Volume 18 Number 16 21 August 2016 Pages 4315–4572

Green
Chemistry
Cutting-edge research for a greener sustainable future
www.rsc.org/greenchem

Themed issue: Molecular Design for Reduced Toxicity

ISSN 1463-9262

PAPER
Nicholas Gathergood, Klaus Kümmerer et al.
On the way to greener ionic liquids: identification of a fully mineralizable
phenylalanine-based ionic liquid
Green Chemistry
PAPER

Synthesis of a series of amino acid derived ionic

Cite this: Green Chem., 2016, 18,
liquids and tertiary amines: green chemistry
4374 metrics including microbial toxicity and
preliminary biodegradation data analysis†
Andrew Jordan,a Annette Haiß,b Marcel Spulak,c Yevgen Karpichev,d
Klaus Kümmerer*b and Nicholas Gathergood*d

Received 12th February 2016,
Accepted 7th June 2016
DOI: 10.1039/c6gc00415f
www.rsc.org/greenchem
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.

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

The overarching concept of the 12 Principles of Green Chem-
istry is to promote the development and application of safer
chemicals, which will therefore lead to a reduced impact on
the environment.1 Whether this is due to a cleaner more
a
School of Chemical Sciences, Dublin City University, Glasnevin, Dublin 9, Ireland
b
Institute of Sustainable and Environmental Chemistry, University of Lüneburg,
Scharnhorststraße 1, D-21335 Lüneburg, Germany.
E-mail: Klaus.Kuemmerer@uni.leuphana.de
c
Department of Inorganic and Organic Chemistry, Faculty of Pharmacy,
Heyrovského 1203, Hradec Kralove, Czech Republic, CZ500 05
d
Department 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
reagents,2,3 solvents4 and catalysts5), avoiding non-biodegrad-
able compounds, an iterative approach can ultimately lead to
significant advances in the field.6–8 A class of chemicals which
has been extensively studied as safer and greener replacements
for a wide range of chemicals is ionic liquids (ILs).9–11 ILs 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
can be based on chemical structure, functionality, and appli-
cations.12 An attractive feature of ILs is the ability to fine-tune
the structure, to tailor the properties for a particular
purpose.9–11 The availability of a series of closely related struc-
tures has led to many research teams determining the
toxicity13–15 and ecotoxicity16–19 of ILs, performing structure
activity relationship (SAR) studies13,20,21 and creating quanti-
tative structure activity relationship (QSAR) models.21–23 Bio-

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Green Chemistry
Fig. 1 One of the first ILs to pass the CO2 Headspace 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.14,20,27 Toxicity
and biodegradation data however is only part of a green chem-
istry assessment of a chemical (e.g. IL).1 The synthetic route to
prepare the compound can also be considered, and a plethora
of parameters can be determined,28–30 with 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.31–33 One of the findings from
this work was that the ILs studied containing amide group(s)
exhibited very low biodegradation in the CO2 Headspace test.
This was in agreement with previous results for amide contain-
ing ILs,33–35 with an exception being 2 and 3 reported by Gath-
ergood in 2012, (Fig. 2).15 This is in stark contrast to ILs
containing an ester functional group, where facile breakdown
to the carboxylic acid and conversion of the alcohol to CO2 is
widely reported.25,36 However, even with these ester containing
ILs, the carboxylic acid transformation product has been shown
to persist after a 28 day CBT.37 The development of minerali-
sable ILs is a significant and challenging research front.
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-
Fig. 2 Readily biodegradable amino acid ILs.15 Biodegradation values
assessed by the CO2 Headspace test ISO 14593.
This journal is © The Royal Society of Chemistry 2016
Paper
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 studies40 were
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
The series of ILs 4–24, Fig. 3–5, were designed with respect
to Boethling’s rules of thumb for designing biodegradable
compounds,36 12 Principles of Green Chemistry,1 and 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, 4,15 5–15). 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 mechanism42 and 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.
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 esterification45 of 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 2546 isolated 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
Green Chem., 2016, 18, 4374–4392 | 4375
Paper
Fig. 3 Series of L-phenylalanine ILs (4–15) targets. Charged headgroup study.
example, 1-methylimidazole), to yield the target ILs (4–15)
(Table 1). Compound 4 has been previously prepared by Gath-
ergood et al. in 98% yield using THF as the solvent,15 however
a slightly lower yield of 4 was obtained using diethyl ether
(entry 1). Excellent yields for the pyridinium and 3-methoxy-
pyridinium ILs 5 and 6 (entries 2 and 3) however were
obtained in diethyl ether. Attempts to prepare IL 7 from ethyl
4376 | Green Chem., 2016, 18, 4374–4392
Green Chemistry
nicotinate in diethyl ether at room temperature after 24 h
failed (entry 4). A fair yield (56%) however was attained for 7
by increasing the temperature, extending the reaction time
and changing to a more polar solvent, THF (entry 5). A high
yield of IL 8 from 4-dimethylaminopyridine in diethyl ether
was observed (entry 6). Aliphatic amines N-methylmorpholine
and dimethylaminoethanol gave considerably different yields
This journal is © The Royal Society of Chemistry 2016
Green Chemistry
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
(34%) was observed for the morpholinium IL 9 (entry 7), while
10 was isolated in a quantitative yield (entry 8). C2-Symmetric
IL 11 was prepared by a two-step procedure from N-trimethyl-
silylimidazole.47 The double alkylation proceeded in excellent
yield (91%) if after 24 h in diethyl ether, the solvent was
changed to ethyl acetate and heated at reflux for 24 h (entry 9).
N-Methyl proline ethyl esters (31 and 33, entries 10 and 11)
This journal is © The Royal Society of Chemistry 2016
Paper
were prepared by reductive amination using formaldehyde
according to a procedure by Lin et al.,48 followed by Fischer–
Speier esterification (see ESI, Scheme S1†).46 Proline derivative
31 required ethyl acetate as solvent for synthesis of IL 12 and
13 in a combined yield of 50% (entry 10 vs. 11). Similar results
were obtained using the D-proline analogue 33 (entry 12). In
both cases (entries 11 and 12) a 5 : 2 ratio of epimers was
established in the crude product by 1H NMR. However, iso-
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Paper Green Chemistry

Scheme 1 Synthesis of 26 from L-phenylalanine.

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

Entry NR1R2R3 Reaction conditionsa IL Yieldb (%)

1 N-Methylimidazole A 4 89
2 Pyridine A 5 98
3 3-Methoxypyridine A 6 96
4 Ethyl nicotinate A 7 0c
5 Ethyl nicotinate B 7 56
6 4-Dimethylaminopyridine A 8 88
7 N-Methylmorpholine A 9 34
8 Dimethylaminoethanol A 10 99
9 N-Trimethylsilylimidazole C 11 91
10 N-Methyl-L-proline-ethyl ester (31) A 12 0c
13 0c
11 N-Methyl-L-proline-ethyl ester (31) D 12 37
13 13
12 N-Methyl-D-proline-ethyl ester (33) D 14 25
15 34
a
A: 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.
b
See Table 4 for more detailed green chemistry metric analysis. c No product detected by TLC 10% MeOH : DCM.

lated yields of each epimer after purification by column
chromatography are shown in Table 1. The absolute stereo-
chemistry of prolinium derivatives (12, 13 and 14, 15) was
determined by 2-dimensional NMR by utilising the nuclear
overhauser effect (i.e. a NOESY experiment). Very good overall
yields (76 to 87%) for the synthesis of ILs 4, 5, 6, 8, 10 and 11
from L-phenylalanine were achieved.
L-Tyrosine ILs (16–20) were synthesised according to the
route outlined in Scheme 2. Initial attempts to selectively
acylate the amino group of 27 with bromoacetyl bromide were
unsuccessful. While a range of phenol protection group strat-
egies (for example, silyl ethers) were considered, the additional
steps required were deemed undesirable. Due to the observed
ease of acylation of the phenol group, and expected higher
reactivity of the formed phenolic ester vs. amide to hydrolysis,
a double acylation of 26 route was proposed. We were pleased
to find that quantitative conversion of 27 to intermediate 28
occurred after 12 h (by TLC), and after acid hydrolysis with
HCl in ethanol, 29 was obtained in an overall yield of 54%.
Potential transesterification problems were avoided by select-
ing ethanol as the solvent. The final alkylation step in reflux-
ing ethyl acetate gave ILs 16–20 in good to quantitative yields,
with the highest yields obtained for ILs 17 and 18. IL 19
required purification by flash chromatography and a lower iso-
lated yield (71%) was obtained.
The neutral derivatives (21–24) were synthesised from alky-
lating reagent (26) in poor to fair yields (34–56%). We propose
that this is due to the higher solubility of the products in
either diethyl ether or THF solvent. This effect was also seen
with the tyrosine series above. The compounds 21–24 were
purified by column chromatography and all isolated as liquids
at room temperature (Table 2).
Very good to quantitative yields were obtained for the final
alkylation step in the synthesis of ILs 4, 5, 6, 8, 10, 11, 17 and

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Green Chemistry Paper

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 N-Trimethylsilylimidazole Diethyl ether 21 55

2 N-Methylaminoethanol Diethyl ether 22 34
3 Morpholine THFa 23 56
b
4 L-Proline-ethyl ester·HCl THF 24 53
a
Reflux. b In situ neutralisation with Na2CO3.

18. The product precipitating from the reaction solvent signifi-
cantly increases the yield compared to examples where chrom-
atography is required.
A wide range of melting points were also observed for the
ILs synthesised. Under the classical definition of an IL existing
as a liquid below 100 °C all phenylalanine examples from
Fig. 3 (4–15) and Fig. 5 (21–24) with the exception of (8, 9 and
11) conform, though (5 and 10) have melting points close to
100 °C. A broad range of melting points (30–32 to 141–143 °C)
were also observed for the tyrosine derivatives with the imida-
zolium IL 16 38–40 °C, and pyridinium IL 17 30–32 °C, the
lowest recorded. Introduction of an ester group to the pyridi-
nium ring gave an increase in melting point to 53–55 °C (17 vs.
19) and significantly higher melting points were observed for
the 3-methoxypyridinium derivative 18 117–119 °C and the
cholinium derivative 20 141–143 °C. The presence of the –OH
group on the tyrosine ring greatly reduces the melting points
of several of the ILs (16, 17 and 19) when compared to their
L-phenylalanine counterparts. A decrease of 40 °C was observed
for the imidazolium IL analogues (16 vs. 4), 68 °C for the pyri-
dinium examples (17 vs. 5), and a slight decrease in melting
point of 7 °C for ethyl nicotinium analogues (19 vs. 7). Excep-
tions to this trend are the tyrosine salts, 3-methoxypyridinium
IL 18 where an increase in melting point of 45 °C was observed

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Paper
and cholinium IL 20, with an increase of 40 °C when com-
pared to their phenylalanine counterparts, 6 and 10
respectively.
Green chemistry metrics
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.
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.15 The 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-
tion49 or 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 Na2CO3 was 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 Na2CO3 was 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)15 with no formation of side products
Table 3 Optimisation of synthesis of 26
Entry Amine Reaction solvent Reaction time (h)
1 25·HCl DCM 5 TEA
2 25·HCl DCM 5 Na2CO3
3 25 DCM 3 Na2CO3
4 25 THF 5 Na2CO3
4380 | Green Chem., 2016, 18, 4374–4392
Green Chemistry
detected by TLC or 1H NMR. The outcome was a reaction
mixture that could be purified by filtration to remove the
Na2CO3 followed 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
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,50 E-factor,7 Andraos reaction mass
efficiency,51 GSK reaction mass efficiency,52 mass intensity,53
solvent intensity,53 stoichiometric factor28 and material recov-
ery parameter.54
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
Base Column chromatography required Yield 26 (%)
Y 6815
Y 61
N 89
N 74
This journal is © The Royal Society of Chemistry 2016
Green Chemistry Paper

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

Atom Sheldon GSK Andraos Mass Solvent Stoichiometric Material recovery Yield
Compound economy50 E-factor7 RME52 RME51 intensity53 intensity53 factor28 parameter54 (%)

L-Phe ILs
4 100% 96.7 0.830 0.010 97.7 96.5 0.920 0.012 90
5 100% 93.4 0.964 0.011 94.4 93.3 0.991 0.011 98
6 100% 108.8 0.689 0.009 109.8 108.3 0.922 0.013 75
7 100% 1081.5 0.523 0.001 1082.5 1080.6 0.934 0.002 56
8 100% 120.7 0.812 0.008 121.7 120.5 0.918 0.010 89
9 100% 126.5 0.335 0.008 127.5 124.5 0.991 0.023 34
10 100% 88.1 0.901 0.011 89.1 88.0 0.917 0.012 98
11 80% 33.0 0.718 0.029 34.0 32.6 0.986 0.041 91
12 100% 1416.6 0.335 0.001 1417.6 1414.7 0.921 0.002 37
13 100% 3869.1 0.123 0.001 3870.1 3862.0 0.921 0.002 13
14 100% 1119.0 0.254 0.001 1120.0 1116.1 1.000 0.004 25
15 100% 828.2 0.343 0.001 829.2 826.2 1.000 0.004 34
L-Tyr ILs
16 100% 294.0 0.819 0.003 295.0 293.8 0.979 0.004 84
17 100% 117.1 0.980 0.008 118.1 117.1 0.982 0.009 99
18 100% 120.7 0.971 0.008 121.7 120.7 0.982 0.008 99
19 100% 420.3 0.699 0.002 421.3 419.9 0.986 0.003 71
20 100% 150.7 0.773 0.007 151.7 150.4 0.985 0.009 79
Neutral derivatives
21 66% 1035.4 0.288 0.001 1036.4 1032.9 0.783 0.003 56
22 79% 2754.1 0.242 0.001 2755.1 2750.9 0.923 0.001 33
23 80% 476.6 0.433 0.002 477.6 474.6 0.958 0.005 57
24 53% 770.3 0.266 0.001 771.3 767.6 0.946 0.005 53
Alkylating reagents
26a 59% 705.1 0.307 0.001 709.4 555.4 0.904 0.005 64
26b 49% 84.2 0.297 0.012 85.2 52.2 1.000 0.040 61
c
26 63% 10.1 0.470 0.090 11.1 7.4 0.843 0.192 89
26d 63% 36.7 0.428 0.027 37.7 7.3 0.918 0.062 74
29 50% 55.1 0.281 0.018 56.1 17.9 0.873 0.063 54
Amino acid esters
25 59% 20.8 0.425 0.046 21.8 11.8 0.735 0.108 99
27 71% 3.7 0.620 0.211 4.7 3.1 1.000 0.340 87
31 53% 14.6 0.372 0.064 15.6 12.0 1.000 0.172 70
33 53% 29.5 0.231 0.033 30.5 24.5 1.000 0.142 43
34 64% 5.6 0.610 0.150 6.6 5.0 0.959 0.247 99
Reductive alkylation
30 89% 11.6 0.837 0.080 12.6 11.4 0.980 0.093 98
32 89% 15.0 0.309 0.062 16.0 12.8 0.980 0.200 36

For conditions see a Table 3, entry 1; b Table 3, entry 2; c Table 3, entry 3; d Table 3, entry 4.

metrics in a similar manner and can be seen for ILs 7, 12–15
and 19 (Table 4). Material recovery parameters for all ILs are
relatively low and this is due to only the product mass contri-
buting to the material recovery and all other materials are in
effect waste.
Andraos RME is low for all ILs as it takes into account all
solvent used in the IL trituration steps and all chemicals that
are not incorporated into the product are considered waste.
The diethyl ether and chromatography solvent should there-
fore be recycled in future studies or if this process were to be
scaled up in order to reduce the mass/solvent intensity,
E-factor, material recovery parameter and Andraos RME.
The metrics generated for the neutral derivatives (21–24)
are less desirable as lower yields and a higher consumption of
solvent due to the purification by column chromatography was
required. The GSK RME decreases (0.266–0.432) and E-factor
increases (476.6–2754.1).
The alkylating reagents (26 and 29) were both synthesised
with moderate atom economy (63% and 50% respectively) and
a generally lower E-factor than calculated for the ILs. The mod-
erate atom economy is due to the use of bromacetyl bromide
which is not fully incorporated into the product. HBr is a by-
product of the substitution reaction. The presence of an auxili-
ary base also reduces the atom economy. The lower E-factor,
which is desirable, is due to the minimum amount of solvent
employed during the reaction and purification steps when
compared to the ILs which required multiple triturations.
Amino acid esterification reactions (25, 27, 31, 33 and 34)
all proceeded with excellent (e.g. 25, 3.7) to moderate E-factors
(e.g. 31, 40.3) and low solvent and mass intensities. The atom
economies are affected by the use of stoichiometric quantities
of SOCl2 during the esterification reaction and ranged between
53–90%.
Lastly, the reductive alkylation of L-proline to give inter-
mediate 30 can be considered as one of the greenest methods
employed during the synthesis reported here, with high atom
economy (89%), low E-factor (11.6), excellent reaction mass
efficiency (0.837) and low solvent and mass intensity (12.6 and

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Paper Green Chemistry

11.4 respectively). The counterpart to this reaction, 32, is less
green due to the lower yield overall and thus lower GSK RME.
Atom economy remains unchanged and the E-factor is only
slightly increased to 15.0.
During the reaction condition optimisation for alkylating
reagent 26 (see Table 3 for optimisation), metric analysis was
also conducted on each of the reaction conditions (entries
1–4). The original conditions reported by Coleman et al.15 for
the formation of 26 involving the use of two equivalents of tri-
ethylamine and purification by column chromatography led to
a poor atom economy of 50% and an E-factor of 705.1. The
solvent and mass intensities of the reaction are similarly
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 Na2CO3 compared 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-
cular weight bases would result in fewer tons of exhausted
base and resulting salts over a longer period. Solvent recycling
from all aspects of the synthesis reported would dramatically
improve the E-factor as the solvents would obviously no longer
be considered as waste. Material recovery parameters for all
steps are low due to the inevitable waste produced during pro-
cesses that do not have a 100% atom economy. Solvent re-
cycling would also aid in improving the material recovery
parameter. Solvent intensity can be improved if reducing reac-
tion solvent can be tolerated while achieving the same yield.
Mass intensity can also be similarly improved.
Microbial screening
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 IC95 values lower than 2000 µM or the solubi-

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

IL IC95 (µM)
a
Microorganism Time (h) 4 5 6 7 8 9 10 11 12 13 14 15

Gram positive
SA 24 2000 2000 >500 1000 >500 2000 2000 500 >2000 1000 >2000 1000
48 >2000 >2000 >500 1000 >500 2000 >2000 500 >2000 1000 >2000 1000
MRSA 24 >2000 >2000 >500 1000 >500 2000 >2000 1000 >2000 >1000 >2000 >2000
48 >2000 >2000 >500 1000 >500 2000 >2000 1000 >2000 >1000 >2000 >2000
SE 24 >2000 >2000 >500 1000 >500 >2000 >2000 500 2000 1000 >2000 1000
48 >2000 >2000 >500 1000 >500 >2000 >2000 500 2000 1000 >2000 1000
EF 24 >2000 >2000 >500 >1000 >500 >2000 >2000 1000 >2000 >1000 >2000 >2000
48 >2000 >2000 >500 >1000 >500 >2000 >2000 1000 >2000 >1000 >2000 >2000
Gram negative
EC 24 >2000 >2000 >500 >1000 >500 >2000 >2000 2000 >2000 >1000 >2000 >2000
48 >2000 >2000 >500 >1000 >500 >2000 >2000 2000 >2000 >1000 >2000 >2000
KP 24 >2000 >2000 >500 >1000 >500 >2000 >2000 >2000 >2000 >1000 >2000 >2000
48 >2000 >2000 >500 >1000 >500 >2000 >2000 >2000 >2000 >1000 >2000 >2000
KP-E 24 >2000 >2000 >500 >1000 >500 >2000 >2000 >2000 >2000 >1000 >2000 >2000
48 >2000 >2000 >500 >1000 >500 >2000 >2000 >2000 >2000 >1000 >2000 >2000
PA 24 >2000 >2000 >500 >1000 >500 >2000 >2000 2000 >2000 >1000 >2000 >2000
48 >2000 >2000 >500 >1000 >500 >2000 >2000 2000 >2000 >1000 >2000 >2000
a
SA, 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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Green Chemistry Paper

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

IL IC95 (µM)
a
Microorganism Time (h) 16 17 18 19 20

SA 24 1000 >2000 >2000 >2000 2000

48 1000 >2000 >2000 >2000 2000
MRSA 24 2000 2000 >2000 >2000 >2000
48 >2000 2000 >2000 >2000 >2000
SE 24 500 >2000 >2000 >2000 1000
48 1000 >2000 >2000 >2000 2000
EF 24 >2000 >2000 >2000 >2000 >2000
48 >2000 >2000 >2000 >2000 >2000
EC 24 >2000 >2000 >2000 >2000 >2000
48 >2000 >2000 >2000 >2000 >2000
KP 24 >2000 >2000 >2000 >2000 >2000
48 >2000 >2000 >2000 >2000 >2000
KP-E 24 >2000 >2000 >2000 >2000 >2000
48 >2000 >2000 >2000 >2000 >2000
PA 24 >2000 >2000 >2000 >2000 >2000
48 >2000 >2000 >2000 >2000 >2000
a
SA, 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.
IC95 values 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 IC50 and 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
IC95 values 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-
pounds 21–24
4 gram negative bacteria strains, 9 yeasts and 3 filamentous
fungi strains (Table 8).
IL IC95 (µM) When compared to microbial screening on amino acid
a derived ILs previously carried out by Coleman et al. the most
Microorganism Time (h) 21 22 23 24
active antifungal ILs were comprised of L-valine (35 and 36),
SA 24 1000 1000 2000 125 (Fig. 6), towards the filamentous fungal strains (AC, TM).15
48 1000 1000 2000 125 IC50 values as low as 62.5 µM were determined for L-valine
MRSA 24 1000 2000 2000 500
48 1000 2000 2000 500 ethyl ester analogue 35.
SE 24 1000 500 >2000 250 Compounds presented within this publication (4–24) were
48 1000 500 >2000 250 determined to be less toxic towards the fungal strains
EF 24 >2000 >2000 >2000 >1000
48 >2000 >2000 >2000 >1000 screened. We propose that L-phenylalanine is a preferable
EC 24 >2000 >2000 >2000 >1000 amino acid when considering the synthesis of low toxicity
48 >2000 >2000 >2000 >1000 amino acid ILs.
KP 24 >2000 >2000 >2000 >1000
48 >2000 >2000 >2000 >1000
KP-E 24 >2000 >2000 >2000 >1000
48 >2000 >2000 >2000 >1000 Preliminary biodegradation studies of ionic liquids and
PA 24 >2000 >2000 >2000 >1000 tertiary amino analogues
48 >2000 >2000 >2000 >1000
The biodegradation screening was carried out according to the
a
SA, Staphylococcus aureus; MRSA, Staphylococcus aureus MRSA; SE, Closed Bottle test (CBT).40,55,57 As can be seen from Table 9, of
Staphylococcus epidermidis; EF, Enterococcus sp.; EC, Escherichia coli;
KP, Klebsiella pneumoniae; KP-E, Klebsiella pneumoniae; PA, Pseudomo- the ILs screened, imidazolium IL 4 did not reach the 60% bio-
nas aeruginosa. degradation threshold required to pass the CBT. Pyridinium IL 5,

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Paper Green Chemistry

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

IL MICb (µM)

Microorganisma Time (h) 4–6, 10, 13–15, 17–23 7, 24 8 9 11 12 16

CA1 24 >2000 >1000 >500 >2000 >2000 >2000 >2000

48 >2000 >1000 >500 >2000 >2000 >2000 >2000
CA2 24 >2000 >1000 >500 >2000 >2000 >2000 >2000
48 >2000 >1000 >500 >2000 >2000 >2000 >2000
CP 24 >2000 >1000 >500 >2000 >2000 >2000 2000
48 >2000 >1000 >500 >2000 >2000 >2000 >2000
CK1 24 >2000 >1000 >500 2000 2000 >2000 >2000
48 >2000 >1000 >500 2000 >2000 >2000 >2000
CK2 24 >2000 >1000 >500 2000 >2000 >2000 >2000
48 >2000 >1000 >500 2000 >2000 >2000 >2000
CT 24 >2000 >1000 >500 >2000 2000 >2000 >2000
48 >2000 >1000 >500 >2000 2000 >2000 >2000
CG 24 >2000 >1000 >500 2000 >2000 >2000 >2000
48 >2000 >1000 >500 2000 >2000 >2000 >2000
CL 24 >2000 >1000 >500 >2000 2000 >2000 >2000
48 >2000 >1000 >500 >2000 2000 >2000 >2000
TA 24 >2000 >1000 >500 2000 >2000 >2000 >2000
48 >2000 >1000 >500 2000 >2000 >2000 >2000
AF 24 >2000 >1000 >500 >2000 >2000 >2000 >2000
48 >2000 >1000 >500 >2000 >2000 >2000 >2000
AC 24 >2000 >1000 >500 2000 >2000 >2000 >2000
48 >2000 >1000 >500 >2000 >2000 >2000 >2000
TM 72 >2000 >1000 >500 2000 2000 2000 >2000
120 >2000 >1000 >500 2000 2000 >2000 >2000
a
CA1, 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. b IC50 values were assessed for AF, AC and TM and IC80 for 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.56,57 This 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 properties24,26 and our result is an exception to
a
this trend. The C2 symmetric imidazolium IL 11 was also Biodegradation data for 5–8, 10–12, 21, 22 and 24 previously reported
in accompanying paper by Haiß et al.39 b One of the two bottles was
shown to be recalcitrant to biodegradation with the lowest leaking. c Not valid (biodegradation values in the replicates differ more
value (17%) recorded. than 20%).

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Green Chemistry Paper

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%
Fig. 7 Carbon atom distribution of amino acid IL (4).
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
plus the ester is considered, then these predicted levels of bio-
antibiotics amoxicillin (with a phenol ring, mineralizable in
degradation suggest that many of the ILs and amino com-
Zahn Wellens test) and piperacillin (even in the ZWT not
pounds (e.g. 4, 5, 10, 11, 16, 17, 20 and 21–23) could achieve
biodegradable).58
>60% biodegradation, based on complete biodegradation of
The similar levels of biodegradability for the L-phenyl-
the aminoacid ester moiety (Table 9). ILs (4–11, and 16–20) are
alanine and L-tyrosine derivatives suggests that the L-phenyl-
observed to have levels of biodegradation greater than only
alanine may not be undergoing conversion into L-tyrosine
ester degradation but less than complete amino acid ester
through enzymatic transformations by hydroxylase enzymes.59
degradation, except 5. However, this rough approximation of
The L-phenylalanine is instead being degraded directly and
carbon distribution and attributable degradation cannot be
may not be entering the homogentisate pathway.60 However it
fully substantiated without a transformation product analysis
cannot be inferred from the data directly and analysis of the
of the CBT. What exactly is breaking down? Is the rate of
metabolic products by a suitable LCMS technique to deter-
biodegradation such that some of the compound in the test
mine the biodegradation pathways and metabolic products
undergoes high levels of mineralisation after a particular bio-
formed is required.39
chemical transformation? Is there some rate limiting step that
None of the tertiary amino derivatives (21–24) CBT results
once completes, allows the IL to biodegrade? Is ester hydro-
can be considered a pass (Table 9). For 21 and 24 valid data
lysis occurring preferentially to amide bond hydrolysis or are
based on two replicates was not obtained in this initial bio-
both happening at the same time? Is there fully intact parent
degradation study. For subsequent CBT investigations and
compound present in the CBT vessel after the 28 day period?
more detailed biodegradation results and discussion see Haiß
To answer these questions a detailed biodegradation assess-
et al.39 22 and 23 did not reach the 60% threshold, within 28
ment, including transformation product identification and
days, and thus does not pass the CBT. However, when the ter-
analysis of the transformation products oxygen demand is
tiary amino derivative 22 was compared to the IL analogue it
required, and is published in the accompanying paper.39
can be seen that tertiary amino derivative 22 undergoes higher
Based on the data presented in Table 9 we cannot state with
levels of biodegradation (+22%) than IL analogue 10. The elev-
absolute certainty that a general trend is biodegradation of all
ated levels of biodegradation suggest that the presence of a
ester groups occurs for 4–24.
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 Transformation product
in agreement with the literature,6 but 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.25,33 Table 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

This journal is © The Royal Society of Chemistry 2016 Green Chem., 2016, 18, 4374–4392 | 4385
Paper
Scheme 3 Selective hydrolysis of ethyl ester of 10.
and biodegradation studies is challenging. Our approach was to
establish new synthetic routes to the proposed transformation pro-
ducts, provide authentic samples, and due to the sufficient
amounts prepared, determine whether these compounds have
high antimicrobial activity. We submit that toxicity data of con-
firmed transformation products assists in the design of safer
chemicals. Of note, due to the collaborative nature of this work, it
is only by detailed LCMS studies that the structure of the trans-
formation products can be confirmed. Therefore, herein we will
classify the transformation products (TP) as proposed transform-
ation products (PTP) and the reader is directed to the accompany-
ing paper for the related LCMS studies.39
The first strategy for the synthesis of proposed transform-
ation products (PTP) 37 and 40–46 was based on facile conver-
sion of ILs already prepared as part of this investigation.
Selective hydrolysis of an ester bond in the presence of an
amide is widely reported in peptide synthesis.61 To this end a
Scheme 4 Benzyl ester hydrogenolysis route to PTP 40.
4386 | Green Chem., 2016, 18, 4374–4392
Green Chemistry
LiOH mediated base hydrolysis of cholinium IL 10 procedure
to furnish PTP 37 was performed in good yield (Scheme 3). We
decided not to use this method for examples were two esters
and an amide were present and the selective hydrolysis of one
ester was required. Future investigations using enzymatic
methods will search for a green solution to this problem.
PTP 40 was synthesised according to a benzyl ester hydroge-
nolysis route via product 39 (Scheme 4). Starting compound
3862 underwent a substitution reaction with 26 in ethyl acetate
based on conditions described in Table 1 for the preparation
of L-phenylalanine ILs. Deprotection of the benzyl ester 39
using Pd/C catalysed hydrogenolysis afforded the carboxylic
acid PTP 40 in 87% yield. Efforts to use the same approach for
the nicotinium PTPs 41 and 42 failed due to reduction of the
pyridinium ring in the final step. Therefore, a different strategy
avoiding the use of a benzyl protecting group was devised. We
were pleased to find that sodium nicotinate was a suitable sub-
This journal is © The Royal Society of Chemistry 2016
Green Chemistry Paper

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,63a albeit in low
yields, this headgroup has not been widely adopted in recent
IL studies.63b The 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- Fig. 8 PTPs 43–47.
alanine according to a procedure by Qin et al.63c Treatment
with ethyl nicotinate in refluxing DMF, then HBr gave PTP 42
Table 10 Antibacterial activity results obtained for PTPs 37 and 40–47
in 33% overall yield.
For PTPs (43, 44 and 45) preparation, the route involved IL IC95 (µM)
synthesis of either an ethyl or a methyl ester of the target a
Microorganism Time (h) 37, 40–47 46
heterocycle followed by acid hydrolysis to give the carboxylic
acid PTP in a two-step reaction. The synthetic procedure is as SA 24 >2000 >500
outlined by Pukitis et al. who originally synthesised 43 from 48 >2000 >500
MRSA 24 >2000 >500
dimethylamino pyridine (DMAP).64 PTP 44 and 45 have been
48 >2000 >500
previously reported by Oldfield et al.65 and Deng et al.,37 SE 24 >2000 >500
respectively. PTP 46 was obtained according to the literature 48 >2000 >500
EF 24 >2000 >500
procedure outline by Farfán et al.66 in which methyl-
48 >2000 >500
ethanolamine is reacted with aqueous glyoxal to form a mor- EC 24 >2000 >500
pholine intermediate which undergoes hydrolysis in situ to 48 >2000 >500
KP 24 >2000 >500
give the carboxylic acid compound 46. PTP 47 was commer-
48 >2000 >500
cially available and purchased from TCI Europe (Fig. 8). KP-E 24 >2000 >500
Antimicrobial activity data shows that none of the PTPs (37 48 >2000 >500
PA 24 >2000 >500
and 40–47) have high toxicity to the 20 strains screened
48 >2000 >500
(Tables 10 and 11). This data is consistent with the results for
a
compounds 1–23 in Tables 5–8. ILs (41 and 43) showed the SA, Staphylococcus aureus; MRSA, Staphylococcus aureus MRSA; SE,
Staphylococcus epidermidis; EF, Enterococcus sp.; EC, Escherichia coli;
highest level of fungal toxicity (Table 11). IL 41 had an IC95 of KP, Klebsiella pneumoniae; KP-E, Klebsiella pneumoniae; PA, Pseudomo-
2000 µM towards the two Candida albicans strains whilst IL 43 nas aeruginosa.

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Paper Green Chemistry

Table 11 Antifungal activity results obtained for PTPs 37 and 40–47 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
IL MICb (µM)
achieved. C2-Symmetric IL 11 was prepared from TMS-imida-
Microorganisma Time (h) 37, 40, 45, 42, 44 41 43 46 zole in a one pot two step method in excellent yield (91%). Syn-
thesis of the L-tyrosine IL derivatives used a simple protection
CA1 24 >2000 2000 >2000 >500
group strategy by using an extra equivalent of the bromoacetyl
48 >2000 2000 >2000 >500
CA2 24 >2000 2000 >2000 >500 bromide reagent. Improvements in the synthesis of α-bromo-
48 >2000 2000 >2000 >500 amide 2615 were made. Reducing the volume of solvent required
CP 24 >2000 >2000 2000 >500
and removing the requirement to run the reaction at low temp-
48 >2000 >2000 2000 >500
CK1 24 >2000 >2000 >2000 >500 erature (−78 °C) lead to a greener method. A procedure repla-
48 >2000 >2000 >2000 >500 cing DCM with THF was also demonstrated, however the yield
CK2 24 >2000 >2000 >2000 >500
decreased by 15%. Green chemistry metrics were determined
48 >2000 >2000 >2000 >500
CT 24 >2000 >2000 >2000 >500 for the synthesis of ILs and their amino derivatives and rec-
48 >2000 >2000 >2000 >500 ommendations for improvements in future studies compiled.
CG 24 >2000 >2000 >2000 >500
We submit that the reactions can be classified into two types.
48 >2000 >2000 >2000 >500
CL 24 >2000 >2000 >2000 >500 First, quantitative to excellent yield has been established and
48 >2000 >2000 >2000 >500 the focus will be on reducing solvent use, using safer solvents
TA 24 >2000 >2000 >2000 >500
and reducing the amount of chemicals required, where poss-
48 >2000 >2000 >2000 >500
AF 24 >2000 >2000 >2000 >500 ible. This will lead to improved outputs for the green chemistry
48 >2000 >2000 >2000 >500 metrics, providing evidence that a greener synthesis has been
AC 24 >2000 >2000 >2000 >500
developed. Second, low to good yield reactions would benefit
48 >2000 >2000 >2000 >500
TM 72 >2000 >2000 2000 >500 from a broader approach to improve the synthesis. Alternative
120 >2000 >2000 2000 >500 green synthetic methodologies need to be trialled in combi-
a
CA1, Candida albicans; CA2, Candida albicans; CP, Candida parapsilo- nation with the aforementioned points. This is the strategy we
sis; CK1, Candida krusei; CK2, Candida krusei; CT, Candida tropicalis; are following for the synthesis of the next generation of ILs as
CG, Candida glabrata; CL, Candida lusitaniae; TA, Trichosporon asahii; part of this body of work.
AF, Aspergillus fumigatus; AC, Absidia corymbifera; TM, Trichophyton
mentagrophytes. b IC50 values were assessed for AF, AC and TM and IC80 Antimicrobial activity testing of L-phenylalanine ILs, L-tyro-
for all other strains. 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,
displayed an IC95 of 2000 µM towards the fungi Candida para- the highest toxicity (IC95 = 125 and 250 µM) were observed
psilosis and Trichophyton mentagrophytes. Even with these ILs towards the two gram positive strains SA and SE respectively.
the general trend is not a high antifungal activity when consid- High antimicrobial activity was not found for the other bac-
ering all 12 strains. teria or fungi strains screened. We were pleased to establish
We submit that this data significantly expands the scope of that the PTPs did not show high toxicity to the microorgan-
compounds in this study which do not show high microbial isms screened. Due to the maximum validated sample con-
toxicity. Not only can this supplement the earlier results centration of the microbial toxicity test (2000 µM, or
(Tables 5–8), assisting the design of greener ILs, but also pro- solubility limit) and the samples not having high toxicity, a
vides fundamental toxicity data of PTPs to aid biodegradation SAR model cannot be established. However, when comparing
studies. the phenylalanine and tyrosine examples with the valine ana-
logue 35, a lower antifungal activity was demonstrated for
4–24.
Preliminary CBT biodegradation results39 for 4–24 gave a
Conclusion wide range of values (17–63%). The highest value, obtained for
pyridinium IL 5, passes the CBT pass 60% threshold, but
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.39 The biodegradation data in
dation studies by the Kümmerer group.39 Antimicrobial 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.67 In that study,
properties. no compound was found to give a biodegradation value higher

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Green Chemistry
than that which can be attributed to ester transformation, so
that assumption was consistent with the data available. This is
not the case with compounds 4–24. A far more complex biodegra-
dation process occurs with this series, and based on Table 9
data alone it would be ill-advised to state that ester hydrolysis
accounts for the biodegradation values observed. For example,
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,
and then design new greener ILs based on this finding, would
be unadvisable without supporting TP data. One conclusion
that can be made from the data is that the presence of the OH
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
20 microbial strains tested), this is useful data for experts in
biodegradation and TP analysis studies. The formation of a TP
which is a potent biocide during biodegradation is obviously
undesirable and all data which can support research to avoid
this eventuality is valuable.
The preferred green IL from this study was pyridinium com-
pound 5. A short and efficient synthesis of 5 was described,
with improvements achieved in reducing solvent waste, the
required for low temperature alleviated and a lower toxicity
solvent replacement option described. Green chemistry metrics
provide evidence to support the statement that a greener syn-
thesis was developed. IL 5 does not have high antimicrobial
activity to the strains screened and has excellent biodegradation
properties. Although from the data in this paper we cannot state
whether 5 is readily biodegradable, passing the 60% threshold
of the CBT test is a significant result for a halide IL contain-
This journal is © The Royal Society of Chemistry 2016
Paper
ing an amide group. The conclusion that 5 is the preferred
green IL from this study is only possible due to the close
collaboration of research groups with expertise in organic
synthesis, biodegradation and toxicity screening. Promoting the
‘benign by design’ approach to the development of low toxicity
and biodegradable chemicals is our mutual goal.68–70
Experimental
Antibacterial activity experimental method
In vitro antibacterial activities71 of the compounds were evalu-
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 × 108 cfu
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.
Antifungal activity experimental method
In vitro antifungal activities of the compounds were evaluated
on a panel of four ATCC strains (Candida albicans ATCC 44859,
Candida albicans ATCC 90028, Candida parapsilosis ATCC
22019, Candida krusei ATCC 6258) and five clinical isolates of
yeasts (Candida krusei E28, Candida tropicalis 156, Candida
glabrata 20/I, Candida lusitaniae 2446/I, Trichosporon asahii
1188) and filamentous fungi (Aspergillus fumigatus 231, Absidia
corymbifera 272, Trichophyton mentagrophytes 445) from the col-
lection of fungal strains deposited at the Department of Bio-
logical and Medical Sciences, Faculty of Pharmacy, Charles
University, Hradec Králové, Czech Republic. Three ATCC
strains were used as the quality control strains. All the isolates
were maintained on Sabouraud dextrose agar prior to being
tested. Minimum inhibitory concentrations (MICs) were deter-
mined by modified CLSI standard of microdilution format of
the M27-A3 and M38-A2 documents.72,73 Dimethyl sulfoxide
Green Chem., 2016, 18, 4374–4392 | 4389
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−1 for the new compounds) and 10 μL of
inoculum suspension. Fungal inoculum in RPMI 1640 was pre-
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-
mined 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.
Biodegradation method
For biodegradation method see Haiß et al.39
General synthetic methods
Compounds 4,15 25,74 26,15 27,75 30,76 31,77 32,76 3378 and 3479
were prepared according to literature methods. Proposed trans-
formation products 43,64 44,65 4537 and 4666 were 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.†
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.
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