Isolation, Structural Elucidation, and Synthesis of Lepteridine from Manuka (Leptospermum scoparium) Honey

Article abstract of DOI:10.1021/acs.jafc.6b01596

Manuka honey, made from the nectar of Leptospermum scoparium, has garnered scientific and economical interest due to its nonperoxide antibacterial activity. Biomarkers for genuine manuka honey are increasingly in demand due to the presence of counterfeit manuka honey. This work reports the identification of a compound previously unreported in manuka honey by HPLC, and determination of the structure of the as 3,6,7-trimethyllumazine using NMR, MS, IR, and UV/vis spectroscopy. This assignment was confirmed by total synthesis. The natural product, renamed lepteridine, was only observed in manuka honeys and could potentially serve as a biomarker for genuine manuka honey.

Full text of DOI:10.1021/acs.jafc.6b01596

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Article
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: +64ꢀ9ꢀ9238389; Fax: +64ꢀ9ꢀ3737422; Eꢀmail:
d.furkert@auckland.ac.nz)
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ABSTRACT: Mānuka honey, made from the nectar of Leptospermum scoparium, has
garnered scientific and economical interest due to its nonꢀperoxide antibacterial activity.
Biomarkers for genuine mānuka honey are increasingly in demand due to the presence of
counterfeit mānuka honey. This work reports the identification of a compound previously
unreported in mānuka honey by HPLC, and determination of the structure of the as
3,6,7ꢀtrimethyllumazine using NMR, MS, IR and UV/Vis spectroscopy. This assignment was
confirmed by total synthesis. The natural product, renamed lepteridine, was only observed in
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
currently a clinical wound treatment.2ꢀ3 New Zealand mānuka honey, derived from the nectar
of Leptospermum scoparium, exhibits nonꢀperoxide based antibacterial activity due to the
presence of methyl glyoxal (MGO).4 Mānuka honey is active against methicillinꢀresistant
Staphylococcus aureus (MRSA)5ꢀ6 and increases the susceptibility of MRSA to rifampicin7
and oxacillin.8
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Currently, genuine mānuka honey is labelled by either reporting the MGO concentration9 or
using the Unique Mānuka Factor (UMF) scale, which equates the bactericidal activity of a
given honey sample with that of a given concentration of phenol.10 This bactericidal activity
is dependent on the concentration of MGO. Since MGO is produced in honey from
dihydroxyacetone during incubation11 the concentration of MGO and the UMF of a given
honey sample can change over time. Furthermore, adulteration of honey via addition of
dihydroxyacetone or MGO gives rise to comparable bactericidal activity,12 meaning that the
current labelling system does not provide consumers or suppliers with the information
required to recognize counterfeit mānuka honeys, which pose a threat to the mānuka honey
market.13 Due to its medical and economic relevance, compounds that could serve as unique
markers for genuine mānuka honey are of scientific and commercial importance.
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The phytochemical profile of mānuka honey has been probed with the aim of elucidating
the mechanism(s) of the bioactivity of the honey and discovering unique biomarkers.14 One
compound, leptosperin, 1, has been proposed as a both a contributor towards the bactericidal
activity of mānuka honey and as a unique mānuka marker (Figure 1).15ꢀ16 Additionally, 2ꢀ
acetylꢀ1ꢀpyrroline (2) has been found to be produced from native proline in the presence of
high (>250 mg/kg) concentrations of MGO, and has also been proposed as a potential
marker.17 Additional markers would provide greater security for honey consumers and further
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choice for regulators. Herein we report the isolation, structural elucidation, and synthesis of
lepteridine, 3, a pteridine derivative with the systematic name 3,6,7ꢀtrimethyllumazine,18ꢀ27
from mānuka honey. We observed lepteridine, 3, exclusively in Leptospermum honeys; hence
3 could serve as a unique marker for mānuka honey.
39
Materials and Methods
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Botanical samples. Nectar samples were obtained by direct sampling from specimens
grown in glasshouses using a micropipette and stored at ꢀ20 °C until analysis. Mānuka honey
samples were collected from hives in the field. Botanical origin was established by floralꢀ
source field site analysis during collection, the presence of diagnostic compounds such as
DHA and MGO, and phenolic composition. Additional honey samples were obtained from
commercial suppliers in New Zealand. Samples were stored at 4 °C until analysis. Honeydew
was collected from wild Fuscospora solandri specimens and stored at ꢀ20 °C until analysis.
Reagents and Solvents. The following chemicals were purchased as analytical reagent grade
and used without further purification: 6ꢀaminouracil and 5,6ꢀdiaminoꢀ1ꢀmethyluracil (AK
Scientific, Union City, CA); CDCl3 and (CD3)2SO (Cambridge Isotope Laboratories,
Tewksbury, MA); AcOH, K2CO3, NaHCO3, NH4OH and NaOH (ECP NZ Ltd, Auckland,
New Zealand); HCOOH (May and Baker Ltd, London, England); MeCN, MeI (Merck,
Auckland, New Zealand); 2,3ꢀbutanedione, hexamethyldisilazane, KMnO4, NaNO2 and
Na2S2O4 (Sigma Aldrich NZ Ltd, Auckland, New Zealand). The following chemicals were
purchased and distilled by rotary evaporation prior to use: EtOAc, MeOH and petroleum
ether (ECP NZ Ltd, Auckland, New Zealand). DMF (ECP NZ Ltd, Auckland, New Zealand)
was degassed and dried using an LC Technical SPꢀ1 solvent purification system (Scitek
Australia, Sydney, Australia). Bidistilled water was generated by a Barnstead NANOpure
Diamond Water Purification System (Thermofisher Scientific, Auckland, New Zealand).
EtOH (ECP NZ Ltd, Auckland, New Zealand) was distilled over Mg(OEt)2 (Sigma Aldrich
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NZ
Ltd,
Auckland,
New
Zealand).
Instrumentation. Solid phase extraction (SPE) was performed using Strata C18 E 70 Å, 55
µm 20 g columns (Phenomenex, Auckland, New Zealand). RPꢀHPLC was performed using
an Agilent 1100 system (Global Science, Auckland, New Zealand). The column used was a
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
same material (Phenomenex, Auckland, New Zealand) at a flow rate of 0.2 mL/min with a
DAD Detector operating at 262, 280 and 320 nm (Global Science, Auckland, New Zealand).
Samples were analyzed in duplicate. Thin layer chromatography (TLC) was performed using
0.2 mm Kieselgel F254 (Merck, Auckland, New Zealand) silica plates and compounds were
visualized using UV irradiation at 254 or 365 nm and/or staining with a solution of potassium
permanganate and potassium carbonate in aqueous sodium hydroxide. Flash chromatography
was carried out using 0.063ꢀ0.1 mm Davisil silica gel (Trajan, Melbourne, Australia) with the
desired solvent. Preparative TLC was performed using 500 µm, 20 × 20 cm Uniplate (John
Morris Scientific, Auckland, New Zealand) silica gel TLC plates and compounds were
visualized using UV irradiation at 254 or 365 nm. High resolution mass spectra were
recorded on a Bruker micrOTOFꢀQ II mass spectrometer with ESI ionization source.
Ultravioletꢀvisible spectra were run as H2O solutions on a Shimadzu UVꢀ2101PC scanning
spectrophotometer. Infrared spectra were obtained using a PerkinꢀElmer Spectrum 100 FTIR
spectrometer on a film ATR sampling accessory. Absorption maxima are expressed in
wavenumbers (cmꢀ1). Melting points were determined on a Stuart Scientific SMPꢀ3 capillary
apparatus (Global Science NZ Ltd, Auckland, New Zealand). NMR spectra were recorded as
1
indicated on either a Bruker Avance 400 spectrometer operating at 400 MHz for H nuclei
and 100 MHz for 13C nuclei, a Bruker Avance AVIIIꢀHD 500 spectrometer operating at 500
MHz for H nuclei, 125 MHz for 13C nuclei or a Bruker Avance 600 spectrometer operating
1
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 13C) or (CD3)2SO (1H and 13C). 15N
chemical shifts were referenced using the unified Ξi scale28 as implemented by the Bruker
1
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library function “xiref.” H 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
and
HMBC
experiments
where
required.
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Analysis of samples by HPLC. Honey (~1 g) was dissolved in H2O containing 0.1%
HCOOH (3 mL), sonicated for 20 min and centrifuged to remove insoluble particles. An
aliquot (1 mL) of each honey sample was applied to a preconditioned (MeOH) and
equilibrated (H2O containing 0.1% HCOOH) SPE column using H2O containing 0.1%
HCOOH and washed with MeOH/H2O (3 mL, 1:9, v/v) containing 0.1% HCOOH. The
desired fraction was then eluted using MeOH/H2O (3 mL, 4:1, v/v) containing 0.1%
HCOOH. A 5 µL aliquot of sample was injected and separated by gradient elution using the
following gradient: 5%ꢀ25% B over 10 min; 25%ꢀ50% B over 10 min; 50%ꢀ100% B over 5
min, where solvent A was H2O containing 0.1% HCOOH and B was A/MeCN (1:4, v/v).
Peaks of interest were collected manually. Nectar samples were diluted 5ꢀfold before a 5 µL
aliquot of sample was analyzed by HPLC as described above. Concentrations were calculated
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using
a
standard
curve
derived
from
synthetic
sample.
Isolation of 3,6,7ꢀtrimethyllumazine, 3, from mānuka honey in preparative quantities.
Raw mānuka honey (51.3 g) was dissolved in H2O containing 0.1% HCOOH (80 mL),
sonicated for 20 min and filtered. The sample was applied to a preconditioned (MeOH) and
equilibrated (H2O containing 0.1% HCOOH) SPE column using H2O containing 0.1%
HCOOH and washed with MeOH/H2O (80 mL, 1:9, v/v) containing 0.1% HCOOH. The
desired fraction was then eluted using MeOH/H2O (80 mL, 4:1, v/v) containing 0.1%
HCOOH and concentrated in vacuo to give the crude extract (0.23 g) which was further
purified by flash chromatography (petroleum etherꢀEtOAc 1:5, v/v) to give purified extract (3
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mg) as a brown solid. Several purified extracts were combined (6 mg total) and further
purified by preparative TLC (petroleum etherꢀEtOAc 1:4, v/v, 4 runs) to give 3 (4 mg) as a
colorless solid. MP: 270ꢀ271 °C (lit19 271ꢀ272 °C); IR νmax (film) cmꢀ1 2921, 1723, 1667,
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1
1435, 1393, 1363, 1281, 1119; UV/Vis λmax 211, 231 and 329nm; H NMR (400 MHz,
CDCl3) δ 2.63 (3H, s, C6ꢀCH3), 2.67 (3H, s, C7ꢀCH3), 3.50 (3H, s, NꢀCH3), 8.42 (1H, s, NH);
13C NMR (100 MHz, CDCl3) δ 21.9 (C7ꢀCH3), 22.8 (C6ꢀCH3), 28.5 (NꢀCH3), 123.7 (Cꢀ4a),
144.8 (Cꢀ8a), 149.9 (Cꢀ2), 150.6 (Cꢀ7), 158.9 (Cꢀ6), 161.1 (Cꢀ4); m/z (+ve ion HRESIꢀMS)
229.0696
[M+Na+],
C9H10N4O2Na+
requires
229.0689.
Synthesis of 3,6,7ꢀTrimethyllumazine, 3. 5,6ꢀDiaminoꢀ3ꢀmethyl uracil (7) (0.48 g, 3.05
mmol) was suspended in EtOH (10 mL). 2,3ꢀButandione (8) (0.29 g, 3.42 mmol) and AcOH
(0.94 g, 15.7 mmol) were added. The mixture was heated at reflux for 24 h before being
cooled to r.t. and concentrated in vacuo. The crude product was purified by flash
chromatography (petroleum etherꢀEtOAc 1:4, v/v) to give the title compound 3 (0.40 g, 63%)
as a colorless solid. MP 268ꢀ269 °C (lit19 271ꢀ272 °C); IR νmax (film) cmꢀ1 2955, 1728, 1664,
1437, 1399, 1368, 1285, 1046; UV λmax 208, 234, and 329 nm; 1H NMR (400 MHz, CDCl3) δ
2.65 (3H, s, C6ꢀCH3), 2.66 (3H, s, C7ꢀCH3), 3.50 (3H, s, NꢀCH3), 9.54 (1H, s, NH); 13C
NMR (100 MHz, CDCl3) δ 21.8 (C7ꢀCH3), 22.6 (C6ꢀCH3), 28.3 (NꢀCH3), 123.6 (Cꢀ4a),
144.9 (Cꢀ8a), 150.4 (Cꢀ2), 150.5 (Cꢀ7), 158.8 (Cꢀ6), 161.0 (Cꢀ4); m/z (+ve ion HRESIꢀMS)
229.0696 [M+Na+], C9H10N4O2Na+ requires 229.0689. 1,6,7ꢀTrimethyllumazine, 4. MP
317ꢀ325 °C (lit19 340ꢀ342 °C); IR νmax (film) cmꢀ1 3040, 1685, 1551, 1495, 1395, 1349, 1283,
1
998; UV λmax, 201, 210, 252, and 334 nm; H NMR (400 MHz, (CD3)2SO) δ 2.53 (3H, s,
C7ꢀCH3), 2.59 (3H, s, C6ꢀCH3), 3.44 (3H, s, NꢀCH3); 13C NMR (100 MHz, (CD3)2SO) δ 21.0
(C7ꢀCH3), 22.5 (C6ꢀCH3), 27.9 (NꢀCH3), 124.9 (Cꢀ4a), 147.2 (Cꢀ7), 147.3 (Cꢀ8a), 150.2 (Cꢀ
2), 156.8 (Cꢀ6), 160.2 (Cꢀ4); m/z (+ve ion HRESIꢀMS) 229.0696 [M+Na+], C9H10N4O2Na+
requires 229.0701.
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Results and Discussion
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Detection and isolation of lepteridine: While using HPLC to examine New Zealand
honeys for the presence of leptosperin, 1, an unexpected UV absorbance 3 was noted at 320
nm (Figure 2A). This absorbance was observed in mānuka honeys from a variety of New
Zealand locations and in kānuka (Kunzea ericoides) honey, but was absent from other New
Zealand honeys including clover (Trifolium repens), rewarewa (Knightia excelsa), and
pohutukawa (Metrosideros excelsa) honeys (Table 1). Mānuka and kānuka plants flower
concurrently and mixing of mānkua and kānuka honey occurs as a result.29 The nectars of
both plants were examined and the absorbance detected only in mānuka nectar. To confirm
that the absorbance did not arise from contamination from honeydew produced by scale
insects known to feed on mānuka,30 honeydew from black beech (Fuscospora solandri) was
also examined and found to lack the distinctive absorbance. Having confirmed that the
absorbance was likely due to a native compound in mānuka nectar, we attempted to isolate
this compound from mānuka honey. SPE followed by reversedꢀphase HPLC enabled isolation
and partial characterization of the compound that exhibited this UV absorbance. Subjection
of mānuka honey to SPE, followed by normalꢀphase flash chromatography and preparative
TLC enabled isolation of the unknown compound as a colorless solid in sufficient quantity to
conduct spectroscopic analysis.
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Structural Elucidation: The compound was soluble in CD3OD and CDCl3; the latter was
used for recording NMR spectra due to the presence of a broad resonance at δ 8.55 ppm (Hꢀ
1) that was absent from spectra recorded in CD3OD (Table 2). This peak was assigned as an
amide proton on the basis of its chemical shift and the absence of a distinctive hydroxyl
absorption in the IR spectrum. Two singlets at δ 2.63 ppm (Hꢀ10) and δ 2.67 ppm (Hꢀ11)
were assigned as heteroaryl methyl groups on the basis of their chemical shift, and the
1
remaining singlet at δ 3.50 ppm (Hꢀ9) was assigned as an Nꢀmethyl group due to H, 13Cꢀ
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HMBC correlations of equal intensity to two quaternary carbonyl C signals (Cꢀ2, Cꢀ4) and
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an HSQC correlation to a carbon signal at δ 28.5 ppm (Cꢀ9, Figure 3). H, 15NꢀHMBC
correlations from Hꢀ10 and Hꢀ11 to Nꢀ5 and Nꢀ8 at δ 292.0 ppm and δ 329.9 ppm
respectively, suggested that these two methyl groups were attached to a pyrazine ring. A
2,3ꢀdimethyl substitution pattern was assigned based on 1H, 13CꢀHMBC correlations from Hꢀ
10 to Cꢀ7 and from Hꢀ11 to Cꢀ6. Given the high degree of unsaturation in the structure and
the presence of a pyrazine ring, a pteridine (pyrazine[2,3ꢀd]pyrimidine) structure was
proposed for the unknown compound.
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Initial Assignment: A similarity was noted between the chemical shifts for carbons Cꢀ2, Cꢀ
4 and Cꢀ4a with shifts reported for analogous carbons in natural products containing
lumazine structures.31 This observation, coupled with HMBC correlations from Hꢀ9 to Cꢀ2
and Cꢀ4 and an additional four bond coupling from Hꢀ10 to Cꢀ4a, led to the tentative
assignment of the structure of the isolated compound as either 3,6,7ꢀtrimethyllumazine, 3, or
1,6,7ꢀtrimethyllumazine, 4 (Figure 3).
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3,6,7ꢀTrimethyllumazine, 3, was first synthesized in 1958. Since then it has been reported
in several studies on related lumazines. Characterization data for lumazine 3 is limited to a
melting point, elemental analysis and UV/Vis peaks; no NMR, MS or IR data have been
reported to date.18ꢀ27 Similarly, characterization data for lumazine 4 is limited to a melting
point, elemental analysis and UV/Vis.32ꢀ41 To date, no reports detailing the isolation of
lumazines 3 or 4 from natural materials have been published.
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The melting point (270ꢀ271 °C) of the unknown compound correlated with the reported
melting point of lumazine 3 (271ꢀ272 °C) rather than that of lumazine 4 (340ꢀ342 °C).19 Both
compounds were synthesized in order to unambiguously determine the structure of the
unknown.
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Total synthesis: Following the work of Gala et al.,42 Nꢀmethylation of 6ꢀaminouracil, 5, at
position 3 was accomplished via silylation of the exocyclic amino and carbonyl groups using
hexamethyldisilazane in the presence of a catalytic amount of sulfuric acid (Figure 4A).
Methylation was then effected using iodomethane. Subsequent desilylation during aqueous
workup afforded 6ꢀaminoꢀ3ꢀmethyluracil, 6, in 78% yield. Amino uracil 6 was then treated
with sodium nitrite and acetic acid, followed by reduction with sodium dithionite in aqueous
ammonia at 70 °C43 to give 5,6ꢀdiaminoꢀ3ꢀmethyluracil, 7, in 31% yield over two steps. The
low yield was attributed to the difficulty in retrieving diamine 7 from the aqueous reaction
medium. Condensation of diaminouracil 7 with 2,3ꢀbutanedione, 8, in ethanol gave
3,6,7ꢀtrimethyllumazine, 3, as a colorless solid (63%). Similarly, condensation of
commercially available 5,6ꢀdiaminoꢀ1ꢀmethyl uracil, 9, with 844 afforded
1,6,7ꢀtrimethyllumazine, 4, as a yellow solid in 60% yield (Figure 4B). Spectroscopic data
1
13
(UV/Vis, IR, H NMR, C NMR) of 3,6,7ꢀtrimethyllumazine, 3, was in excellent agreement
1
with that of the isolated natural product. Furthermore, the H NMR spectrum of combined
1
natural and synthetic products was identical to the H NMR spectra of separate natural and
1
synthetic material. The H NMR shifts of the methyl protons of lumazine 4 in CD3OD (δ
2.62, δ 2.67 and δ 3.61 ppm) did not correlate strongly with those of the unknown compound
in CD3OD (δ 2.60, δ 2.61 and δ 3.40 ppm). The melting point of lumazine 4 (324ꢀ325 °C)
was also distinct from that of the unknown (270ꢀ271 °C). Thus the structure of the unknown
was definitively established as 3,6,7ꢀtrimethyllumazine, 3. Given that lumazine 3 is a
pteridine derivative isolated from Leptospermum honey the isolated compound was named
lepteridine.
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It is noteworthy that lepteridine was detected at a wavelength (320 nm) longer than those
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.
Lepteridine is thus a valuable addition to the chemical “fingerprint” of mānuka honey.
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In summary, 3,6,7ꢀtrimethyllumazine, 3, was isolated from New Zealand mānuka honey,
and this structural assignment was confirmed by synthesis. Lumazine 3, named lepteridine,
has only been observed in mānuka honey and thus offers potential utility as a unique marker
for identification of genuine mānuka honey.
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Associated Content
1
215
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Supporting Information: Experimental procedures detailing the preparation of and H and
13C NMR spectra of compounds 4, 6 and 7. This material is available free of charge via the
Internet at http://pubs.acs.org.
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Author Information
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220
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*(D.P.F.) Phone +64ꢀ9ꢀ9238389. Eꢀmail: d.furkert@auckland.ac.nz.
*(M.A.B.) Phone +64ꢀ9ꢀ9238259. Eꢀmail: m.brimble@auckland.ac.nz.
Funding
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This work was financially supported by Callaghan Innovation Ltd and Comvita NZ Ltd.
Notes
Gordana Prijic, Jonathan Stephens and Ralf Schlothauer are employees of Comvita NZ Ltd.
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References
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2.
3.
Guthrie, D., A History of Medicine. Revised ed.; Thomas Nelson and Sons Ltd: New
York, 1960.
Molan, P. C., Reꢀintroducing honey in the management of wounds and ulcers ꢀ theory
and practice. Ostomy Wound Manag. 2002, 48, 28ꢀ40.
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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354
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FIGURE CAPTIONS
Figure 1. Leptosperin, 1, 2ꢀacetylꢀ1ꢀpyrroline, 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, 13CꢀHMBC (red) and 1H, 15NꢀHMBC (blue) correlations in 3; 1,6,7ꢀ
trimethyllumazine ,4, an isomer of 3.
Figure 4. Synthesis of (A) 3,6,7ꢀtrimethyllumazine, 3 and (B) 1,6,7ꢀtrimethyllumazine, 4.
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Journal of Agricultural and Food Chemistry
TABLES
Table 1. Detection of 3 in Various Botanical Samples.
# of
Floral Source (Sample Type)
samples
Concentration of
lepteridine (µg/g)
38.35
Location
L. scoparium (mānuka honey)
L. scoparium (mānuka honey)
L. scoparium (mānuka honey)
L. scoparium (mānuka honey)
L. scoparium (mānuka honey)
Kunzea ericoides (kānuka honey)
2
1
1
1
1
1
Northland
Waikato
38.40
39.29
5.23
10.00
Eastland
Wairarapa
South Island
Northland
Bay of
3.55
Knightia excelsa (rewarewa honey)
2
n.d.
Plenty
Trifolium repens (clover honey)
Metrosideros robusta (rata honey)
2
2
South Island
West Coast
Bay of
n.d.
n.d.
Weinmania racemosa (kamahi honey)
Ixerbia brexioides (tawari honey)
2
1
n.d.
n.d.
Plenty
Bay of
Plenty
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)
1
1
1
2
2
2
2
2
Northland
Auckland
Otago
Glasshouse
Glasshouse
Glasshouse
Glasshouse
Canterbury
n.d.
n.d.
n.d.
35.00
79.93
43.70
n.d.
Fuscospora solandri (honeydew)
n.d.
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Table 2. 1H NMR, 13C NMR and 15N NMR Shifts for 3a.
Position
δC/δN , type
NH
δH
HMBCb
1
8.42
2
149.9, C
154.1, N
161.1, C
123.7, C
292.0, N
158.9, C
150.6, C
329.9, N
144.8, C
28.5, CH3
22.8, CH3
21.9, CH3
3
4
4a
5
6
7
8
8a
9
3.50, s
2.63, s
2.67, s
2, 3, 4
10
4a, 5, 6, 7
6, 7, 8
11
a
1H (400 MHz); 13C (100 MHz); 15N (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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Figure 3
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A
O
O
1) HMDS, H2SO4 (cat)
reflux
1) NaNO2, AcOH
Me
O
H2O
HN
N
2) MeI, DMF,
aqueous workup
71% (2 steps)
2) Na2S2O4
NH3(aq)
31% (2 steps)
O
N
H
NH2
N
H
NH2
5
6
O
O
O
Me
8
Me
Me
O
NH2
Me
O
N
N
Me
Me
O
N
N
AcOH, EtOH
reflux
N
H
NH2
N
H
63%
7
3
B
O
O
Me
O
8
Me
NH2
NH2
N
N
Me
Me
O
HN
HN
AcOH, H2O
reflux
O
N
O
N
Me
Me
60%
9
4
Figure 4
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Journal of Agricultural and Food Chemistry
TABLE OF CONTENTS GRAPHIC
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