Isolation, Structural Elucidation, and Synthesis of Lepteridine From

May 22, 2016 - While using HPLC to examine New Zealand honeys for the presence of leptosperin 1, an unexpected UV absorbance 3 was noted at 320 nm (Fi...
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Isolation, Structural Elucidation and Synthesis of Lepteridine from M#nuka Honey Benjamin J Daniels, Gordana Prijic, Sarah Meidinger, Kerry M. Loomes, Jonathan M. Stephens, Ralf C. Schlothauer, Daniel P. Furkert, and Margaret A. Brimble J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b01596 • Publication Date (Web): 22 May 2016 Downloaded from http://pubs.acs.org on May 31, 2016

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Journal of Agricultural and Food Chemistry

Isolation, Structural Elucidation and Synthesis of Lepteridine From Mānuka (Leptospermum scoparium) Honey Benjamin J. Daniels,† Gordana Prijic, ‡§ Sarah Meidinger,§ Kerry M. Loomes,§ # Jonathan M. Stephens,‡§ Ralf C. Schlothauer,‡ Daniel P. Furkert,† #* Margaret A. Brimble.† #* †

School of Chemical Sciences, The University of Auckland, 23 Symonds Street, Auckland, New

Zealand. ‡

Comvita NZ Limited, 23 Wilson South Road, Te Puke, New Zealand.

§

School of Biological Sciences and Institute for Innovation in Biotechnology, The University of

Auckland, 3A Symonds Street, Auckland, New Zealand. #

Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland, 3 Symonds Street,

Auckland, New Zealand. *Corresponding author (Tel: +64-9-9238389; Fax: +64-9-3737422; E-mail: [email protected])

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ABSTRACT: Mānuka honey, made from the nectar of Leptospermum scoparium, has

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garnered scientific and economical interest due to its non-peroxide antibacterial activity.

3

Biomarkers for genuine mānuka honey are increasingly in demand due to the presence of

4

counterfeit mānuka honey. This work reports the identification of a compound previously

5

unreported in mānuka honey by HPLC, and determination of the structure of the as

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3,6,7-trimethyllumazine using NMR, MS, IR and UV/Vis spectroscopy. This assignment was

7

confirmed by total synthesis. The natural product, renamed lepteridine, was only observed in

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mānuka honeys and could potentially serve as a biomarker for genuine mānuka honey.

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KEYWORDS Leptospermum, mānuka honey, biomarker, lepteridine, lumazine.

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Introduction

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Honey was first used to augment the healing of wounds by the ancient Egyptians1 and is

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currently a clinical wound treatment.2-3 New Zealand mānuka honey, derived from the nectar

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of Leptospermum scoparium, exhibits non-peroxide based antibacterial activity due to the

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presence of methyl glyoxal (MGO).4 Mānuka honey is active against methicillin-resistant

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Staphylococcus aureus (MRSA)5-6 and increases the susceptibility of MRSA to rifampicin7

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and oxacillin.8

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Currently, genuine mānuka honey is labelled by either reporting the MGO concentration9 or

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using the Unique Mānuka Factor (UMF) scale, which equates the bactericidal activity of a

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given honey sample with that of a given concentration of phenol.10 This bactericidal activity

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is dependent on the concentration of MGO. Since MGO is produced in honey from

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dihydroxyacetone during incubation11 the concentration of MGO and the UMF of a given

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honey sample can change over time. Furthermore, adulteration of honey via addition of

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dihydroxyacetone or MGO gives rise to comparable bactericidal activity,12 meaning that the

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current labelling system does not provide consumers or suppliers with the information

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required to recognize counterfeit mānuka honeys, which pose a threat to the mānuka honey

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market.13 Due to its medical and economic relevance, compounds that could serve as unique

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markers for genuine mānuka honey are of scientific and commercial importance.

28

The phytochemical profile of mānuka honey has been probed with the aim of elucidating

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the mechanism(s) of the bioactivity of the honey and discovering unique biomarkers.14 One

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compound, leptosperin, 1, has been proposed as a both a contributor towards the bactericidal

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activity of mānuka honey and as a unique mānuka marker (Figure 1).15-16 Additionally, 2-

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acetyl-1-pyrroline (2) has been found to be produced from native proline in the presence of

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high (>250 mg/kg) concentrations of MGO, and has also been proposed as a potential

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marker.17 Additional markers would provide greater security for honey consumers and further 3 ACS Paragon Plus Environment

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choice for regulators. Herein we report the isolation, structural elucidation, and synthesis of

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lepteridine, 3, a pteridine derivative with the systematic name 3,6,7-trimethyllumazine,18-27

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from mānuka honey. We observed lepteridine, 3, exclusively in Leptospermum honeys; hence

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3 could serve as a unique marker for mānuka honey.

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Materials and Methods

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Botanical samples. Nectar samples were obtained by direct sampling from specimens

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grown in glasshouses using a micropipette and stored at -20 °C until analysis. Mānuka honey

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samples were collected from hives in the field. Botanical origin was established by floral-

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source field site analysis during collection, the presence of diagnostic compounds such as

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DHA and MGO, and phenolic composition. Additional honey samples were obtained from

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commercial suppliers in New Zealand. Samples were stored at 4 °C until analysis. Honeydew

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was collected from wild Fuscospora solandri specimens and stored at -20 °C until analysis.

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Reagents and Solvents. The following chemicals were purchased as analytical reagent grade

48

and used without further purification: 6-aminouracil and 5,6-diamino-1-methyluracil (AK

49

Scientific, Union City, CA); CDCl3 and (CD3)2SO (Cambridge Isotope Laboratories,

50

Tewksbury, MA); AcOH, K2CO3, NaHCO3, NH4OH and NaOH (ECP NZ Ltd, Auckland,

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New Zealand); HCOOH (May and Baker Ltd, London, England); MeCN, MeI (Merck,

52

Auckland, New Zealand); 2,3-butanedione, hexamethyldisilazane, KMnO4, NaNO2 and

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Na2S2O4 (Sigma Aldrich NZ Ltd, Auckland, New Zealand). The following chemicals were

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purchased and distilled by rotary evaporation prior to use: EtOAc, MeOH and petroleum

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ether (ECP NZ Ltd, Auckland, New Zealand). DMF (ECP NZ Ltd, Auckland, New Zealand)

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was degassed and dried using an LC Technical SP-1 solvent purification system (Scitek

57

Australia, Sydney, Australia). Bidistilled water was generated by a Barnstead NANOpure

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Diamond Water Purification System (Thermofisher Scientific, Auckland, New Zealand).

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EtOH (ECP NZ Ltd, Auckland, New Zealand) was distilled over Mg(OEt)2 (Sigma Aldrich 4 ACS Paragon Plus Environment

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NZ

Ltd,

Auckland,

New

Zealand).

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Instrumentation. Solid phase extraction (SPE) was performed using Strata C18 E 70 Å, 55

62

µm 20 g columns (Phenomenex, Auckland, New Zealand). RP-HPLC was performed using

63

an Agilent 1100 system (Global Science, Auckland, New Zealand). The column used was a

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250 mm × 2.0 mm i.d., 5 µm, Jupiter 300 C18, with a 4 mm x 4 mm i.d. guard column of the

65

same material (Phenomenex, Auckland, New Zealand) at a flow rate of 0.2 mL/min with a

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DAD Detector operating at 262, 280 and 320 nm (Global Science, Auckland, New Zealand).

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Samples were analyzed in duplicate. Thin layer chromatography (TLC) was performed using

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0.2 mm Kieselgel F254 (Merck, Auckland, New Zealand) silica plates and compounds were

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visualized using UV irradiation at 254 or 365 nm and/or staining with a solution of potassium

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permanganate and potassium carbonate in aqueous sodium hydroxide. Flash chromatography

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was carried out using 0.063-0.1 mm Davisil silica gel (Trajan, Melbourne, Australia) with the

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desired solvent. Preparative TLC was performed using 500 µm, 20 × 20 cm Uniplate (John

73

Morris Scientific, Auckland, New Zealand) silica gel TLC plates and compounds were

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visualized using UV irradiation at 254 or 365 nm. High resolution mass spectra were

75

recorded on a Bruker micrOTOF-Q II mass spectrometer with ESI ionization source.

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Ultraviolet-visible spectra were run as H2O solutions on a Shimadzu UV-2101PC scanning

77

spectrophotometer. Infrared spectra were obtained using a Perkin-Elmer Spectrum 100 FTIR

78

spectrometer on a film ATR sampling accessory. Absorption maxima are expressed in

79

wavenumbers (cm-1). Melting points were determined on a Stuart Scientific SMP-3 capillary

80

apparatus (Global Science NZ Ltd, Auckland, New Zealand). NMR spectra were recorded as

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indicated on either a Bruker Avance 400 spectrometer operating at 400 MHz for 1H nuclei

82

and 100 MHz for 13C nuclei, a Bruker Avance AVIII-HD 500 spectrometer operating at 500

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MHz for 1H nuclei, 125 MHz for 13C nuclei or a Bruker Avance 600 spectrometer operating

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at 600 MHz for 1H nuclei, 150 MHz for 13C nuclei. 1H and 13C chemical shifts are reported in

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parts per million (ppm) relative to CDCl3 (1H and

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chemical shifts were referenced using the unified Ξi scale28 as implemented by the Bruker

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library function “xiref.” 1H NMR data is reported as chemical shift, relative integral,

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multiplicity (s, singlet; assignment). Assignments were made with the aid of COSY, NOESY,

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HSQC

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Analysis of samples by HPLC. Honey (~1 g) was dissolved in H2O containing 0.1%

91

HCOOH (3 mL), sonicated for 20 min and centrifuged to remove insoluble particles. An

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aliquot (1 mL) of each honey sample was applied to a preconditioned (MeOH) and

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equilibrated (H2O containing 0.1% HCOOH) SPE column using H2O containing 0.1%

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HCOOH and washed with MeOH/H2O (3 mL, 1:9, v/v) containing 0.1% HCOOH. The

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desired fraction was then eluted using MeOH/H2O (3 mL, 4:1, v/v) containing 0.1%

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HCOOH. A 5 µL aliquot of sample was injected and separated by gradient elution using the

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following gradient: 5%-25% B over 10 min; 25%-50% B over 10 min; 50%-100% B over 5

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min, where solvent A was H2O containing 0.1% HCOOH and B was A/MeCN (1:4, v/v).

99

Peaks of interest were collected manually. Nectar samples were diluted 5-fold before a 5 µL

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aliquot of sample was analyzed by HPLC as described above. Concentrations were calculated

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using

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Isolation of 3,6,7-trimethyllumazine, 3, from mānuka honey in preparative quantities.

103

Raw mānuka honey (51.3 g) was dissolved in H2O containing 0.1% HCOOH (80 mL),

104

sonicated for 20 min and filtered. The sample was applied to a preconditioned (MeOH) and

105

equilibrated (H2O containing 0.1% HCOOH) SPE column using H2O containing 0.1%

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HCOOH and washed with MeOH/H2O (80 mL, 1:9, v/v) containing 0.1% HCOOH. The

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desired fraction was then eluted using MeOH/H2O (80 mL, 4:1, v/v) containing 0.1%

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HCOOH and concentrated in vacuo to give the crude extract (0.23 g) which was further

109

purified by flash chromatography (petroleum ether-EtOAc 1:5, v/v) to give purified extract (3

and

a

standard

HMBC

curve

13

C) or (CD3)2SO (1H and

experiments

derived

where

from

synthetic

13

C).

15

N

required.

sample.

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mg) as a brown solid. Several purified extracts were combined (6 mg total) and further

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purified by preparative TLC (petroleum ether-EtOAc 1:4, v/v, 4 runs) to give 3 (4 mg) as a

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colorless solid. MP: 270-271 °C (lit19 271-272 °C); IR νmax (film) cm-1 2921, 1723, 1667,

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1435, 1393, 1363, 1281, 1119; UV/Vis λmax 211, 231 and 329nm; 1H NMR (400 MHz,

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CDCl3) δ 2.63 (3H, s, C6-CH3), 2.67 (3H, s, C7-CH3), 3.50 (3H, s, N-CH3), 8.42 (1H, s, NH);

115

13

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144.8 (C-8a), 149.9 (C-2), 150.6 (C-7), 158.9 (C-6), 161.1 (C-4); m/z (+ve ion HRESI-MS)

117

229.0696

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Synthesis of 3,6,7-Trimethyllumazine, 3. 5,6-Diamino-3-methyl uracil (7) (0.48 g, 3.05

119

mmol) was suspended in EtOH (10 mL). 2,3-Butandione (8) (0.29 g, 3.42 mmol) and AcOH

120

(0.94 g, 15.7 mmol) were added. The mixture was heated at reflux for 24 h before being

121

cooled to r.t. and concentrated in vacuo. The crude product was purified by flash

122

chromatography (petroleum ether-EtOAc 1:4, v/v) to give the title compound 3 (0.40 g, 63%)

123

as a colorless solid. MP 268-269 °C (lit19 271-272 °C); IR νmax (film) cm-1 2955, 1728, 1664,

124

1437, 1399, 1368, 1285, 1046; UV λmax 208, 234, and 329 nm; 1H NMR (400 MHz, CDCl3) δ

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2.65 (3H, s, C6-CH3), 2.66 (3H, s, C7-CH3), 3.50 (3H, s, N-CH3), 9.54 (1H, s, NH);

126

NMR (100 MHz, CDCl3) δ 21.8 (C7-CH3), 22.6 (C6-CH3), 28.3 (N-CH3), 123.6 (C-4a),

127

144.9 (C-8a), 150.4 (C-2), 150.5 (C-7), 158.8 (C-6), 161.0 (C-4); m/z (+ve ion HRESI-MS)

128

229.0696 [M+Na+], C9H10N4O2Na+ requires 229.0689. 1,6,7-Trimethyllumazine, 4. MP

129

317-325 °C (lit19 340-342 °C); IR νmax (film) cm-1 3040, 1685, 1551, 1495, 1395, 1349, 1283,

130

998; UV λmax, 201, 210, 252, and 334 nm; 1H NMR (400 MHz, (CD3)2SO) δ 2.53 (3H, s,

131

C7-CH3), 2.59 (3H, s, C6-CH3), 3.44 (3H, s, N-CH3); 13C NMR (100 MHz, (CD3)2SO) δ 21.0

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(C7-CH3), 22.5 (C6-CH3), 27.9 (N-CH3), 124.9 (C-4a), 147.2 (C-7), 147.3 (C-8a), 150.2 (C-

133

2), 156.8 (C-6), 160.2 (C-4); m/z (+ve ion HRESI-MS) 229.0696 [M+Na+], C9H10N4O2Na+

134

requires 229.0701.

C NMR (100 MHz, CDCl3) δ 21.9 (C7-CH3), 22.8 (C6-CH3), 28.5 (N-CH3), 123.7 (C-4a),

[M+Na+],

C9H10N4O2Na+

requires

229.0689.

13

C

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Results and Discussion

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Detection and isolation of lepteridine: While using HPLC to examine New Zealand

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honeys for the presence of leptosperin, 1, an unexpected UV absorbance 3 was noted at 320

138

nm (Figure 2A). This absorbance was observed in mānuka honeys from a variety of New

139

Zealand locations and in kānuka (Kunzea ericoides) honey, but was absent from other New

140

Zealand honeys including clover (Trifolium repens), rewarewa (Knightia excelsa), and

141

pohutukawa (Metrosideros excelsa) honeys (Table 1). Mānuka and kānuka plants flower

142

concurrently and mixing of mānkua and kānuka honey occurs as a result.29 The nectars of

143

both plants were examined and the absorbance detected only in mānuka nectar. To confirm

144

that the absorbance did not arise from contamination from honeydew produced by scale

145

insects known to feed on mānuka,30 honeydew from black beech (Fuscospora solandri) was

146

also examined and found to lack the distinctive absorbance. Having confirmed that the

147

absorbance was likely due to a native compound in mānuka nectar, we attempted to isolate

148

this compound from mānuka honey. SPE followed by reversed-phase HPLC enabled isolation

149

and partial characterization of the compound that exhibited this UV absorbance. Subjection

150

of mānuka honey to SPE, followed by normal-phase flash chromatography and preparative

151

TLC enabled isolation of the unknown compound as a colorless solid in sufficient quantity to

152

conduct spectroscopic analysis.

153

Structural Elucidation: The compound was soluble in CD3OD and CDCl3; the latter was

154

used for recording NMR spectra due to the presence of a broad resonance at δ 8.55 ppm (H-

155

1) that was absent from spectra recorded in CD3OD (Table 2). This peak was assigned as an

156

amide proton on the basis of its chemical shift and the absence of a distinctive hydroxyl

157

absorption in the IR spectrum. Two singlets at δ 2.63 ppm (H-10) and δ 2.67 ppm (H-11)

158

were assigned as heteroaryl methyl groups on the basis of their chemical shift, and the

159

remaining singlet at δ 3.50 ppm (H-9) was assigned as an N-methyl group due to 1H,

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C8

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HMBC correlations of equal intensity to two quaternary carbonyl 13C signals (C-2, C-4) and

161

an HSQC correlation to a carbon signal at δ 28.5 ppm (C-9, Figure 3). 1H,

162

correlations from H-10 and H-11 to N-5 and N-8 at δ 292.0 ppm and δ 329.9 ppm

163

respectively, suggested that these two methyl groups were attached to a pyrazine ring. A

164

2,3-dimethyl substitution pattern was assigned based on 1H, 13C-HMBC correlations from H-

165

10 to C-7 and from H-11 to C-6. Given the high degree of unsaturation in the structure and

166

the presence of a pyrazine ring, a pteridine (pyrazine[2,3-d]pyrimidine) structure was

167

proposed for the unknown compound.

15

N-HMBC

168

Initial Assignment: A similarity was noted between the chemical shifts for carbons C-2, C-

169

4 and C-4a with shifts reported for analogous carbons in natural products containing

170

lumazine structures.31 This observation, coupled with HMBC correlations from H-9 to C-2

171

and C-4 and an additional four bond coupling from H-10 to C-4a, led to the tentative

172

assignment of the structure of the isolated compound as either 3,6,7-trimethyllumazine, 3, or

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1,6,7-trimethyllumazine, 4 (Figure 3).

174

3,6,7-Trimethyllumazine, 3, was first synthesized in 1958. Since then it has been reported

175

in several studies on related lumazines. Characterization data for lumazine 3 is limited to a

176

melting point, elemental analysis and UV/Vis peaks; no NMR, MS or IR data have been

177

reported to date.18-27 Similarly, characterization data for lumazine 4 is limited to a melting

178

point, elemental analysis and UV/Vis.32-41 To date, no reports detailing the isolation of

179

lumazines 3 or 4 from natural materials have been published.

180

The melting point (270-271 °C) of the unknown compound correlated with the reported

181

melting point of lumazine 3 (271-272 °C) rather than that of lumazine 4 (340-342 °C).19 Both

182

compounds were synthesized in order to unambiguously determine the structure of the

183

unknown.

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Total synthesis: Following the work of Gala et al.,42 N-methylation of 6-aminouracil, 5, at

185

position 3 was accomplished via silylation of the exocyclic amino and carbonyl groups using

186

hexamethyldisilazane in the presence of a catalytic amount of sulfuric acid (Figure 4A).

187

Methylation was then effected using iodomethane. Subsequent desilylation during aqueous

188

workup afforded 6-amino-3-methyluracil, 6, in 78% yield. Amino uracil 6 was then treated

189

with sodium nitrite and acetic acid, followed by reduction with sodium dithionite in aqueous

190

ammonia at 70 °C43 to give 5,6-diamino-3-methyluracil, 7, in 31% yield over two steps. The

191

low yield was attributed to the difficulty in retrieving diamine 7 from the aqueous reaction

192

medium. Condensation of diaminouracil 7 with 2,3-butanedione, 8, in ethanol gave

193

3,6,7-trimethyllumazine, 3, as a colorless solid (63%). Similarly, condensation of

194

commercially

195

1,6,7-trimethyllumazine, 4, as a yellow solid in 60% yield (Figure 4B). Spectroscopic data

196

(UV/Vis, IR, 1H NMR, 13C NMR) of 3,6,7-trimethyllumazine, 3, was in excellent agreement

197

with that of the isolated natural product. Furthermore, the 1H NMR spectrum of combined

198

natural and synthetic products was identical to the 1H NMR spectra of separate natural and

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synthetic material. The 1H NMR shifts of the methyl protons of lumazine 4 in CD3OD (δ

200

2.62, δ 2.67 and δ 3.61 ppm) did not correlate strongly with those of the unknown compound

201

in CD3OD (δ 2.60, δ 2.61 and δ 3.40 ppm). The melting point of lumazine 4 (324-325 °C)

202

was also distinct from that of the unknown (270-271 °C). Thus the structure of the unknown

203

was definitively established as 3,6,7-trimethyllumazine, 3. Given that lumazine 3 is a

204

pteridine derivative isolated from Leptospermum honey the isolated compound was named

205

lepteridine.

available

5,6-diamino-1-methyl

uracil,

9,

with

844

afforded

206

It is noteworthy that lepteridine was detected at a wavelength (320 nm) longer than those

207

used to find other biomarkers (262 nm,16 283 nm,17 272 nm14). Its absorbance at this

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wavelength and pteridine structure readily distinguishes lepteridine from other biomarkers.

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Lepteridine is thus a valuable addition to the chemical “fingerprint” of mānuka honey.

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In summary, 3,6,7-trimethyllumazine, 3, was isolated from New Zealand mānuka honey,

211

and this structural assignment was confirmed by synthesis. Lumazine 3, named lepteridine,

212

has only been observed in mānuka honey and thus offers potential utility as a unique marker

213

for identification of genuine mānuka honey.

214

Associated Content

215

Supporting Information: Experimental procedures detailing the preparation of and 1H and

216

13

217

Internet at http://pubs.acs.org.

218

Author Information

219

*(D.P.F.) Phone +64-9-9238389. E-mail: [email protected].

220

*(M.A.B.) Phone +64-9-9238259. E-mail: [email protected].

221

Funding

222

This work was financially supported by Callaghan Innovation Ltd and Comvita NZ Ltd.

223

Notes

224

Gordana Prijic, Jonathan Stephens and Ralf Schlothauer are employees of Comvita NZ Ltd.

225

References

226

1.

C NMR spectra of compounds 4, 6 and 7. This material is available free of charge via the

York, 1960.

227 228

2.

231

Molan, P. C., Re-introducing honey in the management of wounds and ulcers - theory and practice. Ostomy Wound Manag. 2002, 48, 28-40.

229 230

Guthrie, D., A History of Medicine. Revised ed.; Thomas Nelson and Sons Ltd: New

3.

Jull, A. B.; Cullum, N.; Dumville, J. C.; Westby, M. J.; Deshpande, S.; Walker, N. Honey as a topical treatment for wounds; John Wiley & Sons, Ltd: 2015.

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4.

Mavric, E.; Wittmann, S.; Barth, G.; Henle, T., Identification and quantification of

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methylglyoxal as the dominant antibacterial constituent of Manuka (Leptospermum

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scoparium) honeys from New Zealand. Mol. Nutr. Food Res. 2008, 52, 483-489.

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Jenkins, R.; Burton, N.; Cooper, R., Manuka honey inhibits cell division in

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methicillin-resistant Staphylococcus aureus. J. Antimicrob. Chemother. 2011, 66,

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2536-2542.

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6.

Henriques, A. F.; Jenkins, R. E.; Burton, N. F.; Cooper, R. A., The intracellular

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effects of manuka honey on Staphylococcus aureus. Eur. J. Clin. Microbiol. Infect.

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Dis. 2010, 29, 45-50.

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Müller, P.; Alber, D. G.; Turnbull, L.; Schlothauer, R. C.; Carter, D. A.; Whitchurch,

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C. B.; Harry, E. J., Synergism between medihoney and rifampicin against methicillin-

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resistant Staphylococcus aureus (mrsa). PLoS ONE 2013, 8, 1-9.

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8.

Jenkins, R. E.; Cooper, R., Synergy between oxacillin and manuka honey sensitizes

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methicillin-resistant Staphylococcus aureus to oxacillin. J. Antimicrob. Chemother.

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2012, 67, 1405-1407.

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properties. Czech J. Food Sci. 2009, 27, S163-S165.

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Allen, K. L.; Molan, P. C.; Reid, G. M., A survey of the antibacterial activity of some New Zealand honeys. J. Pharm. Pharmacol. 1991, 43, 817-822.

250 251

Atrott, J.; Henle, T., Methylglyoxal in manuka honey - correlation with antibacterial

11.

Adams, C. J.; Manley-Harris, M.; Molan, P. C., The origin of methylglyoxal in New

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Zealand manuka (Leptospermum scoparium) honey. Carbohydr. Res. 2009, 344,

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1050-1053.

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Jervis-Bardy, J.; Foreman, A.; Bray, S.; Tan, L.; Wormald, P.-J., Methylglyoxalinfused honey mimics the anti-Staphylococcus aureus biofilm activity of manuka

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FIGURE CAPTIONS Figure 1. Leptosperin, 1, 2-acetyl-1-pyrroline, 2, lepteridine 3. Figure 2. HPLC chromatograms of (A) mānuka (L. scoparium), (B) rewarewa (Knightia excelsa), (C) clover (Trifoilum repens) and (D) pohutukawa (Metrosideros excelsa) honeys at 320 nm. Figure 3. 1H, 13C-HMBC (red) and 1H, 15N-HMBC (blue) correlations in 3; 1,6,7trimethyllumazine ,4, an isomer of 3. Figure 4. Synthesis of (A) 3,6,7-trimethyllumazine, 3 and (B) 1,6,7-trimethyllumazine, 4.

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TABLES

Table 1. Detection of 3 in Various Botanical Samples. Floral Source (Sample Type) L. scoparium (mānuka honey) L. scoparium (mānuka honey) L. scoparium (mānuka honey) L. scoparium (mānuka honey) L. scoparium (mānuka honey) Kunzea ericoides (kānuka honey)

# of samples 2 1 1 1 1 1

Knightia excelsa (rewarewa honey)

2

Trifolium repens (clover honey) Metrosideros robusta (rata honey)

2 2

Weinmania racemosa (kamahi honey)

2

Ixerbia brexioides (tawari honey)

1

Weinmannia silvicola (towai honey) Metrosideros excelsa (pohutukawa honey) Thymus vulgaris (thyme honey) L. scoparium var incarnum (mānuka nectar) L. scoparium var scoparium (mānuka nectar) L. scoparium var myrtifolium (mānuka nectar) Kunzea ericoides (kānuka nectar) Fuscospora solandri (honeydew)

1 1 1 2 2 2 2 2

Location Northland Waikato Eastland Wairarapa South Island Northland Bay of Plenty South Island West Coast Bay of Plenty Bay of Plenty Northland Auckland Otago Glasshouse Glasshouse Glasshouse Glasshouse Canterbury

Concentration of lepteridine (µg/g) 38.35 38.40 39.29 5.23 10.00 3.55 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 35.00 79.93 43.70 n.d. n.d.

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Table 2. 1H NMR, 13C NMR and 15N NMR Shifts for 3a. HMBCb

Position

δC/δN , type

δH

1

NH

8.42

2

149.9, C

3

154.1, N

4

161.1, C

4a

123.7, C

5

292.0, N

6

158.9, C

7

150.6, C

8

329.9, N

8a

144.8, C

9

28.5, CH3

3.50, s

2, 3, 4

10

22.8, CH3

2.63, s

4a, 5, 6, 7

11

21.9, CH3

2.67, s

6, 7, 8

a 1

13

15

H (400 MHz); C (100 MHz); N (60.8 MHz), chemical shift indirectly determined from 1H, 15N HMBC NMR data. b HMBC correlations are from protons stated to the indicated carbon or nitrogen.

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FIGURES

Figure 1

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mAU

lepteridine (3)

A, mānuka

mAU

B, rewarewa

mAU

C, clover

mAU

D, pohutukawa

time

Figure 2

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Journal of Agricultural and Food Chemistry

Figure 3

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A

O HN

O

N H

O

1) HMDS, H2SO4 (cat) reflux

Me

2) MeI, DMF, aqueous workup 71% (2 steps)

NH2

O

5

Page 24 of 25

1) NaNO 2, AcOH H2O

N N H

2) Na2S2O4 NH3(aq) 31% (2 steps)

NH2

6 O

O Me O

NH2

N N H

NH2

O

Me

8 Me

Me

O

AcOH, EtOH reflux 63%

N

O

N H

N

Me

N

Me

N

Me

N

Me

3

7 B

O

O NH2

HN O

N Me 9

NH2

Me

8 Me O

AcOH, H2O reflux 60%

O HN O

N Me 4

Figure 4

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Journal of Agricultural and Food Chemistry

TABLE OF CONTENTS GRAPHIC

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