Synthesis of a series of amino acid derived ionic liquids and tertiary amines: Green chemistry metrics including microbial toxicity and preliminary biodegradation data analysis

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Synthesis of a series of amino acid derived ionic

liquids and tertiary amines: green chemistry

metrics including microbial toxicity and

preliminary biodegradation data analysis†

Cite this: DOI: 10.1039/c6gc00415f

Andrew Jordan,^aAnnette Haiß,^bMarcel Spulak,^cYevgen Karpichev,^d

Klaus Kümmerer*^band Nicholas Gathergood*^d

A series of L-phenylalanine ionic liquids (ILs), L-tyrosine ILs, tertiary amino analogues and proposed trans-

formation products (PTPs) have been synthesised. Antimicrobial toxicity data, as part of the green chem-

istry metrics evaluation and to supplement preliminary biodegradation studies, was determined for ILs,

tertiary amino analogues and PTPs. Good to very good overall yields (76 to 87%) for the synthesis of 6 ILs

from ^L-phenylalanine were achieved. A C₂-symmetric IL was prepared from TMS-imidazole in a one-pot

two-step method in excellent yield (91%). Synthesis of the ^L-tyrosine IL derivatives utilised a simple pro-

tection group strategy by using an extra equivalent of the bromoacetyl bromide reagent. Improvements in

the synthesis of the α-bromoamide alkylating reagent from ^L-phenylalanine were achieved, directed by

green chemistry metric analysis. A solvent switch from dichloromethane to THF is described, however the

yield was 15% lower. Antimicrobial activity testing of ^L-phenylalanine ILs, L-tyrosine ILs, tertiary amino ana-

logues and PTPs, against 8 bacteria and 12 fungi strains, showed that no compound had a high anti-

microbial activity, apart from an ^L-proline analogue. In this exceptional case, the highest toxicity (IC₉₅

=

125 and 250 µM) was observed towards the two Gram positive strains Staphylococcus aureus and Staphy-

lococcus epidermidis respectively. High antimicrobial activity was not found for the other bacteria or

fungi strains screened. The limitations of the antimicrobial activity study is discussed in relation to SAR

studies. Preliminary analysis of biodegradation data (Closed Bottle Test, OECD 301D) is presented. The

pyridinium IL derivative is the preferred green IL of the series based on synthesis, toxicity and biodegrada-

tion considerations. This work is a joint study with Kümmerer and co-workers and the PTPs were selected

as target compounds based on concurrent biodegradation studies by the Kümmerer group. For the com-

prehensive biodegradation and transformation product analysis see the accompanying paper.

Received 12th February 2016,

Accepted 7th June 2016

DOI: 10.1039/c6gc00415f

www.rsc.org/greenchem

eﬃcient synthesis, utilising lower toxicity chemicals (e.g.

Introduction

reagents,^2,3solvents⁴and catalysts⁵), avoiding non-biodegrad-

The overarching concept of the 12 Principles of Green Chem- able compounds, an iterative approach can ultimately lead to

istry is to promote the development and application of safer significant advances in the field.^6–8A class of chemicals which

chemicals, which will therefore lead to a reduced impact on has been extensively studied as safer and greener replacements

the environment.¹Whether this is due to a cleaner more for a wide range of chemicals is ionic liquids (ILs).^9–11ILs have

been previously defined as salts comprised of a cation and an

anion with melting points <100 °C, however in recent years

this arbitrary threshold is less often cited, and classification

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

^bInstitute of Sustainable and Environmental Chemistry, University of Lüneburg,

can be based on chemical structure, functionality, and appli-

Scharnhorststraße 1, D-21335 Lüneburg, Germany.

cations.¹²An attractive feature of ILs is the ability to fine-tune

E-mail: Klaus.Kuemmerer@uni.leuphana.de

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

the structure, to tailor the properties for

a particular

purpose.^9–11The availability of a series of closely related struc-

tures has led to many research teams determining the

toxicity^13–15and ecotoxicity^16–19of ILs, performing structure

activity relationship (SAR) studies^13,20,21and creating quanti-

tative structure activity relationship (QSAR) models.^21–23Bio-

Heyrovského 1203, Hradec Kralove, Czech Republic, CZ500 05

^dDepartment of Chemistry, Chair of Green Chemistry, Tallinn University of

Technology, Akadeemia tee 15, 12618 Tallinn, Estonia.

E-mail: nicholas.gathergood@ttu.ee

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

c6gc00415f

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city studies and biodegradation analysis. A detailed biodegra-

dation and transformation product evaluation is reported in

the accompanying paper by Haiß et al.³⁹

Results and discussion

The goal of this investigation was to design and develop a green

synthesis of phenylalanine ethyl ester derived ILs in tandem

with toxicity and biodegradation studies. Working closely with

Kümmerer’s group from the outset, concurrent synthesis, green

chemistry metrics, toxicity and biodegradation studies⁴⁰were

completed. Through this collaboration, improvements in the

synthesis of the novel target ILs are potentially attainable, at the

same time as their toxicity and biodegradation assessment. The

detailed biodegradation evaluation of compounds presented

herein is presented in the previous paper.³⁹

Fig. 1 One of the ﬁrst ILs to pass the CO₂Headspace test (ISO 14592)

and be classed as readily biodegradable.³⁸

degradation of ILs has also been investigated (Fig. 1, 1),^24–26

although not as extensively as toxicity studies.^14,20,27Toxicity

and biodegradation data however is only part of a green chem-

istry assessment of a chemical (e.g. IL).¹The synthetic route to

prepare the compound can also be considered, and a plethora

of parameters can be determined,^28–30with the objective of

reducing the environmental impact of the process. In 2013,

Gathergood and Connon published a combined microbial toxi-

city, biodegradation, green chemistry metrics and catalyst per-

formance study of a series of ILs.^31–33One of the findings from

this work was that the ILs studied containing amide group(s)

exhibited very low biodegradation in the CO₂Headspace test.

This was in agreement with previous results for amide contain-

ing ILs,^33–35with an exception being 2 and 3 reported by Gath-

ergood in 2012, (Fig. 2).¹⁵This is in stark contrast to ILs

containing an ester functional group, where facile breakdown

to the carboxylic acid and conversion of the alcohol to CO₂is

widely reported.^25,36However, even with these ester containing

ILs, the carboxylic acid transformation product has been shown

to persist after a 28 day CBT.³⁷The development of minerali-

sable ILs is a significant and challenging research front.

The series of ILs 4–24, Fig. 3–5, were designed with respect

to Boethling’s rules of thumb for designing biodegradable

compounds,³⁶12 Principles of Green Chemistry,¹and the

preceding generations of IL research conducted within the

Gathergood research group.^{15,38,41–44}A selection of common IL

cation headgroups were chosen allowing this study to demon-

strate the eﬀect of headgroup on IL synthesis, toxicity and bio-

degradability, (Fig. 3, 4,¹⁵5–15). L-tyrosine ILs analogues were

prepared to examine the eﬀects of the presence of a phenolic

–OH on the amino acid sidechain, (Fig. 4, 16–20). To investi-

gate how the –OH functionality would aﬀect IL synthesis, tox-

icity and biodegradability. It is known that the enzyme

phenylalanine hydroxylase (PheOH) converts phenylalanine

into tyrosine by an oxidative insertion mechanism⁴²and there

exists the possibility that the phenylalanine ILs could be

converted in vivo to the tyrosine derivatives before undergoing

biodegradation. The presence of the hydroxyl group is also

expected to increase water solubility of the tyrosine IL com-

pared to the phenylalanine analogue, and microbial activity can

be compared. Lastly, a series of tertiary amino derivatives,

(Fig. 5, 21–24) were prepared to investigate the influence of a

positive charge due to a quaternary nitrogen on the compounds

examined. The ester chain length (ethyl) and counter-ion

(bromide) were not varied in this study. These were selected as

the focus of this study was the nitrogen headgroup of the IL.

Furthermore, the information obtained from this investigation

is expected to aid the rational design of “next generation” ILs

based on any observed structure activity relationships.

Herein the authors describe their progress towards develop-

ing a green synthesis for biodegradable and mineralisable ILs.

The investigation includes synthesis of a series of analogues of

2, green chemistry metrics analysis, preliminary microbial toxi-

A series of ^L-phenylalanine ILs (4–15) were prepared in a

high yielding, three step synthesis. The synthesis is outline

below in Scheme 1 and Table 1.

First, Fischer–Speier esterification⁴⁵of L-phenylalanine

using thionyl chloride and excess ethanol to produce dry HCl

in situ was completed. The hydrochloride salt intermediate

(25·HCl) was neutralised with base and 25⁴⁶isolated in excel-

lent yield. Second, acylation with bromoacetyl bromide gave

the α-bromoamide of ^L-phenyalanine ethyl ester (26) (see

metrics section for optimisation details). Lastly, the α-bromo-

amide alkylating reagent (26) was reacted with an amine (for

Fig. 2 Readily biodegradable amino acid ILs.¹⁵Biodegradation values

assessed by the CO₂Headspace test ISO 14593.

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Fig. 3 Series of L-phenylalanine ILs (4–15) targets. Charged headgroup study.

example, 1-methylimidazole), to yield the target ILs (4–15) nicotinate in diethyl ether at room temperature after 24 h

(Table 1). Compound 4 has been previously prepared by Gath- failed (entry 4). A fair yield (56%) however was attained for 7

ergood et al. in 98% yield using THF as the solvent,¹⁵however by increasing the temperature, extending the reaction time

a slightly lower yield of 4 was obtained using diethyl ether and changing to a more polar solvent, THF (entry 5). A high

(entry 1). Excellent yields for the pyridinium and 3-methoxy- yield of IL 8 from 4-dimethylaminopyridine in diethyl ether

pyridinium ILs 5 and 6 (entries 2 and 3) however were was observed (entry 6). Aliphatic amines N-methylmorpholine

obtained in diethyl ether. Attempts to prepare IL 7 from ethyl and dimethylaminoethanol gave considerably diﬀerent yields

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Fig. 4 Series of L-tyrosine ILs (16–20) targets. Phenol study.

Fig. 5 Series of neutral L-phenylalanine IL analogues (21–24).

for corresponding IL synthesis in diethyl ether. A poor yield were prepared by reductive amination using formaldehyde

(34%) was observed for the morpholinium IL 9 (entry 7), while according to a procedure by Lin et al.,⁴⁸followed by Fischer–

10 was isolated in a quantitative yield (entry 8). C₂-Symmetric Speier esterification (see ESI, Scheme S1†).⁴⁶Proline derivative

IL 11 was prepared by a two-step procedure from N-trimethyl- 31 required ethyl acetate as solvent for synthesis of IL 12 and

silylimidazole.⁴⁷The double alkylation proceeded in excellent 13 in a combined yield of 50% (entry 10 vs. 11). Similar results

yield (91%) if after 24 h in diethyl ether, the solvent was were obtained using the ^D-proline analogue 33 (entry 12). In

changed to ethyl acetate and heated at reflux for 24 h (entry 9). both cases (entries 11 and 12) a 5 : 2 ratio of epimers was

N-Methyl proline ethyl esters (31 and 33, entries 10 and 11) established in the crude product by ¹H NMR. However, iso-

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Scheme 1 Synthesis of 26 from L-phenylalanine.

Table 1 Alkylation of tertiary amines with 26 to give ILs (4–15)

Entry

NR₁R₂R₃

Reaction conditions^a

IL

Yield^b(%)

1

2

3

4

5

6

7

8

9

N-Methylimidazole

Pyridine

3-Methoxypyridine

Ethyl nicotinate

4-Dimethylaminopyridine

N-Methylmorpholine

Dimethylaminoethanol

N-Trimethylsilylimidazole

N-Methyl-^L-proline-ethyl ester (31)

A

B

A

C

A

4

5

6

7

8

9

10

11

12

13

12

13

14

15

89

98

96

0^c

56

88

34

99

91

0^c

10

0^c

11

12

N-Methyl-L-proline-ethyl ester (31)

N-Methyl-^D-proline-ethyl ester (33)

D

37

13

25

34

^aA: Diethyl ether, rt, 24 h; B: THF, reflux, 48 h; C: diethyl ether, rt, 24 h then ethyl acetate, reflux, 24 h, 2 eq. 26; D ethyl acetate, reflux, 48 h.

^bSee Table 4 for more detailed green chemistry metric analysis. ^cNo product detected by TLC 10% MeOH : DCM.

lated yields of each epimer after purification by column occurred after 12 h (by TLC), and after acid hydrolysis with

chromatography are shown in Table 1. The absolute stereo- HCl in ethanol, 29 was obtained in an overall yield of 54%.

chemistry of prolinium derivatives (12, 13 and 14, 15) was Potential transesterification problems were avoided by select-

determined by 2-dimensional NMR by utilising the nuclear ing ethanol as the solvent. The final alkylation step in reflux-

overhauser eﬀect (i.e. a NOESY experiment). Very good overall ing ethyl acetate gave ILs 16–20 in good to quantitative yields,

yields (76 to 87%) for the synthesis of ILs 4, 5, 6, 8, 10 and 11 with the highest yields obtained for ILs 17 and 18. IL 19

from ^L-phenylalanine were achieved.

required purification by flash chromatography and a lower iso-

L-Tyrosine ILs (16–20) were synthesised according to the lated yield (71%) was obtained.

route outlined in Scheme 2. Initial attempts to selectively

The neutral derivatives (21–24) were synthesised from alky-

acylate the amino group of 27 with bromoacetyl bromide were lating reagent (26) in poor to fair yields (34–56%). We propose

unsuccessful. While a range of phenol protection group strat- that this is due to the higher solubility of the products in

egies (for example, silyl ethers) were considered, the additional either diethyl ether or THF solvent. This eﬀect was also seen

steps required were deemed undesirable. Due to the observed with the tyrosine series above. The compounds 21–24 were

ease of acylation of the phenol group, and expected higher purified by column chromatography and all isolated as liquids

reactivity of the formed phenolic ester vs. amide to hydrolysis, at room temperature (Table 2).

a double acylation of 26 route was proposed. We were pleased

Very good to quantitative yields were obtained for the final

to find that quantitative conversion of 27 to intermediate 28 alkylation step in the synthesis of ILs 4, 5, 6, 8, 10, 11, 17 and

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Scheme 2 General synthetic route employed for L-tyrosine bromide ILs (16–20).

Table 2 Alkylation of secondary amines with 26 to give ILs (21–24)

Entry

HNR₁R₂

Solvent

IL

Yield (%)

1

2

3

4

N-Trimethylsilylimidazole

N-Methylaminoethanol

Morpholine

Diethyl ether

THF^a

21

22

23

24

55

34

56

53

L-Proline-ethyl ester·HCl^b

THF

^aReflux. ^bIn situ neutralisation with Na₂CO₃.

18. The product precipitating from the reaction solvent signifi- nium ring gave an increase in melting point to 53–55 °C (17 vs.

cantly increases the yield compared to examples where chrom- 19) and significantly higher melting points were observed for

atography is required.

the 3-methoxypyridinium derivative 18 117–119 °C and the

A wide range of melting points were also observed for the cholinium derivative 20 141–143 °C. The presence of the –OH

ILs synthesised. Under the classical definition of an IL existing group on the tyrosine ring greatly reduces the melting points

as a liquid below 100 °C all phenylalanine examples from of several of the ILs (16, 17 and 19) when compared to their

Fig. 3 (4–15) and Fig. 5 (21–24) with the exception of (8, 9 and L-phenylalanine counterparts. A decrease of 40 °C was observed

11) conform, though (5 and 10) have melting points close to for the imidazolium IL analogues (16 vs. 4), 68 °C for the pyri-

100 °C. A broad range of melting points (30–32 to 141–143 °C) dinium examples (17 vs. 5), and a slight decrease in melting

were also observed for the tyrosine derivatives with the imida- point of 7 °C for ethyl nicotinium analogues (19 vs. 7). Excep-

zolium IL 16 38–40 °C, and pyridinium IL 17 30–32 °C, the tions to this trend are the tyrosine salts, 3-methoxypyridinium

lowest recorded. Introduction of an ester group to the pyridi- IL 18 where an increase in melting point of 45 °C was observed

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and cholinium IL 20, with an increase of 40 °C when com- detected by TLC or ¹H NMR. The outcome was a reaction

pared to their phenylalanine counterparts,

respectively.

6

and 10 mixture that could be purified by filtration to remove the

Na₂CO₃followed by aqueous bicarbonate solution washings of

the organic layer to remove any HBr and bromoacetic acid.

Removal of the organic solvent by rotary evaporator furnished

Green chemistry metrics

the desired alkylating reagent (26) in high yield (89%) as a

white solid with no further purification required (entry 3). Other

solvents investigated as green replacements for DCM included

ethyl acetate and THF. A mixture of decomposition products

was observed using ethyl acetate, although a good yield was

obtained of 26 (74%, entry 4) using THF. This solvent oﬀers a

viable non-chlorinated solvent alternative to DCM, albeit with a

lower isolated yield (89% vs. 74%, entry 2 vs. entry 4).

The following metrics were used to evaluate the synthetic

methods used in producing the esters, alkylating reagents and

ILs: atom economy,⁵⁰E-factor,⁷Andraos reaction mass

eﬃciency,⁵¹GSK reaction mass eﬃciency,⁵²mass intensity,⁵³

solvent intensity,⁵³stoichiometric factor²⁸and material recov-

ery parameter.⁵⁴

We decided to adopt a two tier approach with the aim of devel-

oping a greener synthesis for the ILs described herein. First, a

preliminary evaluation of solvent selection would be under-

taken. This would be based guidelines proposed by Prat et al.⁴

and prioritise replacement of toxic solvents and reduce the

volume used while increasing yields. Second, a more detailed

metric analysis would be completed for all reaction steps

reported herein. This tiered approach to green chemistry

metrics has been proposed before by Clark et al.³⁰

Comparing the reaction solvents used and yields attained

in each of the three main steps in the IL synthesis: (1) ester for-

mation (ethanol), (2) α-bromoamide formation (DCM) and (3)

alkylation (diethyl ether, THF and ethyl acetate), the second

step was selected for optimisation.

It can be seen from the generated metrics, Table 4 and

ESI,† that the final step in the synthesis of nearly all the ILs

had a 100% atom economy, IL 11 (80%) being the only excep-

tion. This is due to the use of the trimethylsilyl protection

group. Atom economy, however, does not take reaction stoi-

chiometry, solvent use or reagents not incorporated into the

final product (e.g. auxiliary bases) into account hence

additional metrics must be calculated. The stoichiometric

factor for all of the ILs ranges from 0.92–1.00, thus illustrating

that less than 10% excess of any one reagent was used in each

of the reactions. The stoichiometry employed in the synthesis

of the ILs leads to a high GSK RME, but only when the reaction

yields are high. Yields above 90% for the ILs show good GSK

RME. Thus, loss in GSK RME for ILs (6, 9 and 12–15) is due to

lower yields.

The eﬀect that column chromatography has on the green-

ness of a synthetic pathway is highlighted when the E-factor,

solvent intensity and mass intensity of a process is calculated.

For ILs 4–6, 8–11, 17, 18 and 20, E-factors of 33–150 were

observed (IL 16 has an E-factor of 294 even though purification

by chromatography was not required and is due to the lower

yield of 83%). However, when IL purification involved column

chromatography (7, 12–15 and 19), the E-factors rose consider-

ably (e.g. IL 13, E-factor 4088) and the greenness of the reac-

tion based on this metric can be considered considerably

lower compared to chromatography free trituration methods

(e.g. IL 11, E-factor 33). The solvent required for column

chromatography also aﬀects the mass and solvent intensity

The synthesis outlined in Scheme 1 is a modification of

that employed by Coleman and Gathergood and presents an

overall improvement in the formation of the alkylating reagent

26 from ^L-phenylalanine.¹⁵The original method for the syn-

thesis of 26 in the literature describes the reaction of amino

acid ester hydrochloride salt with trimethylamine (Table 3,

entry 1), followed by addition of bromoacetyl bromide at

−78 °C. This method results in the formation of coloured

impurities if using TEA, possibly due to the Menschutkin reac-

tion⁴⁹or triethylamine promotion of trace by-products having

partial solubility in the organic layer that was not removed

during an aqueous workup step. Impurities were also present

if a heterogeneous base such as Na₂CO₃was employed (entry

2). The neutralisation process of the ester hydrochloride salt

we suggest was too slow, thus leading to the formation of side

products. Purification of 26 by column chromatography was

therefore necessary before progressing to the final alkylation

step (entries 1 and 2). Using these methods 26 was isolated in

yields of 61 and 68% (entries 1 and 2).¹⁵

The synthesis route was modified by neutralising the amino

acid ester·HCl to the free base form, 25, before being used in

the reaction. An excess of Na₂CO₃was used as an auxiliary base

to remove the HBr formed during the reaction. This pre-neutral-

isation step allowed the primary amine group of the amino acid

ester to rapidly and more eﬃciently react with the bromoacetyl

bromide. This significantly reduced the formation of unwanted

side products. The reaction could also be run at room tempera-

ture (cf. −78 °C, entry 1)¹⁵with no formation of side products

Table 3 Optimisation of synthesis of 26

Entry

Amine

Reaction solvent

Reaction time (h)

Base

Column chromatography required

Yield 26 (%)

1

2

3

4

25·HCl

25

DCM

THF

5

3

5

TEA

Y

N

68¹⁵

61

89

Na₂CO₃

25

74

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Table 4 Summary of green chemistry metrics for all compounds synthesised (see ESI for worksheet and calculations)

Atom

Sheldon

GSK

Andraos

RME⁵¹

Mass

Solvent

Stoichiometric

factor²⁸

Material recovery

Yield

(%)

Compound

economy⁵⁰

E-factor⁷

RME⁵²

intensity⁵³

parameter⁵⁴

L-Phe ILs

4

5

6

7

8

9

10

11

12

13

14

15

L-Tyr ILs

16

17

18

19

20

100%

80%

100%

96.7

93.4

108.8

1081.5

120.7

126.5

88.1

0.830

0.964

0.689

0.523

0.812

0.335

0.901

0.718

0.335

0.123

0.254

0.343

0.010

0.011

0.009

0.001

0.008

0.011

0.029

0.001

97.7

94.4

109.8

1082.5

121.7

127.5

89.1

96.5

93.3

108.3

1080.6

120.5

124.5

88.0

0.920

0.991

0.922

0.934

0.918

0.991

0.917

0.986

0.921

1.000

0.012

0.011

0.013

0.002

0.010

0.023

0.012

0.041

0.002

0.004

90

98

75

56

89

34

98

91

37

13

25

34

33.0

34.0

32.6

1416.6

3869.1

1119.0

828.2

1417.6

3870.1

1120.0

829.2

1414.7

3862.0

1116.1

826.2

100%

294.0

117.1

120.7

420.3

150.7

0.819

0.980

0.971

0.699

0.773

0.003

0.008

0.002

0.007

295.0

118.1

121.7

421.3

151.7

293.8

117.1

120.7

419.9

150.4

0.979

0.982

0.986

0.985

0.004

0.009

0.008

0.003

0.009

84

99

71

79

Neutral derivatives

21

22

23

24

66%

1035.4

2754.1

476.6

0.288

0.242

0.433

0.266

0.001

0.002

0.001

1036.4

2755.1

477.6

1032.9

2750.9

474.6

0.783

0.923

0.958

0.946

0.003

0.001

0.005

56

33

57

53

79%

80%

53%

770.3

771.3

767.6

Alkylating reagents

26^a

26^b

26^c

26^d

29

59%

705.1

84.2

10.1

36.7

55.1

0.307

0.297

0.470

0.428

0.281

0.001

0.012

0.090

0.027

0.018

709.4

85.2

11.1

37.7

56.1

555.4

52.2

7.4

7.3

17.9

0.904

1.000

0.843

0.918

0.873

0.005

0.040

0.192

0.062

0.063

64

61

89

74

54

49%

63%

50%

20.8

3.7

14.6

29.5

5.6

0.425

0.620

0.372

0.231

0.610

0.046

0.211

0.064

0.033

0.150

21.8

4.7

15.6

30.5

6.6

11.8

3.1

12.0

24.5

5.0

0.735

1.000

0.959

0.108

0.340

0.172

0.142

0.247

99

87

70

43

99

Reductive alkylation

30

32

89%

11.6

15.0

0.837

0.309

0.080

0.062

12.6

16.0

11.4

12.8

0.980

0.093

0.200

98

36

For conditions see ^aTable 3, entry 1; ^bTable 3, entry 2; ^cTable 3, entry 3; ^dTable 3, entry 4.

metrics in a similar manner and can be seen for ILs 7, 12–15 a generally lower E-factor than calculated for the ILs. The mod-

and 19 (Table 4). Material recovery parameters for all ILs are erate atom economy is due to the use of bromacetyl bromide

relatively low and this is due to only the product mass contri- which is not fully incorporated into the product. HBr is a by-

buting to the material recovery and all other materials are in product of the substitution reaction. The presence of an auxili-

eﬀect waste.

ary base also reduces the atom economy. The lower E-factor,

Andraos RME is low for all ILs as it takes into account all which is desirable, is due to the minimum amount of solvent

solvent used in the IL trituration steps and all chemicals that employed during the reaction and purification steps when

are not incorporated into the product are considered waste. compared to the ILs which required multiple triturations.

The diethyl ether and chromatography solvent should there-

Amino acid esterification reactions (25, 27, 31, 33 and 34)

fore be recycled in future studies or if this process were to be all proceeded with excellent (e.g. 25, 3.7) to moderate E-factors

scaled up in order to reduce the mass/solvent intensity, (e.g. 31, 40.3) and low solvent and mass intensities. The atom

E-factor, material recovery parameter and Andraos RME.

economies are aﬀected by the use of stoichiometric quantities

The metrics generated for the neutral derivatives (21–24) of SOCl₂during the esterification reaction and ranged between

are less desirable as lower yields and a higher consumption of 53–90%.

solvent due to the purification by column chromatography was

required. The GSK RME decreases (0.266–0.432) and E-factor mediate 30 can be considered as one of the greenest methods

increases (476.6–2754.1). employed during the synthesis reported here, with high atom

Lastly, the reductive alkylation of ^L-proline to give inter-

The alkylating reagents (26 and 29) were both synthesised economy (89%), low E-factor (11.6), excellent reaction mass

with moderate atom economy (63% and 50% respectively) and eﬃciency (0.837) and low solvent and mass intensity (12.6 and

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11.4 respectively). The counterpart to this reaction, 32, is less cular weight bases would result in fewer tons of exhausted

green due to the lower yield overall and thus lower GSK RME. base and resulting salts over a longer period. Solvent recycling

Atom economy remains unchanged and the E-factor is only from all aspects of the synthesis reported would dramatically

slightly increased to 15.0.

improve the E-factor as the solvents would obviously no longer

During the reaction condition optimisation for alkylating be considered as waste. Material recovery parameters for all

reagent 26 (see Table 3 for optimisation), metric analysis was steps are low due to the inevitable waste produced during pro-

also conducted on each of the reaction conditions (entries cesses that do not have a 100% atom economy. Solvent re-

1–4). The original conditions reported by Coleman et al.¹⁵for cycling would also aid in improving the material recovery

the formation of 26 involving the use of two equivalents of tri- parameter. Solvent intensity can be improved if reducing reac-

ethylamine and purification by column chromatography led to tion solvent can be tolerated while achieving the same yield.

a poor atom economy of 50% and an E-factor of 705.1. The Mass intensity can also be similarly improved.

solvent and mass intensities of the reaction are similarly

Microbial screening

aﬀected. Reaction mass eﬃciency was calculated to 0.307 due

to the moderate yield and excess trimethylamine required in

the reaction. When the TEA was replaced with Na₂CO₃(entry

2), the atom economy was reduced to 49% due to the higher

molecular weight of Na₂CO₃compared to that of triethyl-

amine. The reaction mass eﬃciency is also reduced to 0.297.

The optimised conditions in DCM (entry 3) and THF (entry 4)

both show improved atom economies of 63% and good

E-factors (DCM 10.1, THF – 36.7). The GSK RME is also the

highest out of all the trialled conditions as shown in entry 3

(0.470). Solvent and mass intensities are also low for the pro-

cedures described in entry 3 and 4 due to the elimination of

chromatographic purification.

The outlook from the optimisation metrics is that elimin-

ation of column chromatography is essential for low E-factor

and high GSK and Andraos RME. Avoiding the use of reagents

in excess, if possible, will improve the RME. The use of lower

molecular weight bases can improve atom economy, however it

is important to note that the number of moles of base required

will remain the same. This type of optimisation is more rele-

vant for large scale production where the use of lower mole-

Microbial toxicity screening was performed to principally

identify any ILs with high activity. This approach enables us to

prioritise ILs which do not have high antimicrobial activity to

be evaluated first for biodegradation studies. One possibility is

that ILs with high antimicrobial activities may inhibit micro-

organisms required for biodegradation to occur. This microbial

toxicity assessment represents a starting point for a more com-

prehensive investigation as part of a study to design safer ILs.

Antibacterial activity results for 4–15 are shown in Table 5,

16–20 in Table 6 and 21–24 in Table 7. We were pleased

to observe that all compounds 4–24 did not show high anti-

bacterial activity, except 24. In this exceptional case, the

highest toxicity (IC₉₅= 125 and 250 µM) were observed towards

the two gram positive strains Staphylococcus aureus (SA) and

Staphylococcus epidermidis (SE) respectively. The toxicity test

method is only validated up to a concentration of 2000 µM

and suits our purpose to identify high activity compounds. We

submit that IC values determined in the range 125–1000 µM,

although do not represent high antimicrobial activity, are sig-

nificant. While IC₉₅values lower than 2000 µM or the solubi-

Table 5 Antibacterial activity results obtained for L-phenylalanine ILs 4–15

IL IC₉₅(µM)

Microorganism^a

Time (h)

4

5

6

7

8

9

10

11

12

13

14

15

Gram positive

SA

24

48

24

48

24

48

24

48

2000

>2000

2000

>2000

>500

1000

>1000

>500

2000

>2000

500

>2000

2000

>2000

1000

>1000

1000

>1000

>2000

1000

>2000

1000

>2000

500

1000

500

1000

MRSA

SE

2000

>2000

EF

>2000

>1000

>2000

Gram negative

EC

24

48

24

48

24

48

24

48

>2000

>500

>1000

>500

>2000

2000

>2000

2000

>2000

>1000

>2000

KP

KP-E

PA

2000

^aSA, Staphylococcus aureus; MRSA, Staphylococcus aureus MRSA; SE, Staphylococcus epidermidis; EF, Enterococcus sp.; EC, Escherichia coli; KP, Kleb-

siella pneumoniae; KP-E, Klebsiella pneumoniae, PA, Pseudomonas aeruginosa.

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Table 6 Antibacterial activity of L-tyrosine ILs 16–20

IL IC₉₅(µM)

Microorganism^a

Time (h)

16

17

18

19

20

SA

24

48

24

48

24

48

24

48

24

48

24

48

24

48

24

48

1000

2000

>2000

500

1000

>2000

2000

>2000

2000

>2000

1000

MRSA

SE

2000

>2000

2000

EF

>2000

EC

KP

KP-E

PA

^aSA, Staphylococcus aureus; MRSA, Staphylococcus aureus MRSA; SE, Staphylococcus epidermidis; EF, Enterococcus sp.; EC, Escherichia coli; KP, Kleb-

siella pneumoniae; KP-E, Klebsiella pneumoniae, PA, Pseudomonas aeruginosa.

lity limit (e.g. 16) are shown in Tables 5–7, no significant SAR 4 gram positive strains is clearly apparent. In addition, all com-

comparisons can be made due to the similarity of values. It pounds 4–24 do not show high antibacterial activity to the four

would be ill advised to draw conclusions based on comparing gram negative strains.

IC₉₅values of 500 µM and 2000 µM for selected strains and

Antifungal activity results for 4–24 are shown in Table 8.

undertaking a SAR study based on bacterial toxicity. Once Although compounds 9, 11, 12 and 16 have IC₅₀and IC₈₀

again the only exception would be with compound 24, where values of 2000 µM for some fungal strains, the data overall

there is nearly an order of magnitude diﬀerence between the supports the finding that 4–24 do not have high antifungal

IC₉₅values for SA and the 4 gram negative strains. This, activity. Based on the antibacterial and antifungal activity

however, represents an isolated trend in the context of all the results all compounds were selected for biodegradation

data presented in Tables 5–7. However, the general trend that studies. Compound 24 was included in the biodegradation

compounds 4–23 do not show high antibacterial activity to the study, despite having the highest antimicrobial toxicity

recorded. This decision was based on the combined antibac-

terial and antifungal data, where high toxicity was only

observed for 4 gram positive bacteria strains and not for

Table 7 Antibacterial activity results obtained for neutral amino com-

4 gram negative bacteria strains, 9 yeasts and 3 filamentous

pounds 21–24

fungi strains (Table 8).

IL IC₉₅(µM)

When compared to microbial screening on amino acid

derived ILs previously carried out by Coleman et al. the most

active antifungal ILs were comprised of ^L-valine (35 and 36),

Microorganism^a

Time (h)

21

22

23

24

SA

24

48

24

48

24

48

24

48

24

48

24

48

24

48

24

48

1000

2000

125 (Fig. 6), towards the filamentous fungal strains (AC, TM).¹⁵

125

500

IC₅₀values as low as 62.5 µM were determined for L-valine

ethyl ester analogue 35.

MRSA

SE

1000

2000

1000

500

>2000

250

>1000

Compounds presented within this publication (4–24) were

determined to be less toxic towards the fungal strains

screened. We propose that ^L-phenylalanine is a preferable

EF

>2000

>1000

EC

>1000 amino acid when considering the synthesis of low toxicity

>1000

amino acid ILs.

KP

>1000

KP-E

PA

Preliminary biodegradation studies of ionic liquids and

tertiary amino analogues

The biodegradation screening was carried out according to the

Closed Bottle test (CBT).^40,55,57As can be seen from Table 9, of

the ILs screened, imidazolium IL 4 did not reach the 60% bio-

degradation threshold required to pass the CBT. Pyridinium IL 5,

^aSA, Staphylococcus aureus; MRSA, Staphylococcus aureus MRSA; SE,

Staphylococcus epidermidis; EF, Enterococcus sp.; EC, Escherichia coli;

KP, Klebsiella pneumoniae; KP-E, Klebsiella pneumoniae; PA, Pseudomo-

nas aeruginosa.

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Table 8 Antifungal activity results for L-phenylalanine, L-tyrosine and 3° amine compounds 4–24

IL MIC^b(µM)

Microorganism^a

Time (h)

4–6, 10, 13–15, 17–23

7, 24

8

9

11

12

16

CA1

CA2

CP

24

48

24

48

24

48

24

48

24

48

24

48

24

48

24

48

24

48

24

48

24

48

72

120

>2000

>1000

>500

>2000

2000

>2000

2000

>2000

2000

>2000

2000

>2000

2000

>2000

CK1

CK2

CT

>2000

2000

CG

CL

>2000

2000

>2000

2000

TA

>2000

2000

AF

>2000

2000

>2000

2000

AC

TM

2000

>2000

^aCA1, Candida albicans; CA2, Candida albicans; CP, Candida parapsilosis; CK1, Candida krusei; CK2, Candida krusei; CT, Candida tropicalis; CG,

Candida glabrata; CL, Candida lusitaniae; TA, Trichosporon asahii; AF, Aspergillus fumigatus; AC, Absidia corymbifera; TM, Trichophyton mentagro-

phytes. ^bIC₅₀values were assessed for AF, AC and TM and IC₈₀for all other strains.

Table 9 Biodegradability and percentage carbon distribution of com-

pounds 4–24, colour coded according to a traﬃc light classiﬁcation³³

Fig. 6 Previously reported amino acid ILs with antifungal activity (IC₅₀

)

by Coleman et al.¹⁵

attained the highest biodegradation of 63%, however cannot

be considered readily biodegradable under OECD guidelines

as biodegradation took more than 10 days after degradation

reached 10% ThOD.^56,57This still represents a significant

breakthrough for high biodegradability of halide IL salts. The

addition of an ether group at the 3-position to give the 3-meth-

oxypyridinium IL 6 reduced the biodegradability to 52%. A

similar observation was made for the nicotinate IL 7 and

DMAP derivative 8, although the decrease to 32% and 33%

respectively is more significant. Pyridinium ILs substituted at

the 3 position with ester groups are known to have good bio-

degradation properties^24,26and our result is an exception to

this trend. The C₂symmetric imidazolium IL 11 was also

shown to be recalcitrant to biodegradation with the lowest

value (17%) recorded.

^aBiodegradation data for 5–8, 10–12, 21, 22 and 24 previously reported

in accompanying paper by Haiß et al.³⁹^bOne of the two bottles was

leaking. ^cNot valid (biodegradation values in the replicates diﬀer more

than 20%).

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The non-aromatic derivatives based on a prolinium (12–15)

scaﬀold gave poor results in the CBT. A narrow range of values

(21–24%) was determined which suggests that for these four

diastereomers, absolute stereochemistry was not a significant

factor.

The presence of an additional –OH group on the ^L-tyrosine

derived ILs 16–20 appears not to promote biodegradation.

When comparing ^L-phenylalanine derivative 4 to L-tyrosine

derivative 16 only a diﬀerence of 7% (40 vs. 47%) was deter-

mined. Indeed, all the ^L-tyrosine analogues of L-phenylalanine

ILs (4–7) and 10 gave similar results within only a 5–13%

diﬀerence, which is not large enough to identify specific

trends. This is in contrast to rules of thumb that phenols are

better biodegradable than phenyl groups as also shown for the

antibiotics amoxicillin (with a phenol ring, mineralizable in

Zahn Wellens test) and piperacillin (even in the ZWT not

biodegradable).⁵⁸

The similar levels of biodegradability for the L-phenyl-

alanine and ^L-tyrosine derivatives suggests that the L-phenyl-

alanine may not be undergoing conversion into ^L-tyrosine

through enzymatic transformations by hydroxylase enzymes.⁵⁹

The L-phenylalanine is instead being degraded directly and

may not be entering the homogentisate pathway.⁶⁰However it

cannot be inferred from the data directly and analysis of the

metabolic products by a suitable LCMS technique to deter-

mine the biodegradation pathways and metabolic products

formed is required.³⁹

None of the tertiary amino derivatives (21–24) CBT results

can be considered a pass (Table 9). For 21 and 24 valid data

based on two replicates was not obtained in this initial bio-

degradation study. For subsequent CBT investigations and

more detailed biodegradation results and discussion see Haiß

et al.³⁹22 and 23 did not reach the 60% threshold, within 28

days, and thus does not pass the CBT. However, when the ter-

tiary amino derivative 22 was compared to the IL analogue it

can be seen that tertiary amino derivative 22 undergoes higher

levels of biodegradation (+22%) than IL analogue 10. The elev-

ated levels of biodegradation suggest that the presence of a

quaternary nitrogen atom, substituted with a methyl group,

can impede the biodegradation process, thus tertiary amine 22

can exhibit higher levels of biodegradation. This observation is

Fig. 7 Carbon atom distribution of amino acid IL (4).

plus the ester is considered, then these predicted levels of bio-

degradation suggest that many of the ILs and amino com-

pounds (e.g. 4, 5, 10, 11, 16, 17, 20 and 21–23) could achieve

>60% biodegradation, based on complete biodegradation of

the aminoacid ester moiety (Table 9). ILs (4–11, and 16–20) are

observed to have levels of biodegradation greater than only

ester degradation but less than complete amino acid ester

degradation, except 5. However, this rough approximation of

carbon distribution and attributable degradation cannot be

fully substantiated without a transformation product analysis

of the CBT. What exactly is breaking down? Is the rate of

biodegradation such that some of the compound in the test

undergoes high levels of mineralisation after a particular bio-

chemical transformation? Is there some rate limiting step that

once completes, allows the IL to biodegrade? Is ester hydro-

lysis occurring preferentially to amide bond hydrolysis or are

both happening at the same time? Is there fully intact parent

compound present in the CBT vessel after the 28 day period?

To answer these questions a detailed biodegradation assess-

ment, including transformation product identification and

analysis of the transformation products oxygen demand is

required, and is published in the accompanying paper.³⁹

Based on the data presented in Table 9 we cannot state with

absolute certainty that a general trend is biodegradation of all

ester groups occurs for 4–24.

Transformation product

in agreement with the literature,⁶but in stark contrast to the To assist in the identification of transformation products

CBT results for 9 vs. 23 (i.e. not a significant diﬀerence; 9, 17% formed during the CBT, synthesis of possible compounds

and 23, 28%) Therefore, a larger sample size of closely related which are consistent with proposed structures based on LCMS

tertiary amino compounds and ILs would need to be examined studies was initiated. While significant progress has been

to identify any general trends.

made developing analytical techniques to identify transform-

An assumption which is often made when interpreting bio- ation products there are several benefits to preparing authentic

degradation data of ILs is that ester group hydrolysis is facile samples by total synthesis. This includes unambiguously con-

and complete conversion (e.g. to CO₂) under the test con- firming the structure of the transformation product on com-

ditions occurs.^25,33Table 9 includes the percentage biodegra- parison with an authentic sample, increasing the scope of

dation attributed to this process and the percentage carbon synthetic chemistry expertise for the preparation of close

content of the aminoacid ester and ester subunits as exempli- derivatives of compounds of interest. A limitation of LCMS

fied in Fig. 7.

studies of biodegradation samples is that due to the low con-

For a number of compounds, the percentage biodegrada- centration used in the test (2–10 mg L⁻¹), and sampling, even

tion observed is greater than that which can be attributed to using preparative HPLC techniques, obtaining suﬃcient amounts

ester degradation. When the carbon content of the amino acid of the transformation product for further antimicrobial toxicity

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Scheme 3 Selective hydrolysis of ethyl ester of 10.

and biodegradation studies is challenging. Our approach was to LiOH mediated base hydrolysis of cholinium IL 10 procedure

establish new synthetic routes to the proposed transformation pro- to furnish PTP 37 was performed in good yield (Scheme 3). We

ducts, provide authentic samples, and due to the suﬃcient decided not to use this method for examples were two esters

amounts prepared, determine whether these compounds have and an amide were present and the selective hydrolysis of one

high antimicrobial activity. We submit that toxicity data of con- ester was required. Future investigations using enzymatic

firmed transformation products assists in the design of safer methods will search for a green solution to this problem.

chemicals. Of note, due to the collaborative nature of this work, it

PTP 40 was synthesised according to a benzyl ester hydroge-

is only by detailed LCMS studies that the structure of the trans- nolysis route via product 39 (Scheme 4). Starting compound

formation products can be confirmed. Therefore, herein we will 38⁶²underwent a substitution reaction with 26 in ethyl acetate

classify the transformation products (TP) as proposed transform- based on conditions described in Table 1 for the preparation

ation products (PTP) and the reader is directed to the accompany- of ^L-phenylalanine ILs. Deprotection of the benzyl ester 39

ing paper for the related LCMS studies.³⁹

using Pd/C catalysed hydrogenolysis aﬀorded the carboxylic

The first strategy for the synthesis of proposed transform- acid PTP 40 in 87% yield. Eﬀorts to use the same approach for

ation products (PTP) 37 and 40–46 was based on facile conver- the nicotinium PTPs 41 and 42 failed due to reduction of the

sion of ILs already prepared as part of this investigation. pyridinium ring in the final step. Therefore, a diﬀerent strategy

Selective hydrolysis of an ester bond in the presence of an avoiding the use of a benzyl protecting group was devised. We

amide is widely reported in peptide synthesis.⁶¹To this end a were pleased to find that sodium nicotinate was a suitable sub-

Scheme 4 Benzyl ester hydrogenolysis route to PTP 40.

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Scheme 5 PTP 41 synthesis from sodium nicotinate.

Scheme 6 PTP 42 synthesis from L-phenylalanine.

strate for the alkylation reaction with 26 (Scheme 5). This gave

directly the transformation product 41, although the yield was

fair, 37% due to challenging purification by column chromato-

graphy. Although α-bromoamides (e.g. N,N-diethyl) have been

used to N-alkylate nicotinic acid previously,^63aalbeit in low

yields, this headgroup has not been widely adopted in recent

IL studies.^63bThe IL 41 was only selected as a target due to the

biodegradation and transformation product joint investigation

with the Kümmerer group.

For the synthesis of PTP 42 a four step method was used

(Scheme 6). ^L-Phenylalanine was converted to the sodium salt

then reacted with bromoacetyl bromide under biphasic con-

ditions to give the alkylating reagent (2-bromoacetyl)-^L-phenyl-

alanine according to a procedure by Qin et al.^63cTreatment

with ethyl nicotinate in refluxing DMF, then HBr gave PTP 42

in 33% overall yield.

Fig. 8 PTPs 43–47.

Table 10 Antibacterial activity results obtained for PTPs 37 and 40–47

For PTPs (43, 44 and 45) preparation, the route involved

synthesis of either an ethyl or a methyl ester of the target

heterocycle followed by acid hydrolysis to give the carboxylic

acid PTP in a two-step reaction. The synthetic procedure is as

outlined by Pukitis et al. who originally synthesised 43 from

dimethylamino pyridine (DMAP).⁶⁴PTP 44 and 45 have been

previously reported by Oldfield et al.⁶⁵and Deng et al.,³⁷

respectively. PTP 46 was obtained according to the literature

procedure outline by Farfán et al.⁶⁶in which methyl-

ethanolamine is reacted with aqueous glyoxal to form a mor-

pholine intermediate which undergoes hydrolysis in situ to

give the carboxylic acid compound 46. PTP 47 was commer-

cially available and purchased from TCI Europe (Fig. 8).

Antimicrobial activity data shows that none of the PTPs (37

and 40–47) have high toxicity to the 20 strains screened

(Tables 10 and 11). This data is consistent with the results for

compounds 1–23 in Tables 5–8. ILs (41 and 43) showed the

highest level of fungal toxicity (Table 11). IL 41 had an IC₉₅of

2000 µM towards the two Candida albicans strains whilst IL 43

IL IC₉₅(µM)

Microorganism^a

Time (h)

37, 40–47

46

SA

24

48

24

48

24

48

24

48

24

48

24

48

24

48

24

48

>2000

>500

MRSA

SE

EF

EC

KP

KP-E

PA

^aSA, Staphylococcus aureus; MRSA, Staphylococcus aureus MRSA; SE,

Staphylococcus epidermidis; EF, Enterococcus sp.; EC, Escherichia coli;

KP, Klebsiella pneumoniae; KP-E, Klebsiella pneumoniae; PA, Pseudomo-

nas aeruginosa.

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Good to very good overall yields (76 to 87%) for the syn-

thesis of ILs 4, 5, 6, 8, 10 and 11 from ^L-phenylalanine were

achieved. C₂-Symmetric IL 11 was prepared from TMS-imida-

zole in a one pot two step method in excellent yield (91%). Syn-

thesis of the ^L-tyrosine IL derivatives used a simple protection

group strategy by using an extra equivalent of the bromoacetyl

bromide reagent. Improvements in the synthesis of α-bromo-

amide 26¹⁵were made. Reducing the volume of solvent required

and removing the requirement to run the reaction at low temp-

erature (−78 °C) lead to a greener method. A procedure repla-

cing DCM with THF was also demonstrated, however the yield

decreased by 15%. Green chemistry metrics were determined

for the synthesis of ILs and their amino derivatives and rec-

ommendations for improvements in future studies compiled.

We submit that the reactions can be classified into two types.

First, quantitative to excellent yield has been established and

the focus will be on reducing solvent use, using safer solvents

and reducing the amount of chemicals required, where poss-

ible. This will lead to improved outputs for the green chemistry

metrics, providing evidence that a greener synthesis has been

developed. Second, low to good yield reactions would benefit

from a broader approach to improve the synthesis. Alternative

green synthetic methodologies need to be trialled in combi-

nation with the aforementioned points. This is the strategy we

are following for the synthesis of the next generation of ILs as

part of this body of work.

Table 11 Antifungal activity results obtained for PTPs 37 and 40–47

IL MIC^b(µM)

Microorganism^aTime (h) 37, 40, 45, 42, 44

41

43

46

CA1

CA2

CP

24

48

24

48

24

48

24

48

24

48

24

48

24

48

24

48

24

48

24

48

24

48

72

120

>2000

2000 >2000 >500

>2000

2000 >500

CK1

CK2

CT

>2000 >2000 >500

CG

CL

TA

AF

AC

TM

>2000

2000 >500

^aCA1, Candida albicans; CA2, Candida albicans; CP, Candida parapsilo-

sis; CK1, Candida krusei; CK2, Candida krusei; CT, Candida tropicalis;

CG, Candida glabrata; CL, Candida lusitaniae; TA, Trichosporon asahii;

AF, Aspergillus fumigatus; AC, Absidia corymbifera; TM, Trichophyton

mentagrophytes. ^bIC₅₀values were assessed for AF, AC and TM and IC₈₀

for all other strains.

Antimicrobial activity testing of ^L-phenylalanine ILs, L-tyro-

sine ILs, tertiary amino analogues and PTPs, against 8 bacteria

and 12 fungi strains, showed that no compound had a high

antimicrobial toxicity, apart from 24. In this exceptional case,

the highest toxicity (IC₉₅= 125 and 250 µM) were observed

towards the two gram positive strains SA and SE respectively.

High antimicrobial activity was not found for the other bac-

teria or fungi strains screened. We were pleased to establish

that the PTPs did not show high toxicity to the microorgan-

isms screened. Due to the maximum validated sample con-

centration of the microbial toxicity test (2000 µM, or

solubility limit) and the samples not having high toxicity, a

SAR model cannot be established. However, when comparing

the phenylalanine and tyrosine examples with the valine ana-

logue 35, a lower antifungal activity was demonstrated for

4–24.

displayed an IC₉₅of 2000 µM towards the fungi Candida para-

psilosis and Trichophyton mentagrophytes. Even with these ILs

the general trend is not a high antifungal activity when consid-

ering all 12 strains.

We submit that this data significantly expands the scope of

compounds in this study which do not show high microbial

toxicity. Not only can this supplement the earlier results

(Tables 5–8), assisting the design of greener ILs, but also pro-

vides fundamental toxicity data of PTPs to aid biodegradation

studies.

Preliminary CBT biodegradation results³⁹for 4–24 gave a

wide range of values (17–63%). The highest value, obtained for

pyridinium IL 5, passes the CBT pass 60% threshold, but

Conclusion

A series of L-phenylalanine ILs, L-tyrosine ILs, tertiary amino further biodegradation studies and HRMS analysis were

analogues and PTPs have been synthesised. The PTPs were required before being able to state whether the compound was

selected as target compounds based on concurrent biodegra- fully biodegradable or not.³⁹The biodegradation data in

dation studies by the Kümmerer group.³⁹Antimicrobial toxi- Table 9 should not be rationalised according to substructures

city data, as part of the green chemistry metrics evaluation of the ILs and neutral amino compounds (e.g. headgroup,

and to supplement biodegradation studies, was determined aminoacid ester and ester). In particular, stating that every com-

for ILs, tertiary amino analogues and PTPs. We propose that pound undergoes complete conversion of the alcohol

this combined study by the Gathergood and Kümmerer groups fragment of the ester, after a hydrolysis step. Although, indeed

has led to advances in the synthesis and design of low anti- this was an approach our group previously used to account for

microbial toxicity ionic liquids with favourable biodegradation low biodegradation of a series of over 20 ILs.⁶⁷In that study,

properties.

no compound was found to give a biodegradation value higher

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than that which can be attributed to ester transformation, so ing an amide group. The conclusion that 5 is the preferred

that assumption was consistent with the data available. This is green IL from this study is only possible due to the close

not the case with compounds 4–24. A far more complex biodegra- collaboration of research groups with expertise in organic

dation process occurs with this series, and based on Table 9 synthesis, biodegradation and toxicity screening. Promoting the

data alone it would be ill-advised to state that ester hydrolysis ‘benign by design’ approach to the development of low toxicity

accounts for the biodegradation values observed. For example, and biodegradable chemicals is our mutual goal.^68–70

ILs 12–15 gave 21–23% biodegradation under CBT conditions

with 19% carbon content accounted for by the ethyl fragment

of the ester group. To assume that these results were linked,

Experimental

and then design new greener ILs based on this finding, would

Antibacterial activity experimental method

be unadvisable without supporting TP data. One conclusion

In vitro antibacterial activities⁷¹of the compounds were evalu-

that can be made from the data is that the presence of the OH

ated on a panel of three ATCC strains (Staphylococcus aureus

ATCC 6538, Escherichia coli ATCC 8739, Pseudomonas aerugi-

nosa ATCC 9027) and five clinical isolates (Staphylococcus

aureus MRSA HK5996/08, Staphylococcus epidermidis HK6966/

08, Enterococcus sp. HK14365/08, Klebsiella pneumoniae

HK11750/08, Klebsiella pneumoniae ESBL HK14368/08) from

the collection of fungal strains deposited at the Department of

Biological and Medical Sciences, Faculty of Pharmacy, Charles

University, Hradec Králové, Czech Republic. The above-men-

tioned ATCC strains also served as the quality control strains.

All the isolates were maintained on Mueller-Hinton agar prior

to being tested. Dimethyl sulfoxide (100%) served as a diluent

for all compounds; the final concentration did not exceed 2%.

Mueller-Hinton agar (MH, HiMedia, Čadersky-Envitek, Czech

Republic) buﬀered to pH 7.4 ( 0.2) was used as the test

medium. The wells of the microdilution tray contained 200 μL

of the Mueller-Hinton medium with 2-fold serial dilutions of

the compounds (2000 to 0.488 μmol L⁻¹) and 10 μL of inocu-

lum suspension. Inoculum in MH medium was prepared to

give a final concentration of 0.5 McFarland scale (1.5 × 10⁸cfu

mL⁻¹). The trays were incubated at 37 °C and MICs were read

visually after 24 h and 48 h. The MICs were defined as 95%

inhibition of the growth of control. MICs were determined

twice and in duplicate. The deviations from the usually

obtained values were no higher than the nearest concentration

value up and down the dilution scale.

group in tyrosine compared to phenylalanine did not lead to

an increase in biodegradation. Due to this result, the tyrosine

examples were not prioritised for biodegradation LCMS

studies/TP identification and the focus was only the study of

phenylalanine compounds prepared.

To assist in the identification of transformation products

formed during the CBT, synthesis of possible compounds

which are consistent with proposed structures based on LCMS

studies was completed. This was by either simple hydrolysis of

IL targets already in hand (37 from 10), from the stockpile of

key alkylating reagent (41 from 26) or total synthesis (40 from

38; 42 from phenylalanine). We submit that the approach of

the first two methods oﬀers a green solution to the preparation

of the required PTPs for this project based on materials and

expertise already in the group. It is advisable that future

investigations should include crucial TP analysis. Thus, devel-

oping robust green synthetic methods to support these studies

is a worthwhile endeavour. Of note also is the case of 41 syn-

thesis from sodium nicotinate. The requirement to prepare 41

as a PTP lead to the discovery that sodium nicotinate is a toler-

ated substrate for the alkylation reaction of 26. This headgroup

can now be included in our ongoing investigations in IL

research. While the microbial toxicity data showed that none

of the PTPs have high activity, which was a desirable result, it

is the evaluation step which is an important aspect. One poss-

ible outcome from this microbial toxicity screening was that

PTPs with high antimicrobial toxicity were found. Although

the significance of this outcome should not be overstated (only

Antifungal activity experimental method

20 microbial strains tested), this is useful data for experts in In vitro antifungal activities of the compounds were evaluated

biodegradation and TP analysis studies. The formation of a TP on a panel of four ATCC strains (Candida albicans ATCC 44859,

which is a potent biocide during biodegradation is obviously Candida albicans ATCC 90028, Candida parapsilosis ATCC

undesirable and all data which can support research to avoid 22019, Candida krusei ATCC 6258) and five clinical isolates of

this eventuality is valuable.

yeasts (Candida krusei E28, Candida tropicalis 156, Candida

The preferred green IL from this study was pyridinium com- glabrata 20/I, Candida lusitaniae 2446/I, Trichosporon asahii

pound 5. A short and eﬃcient synthesis of 5 was described, 1188) and filamentous fungi (Aspergillus fumigatus 231, Absidia

with improvements achieved in reducing solvent waste, the corymbifera 272, Trichophyton mentagrophytes 445) from the col-

required for low temperature alleviated and a lower toxicity lection of fungal strains deposited at the Department of Bio-

solvent replacement option described. Green chemistry metrics logical and Medical Sciences, Faculty of Pharmacy, Charles

provide evidence to support the statement that a greener syn- University, Hradec Králové, Czech Republic. Three ATCC

thesis was developed. IL 5 does not have high antimicrobial strains were used as the quality control strains. All the isolates

activity to the strains screened and has excellent biodegradation were maintained on Sabouraud dextrose agar prior to being

properties. Although from the data in this paper we cannot state tested. Minimum inhibitory concentrations (MICs) were deter-

whether 5 is readily biodegradable, passing the 60% threshold mined by modified CLSI standard of microdilution format of

of the CBT test is a significant result for a halide IL contain- the M27-A3 and M38-A2 documents.^72,73Dimethyl sulfoxide

Green Chem.

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Paper

(100%) served as a diluent for all compounds; the final con-

centration did not exceed 2%. RPMI 1640 (Sevapharma,

Prague) medium supplemented with ^L-glutamine and buﬀered

with 0.165 M morpholinepropanesulfonic acid (Serva) to pH

7.0 by 10 M NaOH was used as the test medium. The wells of

the microdilution tray contained 200 μL of the RPMI

1640 medium with 2-fold serial dilutions of the compounds

(2000 to 0.488 μmol L⁻¹for the new compounds) and 10 μL of

inoculum suspension. Fungal inoculum in RPMI 1640 was pre-

3 X. Liu, S. S. Li, Y. M. Liu and Y. Cao, Chin. J. Catal., 2015,

36, 1461–1475.

4 D. Prat, A. Wells, J. Hayler, H. Sneddon, C. R. McElroy,

S. Abou-Shehada and P. J. Dunn, Green Chem., 2016, 18,

288–296.

5 R. A. Sheldon, I. Arends, G. J. Ten Brink and A. Dijksman,

Acc. Chem. Res., 2002, 35, 774–781.

6 R. S. Boethling, E. Sommer and D. DiFiore, Chem. Rev.,

2007, 107, 2207–2227.

7 R. A. Sheldon, C. R. Acad. Sci., Ser. IIc: Chim., 2000, 3, 541–

551.

8 P. Anastas and N. Eghbali, Chem. Soc. Rev., 2010, 39, 301–

312.

9 J. P. Hallett and T. Welton, Chem. Rev., 2011, 111, 3508–

3576.

pared to give a final concentration of 5 × 103 0.2 cfu mL⁻¹

.

The trays were incubated at 35 °C and MICs were read visually

after 24 h and 48 h. The MIC values for the dermatophytic

strain (T. mentagrophytes) were determined after 72 h and

120 h. The MICs were defined as 80% inhibition (IC₈₀) of the

growth of control for yeasts and as 50% inhibition (IC₅₀) of the

growth of control for filamentous fungi. MICs were deter- 10 V. I. Pârvulescu and C. Hardacre, Chem. Rev., 2007, 107,

mined twice and in duplicate. The deviations from the usually 2615–2665.

obtained values were no higher than the nearest concentration 11 N. V. Plechkova and K. R. Seddon, Chem. Soc. Rev., 2008,

value up and down the dilution scale.

37, 123–150.

12 K. Seddon, presented in part at the COIL-6, Jéju, South

Korea, 2015.

Biodegradation method

For biodegradation method see Haiß et al.³⁹

13 L. Carson, P. K. W. Chau, M. J. Earle, M. A. Gilea,

B. F. Gilmore, S. P. Gorman, M. T. McCann and

K. R. Seddon, Green Chem., 2009, 11, 492–497.

14 K. M. Docherty and C. F. Kulpa, Green Chem., 2005, 7, 185–

189.

15 D. Coleman, M. Spulak, M. T. Garcia and N. Gathergood,

Green Chem., 2012, 14, 1350–1356.

16 A. S. Wells and V. T. Coombe, Org. Process Res. Dev., 2006,

10, 794–798.

General synthetic methods

Compounds 4,¹⁵25,⁷⁴26,¹⁵27,⁷⁵30,⁷⁶31,⁷⁷32,⁷⁶33⁷⁸and 34⁷⁹

were prepared according to literature methods. Proposed trans-

formation products 43,⁶⁴44,⁶⁵45³⁷and 46⁶⁶were prepared by

literature methods. Commercially available PTP 47 was pur-

chased from TCI Europe. Experimental procedures and charac-

terisation of all novel compounds, including NMR spectra, are

available in the ESI.†

17 S. P. M. Ventura, C. S. Marques, A. A. Rosatella,

C. A. M. Afonso, F. Goncalves and J. A. P. Coutinho, Ecotoxi-

col. Environ. Saf., 2012, 76, 162–168.

18 C. W. Cho, Y. C. Jeon, T. P. T. Pham, K. Vijayaraghavan and

Y. S. Yun, Ecotoxicol. Environ. Saf., 2008, 71, 166–171.

19 J. I. Santos, A. M. M. Goncalves, J. L. Pereira, B. F. H.

Acknowledgements

The authors would like to thank the EPA in Ireland for finan-

cial support. This project has also received funding from the

European Union’s Seventh Framework Programme for

research, technological development and demonstration under

grant agreement No. 621364 (TUTIC-Green). The antibacterial

and antifungal screening was supported by the Czech Science

Foundation (project No. 15-07332S). We also thank COST

Actions CM1206 and TD1203 for their contribution to this

study. We acknowledge Hannah Prydderch for her contri-

butions to PTP synthesis and Dr Andrew Kellett for his assist-

ance with the project at Dublin City University. We thank

Prof. A. Lapkin at the University of Cambridge and BRITEST

Ltd for providing the Green Metrics Lab Book spreadsheet.

T. Figueiredo, F. A.

e

Silva, J. A. P. Coutinho,

S. P. M. Ventura and F. Goncalves, Green Chem., 2015, 17,

4657–4668.

20 B. Jastorﬀ, R. Stormann, J. Ranke, K. Molter, F. Stock,

B. Oberheitmann, W. Hoﬀmann, J. Hoﬀmann, M. Nuchter,

B. Ondruschka and J. Filser, Green Chem., 2003, 5, 136–142.

21 S. Stolte, J. Arning, U. Bottin-Weber, M. Matzke, F. Stock,

K. Thiele, M. Uerdingen, U. Welz-Biermann, B. Jastorﬀ and

J. Ranke, Green Chem., 2006, 8, 621–629.

22 P. Luis, I. Ortiz, R. Aldaco and A. Irabien, Ecotoxicol.

Environ. Saf., 2007, 67, 423–429.

23 C. W. Cho, J. Ranke, J. Arning, J. Thoeming, U. Preiss,

C. Jungnickel, M. Diedenhofen, I. Krossing and S. Stolte,

SAR QSAR Environ. Res., 2013, 24, 863–882.

24 D. Coleman and N. Gathergood, Chem. Soc. Rev., 2010, 39,

600–637.

References

1 P. T. Anastas, Green Chemistry: Theory and Practice, ed. 25 S. Stolte, S. Steudte, A. Igartua and P. Stepnowski, Curr.

P. T. Anastas and J. C. Warner, Oxford University Press,

New York, 1998.

2 P. Tundo and M. Selva, Acc. Chem. Res., 2002, 35, 706–716.

Org. Chem., 2011, 15, 1946–1973.

26 A. Jordan and N. Gathergood, Chem. Soc. Rev., 2015, 44,

8200–8237.

Green Chem.

View Article Online

Paper

Green Chemistry

27 M. Petkovic, K. R. Seddon, L. P. N. Rebelo and C. S. Pereira, 53 C. Jiménez-González, D. Constable, A. Curzons and

Chem. Soc. Rev., 2011, 40, 1383–1403.

V. Cunningham, Clean Technol. Environ. Policy, 2002, 4, 44–

28 J. Andraos, Org. Process Res. Dev., 2005, 9, 149–163.

29 B. M. Trost, Angew. Chem., Int. Ed. Engl., 1995, 34, 259–281.

53.

54 J. Andraos, Pure Appl. Chem., 2011, 83, 1361–1378.

30 C. R. McElroy, A. Constantinou, L. C. Jones, L. Summerton 55 S. Sasu, J. W. Metzger, M. Kranert and K. Kümmerer, Clean:

and J. H. Clark, Green Chem., 2015, 17, 3111–3121. Soil, Air, Water, 2015, 43, 166–172.

31 L. Myles, R. G. Gore, N. Gathergood and S. J. Connon, 56 OECD, OECD Guideline for Testing of Chemicals, 1992.

Green Chem., 2013, 15, 2740–2746.

57 OECD, Test No. 301: Ready Biodegradability, OECD Publish-

ing, 1992.

58 A. Längin, R. Alexy, A. König and K. Kümmerer, Chemo-

sphere, 2009, 75, 347–354.

32 R. G. Gore, T.-K.-T. Truong, M. Pour, L. Myles, S. J. Connon

and N. Gathergood, Green Chem., 2013, 15, 2727–2739.

33 R. G. Gore, L. Myles, M. Spulak, I. Beadham, T. M. Garcia,

S. J. Connon and N. Gathergood, Green Chem., 2013, 15, 59 P. F. Fitzpatrick, Annu. Rev. Biochem., 1999, 68, 355–381.

2747–2760.

60 E. Arias-Barrau, E. R. Olivera, J. M. Luengo, C. Fernandez,

B. Galan, J. L. Garcia, E. Diaz and B. Minambres, J. Bacter-

iol., 2004, 186, 5062–5077.

34 J. R. Harjani, R. D. Singer, M. T. Garciac and

P. J. Scammells, Green Chem., 2009, 11, 83–90.

35 L. Myles, R. Gore, M. Spulak, N. Gathergood and 61 V. M. Krishnamurthy, V. Semetey, P. J. Bracher, N. Shen

S. J. Connon, Green Chem., 2010, 12, 1157–1162.

36 A. Jordan and N. Gathergood, Chem. Soc. Rev., 2015, 44,

8200–8237.

and G. M. Whitesides, J. Am. Chem. Soc., 2007, 129, 1312–

1320.

62 P. Zaderenko, M. S. Gil, P. Ballesteros and S. Cerdan, J. Org.

Chem., 1994, 59, 6268–6273.

37 Y. Deng, I. Beadham, M. Ghavre, M. F. Costa Gomes,

N. Gathergood, P. Husson, B. Legeret, B. Quilty, 63 (a) B. Potter, J. Dowden, A. Galione, A. Guse and A. Flugel,

M. Sancelme and P. Besse-Hoggan, Green Chem., 2015, 17,

1479–1491.

38 N. Gathergood, P. J. Scammells and M. T. Garcia, Green

Chem., 2006, 8, 156–160.

US Pat. Appl, US20070105810, 2007; (b) Z. Wang, C. Wang,

W. Bao and T. Ying, J. Chem. Res., 2005, 6, 388–390;

(c) H.-P. Li, S. Li, Z.-D. Wang and L. Qin, J. Korean Chem.

Soc., 2011, 55, 978–982.

39 A. Haiß, A. Jordan, J. Westphal, E. Logunova, N. Gathergood 64 O. Y. Neilands, E. V. Shebenina and G. G. Pukitis, Int. Arch.

and K. Kümmerer, Green Chem., 2016, DOI: 10.1039/

C6GC00417B.

40 J. Friedrich, A. Längin and K. Kümmerer, Clean: Soil, Air,

Water, 2013, 41, 251–257.

41 Y. Deng, S. Morrissey, N. Gathergood, A. M. Delort, P. Husson

and M. F. C. Gomes, ChemSusChem, 2010, 3, 377–385.

42 S. Morrissey, B. Pegot, D. Coleman, M. T. Garcia,

Allergy Appl. Immunol., 1999, 35, 1443–1450.

65 J. M. Sanders, Y. Song, J. M. W. Chan, Y. Zhang,

S. Jennings, T. Kosztowski, S. Odeh, R. Flessner,

C. Schwerdtfeger, E. Kotsikorou, G. A. Meints, A. O. Gómez,

D. González-Pacanowska, A. M. Raker, H. Wang, E. R. van

Beek, S. E. Papapoulos, C. T. Morita and E. Oldfield, J. Med.

Chem., 2005, 48, 2957–2963.

D. Ferguson, B. Quilty and N. Gathergood, Green Chem., 66 N. Farfán, L. Cuéllar, J. M. Aceves and R. Contreras, Syn-

2009, 11, 475–483. thesis, 1987, 927–929.

43 M. T. Garcia, N. Gathergood and P. J. Scammells, Green 67 R. G. Gore, L. Myles, M. Spulak, I. Beadham, M. T. Garcia,

Chem., 2005, 7, 9–14.

44 N. Gathergood, M. T. Garcia and P. J. Scammells, Green

Chem., 2004, 6, 166–175.

S. J. Connon and N. Gathergood, Green Chem., 2013, 15,

2747–2760.

68 K. Kümmerer, Green Chem., 2007, 9, 899–907.

45 E. Fischer and A. Speier, Ber. Dtsch. Chem. Ges., 1895, 28, 69 I. Beadham, M. Gurbisz and N. Gathergood, Handbook

3252–3258.

of Green Chemistry, Chapter 6, Volume 9: Designing

Safer Chemicals, ed. R. Boethling and A. Voutchkova, Wiley-

VCH Verlag GmbH & Co. KGaA, 1st edn, 2012, pp. 137–

158.

46 E. Kumazaki and H. Nagano, Tetrahedron, 2013, 69, 3486–

3494.

47 Z. Fei, D. Zhao, T. J. Geldbach, R. Scopelliti and P. J. Dyson,

Chem. – Eur. J., 2004, 10, 4886–4893.

48 Y. H. L. Nan-horng, R. L. Elliott, M. S. Chorghade,

S. J. Wittenberger, W. H. Bunnelle, B. A. Narayanan,

P. Singam, T. K. J. Esch, D. O. Beer, C. C. Witzig,

T. C. Herzig and A. R. Rao, Eur. Pat, EP0717741, 2001.

49 Z. Wang, in Comprehensive Organic Name Reactions and

Reagents, John Wiley & Sons, Inc., 2010, vol. 426, pp.

1897–1900.

70 I. Beadham, M. Gurbisz and N. Gathergood, Handbook of

Green Chemistry, Chapter 7, Volume 9: Designing Safer

Chemicals, ed. R. Boethling and A. Voutchkova, Wiley-VCH

Verlag GmbH & Co. KGaA, 1st edn, 2012, pp. 159–226.

71 Reference Method for Broth Dilution Antifungal Suscepti-

bility Testing of Yeasts. Approved standard. Document

M27-A3. Clinical Laboratory Standard Institute, Wayne, PA,

2008.

50 B. M. Trost, Science, 1991, 254, 1471–1477.

51 J. Andraos, Org. Process Res. Dev., 2006, 10, 212–240.

52 A. D. Curzons, D. J. C. Constable, D. N. Mortimer and

V. L. Cunningham, Green Chem., 2001, 3, 1–6.

72 Reference Method for Broth Dilution Antifungal Suscepti-

bility Testing of Filamentous Fungi. Approved standard.

Document M38-A2. Clinical Laboratory Standard Institute,

Wayne, PA, 2008.

Green Chem.

View Article Online

Green Chemistry

Paper

73 Methods for Dilution Antimicrobial Susceptibility Tests for 76 K. D. Daughtry, Y. Xiao, D. Stoner-Ma, E. Cho,

Bacteria that Grow Aerobically. Approved Standard

Seventh Edition. Document M07-A7. Clinical Laboratory

Standard Institute, Wayne, PA, 2006.

–

A. M. Orville, P. Liu and K. N. Allen, J. Am. Chem. Soc.,

2012, 134, 2823–2834.

77 D. M. Evans, W.O. Pat, 2011051673, 2011.

74 J. Hartwig, J. B. Metternich, N. Nikbin, A. Kirschning and 78 A. M. Likhosherstov, A. S. Lebedeva, V. N. Kulakov,

S. V. Ley, Org. Biomol. Chem., 2014, 12, 3611–3615.

75 M. Ousmer, N. A. Braun, C. Bavoux, M. Perrin and

A. M. Kritsyn, D. A. Kharkevich and A. P. Skoldinov, Pharm.

Chem. J., 1973, 7, 274–277.

M. A. Ciufolini, J. Am. Chem. Soc., 2001, 123, 7534– 79 R. Almansa, D. Guijarro and M. Yus, Tetrahedron: Asymme-

7538.

try, 2007, 18, 2828–2840.

Green Chem.

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Synthesis of a series of amino acid derived ionic liquids and tertiary amines: Green chemistry metrics including microbial toxicity and preliminary biodegradation data analysis

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