Sustainable Straightforward Synthesis and Evaluation of the Antioxidant and Antimicrobial Activity of Sinapine and Analogues

Article abstract of DOI:10.1021/acs.jafc.0c02183

Naturally occurring sinapine was successfully synthesized through a proline-mediated Knoevenagel-Doebner condensation in ethanol. This synthetic process involving biobased syringaldehyde, Meldrum's acid, and choline chloride offers a sustainable alternative to the existing low-yield pathways. This two-step strategy gives access to sinapine in a 52% overall yield and has been implemented in the synthesis of sinapine analogues, using 4-hydroxybenzaldehyde, 3,4-dihydroxybenzaldehyde, and vanillin as precursors, giving target molecules with 34-61% overall isolated yields. The purity of synthetic sinapine and its analogues (ca. 95%) was assessed by NMR and high-performance liquid chromatography-mass spectrometry analyses. Furthermore, the antioxidant and antimicrobial activities were assessed, and the potential of this series of molecules was confirmed.

Full text of DOI:10.1021/acs.jafc.0c02183

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Antimicrobial activity of
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Aurelien Peru, Matthieu
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Page J1ooufr2n3al of Agricultural and Food Chemistry
Cl-
N+
HO
Cl-
HO
O
ACN
N+
+
O
O
Cl-
N+
O
O
O
O
O
O
EtOH, proline
R1
R2
O
OH
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R
1 = H, OH, OMe
R2 = H, OH, OMe
R1
R2
OH

Journal of Agricultural and Food Chemistry
Page 2 of 23
1
2
3
Sustainable Straightforward Synthesis and
Evaluation of the Antioxidant and Antimicrobial
activity of Sinapine and Analogs
4
5
Louis M. M. Mouterde*, Aurélien A. M. Péru, Matthieu Mention,
Fanny Brunissen, Florent Allais*
6 URD Agro-Biotechnologies Industrielles (ABI), CEBB, AgroParisTech,
7 51110, Pomacle, France
8
9
Phone +33 3 52 62 04 65, E-mail louis.mouterde@agroparistech.fr, florent.allais@agroparistech.fr
*Authors to whom correspondence should be addressed
10
11
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Journal of Agricultural and Food Chemistry
12 Abstract
13 Naturally occurring sinapine was successfully synthesized through
14 a proline-mediated Knoevenagel-Doebner condensation in ethanol.
15 This synthetic process involving bio-based syringaldehyde,
16 Meldrum’s acid and choline chloride, offers a sustainable
17 alternative to the existing low yield pathways. This two-step
18 strategy gives access to sinapine in a 52% overall yield, and has
19 been implemented to the synthesis of sinapine analogs using 4-
20 hydroxybenzaldehyde, 3,4-dihydroxybenzaldehyde and vanillin as
21 precursors giving target molecules with 34-61% overall isolated
22 yields. Purity of synthetic sinapine and that of its analogs (ca.
23 95%) was assessed by NMR and HPLC-MS analysis. Furthermore,
24 antioxidant and antimicrobial activities were assessed and
25 confirmed the potential of this series of molecules.
26 Keywords: Sinapine, Knoevenagel-Doebner, Meldrum's acid, Choline chloride, Choline
27 phenolic esters
28
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29
30 Introduction
31 Naturally occurring sinapic acid (1) and its main esters (e.g., sinapoyl-glucose, sinapoyl-malate
32 and sinapoyl-choline aka sinapine) have shown great potential in a wild range of applications
33 thanks to their potent anti-UV, antioxidant, anti-inflammatory, anti-cancer and/or anti-microbial
34 properties (Scheme 1).1-9 These compounds can be found in Nature in a wide variety of products
35 such as fruits, vegetables, cereals or Brassicaceae seeds.10 The latter are particularly of interest
36 due to their relatively high contents in sinapic analogs and their availability, especially rapeseeds
37 that can contain up to 18,000 µg of sinapic derivatives per gram of seed.11, 12 After obtaining the
38 oil by cold pressure, the resulting seed cake can contain up to 10,000 µg of sinapic analogs per
39 gram of dry weight.12, 13 Moreover, the global production of rapeseed cake was at an average of 69
40 millions of tons between 2013 and 2016, which represents a capacity of 69 kT sinapic acid per
41 year.14 This potential source of sinapic derivatives has generated many research works aimed at
42 efficiently extracting sinapic compounds from this agricultural byproduct.15-18 The same
43 observation can be made for mustard bran that contains up to 125,000 µg of sinapic derivatives
44 per gram of dry weight.4, 19, 20 Although great advances have been made in this area, no viable
45 industrial process has been developed yet. Therefore, alternatives must be found to access these
46 valuable molecules.
HO
O
O
OH
OH
OH
O
X
O
O
O
O
O
O
O
O
N
HO
OH
O
O
O
O
OH
OH
OH
Sinapoyl-glucose
Sinapoyl-malate
Sinapine
47
48
Scheme 1. Wathelet et al. synthesis strategy to sinapine
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49
50 While straightforward synthesis of sinapoyl-malate and sinapoyl-glucose have been described in
51 the literature, it is more ambiguous in the case of sinapine.21, 22 A method has been reported by
52 Wathelet et al., based on the work of Clausen et al., in a communication poster involving sinapoate
53 (2) and bromocholine bromide (3) (Scheme 2).23, 24 This method presents several disadvantages
54 because of (1) the relative low availability and high price of sinapic acid (1), (2) the significant
55 toxicity as well as the corrosivity of bromocholine bromide (3), and (3) the necessity of performing
56 a precise titration for the formation of 2.25 Moreover, this method has been developed on a ~50 mg
57 scale and the yields are not clearly reported. It is noteworthy to mention that the synthesis of
58 sinapine analogs through this method has been reported in the literature using chlorocholine
59 chloride as precursor.26 Another strategy consisted in an esterification of sinapic acid, via an acyl
60 chloride and using dimethyl-ethanolamine, followed by a methylation of the resulting
61 intermediate.27, 28
Br
O
OH
O
O
O
O
Na
Br
N
Br
N
NaOH
MeOH
3
MeOH
O
O
O
O
O
O
OH
OH
OH
1
2
Sinapine bromide
62
63
Scheme 2. Synthetic strategy to sinapine from Wathelet et al. [9a]
64 Under such consideration, a sustainable and straightforward strategy to access sinapine remained
65 to be developed. Herein, we report a convergent retrosynthetic approach starting with the
66 transesterification of Meldrum’s acid (4) with choline halide (5) followed by a Knoevenagel-
67 Doebner condensation of the resulting choline chloride malonate monoester (6) with lignin-derived
68 and readily available syringaldehyde (7) (Scheme 3). This approach was then successfully
69 implemented to other p-hydroxybenzaldehydes and the antioxidant and antimicrobial properties of
70 the resulting choline esters were assessed.
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O
X
O
O
O
N
O
O
OH
X
O
O
N
7
X
O
O
HO
O
+
O
N
O
O
OH
6
4
5
OH
Sinapine
71
72
Scheme 3. Retrosynthetic approach for the synthesis of sinapine
73 Materials and Methods
74 General: Evaporations were conducted under reduced pressure at temperatures below 40 °C
1
75 unless otherwise noted. H NMR spectra were recorded at 300 MHz at 25 °C in the indicated
76 solvent and referenced to residual protons (CD3OD, 4.87 ppm). 13C NMR spectra were recorded
77 at 75 MHz at 25 °C in the indicated solvent and referenced to solvent (CD3OD, 49.2 ppm).
78 HPLC/MS method: LC-MS analyzes were performed on an Agilent 1290 system, equipped with
79 a PDA UV detector, and a 6545 Q-ToF mass spectrometer (Wilmington, DE, USA). The source is
80 equipped with a JetStream ESI probe operating at atmospheric pressure. The spectrometer was
81 configured according to the following settings: mass range m/z 50–1000, gas temperature 325 °C,
82 gas flow 8 L/min, nebulizer 35 psi, sheath gas temperature 350 °C, sheath gas flow 11 L/min.
83 Results were recorded and processed with Mass Hunter B.08.000 software. Elution was performed
84 using a Zorbax Eclipse plus C18 (1.8 µm, 50 x 2.1 mm; Agilent) with the column heated at 40 °C.
85 The mobile phases were 0.1% formic acid in water (solvent A) and acetonitrile (solvent B), the
86 flow rate was set at 0.4 mL/min and followed the gradient: 0-3 min at 5% of B, 3-4 min from 5%
87 to 10% B, 4-13 min from 10% to 99% B, 13-16 min at 99% B, 16-18 min from 99% to 5% B. The
88 sample injection volume was 1 µL and the autosampler was tempered at 10 °C. The UV acquisition
89 was carried out at 250 nm and 320 nm with a reference set at 360 nm.
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90 Method for the synthesis of Sinapine and its analogs: Meldrum’s acid (1.51 g, 10.5 mmol) and
91 choline chloride (980 mg, 7 mmol) were mixed together in acetonitrile (3.5 mL, 2M) and heated
92 at reflux for 5 hours. The resulting mixture was dried in vacuo and dissolved in ethanol (630 mM,
93 10 mL). The corresponding aldehyde (6.9 mmol) and proline (725 mg, 6.9 mmol) were then added
94 and the solution was refluxed overnight. The crude mixture was concentrated in vacuo, dissolved
95 in deionized water (10 mL) and directly applied to a C18 reversed phase flash (95/5,
96 water/methanol). The fraction containing the desired product were combined and dried in vacuo
97 to yield the pure product.
1
98 Coumaroyl-Choline: 34% isolated yield. M.p. 240-242 °C. H NMR (300 MHz, CD3OD): δ =
99 7.70 (d, J = 15.9 Hz, 1H, H-3), 7.50 (d, J = 8.4 Hz, 1H, H-5 and H-9), 6.83 (d, J = 8.4 Hz, 2H, H-6
100 and H-8), 6.39 (d, J = 15.9 Hz, 1H, H-2), 4.67 (m, 2H, H-10), 3.81 (m, 2H, H-11), 3.30 (s, 9H, H-
101 12). 13C NMR (75 MHz, CD3OD): δ = 167.0 (s, C-1), 160.7 (s, C-7), 146.8 (d, C-3), 130.5 (d,C-5
102 and C-9), 125.9 (s, C-4), 116.0 (d, C-6 and C-8), 113.1 (d, C-2), 65.3 (t, C-11), 57.9 (t, C-10), 53.6
+
103 (q, C-12). TOF MS ES+: [M]+ for C14H20NO3 : m/z 250.1438; found: m/z 250.1437.
104 Caffeoyl-Choline: 61% isolated yield. M.p. 183-185 °C. 1H NMR (300 MHz, CD3OD): δ = 7.61
105 (d, J = 15.9 Hz, 1H, H-3), 7.09 (d, J = 1.8 Hz, 1H, H-9), 6.98 (dd, J = 1.8 and 8.4 Hz, 1H, H-8),
106 6.80 (d, J = 8.4 Hz, 1H, H-5), 6.31 (d, J = 15.9 Hz, 1H, H-2), 4.65 (m, J = Hz, 1H, H-10), 3.80 (m,
107 5H, H-11 and H-13), 3.27 (s, 9H, H-12). 13C NMR (75 MHz, CD3OD): δ = 166.5 (s, C-1), 148.5
108 (s, C-7), 146.7 (s, C-6), 145.5 (d, C-3), 126.1 (s,C-4), 121.9 (d, C-9), 115.2 (d, C-8), 114.0 (d, C-
109 2), 112.6 (d, C-5), 64.8 (t, C-11), 57.4 (t, C-10), 53.1 (q, C-12). TOF MS ES+: [M]+ for
+
110 C14H20NO4 : m/z 266.1387; found: m/z 266.1385.
111 Feruloyl-Choline: 50% isolated yield. M.p. 109-111 °C. 1H NMR (300 MHz, CD3OD): δ = 7.50
112 (d, J = 15.9 Hz, 1H, H-3), 7.06 (d, J = 1.8 Hz, 1H, H-9), 6.96 (dd, J = 1.8 and 8.1 Hz, 1H, H-6),
113 6.76 (d, J = 8.1 Hz, 1H, H-5), 6.27 (d, J = 15.9 Hz, 1H, H-2), 4.57 (m, J = Hz, 1H, H-10), 3.76 (m,
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114 5H, H-11 and H-13), 3.23 (s, 9H, H-12). 13C NMR (75 MHz, CD3OD): δ = 167.9 (s, C-1), 150.6
115 (s, C-8), 149.2 (s, C-7), 147.7 (d, C-3), 127.3 (s,C-4), 124.4 (d, C-5), 116.5 (d, C-6), 114.3 (d, C-
116 2), 111.8 (d, C-9), 66.0 (t, C-11), 58.9 (t, C-10), 56.5 (q, C-13), 54.6 (q, C-12). TOF MS ES+: [M]+
+
117 for C15H22NO4 : m/z 280.1543; found: m/z 280.1545.
118 Sinapine: 48% isolated yield. M.p. 82-84 °C. 1H NMR (300 MHz, CD3OD): δ = 7.44 (d, J = 15.9
119 Hz, 1H, H-3), 6.74 (s, 2H, H-5 and 9), 6.24 (d, J = 15.9 Hz, 1H, H-2), 4.56 (m, 2H, H-10), 3.78
120 (m, 2H, H-11), 3.74 (s, 6H, H-13), 3.24 (s, 9H, H-12). 13C NMR (75 MHz, CD3OD): δ = 167.7 (s,
121 C-1), 149.2 (s, C-6 and 8), 147.8 (d, C-3), 139.5 (s, C-7), 126.2 (s, C-4), 114.7 (d, C-2), 106.9 (d,
122 C-5 and 9), 66.1 (t, C-11), 59.9 (t, C-10), 56.8 (q, C-12), 54.6 (q, C-13). TOF MS ES+: [M]+ for
+
123 C16H24NO5 : m/z 310.1649; found: m/z 310.1648.
124 Antioxidant assay: 190 μL of homogeneous DPPH solution (200 μM, 2,2-DiPhenyl-1-
125 PicrylHydrazyl) in ethanol was added to a well containing 10 μL of coumaroyl-, caffeoyl-,
126 feruloyl-choline or sinapine in ethanol at different concentrations (from 400 μM to 12.5 μM). The
127 reaction was followed by a microplate Multiskan FC, performing 1 scan every 5 min for 7.5 h at
128 515 nm. The use of different amounts of substrate give the EC50 value, which is describe as the
129 efficient concentration needed to reduce the initial population of DPPH by half.
130 Antimicrobial assay: Overnight cultures of Escherichia coli K12 strain were diluted to an OD600
131 = 0.1 in a sterile 96-well microplate with fresh LB medium with or without the addition of
132 coumaroyl-, caffeoyl-, feruloyl-choline or sinapine at a final concentration of 10, 5, 2.5, 1.25, 0.630,
133 0.320, 0.160, 0.080, 0.040 or 0.020 %w, and the optical density at 600 nm was measured every 15
134 min. Proper blank controls were used in each microplate. Wells containing the MBCs and MICs
135 were streaked onto Agar plates and incubated for 24 hours.
136
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137 Results and Discussion
138 Synthesis of sinapine and its analogs. The first step of this study dealt with the formation of the
139 choline chloride malonate monoester (9). Unfortunately, the latter cannot be readily obtained
140 through classical Fischer esterification. Indeed, under such conditions, the desymmetrization of
141 malonic acid with choline chloride is difficult due to the even reactivity of the two carboxylic acid
142 functions. Therefore, an alternative strategy consisting in performing a transesterification on
143 Meldrum’s acid with choline chloride was investigated (Scheme 4).29
Cl
Cl
O
O
O
HO
HO
O
+
+
N
N
O
O
O
O
5
8
9
144
145
146
Scheme 4. Formation of the choline chloride malonate monoester (9)
147 Preliminary experiments with an equimolar mixture of choline chloride and Meldrum’s acid with
148 or without solvent (acetonitrile 2M) showed the degradation of Meldrum's acid into malonic acid,
149 and further into acetic acid (Table 1, entries 1 and 4). In order to increase the conversion and to
150 find the optimal conditions, the reaction was carried out with or without solvent at different choline
151 chloride/Meldrum’s acid ratio over a 3-hour period. The reactions without solvent were performed
152 at 90 °C, melting point of Meldrum's acid, and the reaction in acetonitrile at 82 °C, boiling
153 temperature of the solvent. A slight excess of Meldrum's acid (1.25 equivalents) resulted in a better
154 conversion with or without solvent (Table 1, entries 2 and 5). Increasing this excess to 1.5
155 equivalents allowed to reach 100% conversion in presence of solvent (Table 1, entry 3), while 85%
156 conversion was observed without solvent (Table 1, entry 6). It seems that acetic acid formed in
157 situ hindered the reaction completion by favorize the degradation of Meldrum's acid if not diluted
158 in a solvent. Therefore, the optimal conditions to obtain the monoester 9 in quantitative conversion
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159 appeared to be in acetonitrile at 82 °C with 1.5 equivalents of Meldrum's acid (characteristic peak
160 at 1.70 ppm, ESI-S5) (Table 1, entry 3). However, in such conditions, residual Meldrum’s acid
161 was observed. This issue was easily overcome by extending the reaction time to 5 hours (Table 1,
162 entry 7).
163 Table 1. Optimization of the choline chloride malonate monoester synthesis.
Ratio choline
Temperature
(°C)
Conversion
(%) [a]
Entry
Solvent
Duration (h)
chloride/Meldrum's
acid
1
2
3
4
5
6
7
Acetonitrile
Acetonitrile
Acetonitrile
None
82
82
82
90
90
90
82
3
3
3
3
3
3
5
1:1
1:1.25
1:1.5
1:1
60
76
100
65
None
1:1.25
1:1.5
1:1.5
79
None
85
Acetonitrile
100
164 [a] Conversion were determined by 1H NMR of the crude reaction mixture.
165 After concentration in vacuo, the crude reaction mixture (Table 1, entry 7) was directly submitted
166 to a proline-mediated Knoevenagel-Doebner condensation with syringaldehyde in ethanol to
167 afford sinapine.30, 31 The mechanism of this reaction, previously described by Peyrot et al., involves
168 the activation of the aldehyde moiety through the formation of the iminium 12 which will then
169 react with the monoester 9. The intermediary 13 thus formed will undergo decarboxylation to yield
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170 14 that rearranges to regenerate proline and provide sinapine (Scheme 5). The latter can be easily
171 purified through C18 reverse phase chromatography giving the pure product in a 48% isolated
172 yield. Such approach avoids the use of the non-sustainable classical pyridine/piperidine system.32
173 It is worth mentioning that transesterification of sinapine by ethanol occurred (up to 20% yield).
174 Polar aprotic solvents such as THF, AcOEt and Cyrene® have been investigated to replace ethanol
175 in order to overcome this issue, unfortunately, the results were not conclusive.
O
O
OH
OH
OH
N
N
O
H2O
O
+
N
OH
Cl
H
O
O
O
O
O
O
HO
O
OH
OH
OH
N
O
O
7
10
11
12
9
Cl
Cl
Cl
O
O
O
O
O
O
N
N
N
N
N
OH
H
O
O
O
HO
HO
O
O
CO2
O
O
O
O
OH
OH
OH
Sinapine
14
13
176
177
Scheme 5. Proline-mediated Knoevenagel-Doebner condensation’s mechanism
178
179 This methodology was then implemented to benzaldehyde, 3,4-dihydroxybenzaldehyde and
180 vanillin in order to yield coumaroyl-, caffeoyl- and feruloyl-choline, respectively.
181
182
183
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184 Table 2. Isolated yields of sinapine analogs.
Substrate
4-hydroxybenzaldehyde
3,4-dihydroxybenzaldehyde
Vanillin
Product
Yield (%) [a]
Coumaroyl-choline
Caffeoyl-choline
Feruloyl-choline
34
61
50
185 [a] Yields were calculated from isolated product after purification.
186 In all cases, quantitative consumption of the aldehyde was observed and isolated yields varied
187 from 34 to 61% (Table 2). It is worth mentioning that these procedures have been validated at the
188 gram-scale.
189 Characterization of sinapine and its analogs. The compounds thus formed and purified were
190 characterized by UHPLC-MS. Mass analysis revealed that in all cases the major peak
191 corresponded to the molecular ion (m/z 250, 266, 280 and 310 for coumaroyl-, caffeoyl-, feruloyl-
192 choline and sinapine, respectively). Additionally, two characteristic fragments corresponding to
193 the loss of trimethylamine and choline were observed for each analog (m/z 59 and 103,
194 respectively). The HPLC UV-detection chromatograms at 320 nm (Figures S-11 and 12) also
195 showed another peak eluting at 0.5 min corresponding to residual choline as confirmed by 1H NMR
196 analysis (ca. 5% mol). Finally, thanks to very different retention time, it was shown that
197 coumaroyl-, caffeoyl-, feruloyl-choline and sinapine can be efficiently separated using reversed
198 phase C18 chromatography, which is a very interesting feature if one wants to use a crude mixture
199 of p-hydroxybenzaldehydes directly obtained after lignin-oxidation (Figure 1).
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200
201
202
Figure 1. HPLC UV-detection chromatograms superposition of coumaroyl (red peak), caffeoyl
(black peak), feruloyl (blue peak) and sinapoyl-choline (green peak)
203 Evaluation of the antioxidant activity of sinapine and analogs. Antioxidant activities of p-
204 hydroxycinnamic acids are well described in the literature.1-9 An efficient method to assess this
205 property is through DPPH assay which determines the capacity of a given molecule to scavenge
206 stable DPPH free radicals, usually expressed as the quantity of molecule needed to reduce half of
207 the initial population of DPPH radicals (aka EC50, the lower the EC50, the higher the antioxidant
208 activity is). In this study, the EC50 value for sinapine was determined as being 18.1 nmol, which is
209 of the same order of magnitude than that of sinapine thiocyanate measured by Wei et al. using the
210 same assay (17.6 nmol).33 This study was extended to coumaroyl-, caffeoyl- and feruloyl-choline,
211 giving EC50 values of >150, 6.26 and 36.73 nmol, respectively. The impact on the antioxidant
212 activity of the substituents nature, number, and position on the aromatic cycle is well known
213 phenomenon34 As already reported, the extra methoxy group of sinapine increases the antiradical
214 activity compared to that of feruloyl-choline (18.1 nmol vs. 36.73 nmol); so does the extra hydroxy
215 group of the caffeoyl-choline (6.26 nmol vs. 36.73 nmol). It is noteworthy to mention that the
216 impact of the hydroxy group on the antioxidant properties is higher than that of the methoxy group
217 (6.26 nmol vs. 18.1 nmol), as it provides an extra phenol moiety able to quench a second radical.
218 Compared to commercially available fossil-based antioxidant such as Irganox 1010 (plastic
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219 industry), Trolox (pharmaceutical industry)BHA (Butylated HydroxyAnisole) and BHT
220 (Butylated HydroxyToluene), sinapine and its analogs have slightly lower free radicals scavenging
221 abilities (Table 3). Nevertheless, these molecules are biobased and show high-water solubility
222 which can be real benefits in the cosmetics and agri-food industry.
223 Table 3. EC50 values for commercially available antioxidants, sinapine and its analogs.
Compound
Irganox 1010
Trolox
EC50 (nmol)
6.89 ± 0.28
4.02 ± 0.34
7.11 ± 0.26
3.67 ± 0.41
13.7 ± 0.66
>150
BHT
BHA
Ethyl-Sinapate
Coumaroyl-choline
Caffeoyl-choline
Feruloyl-choline
Sinapine
6.26 ± 0.11
36.73 ± 1.52
18.1 ± 0.57
224
225 Antimicrobial activity of sinapine and analogs. The antimicrobial potential of sinapine and its
226 analogs has also been assessed using Escherichia coli K12 strain. The microorganism was put in
227 presence of different concentrations of the studied compounds and the growth was measured by
228 optical density at 600 nm (OD600). While sinapine showed minimal inhibitory concentration (MIC)
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229 at 10%w, feruloyl-, caffeoyl- and coumaroyl-choline gave better results at 5, 1.25 and 1.25%w,
230 respectively. The minimal bactericidal concentration (MBC) was also observed at 10, 5, 2.5 and
231 2.5%w for sinapine, feruloyl-, caffeoyl- and coumaroyl-choline, respectively (Figure 2). It has to
232 be noted that, as previously observed for phenolic acids.35 the more methoxy groups on the
233 aromatic ring, the lower the antimicrobial activity. As a comparison, natural antibacterial thymol
234 has a MBC of 0.2%w.36 MICs and MBCs were confirmed by streaking the culture media on agar
235 plates followed by an incubation at 37 °C for 24 hours. MICs showed colonies in the agar plates,
236 where MBCs showed no colonies. It is important to note that the augmentation of the absorbance
237 at the MBCs of caffeoyl-choline and sinapine are due to strong coloration of the culture media
238 (most likely due to degradation products).
239
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240
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242
243
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247
Figure 2. Growth pattern Escherichia coli K12 strain in the presence of coumaroyl-, caffeoyl-,
feruloyl-choline and sinapine. E. coli was grown in LB without (0) or with addition of the
indicated concentrations of studied compounds (in %w), and the OD600 was measured at the
indicated time points.
248 Herein, we have developed and optimized a sustainable synthetic approach to sinapine, feruloyl-,
249 caffeoyl- and coumaroyl-choline through a Knoevenagel-Doebner condensation of the
250 corresponding p-hydroxybenzaldehydes and the asymmetric choline chloride malonate monoester
251 obtained from Meldrum’s acid. This two-step strategy provides the desired products in isolated
252 yields from 34 to 61%. The compounds thus formed were characterized through UHPLC-MS and
253 NMR analysis evaluating their purities being ~95%. Finally, their antioxidant and antimicrobial
254 activities were assessed, and data confirmed their potential as (1) alternatives to current fossil-
255 based antioxidant, and (2) antimicrobial.
256 Abbreviations used
257 AcOEt: Ethyl Acetate
258 BHA: Butylated HydroxyAnisole
259 BHT: Butylated HydroxyToluene
260 DPPH: 2,2-DiPhenyl-1-PicrylHydrazyl
261 EC50: half maximal Effective Concentration
262 HPLC: High-Performance Liquid Chromatography
263 LC-MS: Liquid Chromatography-Mass Spectrometry
264 LB medium: Lysogeny Broth medium
265 MBC: Minimum Bactericidal Concentration
266 MIC: Minimum Inhibition Concentration
267 NMR: Nuclear Magnetic Resonance
268 OD600: Optical Density at 600 nm
269 Q-ToF: Quadrupole-Time of Flight
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270 THF: TetraHydroFuran
271 UHPLC: Ultra High-Performance Chromatography
272 Acknowledgements
273 This work was supported by the Région Grand Est, Conseil Départemental de la Marne and Grand
274 Reims.
275 Supporting Information description
276 A separate Electronic Supporting Information file containing all 1H and 13C NMR spectra for all
277 compounds, HPLC chromatograms as well as DPPH and growth inhibition curves for sinapine and
278 analogs.
279 References
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