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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
2
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
6
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
8
mānuka honeys and could potentially serve as a biomarker for genuine mānuka honey.
9
KEYWORDS Leptospermum, mānuka honey, biomarker, lepteridine, lumazine.
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Introduction
11
Honey was first used to augment the healing of wounds by the ancient Egyptians1 and is
12
currently a clinical wound treatment.2-3 New Zealand mānuka honey, derived from the nectar
13
of Leptospermum scoparium, exhibits non-peroxide based antibacterial activity due to the
14
presence of methyl glyoxal (MGO).4 Mānuka honey is active against methicillin-resistant
15
Staphylococcus aureus (MRSA)5-6 and increases the susceptibility of MRSA to rifampicin7
16
and oxacillin.8
17
Currently, genuine mānuka honey is labelled by either reporting the MGO concentration9 or
18
using the Unique Mānuka Factor (UMF) scale, which equates the bactericidal activity of a
19
given honey sample with that of a given concentration of phenol.10 This bactericidal activity
20
is dependent on the concentration of MGO. Since MGO is produced in honey from
21
dihydroxyacetone during incubation11 the concentration of MGO and the UMF of a given
22
honey sample can change over time. Furthermore, adulteration of honey via addition of
23
dihydroxyacetone or MGO gives rise to comparable bactericidal activity,12 meaning that the
24
current labelling system does not provide consumers or suppliers with the information
25
required to recognize counterfeit mānuka honeys, which pose a threat to the mānuka honey
26
market.13 Due to its medical and economic relevance, compounds that could serve as unique
27
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
29
the mechanism(s) of the bioactivity of the honey and discovering unique biomarkers.14 One
30
compound, leptosperin, 1, has been proposed as a both a contributor towards the bactericidal
31
activity of mānuka honey and as a unique mānuka marker (Figure 1).15-16 Additionally, 2-
32
acetyl-1-pyrroline (2) has been found to be produced from native proline in the presence of
33
high (>250 mg/kg) concentrations of MGO, and has also been proposed as a potential
34
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
36
lepteridine, 3, a pteridine derivative with the systematic name 3,6,7-trimethyllumazine,18-27
37
from mānuka honey. We observed lepteridine, 3, exclusively in Leptospermum honeys; hence
38
3 could serve as a unique marker for mānuka honey.
39
Materials and Methods
40
Botanical samples. Nectar samples were obtained by direct sampling from specimens
41
grown in glasshouses using a micropipette and stored at -20 °C until analysis. Mānuka honey
42
samples were collected from hives in the field. Botanical origin was established by floral-
43
source field site analysis during collection, the presence of diagnostic compounds such as
44
DHA and MGO, and phenolic composition. Additional honey samples were obtained from
45
commercial suppliers in New Zealand. Samples were stored at 4 °C until analysis. Honeydew
46
was collected from wild Fuscospora solandri specimens and stored at -20 °C until analysis.
47
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,
51
New Zealand); HCOOH (May and Baker Ltd, London, England); MeCN, MeI (Merck,
52
Auckland, New Zealand); 2,3-butanedione, hexamethyldisilazane, KMnO4, NaNO2 and
53
Na2S2O4 (Sigma Aldrich NZ Ltd, Auckland, New Zealand). The following chemicals were
54
purchased and distilled by rotary evaporation prior to use: EtOAc, MeOH and petroleum
55
ether (ECP NZ Ltd, Auckland, New Zealand). DMF (ECP NZ Ltd, Auckland, New Zealand)
56
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
58
Diamond Water Purification System (Thermofisher Scientific, Auckland, New Zealand).
59
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).
61
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
64
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
66
DAD Detector operating at 262, 280 and 320 nm (Global Science, Auckland, New Zealand).
67
Samples were analyzed in duplicate. Thin layer chromatography (TLC) was performed using
68
0.2 mm Kieselgel F254 (Merck, Auckland, New Zealand) silica plates and compounds were
69
visualized using UV irradiation at 254 or 365 nm and/or staining with a solution of potassium
70
permanganate and potassium carbonate in aqueous sodium hydroxide. Flash chromatography
71
was carried out using 0.063-0.1 mm Davisil silica gel (Trajan, Melbourne, Australia) with the
72
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
74
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.
76
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
81
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
83
MHz for 1H nuclei, 125 MHz for 13C nuclei or a Bruker Avance 600 spectrometer operating
84
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
86
chemical shifts were referenced using the unified Ξi scale28 as implemented by the Bruker
87
library function “xiref.” 1H NMR data is reported as chemical shift, relative integral,
88
multiplicity (s, singlet; assignment). Assignments were made with the aid of COSY, NOESY,
89
HSQC
90
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
92
aliquot (1 mL) of each honey sample was applied to a preconditioned (MeOH) and
93
equilibrated (H2O containing 0.1% HCOOH) SPE column using H2O containing 0.1%
94
HCOOH and washed with MeOH/H2O (3 mL, 1:9, v/v) containing 0.1% HCOOH. The
95
desired fraction was then eluted using MeOH/H2O (3 mL, 4:1, v/v) containing 0.1%
96
HCOOH. A 5 µL aliquot of sample was injected and separated by gradient elution using the
97
following gradient: 5%-25% B over 10 min; 25%-50% B over 10 min; 50%-100% B over 5
98
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
100
aliquot of sample was analyzed by HPLC as described above. Concentrations were calculated
101
using
102
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%
106
HCOOH and washed with MeOH/H2O (80 mL, 1:9, v/v) containing 0.1% HCOOH. The
107
desired fraction was then eluted using MeOH/H2O (80 mL, 4:1, v/v) containing 0.1%
108
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
111
purified by preparative TLC (petroleum ether-EtOAc 1:4, v/v, 4 runs) to give 3 (4 mg) as a
112
colorless solid. MP: 270-271 °C (lit19 271-272 °C); IR νmax (film) cm-1 2921, 1723, 1667,
113
1435, 1393, 1363, 1281, 1119; UV/Vis λmax 211, 231 and 329nm; 1H NMR (400 MHz,
114
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
116
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
118
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
125
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
132
(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
136
Detection and isolation of lepteridine: While using HPLC to examine New Zealand
137
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,
13
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
173
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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184
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
199
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.
209
Lepteridine is thus a valuable addition to the chemical “fingerprint” of mānuka honey.
210
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.
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2.
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Molan, P. C., Re-introducing honey in the management of wounds and ulcers - theory and practice. Ostomy Wound Manag. 2002, 48, 28-40.
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Guthrie, D., A History of Medicine. Revised ed.; Thomas Nelson and Sons Ltd: New
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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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Jenkins, R.; Burton, N.; Cooper, R., Manuka honey inhibits cell division in
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Henriques, A. F.; Jenkins, R. E.; Burton, N. F.; Cooper, R. A., The intracellular
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Dis. 2010, 29, 45-50.
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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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Jenkins, R. E.; Cooper, R., Synergy between oxacillin and manuka honey sensitizes
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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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Journal of Agricultural and Food Chemistry
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
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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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