecule occupied by each surfactant

molecule was also determined, using the Gibb’s equation:

ꢀ

ꢁ

1

dγ

Γ_max¼ ꢀ

ꢁ

ð6Þ

2:30 ꢁ nRT

d log_C

T

ꢀ

ꢁ

dγ

dlog_C

where Γ is the surface excess concentration,

is the

T

slope of the plot of the surface tension γ vs. log surfactant con-

centration; R = 8.32 J K⁻¹mol⁻¹and T = 298.15 K; n is the

number of ionic species in solution and a value of two is used

for the cation surfactants.

The surface excess concentration Γ is related to the area per

surfactant molecule A_minusing the following equation:

From the surface pressure isotherms, the eﬀectiveness of

the surfactant to reduce the water surface tension (π_CMC) and

the adsorption eﬃciency (pC₂₀) was also estimated. The π_CMC

values were determined using the following equation:

π_cmc¼ γ₀ꢀ γ_cmc

ð4Þ

1

A ¼

;

ð7Þ

N_Aꢁ Γ

where N_Ais Avogadro’s constant.

where γ₀is the surface tension of pure water and γ_CMCis the

surface tension observed at the CMC.⁷¹

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The resulting A_minvalues are given in Table 5, although the the alkyl chain length is increased, the energy required to

A_minvalues must be considered as rough estimates given the form a micelle is decreased. It occurs due to the increase in

minimum obtained in the surface pressure isotherms. hydrophobic repulsion interactions present with longer alkyl

However, while acknowledging this issue, A_minvalues should chains.

be taken into account as useful estimation data, as some

The dependence of the thermodynamic parameters of

general tendency can be pointed out. As expected, the A_minmicelle formation on the surfactant structure shows that a

decreases as a function of the increase of the length of the possible interplay of additional dispersion interactions of the

alkyl chain due to concomitant closer packing of the phenyl ring and possible polar interactions of amide hydrogen

monomer at the air/water interface. The values for C₄ILs are places the novel SAILs among the remarkable surface active

about 1.1–1.3 nm², which are comparable with those of con- ILs as compared to other surfactants, e.g. conventional imida-

ventional surfactants C_nTAB.⁷⁵Elongation of the chain length zolium, pyridinium, and benzyl (2-acylaminoethyl)dimethyl-

results in lower values of A_minthat is 0.6–0.7 nm²for C₈deriva- ammonium counterparts.⁷⁶The insertion of the amino acid

tives which are similar to those of various pyridinium and imi- moiety to link the cationic headgroup and the n-alkyl ester

dazolium SAILs reported in the literature⁴⁵or pyridinium and chain aﬀects both micellization in the bulk and the adsorption

imidazolium pH-responsive functionalized surfactants.⁹⁵

of the SAILs at the interface. The ratio cmc/C₂₀⁷¹is a parameter

The CMC values of these SAILs were also evaluated from that may shed light on what extent the adsorption is more

the conductivity. The conductivity curves are also characteristic favoured as compared to the micellization process. The cmc/

of ionic surfactants. The obtained conductivity values fit into C₂₀values (see Table 5) are high for short chain (C₄and C₆)

two straight lines of diﬀerent slopes. The CMC can be deter- SAILs irrespective of the headgroup, they decrease significantly

mined from the intersection of the two linear dependence of κ for the C₈derivatives, and do not change considerably when

against the concentration of the SAIL with distinct slopes. The the side chain is >C₈. This behaviour is similar to that of con-

breakpoint is caused by either the lower mobility of the ventional ILs^79,97and diﬀerent from that of carbonate-contain-

micelles that contribute to the charge transport or the loss of ing SAILs reported⁴¹to demonstrate the augmentation of the

ionic charges because of the binding of a fraction of the coun- cmc/C₂₀values with the extension of the chain length.

terions to the micellar surface.^15,45The CMC was also evalu-

The CMC value appears to be not the only parameter

ated by taking the first and second derivatives of κ plots;⁹⁶see explaining the toxicity of the SAILs, since the antimicrobial

Fig. S1.† A derivative curve shows an instantaneous decrease eﬀects occur at concentrations far below the CMC (it is worth

in the CMC region and, hence, the center (inflection point) of noting that the CMC has been determined in water, and it is

the reversed sigmoidal curve of the first derivative gives the expected that the CMC in the medium used to determine the

value of the CMC supported by the minimum of the function MICs is much lower) and this parameter does not demonstrate

of the second derivative. A comparison of such a plot with that sensitivity to the “cut-oﬀ” eﬀect in bacterial/fungal toxicities;

of conventional conductivity has an advantage particularly compare Fig. 3 and 7. Moreover, the “cut-oﬀ” eﬀect (increasing

when the break point in the κ curve is not sharp. The CMC MIC₉₅values, which correspond to lower toxicities) observed

values obtained with this technique are consistent with those in the biological activity of the longest chain length SAILs of

obtained from the surface tension.

the series could be attributed to the aggregation process of

Another important parameter that can be evaluated from molecules in aqueous solution.⁴³This is because the tendency

conductivity is the degree of counterion binding (β) that to form molecular aggregates at lower concentrations limits

corresponds to an average number of counterions per surfac- the rate of diﬀusion to the cell surface. An elegant attempt to

tant ion in the micelle and can be estimated by comparing the overcome the problems related to using the CMC or the par-

ratio of the two slopes. The results (Table 5) show that the tition coeﬃcient of octanol/water K_ow(the latter is often

values of β cover the range of 0.5–0.6 and do not depend diﬃcult to determine experimentally for the surfactants) with

remarkably on the chain length as has been reported else- regard to toxicity has been proposed by Rosen and co-

where for other classes of conventional cationic surfactants⁷¹workers.^39,71They introduced the parameter ΔG_a^°_d=A_min, where

and SAILs.^45,51,79This may denote that the micellar surfaces of ΔG_a^°_dis the standard free energy of adsorption of a surfactant

all studied ILs have very similar cationic charges.

to the air/water interface (tendency of the surfactant to adsorb

The standard molar Gibbs energy of micellization, ΔG^°_mic, of onto the membrane of the (micro)organism) and A_minis a

these SAILs was calculated using the following equation:⁸⁹

measure of the ability of the surfactant to penetrate the mem-

brane. This parameter is a hydrophobicity measure, which has

been shown³⁹to correlate well with aquatic toxicity values for

algae and rotifers across the investigated cationic, nonionic,

ΔG^°_mic¼ ð1 þ βÞRT ln x_cmc

;

ð8Þ

where β is the previously discussed counter-ion binding para- and anionic surfactants. It can be calculated experimentally

meter (degree of association) and x_cmcis the critical micelle based on surface tension measurements at various surfactant

concentration expressed as the mole fraction. ΔG_m^°_iccorres- concentrations up to C₂₀rather than to determine the CMC,

ponds to the free energy of transfer of one surfactant mole eqn (9).⁷¹

from the aqueous phase to the hydrophobic micellar pseudo-

phase. Results of the investigation on the ΔG^°_micshow that as

ΔG^°_ad¼ RT ln C_π=ω ꢀ A_π;

ð9Þ

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where R = 8.31 J mol⁻¹K⁻¹, T is the absolute temperature, C_πis metrics. The ILs reported in this paper have been confirmed to

the molar concentration of the surfactant in the aqueous be SAILs, showing a wide variety of properties strictly depen-

phase at a surface pressure of π, and ω is the number of moles dent on the side chain length. This includes their bacterial

of water per liter of water (55.5 mol L⁻¹, at 25 °C). The values and fungal toxicities, biodegradation (C₂vs. C₁₀) and aggrega-

for ΔG_a^°_dand ΔG_a^°_d=A_minfound according to Rosen^39,98are pro- tion properties.

vided in Table 5. The ΔG_a^°_d=A_minis linearly correlated with the

The short chain length (C₂and C₄) and medium chain

chain length (C_n) for the whole series of the SAILs, from C₂to length (C₆and C₈) SAILs generally show low toxicity against

C₁₆(Fig. S2a†), whereas the dependence of log MIC₉₅vs. the microorganisms screened. The MIC₉₅decreases with the

ΔG^°_ad=A_minis linear only for the chain length up to C₁₄–C₁₆; see elongation of the chain length to reach the lowest value

the data for SAILs 17–24 against S. aureus in Fig. S2b.† One of (respectively, the highest levels of bacterial and fungal toxici-

the possible reasons authors³⁹point out is that the adsorption ties) for the C₁₀to C₁₄derivatives, irrespective of the cationic

data at the air/aqueous solution interface might not be repre- head group structure. Depending on the bacterial strain and

sentative of the adsorption at the cell membrane. Even taking type (Gram-positive or Gram-negative), the cut-oﬀ eﬀect

into account this factor, the longest chain length members of appears when the ester side chain is C₁₄to C₁₆. This phenom-

the series (C₁₄and C₁₆) can be considered with precaution as enon is connected to the adsorption and aggregation (CMC)

the green surfactants, in spite of lower microbial toxicity, as properties of the SAILs studied.

compared to their C₁₀and C₁₂counterparts. The high bac-

Evaluating the eﬀect of adding the amino acid linker with a

terial/fungal toxicity observed for the moderately long chain lipophilic side group has resulted in the finding that the

C₁₀and C₁₂SAILs makes it diﬃcult to label them ‘green’, phenyl moiety of ^L-phenylalanine contributes to the self-

although a key finding is that their surface activity (in terms of assembling properties. A comprehensive study of colloid pro-

the CMC, in essence) is comparable to that of conventional perties allows us to conclude that the interplay of dispersion

cationic surfactants with C₁₆–C₁₈chain lengths (those with a interactions of the phenyl ring in addition to the side chain

low CMC but a high T_Krestricting their practical use in hydrophobicity enables us to rank the novel SAILs as remark-

suﬃcient concentrations). Thus, these longer chain SAILs can able compared to other surfactants. The CMC values for the

be considered as greener disinfectants with relatively mild SAILs reported in this study are significantly (up to 10 times)

action and potential biodegradability.

lower than those for conventional surfactants with the same

The high microbial toxicity of the C₁₀SAILs (Table 4) may lengths of the side chains. Substituting Phe with Ala in the

be the main factor responsible for their low degradability SAIL molecule (i.e. replacing CH₂Ph with CH₃) results in the

(Table 4). Therefore, the most promising surfactants showing loss of the remarkable self-aggregation properties. At the same

suﬃcient surface activity along with reasonably low toxicity time, the linear plots of the critical micelle concentrations

(and designed for desirably high biodegradability⁵⁰) are the (log CMC) of the SAILs (17–40) demonstrate about the same

medium chain length members of the studied series. The sensitivity to the ester chain length as those of conventional

SAILs with hexyl (19, 27, and 35) and octyl (20, 28, and 36) cationic surfactants or SAILs with no amino acid fragment,

chain lengths demonstrate CMC values comparable to those and the micellar surface is not significantly dependent on the

for C₁₂TAB⁷⁵or C₁₂PyBr⁹⁹(in the case of C₆SAILs) chain length. The adsorption is among the main factors

and C₁₄TABr⁷⁵(in the case of C₈SAILs), which are widely used aﬀecting the toxicity of SAILs with shorter chains whereas

surfactants. In addition, low to moderate toxicities towards micellization is important in the case of the longer chain ILs

microorganisms for these SAILs may lead to their causing the “cut-oﬀ” eﬀect.

biodegradability.

The C₁₀ILs were found to have poor biodegradability in

CBT compared to the mineralisable analogue C₂IL 17. This

unexpected result warranted a detailed investigation of the bio-

degradability of the series of Phe-derived SAILs and is pre-

sented in a separate publication of Kümmerer et al. ⁵⁰

Conclusions

This study presents the first comprehensive study devoted to

The L-phenylalanine derived SAILs with medium chain

designing green surface-active ionic liquids (SAILs) based on lengths (C₆to C₈) can be recommended as the most green

the ‘benign-by-design’ approach for the preparation of minera- ones in the series with CMC values comparable to those for

lizable pyridinium ILs developed recently by Gathergood and C₁₂- and C₁₄-quaternary ammonium surfactants, respectively.

Kümmerer.^48,49Since the employment of identified readily bio- A crucial diﬀerence is that the medium chain SAILs are not

degradable building blocks is an integral part of the targeted very toxic towards the studied bacterial and fungal strains and

design of green SAILs, a prospective direction consists in the are potentially biodegradable (especially pyridinium deriva-

extension of this platform by means of side chain length vari- tives). Therefore, SAILs 19–20, 27–28 and 35–36 can be con-

ation. A series of 24 novel ^L-phenylalanine derived ionic sidered as a favourable group of green alternatives for conven-

liquids with variable lengths of the n-alkyl ester chain (from C₂tional cationic surfactants. These findings can contribute

to C₁₆) and quaternary ammonium (pyridinium, imidazolium, towards designing new eﬃcient amphiphiles with optimized

and cholinium) head group has been prepared, and the syn- antimicrobial activities and employing them as potential envir-

thetic methods have been assessed using green chemistry onmentally benign mineralisable surfactants.

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dilution broth method according to the EUCAST instruc-

tions.^101,102The details of the procedure are described in

the ESI.†

Experimental section

Materials

All chemicals and solvents were purchased from Sigma

Biodegradation was studied using the methods described

Aldrich, Alfa Aesar, or TCI Europe and used without further in the previous papers.^48,49

purification. Deionized water from a Milli-Q system was used

Synthesis of compounds (1–40) and their characterisation

in all sample preparation. Silica gel 60 F254 plates were used data are presented in the ESI.†

for TLC.

Characterization of products

Conﬂicts of interest

Melting points were determined with Stuart SMP40 apparatus

There are no conflicts of interest to declare.

with parameters for the melting point analysis set at a 2 °C per

minute ramp; values are expressed in °C. The HRMS identifi-

cation of compounds was performed on an Agilent 6540 UHD

Accurate-Mass Q-TOF LC/MS G6540A Mass Spectrometer. IR

spectra were collected with a Tensor 27 FT-IR spectrometer.

Optical rotations were measured using an Anton Paar MCP 500

polarimeter in methanol or chloroform at 20 °C and the values

are expressed in degrees. The NMR spectra were recorded on a

Bruker Avance III 400 MHz spectrometer operating at 400 MHz

Acknowledgements

The authors would like to thank the European Union’s 7th

Framework Programme for research, technological develop-

ment and demonstration under grant agreement No. 621364

(TUTIC-Green) and Estonian Research Council grant PUT

1656. The antibacterial and antifungal screening was sup-

ported by the Czech Science Foundation (project no. 18-

17868S). NG, AJ and AK thank the EPA in Ireland for financial

support under grant [2012-WRM-PhD-6]. NG, LP and AJ also

thank COST Actions CM1206 and TD1203 for their financial

contributions (STSMs) to the study. LP thanks MINECO of

Spain (CTQ2017-88948-P) for funding. Authors thank Maxime

Naudé for surface tension measurements.

1

for H-NMR and 101 MHz for ¹³C-NMR. Samples were run in

deuterated chloroform (CDCl₃) or deuterated dimethyl sulfox-

ide (DMSO-d₆) where appropriate.

Conductivity measurements

The conductivity was measured using a Lab 875 pH set

equipped with an LF413 T conductivity measuring cell at 25 °C

maintained with a cooling thermostat Huber Ministat 125. The

cell constant was calibrated with NaCl solutions of known con-

ductivities and was used for calculating the conductivity of the

surfactant solution. The conductivity of water was subtracted

from the measured conductivity of each sample.

Measurements were carried out at increasing concentrations to

minimise errors from possible contamination of the electrode.

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Article Doi

Synthesis, self-assembly, bacterial and fungal toxicity, and preliminary biodegradation studies of a series of l-phenylalanine-derived surface-active ionic liquids

DOI: 10.1039/c9gc00030e

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