Metabolism of Food-borne Heterocyclic Aromatic Amines by

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Metabolism of Food-borne Heterocyclic Aromatic Amines by Lactobacillus reuteri DSM 20016 Falco Beer, Felix Urbat, Jan Steck, Melanie Huch, Diana Bunzel, Mirko Bunzel, and Sabine E. Kulling J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b01663 • Publication Date (Web): 05 Jul 2017 Downloaded from http://pubs.acs.org on July 7, 2017

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Journal of Agricultural and Food Chemistry

Metabolism of Food-borne Heterocyclic Aromatic Amines by Lactobacillus reuteri DSM 20016

Falco Beer,† Felix Urbat,# Jan Steck,†,# Melanie Huch,† Diana Bunzel,†,* Mirko Bunzel,# Sabine E. Kulling†

†

Department of Safety and Quality of Fruit and Vegetables, Max Rubner-Institut (MRI), Federal

Research Institute of Nutrition and Food, Haid-und-Neu-Straße 9, 76131 Karlsruhe, Germany #

Department of Food Chemistry and Phytochemistry, Karlsruhe Institute of Technology (KIT),

Adenauerring 20a, 76131 Karlsruhe, Germany

*Corresponding author, phone: +49 (0)721 6625 489; fax: +49 (0)721 6625 453. E-mail: [email protected]

ACS Paragon Plus Environment


1

Abstract

2

The heterocyclic aromatic amine (HAA) 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)

3

is converted into 7-hydroxy-5-methyl-3-phenyl-6,7,8,9-tetrahydropyrido[3′,2′:4,5]imidazo[1,2-

4

a]pyrimidin-5-ium chloride (PhIP-M1) via a chemical reaction with 3-hydroxypropionaldehyde or

5

acrolein derived from glycerol by reuterin producing gut bacteria. Because it is unknown whether

6

this reaction is also relevant for other HAAs, seven food-borne HAAs (2-amino-9H-pyrido[2,3-

7

b]indole (AαC), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 2-amino-3-methyl-3H-

8

imidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethyl-3H-imidazo[4,5-f]quinoline (MeIQ), 2-amino-

9

3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 9H-pyrido[3,4-b]indole (norharman), and 1-

10

methyl-9H-pyrido[3,4-b]indole (harman)) were anaerobically incubated with Lactobacillus reuteri

11

DSM 20016 in the presence of glycerol. The extent of conversion, as analyzed by HPLC-

12

DAD/FLD, was dependent on both the studied HAAs and the glucose/glycerol ratio, indicating

13

reuterin to be involved in HAA metabolism. Based on HRMS analyses, PhIP-M1-type metabolites

14

were detected for AαC, Trp-P-1, IQ, MeIQ, MeIQx, harman and norharman. In the case of AαC,

15

this was confirmed by metabolite isolation (AαC-M8, 2,3,4,10-tetrahydro-1H-indolo[2,3-

16

b][1,8]naphthyridin-2-ol) and one- (1H) and two-dimensional (HSQC, HMBC, COSY, DOSY)

17

NMR spectroscopy. In addition, based on HRMS and/or NMR spectroscopy, a new type of HAA

18

metabolite, resulting from the reaction with two molecules of 3-hydroxypropionaldehyde or

19

acrolein, is hypothesized for AαC, Trp-P-1, IQ, MeIQ, and MeIQx.

20 21 22 23 24

KEYWORDS: heterocyclic aromatic amines; bacteria; glycerol; metabolites; Lactobacillus reuteri;

25

reuterin; HPLC; HRMS; NMR. 1


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26

Introduction

27

Based on data from 2012, colorectal cancer was the second most common cause of cancer deaths in

28

Europe.1 There is evidence that higher susceptibility to colorectal cancer in the Western population

29

is associated with dietary habits (“Western diet”) such as a higher consumption of meat.2 Recently,

30

the International Agency for Research on Cancer classified processed meat as “carcinogenic for

31

humans” (group 1) based on sufficient evidence with respect to colorectal cancer.3 However, this

32

evaluation has also been criticized.4 The risk associated with the consumption of large amounts of

33

red or processed meat may be due to genotoxic substances such as heterocyclic aromatic amines

34

(HAAs), N-nitroso compounds and/or heme iron.

35

HAAs are formed during heating of protein-rich food, especially meat and fish. According to their

36

chemical structures, HAAs can be divided into two main classes: aminoimidazoazarenes (IQ type)

37

and amino-carbolines (carboline type) (Figure 1).5 HAA concentrations in meat are only in the ppb

38

range, but depend on the type and quality of the raw meat used6-9 and on the preparation conditions,

39

e. g. temperature, heating time, and cooking method.6,8-13

40

Although the dietary exposure is expected to be very low (several ng to a few µg per capita per

41

day),14 HAAs may be detrimental for human health because of their highly mutagenic and/or

42

carcinogenic potential.15 In vivo, HAAs are partially absorbed, followed by complex

43

biotransformation reactions.16 Initial hydroxylation by cytochrome P450 enzymes is regarded as a

44

key step occurring either at the heterocyclic ring system followed by glucuronidation and renal

45

excretion (detoxification) or at the exocyclic amino group leading to the formation of an N-

46

hydroxylamine which is further metabolized by releasing the aryl nitrenium ion (toxification). Due

47

to its high reactivity, the aryl nitrenium ion is able to form DNA adducts that can cause mutations

48

and, as a result, induction of cancer.

49

In contrast to detailed studies on the human xenobiotic metabolism, only limited information is

50

available regarding the impact of human intestinal bacteria. Research has been mainly focused on 2



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two aspects, namely the role of bacterial β-glucuronidases able to release the critical N-hydroxyl

52

metabolite17 and the ability of intestinal bacteria to bind HAAs in vitro under physiological

53

conditions.18,19 However, it has also been shown that HAAs can be metabolized by human intestinal

54

microbiota. First studies were performed by Bashir et al.20 and Humblot et al.21 who identified the

55

monohydroxylated derivative 7-OH-IQ as a bacterial metabolite of IQ. More recently, another type

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of microbial metabolite, PhIP-M1 (Figure 1), was isolated from anaerobic batch fermentations with

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human fecal suspensions22 and subsequently shown to be formed by the action of reuterin producing

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bacteria such as Lactobacillus reuteri and Eubacterium hallii.23,24 More precisely, PhIP-M1 has

59

been proposed to be formed in a chemical reaction between PhIP and the microbial glycerol

60

degradation

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3-hydroxypropionaldehyde is known to exist within a dynamic, pH- and concentration-dependent

62

equilibrium with its dimer and its hydrate.25,26 This multi-compound system is called reuterin. In

63

addition, acrolein, the dehydration product of 3-hydroxypropionaldehyde, has been proposed to be

64

included in the term reuterin.27 Its reversible formation from 3-hydroxypropionaldehyde was shown

65

to occur under physiologically relevant conditions.27 PhIP-M1 is also formed in vivo, as

66

demonstrated by a human study in which six participants consumed cooked chicken containing 0.9–

67

4.7 µg PhIP.28 These results proved that the amount of reuterin commonly produced in the intestinal

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tract is sufficiently high to play a role in the overall metabolism of PhIP in humans.

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Whether PhIP-M1 formation has to be considered as detoxification or toxification is still a matter of

70

debate because the metabolite was, in contrast to the parent compound, not mutagenic in the Ames

71

assay28 but showed cytotoxic properties such as the induction of apoptosis or cell cycle arrest in

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Caco-2 cells.29 However, based on BALB/c 3T3 cell transformation assay results, Nicken et al.30

73

discussed that the in vivo concentration is probably not sufficient to induce mucosal carcinogenicity,

74

even if 100% of the amount of PhIP ingested daily would be converted into PhIP-M1.

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We hypothesized that, by analogy to PhIP, other HAAs are able to react with

products

3-hydroxypropionaldehyde

or

acrolein.23

3


In

aqueous

solution,

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3-hydroxypropionaldehyde or acrolein because of structural similarities such as the primary amino

77

group and/or an imidazo moiety. Therefore, the aim of this study was to elucidate whether or not

78

PhIP-M1 type metabolites are also formed from other HAAs applying a simple in vitro model. In

79

this model, we investigated the glycerol dependent metabolism of seven food-borne HAAs (Figure

80

1) by L. reuteri DSM 20016. L. reuteri was selected as a representative for reuterin producing gut

81

microbes, because it previously showed high PhIP conversion rates in vitro.28 The selected HAAs

82

are among the most frequently found representatives in cooked food10,31 and include both, IQ type

83

(IQ, MeIQ, and MeIQx) and carboline type (AαC, Trp-P-1, harman, and norharman) HAAs. This

84

selection allows some general conclusions about the effect of certain structural features of HAAs on

85

the formation of reuterin dependent metabolites.

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Material and Methods

87

Chemicals. AαC, IQ, MeIQ, MeIQx, and Trp-P-1 acetate were purchased from Toronto Research

88

Chemicals (Toronto, Canada), norharman and PhIP from ABCR (Karlsruhe, Germany) and harman

89

from Sigma-Aldrich (Steinheim, Germany). HAA purity was at least 95%, as confirmed by HPLC-

90

MS and 1H-NMR analyses. Stock solutions were prepared in DMSO obtained from Merck

91

(Darmstadt, Germany). DMSO-d6 (for NMR analysis),

92

manganese sulfate monohydrate, ammonium citrate, ammonium acetate, and resazurin sodium salt

93

were obtained from Sigma-Aldrich. For HPLC analysis, acetonitrile (super gradient grade) and

94

triethylamine were applied from VWR (Darmstadt, Germany). Formic acid was purchased from

95

Fluka (99–100%) (St. Gallen, Switzerland) and glycerol from Roth (Karlsruhe, Germany). The

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water used for buffers and solvents was purified by a LaboStar UV2 system from Siemens (Munich,

97

Germany) using a 0.2 µm filter. All other chemicals were obtained from Merck.

98

Incubation of HAAs with L. reuteri DSM 20016 under Strict Anaerobic Conditions. Batch

99

incubations were performed in the presence of L. reuteri, a facultative anaerobe known to be a

100

natural inhabitant of the human colon.32 The type strain L. reuteri DSM 20016 used was obtained

L-cysteine

4


hydrochloride monohydrate,


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from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). All

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media were autoclaved (121 °C, 15 min) before use. L. reuteri was pre-cultured in standard de Man,

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Rogosa and Sharpe (MRS) medium consisting of peptone (10 g/L), yeast extract (8 g/L), meat

104

extract (6 g/L), potassium dihydrogen phosphate (2 g/L), sodium acetate (5 g/L), ammonium citrate

105

(2 g/L), magnesium sulfate (0.2 g/L), manganese sulfate (0.04 g/L), glucose (2 g/L), and Tween 80

106

(1 g/L), dissolved in tap water. The pH was adjusted to 5.7 with 1 M HCl. Cryopreserved L. reuteri

107

cells were used to inoculate 10 mL of MRS medium and incubated at 37 °C for 24 h aerobically. An

108

aliquot (100 µL) of the resulting pre-culture suspension was transferred into a Hungate tube filled

109

with anaerobic, nitrogen-flushed MRS medium.

110

Batch incubations were performed under anaerobic conditions applying the Hungate technique.

111

Two different types of media were used differing in their initial glucose/glycerol ratio (nGlc/nGly)

112

(0.05

113

3-hydroxypropionaldehyde.33 For modified MRS medium, single components of standard MRS

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were given into a Schott flask and appropriate amounts of glycerol (200 mM), glucose (10 mM or

115

111 mM, for nGlc/nGly = 0.05 or 0.55, respectively), and L-cysteine monohydrochloride (0.5 g/L)

116

were added. After dissolution in 500 mL of tap water, 50 µL of a 10 g/L stock solution of resazurin

117

(1 g/L) was added. The pH was adjusted to 5.7 with 1 M HCl. The medium was gas-flushed with

118

nitrogen until the absence of dissolved oxygen was indicated. Anaerobic medium (10 mL) was

119

filled into Hungate tubes, gas-flushed for 1 min, and finally sealed airtight with a butyl rubber

120

septum. Inoculation and addition of HAAs were carried out under an atmosphere of N2/H2/CO2

121

(80:10:10, v/v/v) in an A45 anaerobic workstation from Don Whitley (Shipley, UK): 25 µL of HAA

122

stock solution (20 mM in DMSO) was injected into the Hungate tube, resulting in a final HAA

123

concentration of 50 µM and about 0.25 vol.-% DMSO. Inoculation was done by adding 100 µL of

124

the overnight culture. Duplicate incubations were performed for each individual HAA. Control

125

incubations were carried out by injection of 100 µL medium instead of bacterial suspension

or

0.55)

because

this

ratio

is

known

to

affect

5


in

situ

production

of

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126

(controls without inoculum) or 25 µL DMSO instead of HAA stock solution (controls without

127

HAA), respectively. The Hungate tubes were incubated on a rotary shaker (37 °C, 80 rpm) for 72 h.

128

Samples for chemical analysis (two aliquots of 0.7 mL) were taken immediately after inoculation (0

129

h) and after 72 h. All samples were directly frozen and stored at –80 °C until analysis.

130

To isolate and identify metabolites, upscaled anaerobic batch incubations were carried out with

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AαC in Schott flasks filled with 200 mL of modified MRS medium (nGlc/nGly = 0.05). AαC stock

132

solution (20 mM, 1.8 mL) was added, resulting in an AαC concentration of 177 µM, followed by

133

inoculation with 2 mL of an overnight culture of L. reuteri. Schott flasks were incubated on a

134

shaking plate (37 °C, 200 rpm) for 72 h. The upscaled incubations were performed in duplicate.

135

Controls without inoculum or without HAA, respectively, were prepared in Hungate tubes.

136

Metabolite formation was confirmed by sampling two aliquots of 0.7 mL each at 0 h and after 72 h

137

and HPLC analysis. Remaining suspensions (about 200 mL) were directly frozen in the Schott

138

flasks used for incubation and stored at –80 °C until further purification.

139

HPLC-DAD/FLD Analyses. Samples were analyzed on a Nexera HPLC system (Shimadzu,

140

Duisburg, Germany) consisting of a DGU-30A5 degasser, an LC-30AD binary high gradient pump,

141

a tempered CTO-20AC column oven, and an SIL-30AC autosampler coupled with an SPD-M20A

142

diode array detector (DAD) and an RF-20A XS fluorescence detector. Amber glass vials and

143

conical glass inserts from Wicom (Heppenheim, Germany) were used. Frozen samples were gently

144

thawed to room temperature and vortex mixed for 2 min. Two different sample preparation

145

protocols were used for carboline type and IQ type HAAs, respectively. Carboline type: sample

146

suspension (50 µL) was diluted with 200 µL of 0.01 M triethylamine (pH 3.4)/acetonitrile (15:85,

147

v/v). After vortex mixing (1 min) and subsequent centrifugation (10.000 rpm, 2 min), 200 µL of the

148

supernatant was evaporated in a vacuum concentrator (0.1 torr, max. 45 °C). The dried residue was

149

re-dissolved in 200 µL of 0.01 M triethylamine (pH 3.4)/acetonitrile (95:5, v/v) and transferred into

150

the vial insert. IQ type: sample suspension (140 µL) was diluted with 560 µL of 0.01 M 6



151

triethylamine (pH 3.4)/acetonitrile (15:85, v/v). Following vortex mixing (1 min) and centrifugation

152

(10.000 rpm, 2 min), 600 µL of the supernatant was evaporated (0.1 torr, max. 45 °C). The dried

153

residue was re-dissolved in 120 µL of 0.01 M triethylamine (pH 3.4)/acetonitrile (95:5, v/v) before

154

pipetting into the insert.

155

During HPLC analyses, samples were stored in the autosampler at 4 °C. AαC, Trp-P-1, harman, and

156

norharman were analyzed by HPLC-DAD/FLD. The column used was a 50 mm x 2.1 mm i.d., 1.7

157

µm, Kinetex XB-C18, with a 4 x 2.1 mm i.d. guard column of the same material (Phenomenex,

158

Aschaffenburg, Germany). Aqueous formic acid (0.1% (v/v), pH 2.8) was used as eluent A and

159

acetonitrile as eluent B. The flow rate was 0.25 mL/min and the column temperature was 25 °C.

160

The injection volume was 1 µL, and the following gradient system was applied: isocratic at 5% B

161

for 4 min, linear increase from 5% B to 22% B (16% B for AαC) within 3.5 min, hold 22% B for

162

2.5 min, increase to 40% B within 2.5 min (4.5 min for AαC), increase to 86% B within 1 min.

163

DAD signals were monitored between 200 and 500 nm. FLD emission/excitation wavelengths were

164

set at 353/404 nm (AαC), 263/410 nm (Trp-P-1), and 300/440 nm (harman and norharman),

165

respectively. To determine the purity of isolated AαC metabolites, the gradient was adapted as

166

follows: 10% B for 4 min, increase to 90% B within 21 min, hold 90% B for 4 min. Purities were

167

estimated based on peak areas monitored at 254 and 345 nm, respectively. MeIQx was analyzed by

168

HPLC-DAD at 268 nm adapting the method described above, by adjusting the flow rate to 0.13

169

mL/min. Due to higher polarity, IQ and MeIQ were analyzed using a 100 mm x 2.1 mm i.d., 2.6

170

µm, Kinetex EVO C18 column, with a 4 mm x 2.6 mm i.d. guard column of the same material

171

(Phenomenex). Aqueous ammonium acetate (5 mM, pH 8.0) (A) and acetonitrile (B) were used as

172

eluents. The flow rate was 0.30 mL/min, the column temperature 25 °C, and the injection volume

173

was 1 µL. The following gradient was applied: 0% B for 6 min, linear increase to 40% B within 14

174

min, and subsequent increase to 100% B within 2 min. LabSolutions software, version 5.32, was

175

used for data analysis. 7


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176

HPLC-HRMS. Samples collected after 72 h of incubation of HAAs with L. reuteri, controls, and a

177

solvent blank were each analyzed by HRMS. The following HPLC-ToF-MS/MS system was used:

178

Infinity 1290 LC system (Agilent Technologies, Böblingen, Germany) consisting of an LC-30AD

179

binary high gradient pump including a degasser, a TCC G1316C column oven, a G4226A

180

autosampler, and a G4212A DAD, coupled with a high-resolution Triple ToF 5600 tandem mass

181

spectrometer (AB Sciex, Darmstadt, Germany) consisting of a DuoSpray ion source, a quadrupole

182

(Q, precursor ion selection), a collision cell (q, for fragmentation) and a ToF mass analyzer. An

183

aliquot of the thawed sample suspension (100 µL) was diluted with 400 µL of 0.01 M triethylamine

184

(pH 3.4)/acetonitrile (15:85, v/v). After vortex mixing (1 min) and subsequent centrifugation

185

(10.000 rpm, 2 min), 450 µL of the supernatant was evaporated in a vacuum concentrator (0.1 torr,

186

max. 45 °C). The dried residue was re-dissolved in 112.5 µL of 0.01 M triethylamine (pH

187

3.4)/acetonitrile (95:5, v/v) and transferred into an insert. The sample (1 µL) was injected and the

188

chromatography was performed as described above. Electrospray ionization was performed in

189

positive mode due to the basic properties of HAAs. A commercially available MS/MS calibration

190

solution was analyzed after every sixth run. Samples were analyzed by a full scan experiment (m/z

191

100-1000) to identify accurate masses, sum formulas, and ring double bond equivalents (RDBE) of

192

unknown metabolites. Simultaneously, an MS/MS experiment (m/z 50-1000) was performed. Mass

193

spectra were recorded by information dependent analysis creating an inclusion list of accurate

194

masses of parent compounds and potential microbial metabolites (e. g. PhIP-M1 analogs or

195

monohydroxylated derivatives). All analyses were conducted in high sensitivity mode. PeakView

196

software, version 1.2.03, was used for data analysis on two ways: mass dependent, by generating

197

extracted ion chromatograms (XIC) with defined accurate masses of expected metabolites and

198

retention time dependent, by searching for accurate masses at the retention times of metabolite

199

peaks in the base peak chromatogram (BPC) generated from the total ion chromatogram after

200

background correction. 8



201

Isolation of the Main AαC Metabolites Formed by L. reuteri DSM 20016. Frozen 72 h

202

suspensions (two Schott flasks à 200 mL) resulting from upscaled batch incubations were gently

203

thawed and pooled. The pH was adjusted to pH 8.0 with 1 M NaOH. Aliquots of the suspension

204

(200 mL) were extracted four times with ethyl acetate (2 x 200 mL, 2 x 100 mL). The pooled

205

organic phases were dried by rotary evaporation (25 mbar, 40 °C), and the remaining liquid was

206

dried in a vacuum concentrator (0.1 torr, 45 °C). The residue was diluted with 2.5 mL of 0.1%

207

formic acid/acetonitrile (80:20, v/v). The slightly turbid solution was treated in an ultrasonic bath

208

and centrifuged (13.000 rpm, 5 min). The clear supernatant was transferred into amber glass vials

209

and stored at –25 °C until semi-preparative HPLC fractionation. Fractionations were conducted on

210

one of the following two Azura preparative HPLC systems from Knauer (Berlin, Germany): either

211

consisting of two P 2.1L pumps, a dynamic mixing chamber, an Optimas autosampler (type 820)

212

from Spark Holland (Emmen, Netherlands) and an UV/Vis detector (UVD 2.1L) comprising a

213

Jetstream Plus Column Thermostat (Beckman Coulter Life Sciences, Brea, CA) and a V2.1S 16-

214

port valve drive for automated fractionation or consisting of a binary pump (P 2.1L), a dynamic

215

mixing chamber, an injection and fractionation module (ASM 2.1L) comprising a 200 µL injection

216

loop coupled to a UV/Vis detector (UVD 2.1L), and a tempered Mistral column oven from Spark

217

Holland. AαC metabolites were separated using a 250 mm x 4.6 mm i.d., 5 µm, Gemini C18

218

column, with a 4 x 4.6 mm i.d. guard column of the same material (Phenomenex). The flow rate

219

was 1.2 mL/min, and the column temperature was 25 °C. Gradient elution was performed with

220

0.1% (v/v) formic acid in water (A) and acetonitrile (B) as follows: 5% B for 3 min, increase to

221

16% B within 5 min, increase to 20% B within 9 min, and increase to 86% B within 3 min followed

222

by an equilibration step. The injection volume was 20 µL, and the absorbance was monitored at 345

223

nm. Two main fractions were collected eluting between 14.2 to 15.2 min (AαC-M8) and 23.8 to

224

24.5 min (AαC-M11).

225

Sample solutions from NMR analyses of previously isolated fractions of AαC-M8 and AαC-M11 9


Page 10 of 45

Page 11 of 45


226

were evaporated to dryness and re-purified as follows: dry residues were re-dissolved in a 95:5 (v/v)

227

mixture of 0.01% formic acid (v/v) and acetonitrile. Less concentrated formic acid (0.01% vs.

228

0.1%) was used in the solvent and eluent due to concerns about the chemical stability of AαC

229

metabolites. The turbid solution was treated in an ultrasonic bath for 5 min and filtered through a

230

regenerated cellulose filter (0.45 µm). The detection wavelength was changed to 254 nm. Before

231

each sample injection, the loop was rinsed with 500 µL of isopropanol and injection solvent. The

232

injection volume was 100 µL. For purification of AαC-M8, the flow rate was increased to 1.8

233

mL/min, and the following linear gradient program was used: 5% B for 4 min, increase to 16% B

234

within 6 min, to 20% B within 9.5 min, to 85% B within 3.5 min. Three fractions eluting between

235

15.2 and 16.8 min (fraction 1, AαC-M8), 18.1 and 19.7 min (fraction 2, degradation product (dp) 1,

236

AαC-M8 dp1), and 23.8 and 24.3 min (fraction 3, AαC-M8 dp2) were collected. To purify AαC-

237

M11, the flow rate was 1.5 mL/min, and the gradient system was as follows: 20% B for 3 min

238

followed by a linear increase to 100% B within 17 min. Single peak fractions (fraction 3, 15.1 –

239

16.0 min; AαC-M11) or mixed fractions (fraction 1, 4.8 – 7.6 min; fraction 2, 11.6 – 13.7 min)

240

were collected.

241

NMR Spectroscopy. NMR spectroscopic analyses were carried out on an Ascend 500 MHz NMR

242

spectrometer (Bruker, Rheinstetten, Germany) equipped with a 5 mm Prodigy cryoprobe. All

243

samples were dissolved in 600 µL of DMSO-d6, and the spectra were calibrated against the DMSO

244

residual signal (1H 2.54 ppm, 13C 40.45 ppm).34 1H spectra were recorded with up to 256 scans with

245

a width of 10 kHz. COSY and TOCSY spectra were acquired using the cosygpmfphpp and

246

mlevphpp pulse sequence, respectively, with a width between 3.5 and 4.7 kHz (2K data points, 256

247

increments and up to 24 scans per increment). Time domain matrices were converted into

248

1024x1024 matrices after applying a squared sine function in both dimensions. H,C-HSQC spectra

249

were generated from the Bruker pulse sequence hsqcedetgp, and for AαC-M8 dp1 the pulse

250

sequence hsqcetgpsisp2.2 was used. The width was set from 3.5 to 6.0 kHz in f2 and from 20.0 to 10



251

20.7 kHz in f1 (1K data points, 256 or 512 increments and up to 64 transients per increment). The

252

H,N-HSQC spectrum was acquired applying the hsqcetgpsi2 pulse program with a width of 7 kHz

253

in f2 and of 20.3 kHz in f1 (2K, 256 increments with 16 scans). After applying a squared sine

254

function in both dimensions HSQC data were transformed into 1024x1024 matrices. HMBC

255

experiments (long range H,C-correlation) were recorded using the hmbcgplpndqf sequence with a

256

width from 3.5 to 4.7 kHz in f2 and 28 kHz in f1 (2K data points, 128 increments and up to 128

257

transients per increment).

258

DOSY data were acquired using the ledbpgp2s pulse program. The optimum diffusion decay curve

259

was determined by the corresponding 1D pulse program ledbpgp2s1d. With a maximum current of

260

10 A provided by the gradient amplifier and a gradient strength of 5.35 G/cmA the optimized

261

diffusion decay curve was found to be between 2-88% of the maximum current. Diffusion time ∆

262

and gradient pulse length δ were set to 50 ms and 3 ms, respectively. The gradient ramp consisted

263

of 32 steps with 32 scans per step. In order to obtain the DOSY 2D pseudo spectrum data was

264

processed on the DOSYToolbox35 for Matlab (version R2016b 9.1) using a biexponential fit

265

algorithm after applying a lorentzian line broadening function in order to simplify the spectra.

266

Images were generated using nmrglue v0.5.36

267 268

Results and Discussion

269

HAA Conversion by L. reuteri DSM 20016. Seven HAAs (Figure 1) were individually incubated

270

with L. reuteri for 72 h in the presence of glycerol and two different glucose concentrations

271

(nGlc/nGly = 0.05 or 0.55). Following HPLC-DAD/FLD analyses, HAA conversion rates (%) were

272

calculated based on HAA peak areas in the chromatograms of the 0 h compared to the 72 h samples.

273

HAA recoveries from uninoculated control samples varied between 72-109% and were used to

274

adjust conversion rates. When incubated at nGlc/nGly = 0.05, AαC and MeIQx were fully

275

metabolized, IQ and MeIQ were largely converted (60 – 74%), and Trp-P-1 and norharman were 11


Page 12 of 45

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276

metabolized to a lesser extent (17 – 31%) (Table 1). In contrast, only minor (nGlc/nGly = 0.05) or no

277

(nGlc/nGly = 0.55) conversion was calculated for harman. Overall, it should be noted that analytical

278

HAA recovery was estimated based on the uninoculated control. However, this cannot mimic

279

adsorption of HAA to bacterial cells, which is likely more pronounced at 72 h of incubation as

280

compared to 0 h and may therefore affect the analyzed conversion rates to some degree.

281

The results show that the heterocyclic unit is a major factor determining HAA reactivity, with IQ

282

type HAAs and AαC being favorably metabolized (>60%), whereas β- and γ-carbolines were less

283

extensively converted (90% reduction) (Table 1). This may be

287

due to a lower amount of 3-hydroxypropionaldehyde produced in situ because it was shown that

288

3-hydroxypropionaldehyde accumulation only occurs at nGlc/nGly ratios lower than 0.33.33

289

Detection and Characterization of HAA Metabolites Produced by L. reuteri DSM 20016. To

290

detect microbial metabolites, HPLC-DAD/FLD chromatograms from 72 h samples were visually

291

compared with those from 0 h samples. Peaks only occurring in the inoculated sample, but not in

292

the controls without HAA or without L. reuteri were deemed metabolites. With the exception of

293

harman, microbial metabolites were detected for all investigated HAAs at both nGlc/nGly ratios by

294

DAD and/or FLD, with AαC showing the most complex metabolite spectrum consisting of ten

295

metabolite peaks when incubated at nGlc/nGly = 0.55 (Figures 2A and B). Although the extent of

296

conversion was reduced, AαC incubation with high initial glucose levels resulted in a more complex

297

metabolite spectrum. AαC-M11 was only detected by DAD and in samples incubated at low initial

298

glucose levels (Figure 2B). The UV spectra of the fluorescent metabolites AαC-M6 to AαC-M9

299

show high similarity with the parent compound, indicating that the heterocyclic aromatic system is

300

still intact. In contrast, the UV spectra of AαC and AαC-M11 exhibit larger differences, suggesting 12



301

that the chemical structure of AαC-M11 is more different from AαC, possibly accompanied by loss

302

of aromaticity/planarity over the course of its formation. Three metabolites were observed for the β-

303

carboline Trp-P-1 and two metabolites for the γ-carboline norharman. However, their signal

304

intensities in the FLD chromatograms were low. Two microbial metabolites of MeIQx (MeIQx-M1

305

and MeIQx-M2), showing similar retention times and UV spectra, were detected by DAD (268 nm)

306

(Figure 2C). Their UV spectra resembled that of the parent compound, suggesting that the imidazo

307

quinoxaline moiety resisted microbial conversion. Metabolism of IQ and MeIQ resulted in two

308

microbial metabolites each, detected as minor peaks in the UV chromatograms (260 and 263 nm,

309

respectively).

310

HRMS was applied to get more information about the unknown microbial metabolites. Metabolites

311

were identified both by an untargeted approach, determining m/z at defined retention times, based

312

on metabolite peaks previously identified by DAD, in the base peak chromatogram and by a

313

targeted approach, searching for specific masses of possible metabolites (PhIP-M1 analogs and their

314

reduced and/or hydroxylated forms). Accurate masses, sum formulas, and RDBE of the HAAs and

315

their respective metabolites are summarized in Table 2. Deviations between the calculated masses

316

of postulated metabolites and the measured masses of found metabolites generally did not exceed 5

317

ppm or 10 ppm for MS and MS/MS experiments, respectively.

318

Eight microbial AαC metabolites were further characterized by HRMS (Table 2). Their mass

319

spectra showed some fragment ions that were also observed within the fragmentation pattern of

320

AαC, e.g. m/z 140.1, 167.1, 183.1 or 184.1, indicating that the metabolites maintained an AαC

321

substructure (pyrido indole). The metabolites AαC-M6, AαC-M7, and AαC-M8 (Figure 3A) had

322

the same accurate mass, the same proposed sum formula, and the same RDBE, suggesting isomers.

323

Compared to the parent compound AαC (Figure 3B), these metabolites are characterized by a mass

324

increase of 56.062 Da and an RDBE increase from 9.0 to 10.0, indicating the incorporation of

325

C3H4O and an additional ring or double bond, respectively. The mass spectra of AαC-M8 (Figure 13


Page 14 of 45

Page 15 of 45


326

3C), AαC-M6, and AαC-M7 (data not shown) display a fragment ion with m/z 222.1 resulting from

327

the pseudo molecular ion following a loss of water as also observed for PhIP-M1.22 The results

328

suggest AαC-M6, AαC-M7, and AαC-M8 to be PhIP-M1 type metabolites, i.e. AαC bearing an

329

additional six-membered ring and a hydroxyl substituent. AαC-M11 showed a pseudo molecular

330

ion with an accurate mass of 278.129. When compared to AαC, this corresponds to a mass increase

331

of 94.094 Da (+C6H6O) and the existence of three additional RDBE (Table 2), suggesting a more

332

complex chemical structure bearing additional ring(s) and/or double bond(s). As highlighted in

333

Figure 3D, the mass spectrum displays two fragment ions with masses m/z 260.1 and 196.1, formed

334

through the losses of H2O and C5H6O from the pseudo molecular ion. These fragments confirmed

335

the existence of one oxygen atom and indicated the presence of a hydroxyl group in AαC-M11.

336

Thus, HRMS data suggested AαC-M11 to be a new type of HAA metabolite, potentially featuring a

337

structure resulting from the incorporation of two molecules 3-hydroxypropionaldehyde or acrolein.

338

AαC-M9 was characterized by its pseudo molecular ion showing the mass 242.129 (C14H14N3O)

339

that may correspond either to the reduced derivative of a PhIP-M1 analog or, more unlikely, a

340

chemical condensation product with 1,3-propanediol, which is formed by enzymatic reduction of 3-

341

hydroxypropionaldehyde, especially when 3-hydroxypropionaldehyde does not accumulate.33

342

However, due to the low reactivity of 1,3-propanediol as compared to 3-hydroxypropionaldehyde or

343

acrolein, the latter reaction is not expected to be favorable. The formation of AαC-M10 may be

344

explained by an additional hydroxylation step occurring before or after PhIP-M1 type metabolism.

345

By analogy to AαC-M6, AαC-M7, and AαC-M8, the metabolites MeIQx-M1 and MeIQx-M2 are

346

proposed to be PhIP-M1 type isomers. They showed peaks at similar retention times in the XIC

347

(Figure 4A) and the same accurate mass with a mass increase of 56.062 Da as compared to the

348

parent compound. Their proposed sum formulas and RDBE support that hypothesis. As displayed in

349

Figures 4B-D, the mass spectra of both metabolites showed fragments that were also observed in

350

the mass spectrum of the parent compound (i.e., m/z 214.1, 199.1, 131.1). Thus, the conservation of 14



351

a MeIQx substructure is likely. Moreover, the mass spectra display fragments at m/z 252.1

352

([M+H]+-H2O) and 226.1 ([M+H]+-C2H4O) due to the loss of water and ethanol, respectively. In the

353

case of MeIQx-M1, the product following ethanol loss represents the main fragment and the

354

product after water loss only occurs as a minor fragment, whereas for MeIQx-M2 it is the other

355

way around. Hypothetical chemical structures are given in Figures 4C and 4D. MeIQx-M2

356

probably represents the PhIP-M1 analog, as concluded by a very similar fragmentation pattern when

357

compared to PhIP-M1. MeIQx-M1 may be its regioisomer (different position of the OH

358

substituent) or stereoisomer (different orientation of the OH substituent). MeIQx-M3 was not

359

detected by UV and was only found as a minor metabolite in the BPC (data not shown). Mass

360

spectral data suggest that MeIQx-M3 is formed by the same transformation as AαC-M11 or one of

361

its isomers.

362

IQ and MeIQ showed a very similar metabolite profile each consisting of five metabolites, which

363

may be explained by their structural similarity. Whereas, based on their accurate masses, MeIQ-

364

M1, MeIQ-M2, and MeIQ-M3 as well as IQ-M1, IQ-M2, and IQ-M3 appear to be PhIP-M1 type

365

metabolites, MeIQ-M4 and MeIQ-M5 as well as IQ-M4 and IQ-M5 may be AαC-M11 type

366

metabolites. This supports the hypothesis that the chemical reaction between HAAs and 3-

367

hydroxypropionaldehyde or acrolein can occur at different sites of the molecule, at least in the case

368

of AαC and the IQ type HAAs.

369

Based on HRMS data, PhIP-M1 type metabolites and their proposed reduced forms were also found

370

for Trp-P-1 (Trp-P-1-M2, Trp-P-1-M3), norharman (norharman-M1, norharman-M3) and

371

harman (harman-M1, harman-M2). In contrast to the β-carbolines, an AαC-M11 analog was

372

detected for Trp-P-1 (Trp-P-1-M4). As already suggested for AαC-M10, there is some evidence

373

that hydroxylation plays a role in the metabolism of norharman and Trp-P-1 by L. reuteri, because

374

accurate masses of hydroxylated forms of PhIP-M1 type metabolites of norharman (norharman-

375

M3, norharman-M4) as well as of one mono hydroxylated derivative (Trp-P-1-M1) were 15


Page 16 of 45

Page 17 of 45


376

identified. Our data suggest that HAAs and/or their 3-hydroxypropionaldehyde or acrolein adducts

377

are potentially further transformed by bacterial enzymes, because accurate masses corresponding to

378

reduced and/or hydroxylated forms were identified for AαC, harman, norharman, and Trp-P-1.

379

However, because most of these metabolites were not detected in the UV chromatograms and

380

showed rather low signal intensities in the MS chromatograms, they appear to be of minor

381

importance. The high reactivity of 3-hydroxypropionaldehyde or acrolein probably results in a very

382

fast conjugation of HAAs, especially in the case of most reactive HAAs, that is expected to happen

383

faster than an enzyme-catalyzed oxidation. In addition, the reuterin system was demonstrated to

384

inhibit bacterial enzymes such as cytochrome P450 under the chosen incubation conditions.37

385

Isolation and Identification of AαC-M8 and AαC-M11. The metabolism of AαC was studied in

386

more detail, because AαC was most strongly converted by L. reuteri. Preparative batch incubations

387

starting with 13.2 mg of AαC in total were performed with the aim to isolate AαC-M8, representing

388

a PhIP-M1 type metabolite, and AαC-M11, representing a new type of HAA metabolite, possibly

389

being formed by the incorporation of two 3-hydroxypropionaldehyde or acrolein molecules, in

390

sufficient amounts for structural identification by NMR spectroscopy. The ratio nGlc/nGly = 0.05 was

391

chosen because this was expected to give higher yields and a less complex metabolite profile

392

(Figure 2A). The metabolite profile observed for samples from upscaled incubation (72 h) by

393

HPLC-FLD was identical to the analytical experiments. A liquid-liquid extraction with ethyl acetate

394

under alkaline conditions was performed prior to semi-preparative HPLC. HPLC fractions were

395

collected and dried, gaining 1.2 mg of the AαC-M8 fraction and 6.1 mg of the AαC-M11 fraction

396

for NMR spectroscopy. Unfortunately, the 1H-NMR spectra of both fractions demonstrated the

397

presence of impurities, which were assumed to be degradation products formed during fractionation

398

and/or sample preparation. Thus, solutions remained after NMR analysis were evaporated and re-

399

fractionated under slightly modified conditions: (i) by using 0.01% instead of 0.1% formic acid in

400

the eluent to prevent chemical degradation, (ii) by changing the detection wavelength from 345 nm 16



401

to 254 nm enhancing the probability to detect UV-active impurity compounds, and (iii) by

402

continuously cooling of the eluates on ice. The chromatogram resulting from re-fractionation of the

403

originally collected single peak fraction of AαC-M8 showed three well separated single peaks

404

(AαC-M8 fraction 1, 2, and 3), the first one containing AαC-M8. Thus, a substantial degradation of

405

AαC-M8 was confirmed with degradation product 1 (AαC-M8 dp1) being more dominant than

406

degradation product 2 (AαC-M8 dp2). In the case of AαC-M11, re-fractionation resulted in one

407

main fraction representing AαC-M11 and two additional mixed fractions containing several

408

impurity compounds.

409

The re-purified fractions were analyzed by NMR spectroscopy and HRMS. Again, the fraction of

410

AαC-M8 was highly impure, the HPLC-HRMS chromatogram now showing three degradation

411

products next to AαC-M8: AαC-M8 dp1, dp2 and dp3 (chromatogram not shown), confirming the

412

instability of this metabolite and precluding structural elucidation by NMR spectroscopy. However,

413

its chemical structure was deduced from the degradation products AαC-M8 dp1 and AαC-M8 dp2

414

(Figure 5), which were sufficiently pure and stable after fractionation. Their NMR data (Table 3)

415

were compared to a set of verified reference spectra from AαC. Spectra of both substances included

416

signals of the AαC substructure. In the case of AαC-M8 dp1, the proton signals of carbons 9, 10,

417

and 16 overlapped in the 1H-NMR spectrum. However, they were identified by different correlation

418

signals in the HSQC spectrum. Because the expected correlation signal for the proton at position 15,

419

which gave a broad singlet signal, was not seen using the hsqcedetgp pulse sequence, the

420

hsqcetgpsisp2.2 program was applied. Proton shifts of overlapped signals (Table 3) were obtained

421

from the HSQC spectrum. The pseudo molecular ion [M+H]+ of AαC-M8 dp1 as analyzed by

422

HRMS showed a mass of 222.087. The corresponding sum formula and RDBE were calculated to

423

be C14H9N3 and 12.0, respectively, indicating that AαC-M8 dp1 is the dehydrated and oxidized

424

form of AαC-M8. Overall, NMR and HRMS data suggest that the reaction of AαC with

425

3-hydroxypropionaldehyde or acrolein results in a new carbon-carbon linkage (AαC-M8). This is in 17


Page 18 of 45

Page 19 of 45


426

contrast to PhIP-M1, where 3-hydroxypropionaldehyde or acrolein are bound across the non-

427

methylated nitrogen and the amino group of the 2-aminoimidazo part of the structure (Figure 1).

428

This was confirmed by structural elucidation of AαC-M8 dp2: In contrast to AαC-M8 dp1, the

429

three signals of additional H and C atoms were not found in the aromatic but in the aliphatic region

430

of the spectra. These were identified via the multiplicity edited HSQC spectrum as three methylene

431

groups. In this case, the added ring is not aromatic, and AαC-M8 dp2 turned out to be a reduced

432

degradation product of the postulated metabolite AαC-M8. The accurate mass of AαC-M8 dp2

433

(224.119) and the resulting sum formula C14H13N3 indicate four additional hydrogen atoms

434

accompanied by a loss of two double bonds when compared to AαC-M8 dp1, confirming AαC-M8

435

dp2 to be its fully reduced derivative. Based on NMR and HRMS analyses of AαC-M8 dp1 and

436

dp2, the chemical structure of AαC-M8 was unequivocally elucidated (Figure 5). In addition, from

437

HRMS data it is hypothesized that the third degradation product AαC-M8 dp3 is an oxidized

438

derivative of AαC-M8.

439

NMR spectra of re-purified AαC-M11 also showed signals of multiple substances, potentially due

440

to chemical instability and/or poor solubility of AαC-M11. However, all signals that belong to

441

AαC-M11 (Table 4) were assigned by using the following approach: Proton signals at 8.18 and

442

5.36 ppm were excluded because they appear at different diffusion constants in the DOSY spectrum

443

(Figure 6A). Because the protons represented by signals at chemical shifts of 2.62, 3.82, 4.27, and

444

7.24 ppm belong to one spin system (TOCSY) (Figure 6B) and all of them appear in the DOSY

445

spectrum at similar diffusion constants, these four proton signals were assigned to AαC-M11. This

446

was confirmed by the long range H,C-correlation between the proton at 3.82 ppm and the carbon at

447

position 2 (158.3 ppm) of the AαC substructure (HMBC, Table 4). The additional signal at 2.21

448

ppm that appears at the same diffusion constant (DOSY) does not belong to AαC-M11 as it is part

449

of the spin system from 1.29 to 5.36 ppm (TOCSY). Therefore it might be an artifact of the

450

biexponential fit. As determined by HRMS, the difference between the sum formula of the 18



451

unequivocally identified AαC substructure (C11H7N3) and the sum formula of AαC-M11

452

(C17H15N3O) is C6H8O. The proton signal at 9.56 ppm was identified as a hydroxyl group because

453

the corresponding proton is not attached to a carbon or a nitrogen atom (H,N- and H,C-HSQC).

454

Signals at 2.62, 3.82, and 4.27 ppm were identified via the multiplicity edited HSQC as methylene

455

groups and the signal at 7.24 ppm as methine group, respectively. Thus, the remaining two carbon

456

atoms must be quarternary. Based on these conclusions, eight possible isomeric structures were

457

postulated for AαC-M11 (Figure 5). However, it was not possible to unambiguously elucidate the

458

structure of the actual metabolite.

459

Potential mechanisms of formation. The formation of HAA metabolites by L. reuteri DSM 20016

460

is due to the interaction with 3-hydroxypropionaldehyde or acrolein. Glycerol is transformed to

461

3-hydroxypropionaldehyde catalyzed by the glycerol dehydratase of L. reuteri.38 Under the applied

462

incubation conditions (37°C, pH 5.7), 3-hydroxypropionaldehyde is supposed to be, at least

463

partially, converted into its more reactive dehydration product acrolein. Recently, Engels et al.27

464

demonstrated that 3-hydroxypropionaldehyde is dehydrated to acrolein under physiological

465

conditions and hypothesized that acrolein is more heavily involved in the formation of PhIP-M1

466

than previously expected.23 Studies of Hidalgo et al.39 demonstrated that PhIP reacts with 2-

467

pentenal, another reactive α,β-carbonyl formed during lipid peroxidation. Acrolein is well

468

established to react with guanosine,40 cytosine, and adenine derivatives41 within Michael type

469

additions by forming adducts that contain a new six-membered ring bearing a hydroxyl group, thus

470

showing similar structural features when compared to PhIP-M122 and AαC-M8. We therefore

471

assume that acrolein, representing the most reactive component within the reuterin system, is

472

responsible for the observed conjugation of HAAs. Whereas AαC-M8 is probably formed due to a

473

single Michael type addition with acrolein, AαC-M11 is supposed to be formed by two consecutive

474

reactions. The different positions of the newly formed six-membered ring in AαC-M8 and AαC-

475

M11 suggest that the first condensation product is formed by alternative reaction mechanisms 19


Page 20 of 45

Page 21 of 45


476

initiated either by the attack of the pyrido-nitrogen (N-1) at C-3 of acrolein following an 1,4-

477

Michael type addition or nucleophilic attack of the exocyclic amino group (N-14) at the aldehyde

478

group of 3-hydroxypropionaldehyde. Subsequent ring formation would lead to an isomer of AαC-

479

M8 containing a six-membered ring between N-1 and N-14 that could be further attacked by a

480

second acrolein molecule resulting in Aα αC-M11. Formation of AαC-M8 and AαC-M11 is

481

potentially driven by the formation of six- and/or five-membered rings. However, metabolites were

482

shown to be quite unstable. As proposed in Figure 7, AαC-M8 was decomposed due to dehydration

483

and subsequent oxidation or reduction reactions resulting in aromatic AαC-M8 dp1 or non-

484

aromatic AαC-M8 dp2 referred to as stable end products. Based on UV intensity, the former is

485

supposed to be the main product, consistent with the potentially favored re-aromatization of the

486

heterocyclic system. Dehydration of AαC-M8 is supposed to be catalyzed under acidic conditions.

487

Critical steps in our protocol are evaporation steps following semi-preparative HPLC fractionation

488

procedures due to concentration of formic acid present in the eluate. For the observed oxidation and

489

reduction reactions certain oxidizing and reducing agents are necessary. Oxidation may have been

490

caused by the presence of oxygen. Reduction could have been induced either by formic acid, which

491

was shown to be involved in the reduction of pyridines to corresponding piperidines and/or

492

tetrahydropiperidines42 or residues of cysteine, which was used to maintain reductive conditions

493

during incubation. However, in first attempts it was not possible to define causes for the observed

494

instability. Future studies need to focus on the question whether the chemical degradation products

495

are of physiological concern, i.e. are formed under physiological conditions.

496

In conclusion, we demonstrated that a broad spectrum of food-borne HAAs, both IQ type (MeIQx,

497

IQ, MeIQ) and non-IQ type (AαC, Trp-P-1, norharman), can form PhIP-M1 type metabolites.

498

Under the conditions chosen for this study (incubation with L. reuteri DSM 20016 in the presence

499

of glycerol), the extent of HAA conversion was shown to be structure-specific. It also depended on

500

the initial nGlc/nGly ratio, because this ratio controls the amount of in situ produced 320



Page 22 of 45

501

hydroxypropionaldehyde. The incubation conditions of this study were chosen to facilitate

502

metabolite detection. Thus, relatively high HAA (50 mM) and glycerol (200 mM) concentrations

503

were used. The latter was previously applied by Lüthi-Peng et al.33, who studied the glycerol

504

bioconversion by L. reuteri. Now that reuterin dependent HAA metabolites have been

505

characterized, subsequent studies should aim at assessing their relevance in vivo. It should also be

506

noted that, although the dietary intake of glycerol was estimated43, physiological glycerol levels in

507

the gut have not yet been reported in the literature and remain to be elucidated.

508

Next to PhIP-M1 type metabolites, which were identified for MeIQx, IQ, MeIQ, AαC, Trp-P-1, and

509

norharman,

510

3-hydroxypropionaldehyde or acrolein molecules, was postulated. This type of metabolism was

511

observed for AαC, Trp-P-1, IQ, MeIQ, and MeIQx. For the largely metabolized AαC, MeIQx, IQ,

512

and MeIQ different isomers of PhIP-M1 type metabolites were observed, suggesting that reactions

513

with 3-hydroxypropionaldehyde or acrolein may occur at different positions of the heterocyclic

514

system. However, more research is needed to unambiguously elucidate the structures of reuterin

515

induced HAA metabolites. Our results are based on a simple model using a single microbial strain

516

that is able to produce reuterin from glycerol. However, even this simple model revealed that the

517

reuterin dependent metabolite profile of HAAs can be quite complex, as different PhIP-M1 type

518

isomers and new types of metabolites, formed by the incorporation of two acrolein or 3-

519

hydroxypropionaldehyde molecules, can occur. In addition, reuterin dependent HAA metabolites

520

show some instability, which can impede their structural elucidation. Our results provide the basis

521

for further studies elucidating the metabolism of HAAs in the presence of complex gut microbiota.

522

Our data suggest that the outcome of these studies will be even more complex. The present results,

523

however, may facilitate their interpretation. The HAA metabolites observed in our model may also

524

occur in vivo, because reuterin producing bacteria such as L. reuteri or E. hallii24 are inhabitants of

525

the human gut and the formation of PhIP-M1 has been shown in vivo.28 In this human study, six

another

type

of

metabolite,

formed

by

21


the

incorporation

of

two

Page 23 of 45


526

male subjects consumed a single meal of cooked chicken containing 0.88–4.7 µg PhIP and,

527

subsequently, the excretion of PhIP-M1 was determined in urine and feces collected in 8 h and 24 h

528

increments, respectively. The rate of PhIP-M1 excretion varied inter-individually, ranging from

529

1.2–15% and 0.9–11% of the ingested dose in urine and feces, respectively. The authors pointed out

530

that the urinary excretion of PhIP-M1 increased over time, whereas the majority of PhIP was

531

excreted in the first 24 h, indicating the role of gut microbiota in the metabolism of PhIP. Based on

532

the data from Vanhaecke et al.28, it may also be hypothesized, that PhIP-M1 can be formed and

533

absorbed both in the small and large intestine. L. reuteri, for example, can be present both in the

534

small and large intestine.43 It has been reported to be an autochthonous microbe in some

535

humans44,45. However, to the best of our knowledge, no comprehensive data is available about the

536

prevalence and intestinal distribution of total reuterin accumulating bacteria in humans.

537

So far, only three studies have looked at the physiological effects of PhIP-M128-30 as detailed in the

538

introduction section. Whether the reuterin dependent metabolism of HAAs increases or decreases

539

their toxicity should be addressed in future studies and taken into account when assessing the risk of

540

HAAs for human health. As we demonstrated for AαC-M8, chemical instability of microbial

541

metabolites may lead to the formation of further degradation products, which need to be taken into

542

account especially when formed under physiologically relevant conditions.

543

Safety aspects. Some heterocyclic aromatic amines are probable (IQ) or possible human

544

carcinogens (MeIQ, MeIQx, AαC, Trp-P-1). For this reason, HAAs, their metabolites and solutions

545

thereof were handled with special precaution by using appropriate protective equipment. An

546

appropriate breathing mask was worn during handling of pure compounds.

547

Abbreviations Used

548

AαC, 2-amino-9H-pyrido[2,3-b]indole; BPC, base peak chromatogram; dp, degradation product;

549

HAA, heterocyclic aromatic amine; IQ, 2-amino-3-methyl-3H-imidazo[4,5-f]quinoline; MeIQ, 2-

550

amino-3,4-dimethyl-3H-imidazo[4,5-f]quinoline;

MeIQx,

2-amino-3,8-dimethylimidazo[4,5-

22



551

f]quinoxaline; MRS, de Man, Rogosa and Sharpe (medium); nGlc/nGly, glucose/glycerol ratio;

552

RDBE, ring double bond equivalent; Trp-P-1, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole; XIC,

553

extracted ion chromatogram.

554 555

Acknowledgment

556

The authors would like to thank Sebastian Soukup (Max Rubner-Institute) and Malin Reller

557

(Karlsruhe Institute of Technology) for their valuable guidance on mass spectrometry analyses and

558

DOSY experiments, respectively. We also thank Janina Krüger and Luisa Martinez (Max Rubner-

559

Institute) for their skillful technical assistance.

560 561

Supporting Information

562

Figures S1-S9: UV spectra of selected HAAs and HAA metabolites; HPLC-DAD/FLD

563

chromatograms of samples obtained from 72 h incubation of MeIQ, IQ, harman, norharman, and

564

Trp-P-1 with L. reuteri DSM 20016; chromatograms from re-purification of AαC-M8 and AαC-

565

M11 after NMR analysis; 1H-NMR spectrum of AαC-M8 dp1; HSQC spectrum of AαC-M8

566

dp1(excerpt); mass spectra of AαC-M8, AαC-M8 dp1, AαC-M8 dp2 and AαC-M8 dp3; 1H-NMR

567

spectrum of AαC-M8 dp2; 1H-NMR spectrum of AαC-M11; multiplicity edited HSQC spectrum of

568

AαC-M11; Tables S1-S3: HPLC-HRMS parameters; 1H and 13C NMR data of AαC; HRMS data of

569

AαC-M8, AαC-M8 dp1, AαC-M8 dp2, AαC-M8 dp3 and AαC-M11 obtained from analyses of

570

isolated fractions.

571

This material is available free of charge via the Internet at http://pubs.acs.org.

572 573

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574

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The

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damage, apoptosis and cell cycle arrest towards Caco-2 cells. Toxicol. Lett. 2008, 178, 61-69.

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(30) Nicken, P.; Willenberg, I.; Keutz, A. V.; Elsner, L. V.; Hamscher, G.; Vanhaecke, L.;

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potential of PhIP-M1, a bacterial metabolite of the heterocyclic aromatic amine 2-amino-1-methyl-

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6-phenylimidazo[4,5-b]pyridine (PhIP). Toxicol. Lett. 2015, 234, 92-98.

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occurrence, and risk assessment. Compr. Rev. Food Sci. Food Saf. 2016, 15, 269-302.

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microbial

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metabolite

7-hydroxy-5-methyl-3-phenyl-6,7,8,9chloride

27


(PhIP-M1)

induces

DNA

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multidimensional NMR data. J. Biomol. NMR 2013, 55, 355-367.

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(37) Fernández-Cruz, M. L.; Martín-Cabrejas, I.; Pérez-del Palacio, J.; Gaya, P.; Díaz-Navarro, C.;

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Navas, J. M.; Medina, M.; Arqués, J. L., In vitro toxicity of reuterin, a potential food

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biopreservative. Food Chem. Toxicol. 2016, 96, 155-159.

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(38) Talarico, T. L.; Dobrogosz, W. J., Chemical characterization of an antimicrobial substance

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produced by Lactobacillus reuteri. Antimicrob. Agents Chemother. 1989, 33, 674-679.

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(39) Hidalgo, F. J.; Alcón, E.; Zamora, R., Reactive carbonyl-scavenging ability of 2-

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aminoimidazoles: 2-amino-1-methylbenzimidazole and 2-amino-1-methyl-6-phenylimidazo[4,5-

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b]pyridine (PhIP). J. Agric. Food Chem. 2014, 62, 12045-12051.

688

(40) Kozekov, I. D.; Turesky, R. J.; Alas, G. R.; Harris, C. M.; Harris, T. M.; Rizzo, C. J.,

689

Formation of deoxyguanosine cross-links from calf thymus DNA treated with acrolein and 4-

690

hydroxy-2-nonenal. Chem. Res. Toxicol. 2010, 23, 1701-1713.

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(41) Sodum, R. S.; Shapiro, R., Reaction of acrolein with cytosine and adenine derivatives. Bioorg.

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Chem. 1988, 16, 272-282.

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(42) Gibson, H. W., The chemistry of formic acid and its simple derivatives. Chem. Rev.

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(Washington, DC, U. S.) 1969, 69, 673-692.

695

(43) Reuter, G., The Lactobacillus and Bifidobacterium microflora of the human intestine:

696

Composition and succession. Current Issues in Intestinal Microbiology 2001, 2, 43-53.

697

(44) Reuter, G., Microecological importance of three autochthonous Lactobacillus species: L.

698

gasseri, sp.nov. (1980), L. ruminis, sp.nov. (1973) and L. reuteri, sp.nov.(1980). Arch.

699

Lebensmittelhyg. 2007, 58, 164-169.

700

(45) Million, M.; Angelakis, E.; Maraninchi, M.; Henry, M.; Giorgi, R.; Valero, R.; Vialettes, B.; 28



701

Raoult, D., Correlation between body mass index and gut concentrations of Lactobacillus reuteri,

702

Bifidobacterium animalis, Methanobrevibacter smithii and Escherichia coli. Int. J. Obes. 2013, 37,

703

1460-1466.

704 705

Funding Statement: This work was part of the project “Heterocyclic aromatic amines: Microbial

706

metabolism and interaction with dietary fibers from fruit and vegetables” funded by the German

707

Research Foundation (DFG), reference numbers KU 1079/11-1, FR 3450/1-1 and BU 2161/2-1.

708 709

Figure Captions

710

Figure 1. Chemical structures of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and the

711

metabolite

712

a]pyrimidin-5-ium chloride (PhIP-M1) and of the heterocyclic aromatic amines (HAAs)

713

investigated in this study. The blue box highlights the microbially derived multi-compound system

714

reuterin, comprising 3-hydroxypropionaldehyde (3-HPA), HPA hydrate, HPA dimer, and/or its

715

dehydration product acrolein. Reuterin is produced from glycerol by certain gut bacteria, e.g.

716

Eubacterium hallii or Lactobacillus reuteri. The chemical reaction of acrolein or 3-HPA with PhIP

717

are proposed mechanisms of PhIP-M1 formation.23,27 IQ, 2-amino-3-methyl-3H-imidazo[4,5-

718

f]quinoline; MeIQ,

719

dimethyl-3H-imidazo[4,5-f]quinoxaline; AαC, 2-amino-9H-pyrido[2,3-b]indole; Trp-P-1, 3-amino-

720

1,4-dimethyl-5H-pyrido[4,3-b]indole; harman, 1-methyl-9H-pyrido[3,4-b]indole; norharman, 9H-

721

pyrido[3,4-b]indole.

7-hydroxy-5-methyl-3-phenyl-6,7,8,9-tetrahydropyrido[3,2:4,5]imidazo[1,2-

2-amino-3,4-dimethyl-3H-imidazo[4,5-f]quinoline; MeIQx, 2-amino-3,8-

722 723

Figure 2. Chromatograms of 72 h samples and respective controls without added HAA from batch

724

incubations with Lactobacillus reuteri DSM 20016 at initial glucose/glycerol ratios of either 0.05

725

(black) or 0.55 (blue). (A) FLD trace of AαC samples recorded at 353/404 nm (B) UV trace of AαC 29


Page 30 of 45

Page 31 of 45


726

samples recorded at 345 nm. (C) UV trace of MeIQx samples recorded at 268 nm. The sequence of

727

HAA metabolite labels proceeds by their retention time. The non-fluorescent metabolite AαC-M11

728

was only detected by UV.

729 730

Figure 3. Results of the HPLC-HRMS analysis of AαC and respective metabolites. (A) Overlaid

731

XICs of m/z 184.1 (AαC) from 72 h (dashed pale line) and 0 h (solid pale line) samples, of m/z

732

240.1 (AαC-M8) as well as of m/z 278.1 (AαC-M11) (dark lines). Mass spectra of (B) AαC, (C)

733

AαC-M8 and (D) AαC-M11. Masses and/or RDBE of pseudo molecular ions [M+H]+ and

734

characteristic fragments are given, and cleavage products are highlighted. *Only one of eight

735

possible structures is shown for AαC-M11.

736 737

Figure 4. Results of the HRMS analysis of MeIQx and respective metabolites. (A) Overlaid XICs

738

of m/z 214.1 (MeIQx) from 72 h (dashed pale line) and 0 h (solid pale line) samples as well as of

739

m/z 270.1 (MeIQx-M1 and -M2, dark line). Mass spectra of (B) MeIQx, (C) MeIQx-M1 and (D)

740

MeIQx-M2. Masses and/or RDBE of pseudo molecular ions [M+H]+ and characteristic fragments

741

are given and cleavage products are highlighted.

742 743

Figure 5. Structures of the compounds AαC, AαC-M8 dp1, AαC-M8 dp2, and AαC-M11, which

744

were analyzed by NMR. Proposed structures of AαC-M11 comprise eight possible isomers.

745 746

Figure 6. (A) Pseudo 2D DOSY spectrum of AαC-M11 in DMSO-d6. Signals that appear to belong

747

to AαC-M11 are inside the red rectangle. (B) TOCSY spectrum of AαC-M11 in DMSO-d6. Spin

748

systems that belong to AαC-M11 are inside red rectangles. The two spin systems in the aromatic

749

region were assigned to the AαC substructure. Signals belonging to the spin system from 2.62 to

750

7.24 ppm appear at the same diffusion constant in the DOSY spectrum as the signals of the AαC 30



751

substructure.

752 753

Figure 7. Proposed pathway of the formation and degradation of main AαC metabolites formed by

754

Lactobacillus reuteri DSM 20016; AαC-M11 could exist in the form of eight different isomers due

755

to two possible positions of the five-membered ring and the hydroxyl substituent including

756

stereoisomers. Grey colored arrows highlight those chemical structures that were unequivocally

757

elucidated by NMR spectroscopy.

31


Page 32 of 45

Page 33 of 45


Tables Table 1: HAA Conversion (%) and Number of Metabolites Detected by HPLC-DAD/FLD after 72 h of Incubation with L. reuteri DSM 20016 in the Presence of 200 mM Glycerol and a Glucose/Glycerol Ratio (nGlc/nGly) of 0.05 or 0.55. HAA

Detection mode nGlc/nGly

HAA recovery (%)a

HAA conversion after 72 h (%)b

Detected microbial metabolitesc (DAD / FLD)

0.05

0.55

0.05

0.55

0.05

0.55

label

MeIQx

λDAD 268 nm

77.4

74.0

100.0 ± 0.0

34.4 ± 6.7

2/-

2/-

M1 to M2

MeIQ

λDAD 263 nm

108.8

99.5

74.1 ± 0.6

35.7 ± 3.2

2/-

1/-

M1 to M2

IQ

λDAD 260 nm

89.9

83.2

60.6 ± 0.8

4.2 ± 6.2

1/-

1/-

M1 to M2

AαC

λFLD 353/404 nm λDAD 345 nm

97.7

91.4

100.0 ± 0.0

67.2 ± 1.1

4/3

4 / 10

M1 to M11

Trp-P-1


105.5

88.1

31.2 ± 9.4

15.9 ± 1.8

3/2

3/2

M1 to M3

norharman


100.4

71.4

17.4 ± 1.8

12.3 ± 3.4

1/1

1/2

M1 to M2

harman


101.9

85.7

3.7 ± 3.2

-9.0 ± 16.3

0/0

0/0

-

a

Recovery (%) was estimated from the HAA peak area ratio 72 h/0 h of the respective controls without inoculum. b Conversion (%) is given as mean ± range/2 and was calculated based on the HAA peak area ratio 72 h/0 h taking into account the HAA recovery, c The number of microbial metabolites was determined by comparing UV and/or fluorescence chromatograms of the 72 h samples with those of respective controls without inoculum and without HAA, respectively, and searching for peaks present in the sample but not in the controls.

32



Page 34 of 45

Table 2: HPLC-HRMS Data of HAAs and HAA Metabolites Formed by L. reuteri DSM 20016 in the Presence of 200 mM Glycerol and Proposed Metabolite Types. Analyte

AαC

tR,XIC

Acc. mass founda (Da)

Sum formula

RDBE

Proposed type of metabolite

(min)

Acc. mass calculated (Da)

(min) 8.43

8.55

184.0869

184.0868

C11H9N3

9.0

-

240.1131

240.1128

C14H13N3O

10.0

PhIP-M1 type

-M6

n. d.

7.11

-M7

7.81

7.90

240.1130

-M8

8.27

8.37

240.1142

-M9

9.76

9.86

242.1288

242.1288

C14H15N3O

9.0

PhIP-M1 type, red.

-M10b

11.13

11.22

256.1081

256.1084

C14H13N3O2

9.0

PhIP-M1 type, +OH

b

n. d.

3.69

278.1288

278.1288

C17H15N3O

12.0

b

-M2

n. d.

4.06

278.1288

-M11c

16.03

16.11

278.1286

adduct with two 3hydroxypropionaldehyde/ acrolein molecules (“AαC-M11 type“)

Trp-P-1

7.93

8.04

-

-M1

n. d.

-M2

n. d.

-M3b c

b

-M1

212.1187

212.1183

C13H13N3

9.0

6.79

228.1131

228.1131

C13H13N3O

9.0

Trp-P-1, +OH

8.12

268.1144

268.1154

C16H17N3O

10.0

PhIP-M1 type

n. d.

8.57

270.1601

270.1623

C16H19N3O

9.0

PhIP-M1 type, red.

9.26

9.36

306.1602

306.1598

C19H19N3O

12.0

AαC-M11 type

5.89

5.98

169.0760

169.0764

C11H8N2

9.0

-

c

n. d.

5.769

225.1022

225.1023

C14H14N2O

10.0

PhIP-M1 type

-M2b

n. d.

6.38

227.1179

227.1182

C14H16N2O

9.0

PhIP-M1 type, red.

-M4

norharman -M1

c

n. d.

6.49

241.0972

241.0977

C14H14N2O2

10.0

PhIP-M1 type, +OH

-M4b

n. d.

6.62

243.1128

243.1132

C14H16N2O

9.0

PhIP-M1 type, red. +OH

harman

6.72

6.82

189.0917

189.0918

C12H10N2

9.0

-

b

-M2

n. d.

7.59

239.1179

239.1170

C15H14N2O

10.0

PhIP-M1 type

-M1b

n. d.

6.90

241.1335

241.1314

C15H16N2O

9.0

PhIP-M1 type, red.

12.59

12.66

199.0978

199.0976

C11H10N4

9.0

-

-M1

n. d.

11.70

255.1240

255.1240

C14H14N4O

10.0

PhIP-M1 type

-M2c,d

12.59

12.67

255.1241

-M3c

14.47

14.57e

255.1244e

-M4c

n. d.

13.05

C17H16N4O

12.0

AαC-M11 type

-M5c

n. d.

16.88

13.66

13.73

213.1135

213.1137

C12H12N4

9.0

-

-M1

n. d.

12.61

269.1397

269.1397

C15H16N4O

10.0

PhIP-M1 type

-M2c,d

13.66

13.81

269.1395

-M3c

15.24

15.35e

269.1390e C18H18N4O

12.0

AαC-M11 type

-M3

IQ

MeIQ

a

tR,DAD

293.1397

293.1388 293.1400

-M4c,d

13.66

13.95

-M5c

17.90

17.97

MeIQx

6.91

7.14

214.1087

214.1080

-M1

7.74

7.93

270.1349

270.1347

-M2

7.99

8.17

-M3c

n. d.

9.18

307.1553

307.1549 307.1556

C14H15N5O

10.0

PhIP-M1 type

C17H17N5O

12.0

AαC-M11 type

270.1359 308.1506

308.1498

Found and calculated accurate (acc.) masses show mass deviations less than 5 ppm (MS) (with exception of

33


Page 35 of 45


harman-M1, which was -8.9 ppm) or less than 10 ppm (MS/MS), found and calculated isotopic patterns were consistent; bmetabolite was only present in batch incubations performed at an initial glucose/glycerol ratio of 0.55; cmetabolite was only present in batch incubations performed at an initial glucose/glycerol ratio of 0.05; d metabolite coeluting with parent HAA; ethis result is not unambiguous because of noise in the chromatogram; tR, retention time; n. d., not detected; red., reduced derivative; +OH, hydroxylated derivative; RDBE, ring double bond equivalent.

34



Page 36 of 45

Table 3: 1H and 13C NMR Data of AαC-M8 dp1 and AαC-M8 dp2 in DMSO-d6. Position

δ ( 13C)a (ppm)

H

δ (1H) (ppm)

Multiplicityb

J (Hz)

COSY

TOCSY

HSQC

HMBC

AαC-M8 dp1 2

155.5 c

-

-

-

-

-

-

-

-

3

-

-

-

-

-

-

-

-

-

4

129.3

4

9.18

s

-

-

-

129.3

C-6,17,2,13

5

112.0

-

-

-

-

-

-

-

-

6

121.0

-

-

-

-

-

-

-

-

7

122.8

7

8.33

d

7.7

H-8

H-8,9,10

122.8

C-10,9,11

8

120.5

8

7.35

t

7.7

H-7,9

H-7,9,10

120.5

C-5,6

d

9

129.1

9

7.61

m (7.54-7.65)

-

H-8,10

H-7,8,10

129.1

C-11,7

10

119.2

10

7.57d

m (7.54-7.65)

-

H-9

H-7,8,9

119.2

C-6

11

142.7

-

-

-

-

-

-

-

-

12-NH

-

NH

12.01

s

-

-

-

-

-c

13

156.0

-

-

-

-

-

-

-

-

15

152.9

15

9.08

br s

-

H-16

H-16,17

152.9

-

14 (N)

-

-

-

-

-

-

-

-

-

16

111.6

16

7.57d

m (7.54-7.65)

-

H-15,17

H-15,17

111.6

-

17

138.6

17

8.62

d

8.0

H-16

H-15,16

138.6

C-4,15,2

AαC-M8 dp2 2

155.5

-

-

-

-

-

-

-

-

3

108.9

-

-

-

-

-

-

-

-

4

129.0

4

7.83

s

-

-

-

129.0

C-17,13,2

e

5

-

-

-

-

-

-

-

-

-

6

122.8

-

-

-

-

-

-

-

-

7

118.7

7

7.78

d

7.6

H-8

H-8,9,10

118.7

C-9,11

8

119.2

8

7.06

td

7.6; 0.6

H-7,9

H-7,9,10

119.2

C-10,6

9

123.2

9

7.17

td

7.6; 1.0

H-8,10

H-7,8,10

123.2

C-6,11

10

110.8

10

7.30

d

7.6

H-9

H-8,9,10

110.8

C-8

11

138.0

-

-

-

-

-

-

-

-

12-NH

-

NH

10.97

s

-

-

-

-

-c

13

151.7

-

-

-

-

-

-

-

14-NH

-

6.60

s

-

H-15

-

ncs

15

41.6

3.35g

-

-

H-14,16

41.6

ncs

16

22.1

1.87

m (1.83-1.90)

-

H-15,17

22.1

C-3

17

27.4

NH 15 (2Hf) 16 (2Hf) 17 (2Hf)

2.84

t

6.2

H-16

H15,16,17 H14,16,17 H14,15,17 H15,16,17

27.4

C-16,15,3,4,2

a

δ of quarternary carbon atoms obtained from HMBC spectra; bs - singlet, br s - broad singlet, d - doublet, t triplet, td - triplet of doublet, m - multiplet; coutside spectral width; doverlapped by two other signals, obtained from HSQC; enot found; fnumber of protons obtained from multiplicity edited HSQC; goverlapped by water signal, obtained from HSQC; ncs, no correlation signals.

35


Page 37 of 45


Table 4: 1H and 13C NMR Data of AαC-M11 in DMSO-d6. Position

δ ( 13C)a (ppm) H

δ (1H) (ppm) Multiplicityb J (Hz)

COSY

TOCSY

HSQC N-HSQC

HMBC

signals that belong to the AαC substructure 2

158.3

-

-

-

-

-

-

-

-

-

3

100.4

3

6.80

d

8.7

H-4

H-4

100.4

-

C-5,2

4

130.9

4

8.26

d

8.7

H-3

H-3

130.9

-

C-6,13,2

5

106.6

-

-

-

-

-

-

-

-

-

6

122.4

-

-

-

-

-

-

-

-

-

7

119.6

7

7.92

d

7.5

H-8

H-8,9,10

119.6

-

C-5,9,11

8

119.6

8

7.13

t

7.5

H-7,9

H-7,9,10

119.6

-

C-10,6

9

124.2

9

7.27c

-d

-d

H-8,10

H-7,8,10

124.2

-

C-7,11

10

111.2

10

7.37

d

7.9

H-9

H-7,8,9

111.2

-

C-6

11

138.3

-

-

-

-

-

-

-

-

12-NH

-

NH

11.43

s

-

-

-

-

N-12 (122.9 ppm)

13

152.4

-

-

-

-

-

-

-

-

-

14-N

-

-

-

-

-

-

-

-

-

-

26.6

-

42.0

-

40.9

-

ncs C26.6,42.9,150 .1,158.3 C42.0,139.5,15 0.3

150.3

-

ncs

-

-

-

signals that belong to the reuterin adduct part of AαC-M11 (not assignable)

-

26.6

2He

2.62

-f

-f

H-3.82

-

42.0

2He

3.82

-f

-f

H-2.62

-

42.9

2He

4.27

-f

-f

-

-g

-g

-

H3.82,4.27, 7.24 H2.62,3.82, 4.27,7.24 H2.62,3.82, 7.24 H2.62,3.82, 4.27

s

-

-h

-h

-

150.3

1H

7.24c (7.21+7.28)

-

-

OH

9.56

a

e

b

-h c

δ of quarternary carbon atoms obtained from HMBC spectrum; s - singlet, d - doublet, t – triplet; obtained from HSQC spectrum; doverlapped by proton signal at 7.24 ppm; enumber of protons obtained from multiplicity edited HSQC spectrum; foverlapping with signals of impurities; goverlapped by proton signal at 7.27 ppm; houtside spectral width; ncs, no correlation signals.



Figure Graphics

Figure 1

1


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Figure 2



Figure 3


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Figure 4



Figure 5


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Figure 6

A

B



Figure 7


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Table of Contents Graphic


Metabolism of Food-borne Heterocyclic Aromatic Amines by

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