Ascopyrone P: Chemical synthesis from D-glucose

Article abstract of DOI:10.1081/CAR-120016855

The pyranone, 1,5-anhydro-4-deoxy-D-glycero-hex-l-en-3-ulose (1) (ascopyrone P), has been synthesised in eight steps from D-glucose. The key steps were deacetylation of 3,6-di-O-acetyl-1,5-anhydro-D-glycero-hex-3-en-2-ulose (8) to give isomers and hydrate

Full text of DOI:10.1081/CAR-120016855

Journal of Carbohydrate Chemistry
ISSN: 0732-8303 (Print) 1532-2327 (Online) Journal homepage: http://www.tandfonline.com/loi/lcar20
ASCOPYRONE P: CHEMICAL SYNTHESIS FROM d-
GLUCOSE
S. M. Andersen , H. M. Jensen & S. Yu
To cite this article: S. M. Andersen , H. M. Jensen & S. Yu (2002) ASCOPYRONE P: CHEMICAL
SYNTHESIS FROM d-GLUCOSE, Journal of Carbohydrate Chemistry, 21:6, 569-578
To link to this article: http://dx.doi.org/10.1081/CAR-120016855
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JOURNAL OF CARBOHYDRATE CHEMISTRY
Vol. 21, No. 6, pp. 569–578, 2002
ASCOPYRONE P: CHEMICAL
SYNTHESIS FROM D-GLUCOSE
3
S. M. Andersen,1 H. M. Jensen,2, and S. Yu
*
1Department of Chemistry, University of Alberta,
Edmonton, Alberta, T6E 2G2, Canada
2Process Development and Pilot Plant, Danisco,
Taarnvej 25, DK-7200 Grindsted, Denmark
3Danisco Innovation Center, Langebrogade 1, DK-1001
Copenhagen K, Denmark
ABSTRACT
The pyranone, 1,5-anhydro-4-deoxy-D-glycero-hex-1-en-3-ulose (1) (asco-
pyrone P), has been synthesised in eight steps from D-glucose. The key steps
were deacetylation of 3,6-di-O-acetyl-1,5-anhydro-D-glycero-hex-3-en-2-
ulose (8) to give isomers and hydrates of 1,5-anhydro-4-deoxy-D-glycero-
hex-3-en-2-ulose (9). Isomerisation of this mixture afforded 1,5-anhydro-4-
deoxy-D-glycero-hex-1-en-3-ulose (1) (ascopyrone P) in a moderate yield.
Key Words: Ascopyrone P; Ascopyrone T; Antioxidant
INTRODUCTION
1,5-Anhydro-4-deoxy-D-glycero-hex-1-en-3-ulose (1) has as early as 1978[1] been
identified as a pyrolysis product of cellulose and characterised by X-ray diffraction and
NMR spectroscopy.[2] Recently, the enone 1 has also been identified as a degradation
product from a-1,4-glucans in Ascomycetes, when subjected to ‘activating’ plasmolytic
treatments.[3] Several Ascomycetes exhibited an enzymatic activity, which degrades a-
*
Corresponding author.
569
DOI: 10.1081/CAR-120016855
0732-8303 (Print); 1532-2327 (Online)
www.dekker.com
Copyright D 2002 by Marcel Dekker, Inc.

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570
ANDERSEN, JENSEN, AND YU
Figure 1. Ascopyrone P and T.
1,4-glucans to 1,5-anhydro-D-fructose, and further into the enone 1 in Pezizales (enone
1 named ascopyrone P) or to a mixture of three hydrated forms of 1 in Tuberales
(named ascopyrone T) (Figure 1).
Degradation products from a-1,4-glucans, such as starch, are easily available
chiral starting materials from renewable resources and therefore of interest for in-
dustrial uses. The lyase responsible for the degradation of a-1,4-glucans to 1,5-an-
hydro-D-fructose has been cloned and expressed in Aspergillus niger,[4,5] whereas the
dehydratase responsible for the dehydration of 1,5-anhydro-D-fructose to 1 in Pezizales
is not easily available at the present. Initial experiments have shown that 1 possesses
some antioxidative properties.[6] Hence, we now wish to report on the chemical syn-
thesis of ascopyrone P and investigate these properties.
RESULTS AND DISCUSSION
Synthesis of Ascopyrone P
The synthesis and chemistry of unprotected 1,5-anhydro-4-deoxyhexo-2,3-diu-
loses and similar compounds have been scarcely studied. Several derivatives, such as
acylated 1,5-anhydro-4-deoxy-D-glycero-hex-3-en-2-ulose,[7–9] acylated ascopyrone P,[1]
benzoylated ‘‘Herzgift-Methylreduktinsa¨ure,’’[10] as well as oximes[11,12] and osazones
of 1,5-anhydro-4-deoxy-D-glycero-hexo-2,3-diulose[13,14] have been prepared, but in no
cases the unprotected compounds.
We have investigated two approaches for the synthesis of ascopyrone P (1), using
D-glucose as starting material (Schemes 1–3). In the first approach (Schemes 1 and 2)
Scheme 1. Synthesis of ascopyrone T.

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ASCOPYRONE P
571
Scheme 2. Isomerisation to ascopyrone P.
the key intermediate was 3,6-di-O-acetyl-1,5-anhydro-D-glycero-hex-3-en-2-ulose (8),
which upon deacetylation, followed by isomerisation, should afford ascopyrone P.
2,3,4,6-Tetra-O-acetyl-1,5-anhydro-D-arabino-hex-1-enitol (5)[15,16] was treated
with hydroxylamine in pyridine to afford the known crystalline oxime 6[17] in good
yield (61%). Subsequent reductive deoximination with 15% TiCl3 and NH4OAc as a
buffer, then afforded 3,4,6-tri-O-acetyl-1,5-anhydro-D-fructose (7).[14] Ketone 7 readily
eliminated acetic acid upon treatment with NaOAc in acetone to yield 3,6-di-O-acetyl-
1,5-anhydro-4-deoxy-D-glycero-hex-3-en-2-ulose (8)[7] which was isolated by column
chromatography in an excellent yield (89%). Neither compound 7 nor 8 were stable
for more than a few days: acetylated 1,5-anhydro-D-fructose 7 eliminated acetic acid to
afford the enone 8, which decomposed to unidentified products. To prevent eli-
mination and rearrangement reactions from deacetylation of 8, aqueous acid was used
for deprotection, resulting in hydration of the liberated carbonyl moiety. We were
pleased to find that the deacetylation of 3,6-di-O-acetyl-1,5-anhydro-4-deoxy-D-gly-
cero-hex-3-en-2-ulose (8) in aqueous HCl, gave the enone 9 together with several
isomeric forms and hydrates, in a quantitative yield. 13C NMR spectroscopy of the
Scheme 3. The osazone approach.

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572
ANDERSEN, JENSEN, AND YU
isolated product showed the dihydrate 2 as the main product. Ascopyrone P (1) was
not detected.
Since 1 has been reported to be stable in aqueous solution, whereas the other
isomers are hydrated,[3] we expected ascopyrone P to be the main product from iso-
merisation of 9 and its isomers in anhydrous base. Initial experiments to isomerise 9 in
pyridine showed the presence of ascopyrone P (1), although in low yield. After opti-
misation of the reaction by varying the concentration of 9, temperature and reaction-
time, 1 could be crystallised from the concentrated reaction mixture in 18% yield.
Column chromatography of the mother liquor increased the total yield to 23% (51%
based on recovered starting material).
HPLC and TLC showed ascopyrone P (1) to be stable in aqueous solution at pH
7, but unstable in acetone, EtOAc and pyridine at ambient temperature, due to iso-
merisation. Because of that 1 was not recrystallised.
The key intermediates in the second approach were osazones of ascopyrone P,
which could be synthesised in a few steps from the acetylated 2-hydroxy-D-glucal 5.
The osazones are attractive precursors for 1 since deprotection should afford the a-
diketone 11 (Scheme 3), thus reducing the length of the total synthesis. The phenyl-
and 2,4-dinitrophenylosazone of ascopyrone P were prepared according to literature
procedures.[13,14,18]
Deacetylation of 5 was performed with ammonia in methanol to afford an oily
intermediate, which was treated with excess 2,4-dinitrophenylhydrazine hydrochloride
(2,4-DNP/HCl) to give 1,5-anhydro-4-deoxy-D-glycero-hexo-2,3-diulose 2,4-dinitrophe-
nylosazone (10a) in 67% yield.[14,18]
Reaction of 5 with phenylhydrazine in acetic acid yielded 6-O-acetyl-1,5-anhy-
dro-4-deoxy-D-glycero-hexo-2,3-diulose phenylosazone (12) in a good yield and subse-
quent deacetylation with NaOMe/MeOH gave 1,5-anhydro-4-deoxy-D-glycero-hexo-
2,3-diulose phenylosazone (10b) in an excellent yield. Both the acetylated osazones, 12
and 10b, are strongly coloured compounds and therefore the measured optical rotations
are not accurate.
Liberation of the dicarbonyl functions from the osazones 10a and 10b turned out
to be difficult. All attempts to deprotect the osazones with benzaldehyde/H + ,[19]
acetone/H + [20] and dimethoxyethane/TiCl3
osazones 10a and 10b. This approach was not further pursued.
failed, due to the high stability of the
[21]
Antioxidant Activity of Ascopyrone P
Ascopyrone P (1) contains a conjugated keto-enol structure, in analogy with com-
mon antioxidants such as butylhydroxyanisole (BHA), dibutylhydroxytoluene (BHT),
ascorbic acid and kojic acid, and might therefore have an antioxidant activity. This
hypothesis was supported by several preliminary experiments: 1 reduces decoloration of
b-carotene when exposed to oxygen and oxidative intermediates of linoleic acid, delays
oxidation of linoleic acid and functions as a food antioxidant in mayonnaise and yogurt
salad dressing.[6] Since 1 is stable for weeks in aqueous solution, we were able to
investigate the antioxidant activity in an aqueous system. The oxidation of linolenic
acid was followed by monitoring the formation of the oxidation products, lipid
hydroperoxide, malonaldehyde and 4-hydroxynonenal. In the presence of 1, 160 ppm,

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ASCOPYRONE P
573
the formation of lipid hydroperoxide was reduced (47%) as well as the formation of
malonaldehyde and 4-hydroxynonenal (59% reduction), compared to experiments per-
formed without addition of ascopyrone P (1).
CONCLUSIONS
Inspired by the initial promising result of ascopyrone P as an antioxidant, we
have successfully developed a chemical synthesis of 1. Starting from D-glucose we
have prepared sufficient amounts of ascopyrone P for further thorough testing of these
properties. The oxidation experiments clearly show that 1 has an antioxidant activity,
which will be further investigated by addition of 1 to food.
EXPERIMENTAL
General procedures. Melting points were determined with a melting point
apparatus (Bu¨chi 510) and are uncorrected. Optical rotations were measured on a
Perkin-Elmer 241 polarimeter at the Department of Organic Chemistry, Technical
1
University of Denmark. H NMR and 13C NMR spectra were recorded with a Varian
Gemini 200 MHz instrument at ambient temperature (Danisco) and a Bruker AC 300
MHz at ambient temperature (Department of Organic Chemistry, Technical University
of Denmark). For NMR spectra the solvent peak was used as a reference.[22] The
microanalyses were carried out at the Chemical Laboratory II, University of Copen-
hagen. The progress of all reactions was monitored by thin-layer chromatography using
aluminium sheets precoated with silica gel 60 F254 to a thickness of 0.2 mm. Com-
pounds were detected with UV light (254 nm) and/or by spraying the sheets with a
solution of 1.5% ammoniummolybdonate, 1% cerium sulfate and 10% sulfuric acid,
followed by heating. Column chromatography was conducted under pressure (2 bar)
with silica gel (0.043–0.063 mm). LC-MS was performed on a Hewlett Packard 1100
series with an APCI detector.
3,4,6-Tri-O-acetyl-1,5-anhydro-D-arabino-hex-2-ulose oxime (6).[17] To a solu-
tion of 2,3,4,6-tetra-O-acetyl-1,5-anhydro-D-arabino-hex-1-enitol (5)[15,16] (7.90 g, 23.9
mmol) in dry pyridine (40 mL, 496 mmol), HONH2ÁHCl (5.85 g, 84.2 mmol) was added
and the mixture was stirred for 24 h. The reaction mixture was concentrated and dissolved
in CHCl3 (300 mL). The organic phase was washed with 1 M HCl (75 mL), saturated
aqueous NaHCO3 (75 mL) and H2O (75 mL), dried (MgSO4) and concentrated to a syrup
of 6 (7.19 g, 99%). Upon addition of a small volume of EtOH the product crystallised (4.43
g, 61%, mp 86–89°C). Two recrystallisations from toluene afforded an analytical sample:
mp 90–92°C; [a]D À 39.4° (c 1.3, CHCl3) [Lit.[17] mp 89–90°C, [a]D À 39.0 (c 0.4,
1
CHCl3)]. H NMR (DMSO-d6 at 2.50, 300 MHz) d 1.99 (s, 3H, OCOCH3) 2.02 (s, 3H,
OCOCH3), 2.03 (s, 3H, OCOCH3), 3.87 (ddd, J5,6 =3.0, J5,6’ =5.5, J4,5 =8.5, 1H, H-5), 4.03
(d, J1,1’ =15.0, 1H, H-1), 4.05 (dd, J6,6’ = 12.0, 1H, H-6), 4.12 (dd, 1H, H-6’), 4.88 (d, 1H,
H-1’), 4.93 (dd, J3,4 =8.0, 1H, H-4), 5.54 (d, 1H, H-3), 11.42 (s, 1H, NOH). 13C NMR
(DMSO-d6 at 39.5, 50.3 MHz) d 20.6 (3 Â OCOCH3), 60.9 (C-1), 62.5 (C-6), 69.3 (C-4),
70.5 (C-3), 74.9 (C-5), 148.9 (C-2), 169.3–170.2 (3 Â OCOCH3).

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574
ANDERSEN, JENSEN, AND YU
Anal. Calcd for C12H17NO8: C, 47.53; H, 5.65; N, 4.62. Found: C, 47.57; H,
5.56; N, 4.50.
3,4,6-Tri-O-acetyl-1,5-anhydro-D-arabino-hex-2-ulose (7).[14,23] 3,4,6-Tri-O-
acetyl-1,5-anhydro-D-arabino-hex-2-ulose oxime (6) (5.00 g, 16.5 mmol) was dissolved
in dioxane (100 mL) and NH4OAc (13.0 g, 169 mmol) was added. The mixture was
cooled on ice, 15% TiCl3 (44 mL, 54 mmol) was added and the reaction mixture was
stirred at rt for 3 h. The mixture was extracted with CHCl3 (5 Â 30 mL) and the
combined organic phase was washed with saturated aqueous NaHCO3 (70 + 50 mL). The
combined aqueous phase was extracted with CHCl3 (30 mL) and the combined organic
phase was washed with H2O (30 mL). The organic phase was dried (MgSO4) and
concentrated to a syrup of 7 (3.54 g, 75%). Upon addition of Et2O, the product
crystallises (1.29 g, 27%, mp 81–85°C). Two recrystallisations from Et2O afforded an
analytical sample: mp 93–94°C; [a]D À 7.2 (c 1.5, CHCl3) [Lit.[23] mp 86–88°C,
1
[a]D À 10 (c 0.5, CHCl3)]. H NMR (CDCl3 at 7.27, 300 MHz) d 2.08 (s, 3H, OCOCH3)
2.10 (s, 3H, OCOCH3), 2.16 (s, 3H, OCOCH3), 3.99 (ddd, J5,6 =2.5, J5,6’ = 5.0, J4,5 =9.0,
1H, H-5), 4.10 (d, J1,1’ =15.5, 1H, H-1), 4.23 (dd, J6,6’ = 12.5, 1H, H-6), 4.27 (d, 1H,
H-1’), 4.32 (dd, 1H, H-6’), 5.34 (t, J3,4 = 10.0, 1H, H-4), 5.42 (d, 1H, H-3). 13C NMR
(CDCl3 at 77.0, 75.5 MHz) d 20.4, 20.7 (3 Â OCOCH3), 62.1 (C-6), 69.4 (C-4), 72.9
(C-1), 76.5 (C-5), 76.8 (C-3), 169.1, 169.8, 170.5 (3 Â OCOCH3), 196.3 (C-2).
Anal. Calcd for C12H16O8: C, 50.00; H, 5.59. Found: C, 49.87; H, 5.56.
3,6-Di-O-acetyl-1,5-anhydro-4-deoxy-D-glycero-hex-3-en-2-ulose (8). To a
solution of 7 (2.21 g, 7.67 mmol) in dry acetone (77 mL), anhydrous NaOAc (2.2 g,
26.8 mmol) was added and the reaction mixture was stirred for 3 h. The salts were
filtered off and washed with acetone. The filtrate was concentrated and purified by
column chromatography (30 g silica, eluted with hexane-EtOAc, 2:1) to give 8 as a
1
syrup (1.56 g, 89%): [a]D À 36.8 (c 2.0, CHCl3) [Lit.[7] [a]D À 43.7 (c 1.7, CHCl3)]. H
and 13C NMR are in accordance with literature.[7]
Anal. Calcd for C10H12O6: C, 52.63; H, 5.30. Found: C, 52.01; H, 5.18.
1,5-Anhydro-D-glycero-hex-3-en-2-ulose (9). Compound 8 (2.98 g, 13.1 mmol)
was dissolved in aqueous 4 M HCl (130 mL) and kept for 24 h. The mixture was
concentrated and co-concentrated with H2O (2 Â 60 mL) to a syrup, which was purified
by chromatography (60 g silica, eluted with EtOAc, then CHCl3-MeOH, 4:1) to give
9 together with isomeric and hydrated forms as an amorphous solid (1.84 g, 97%).
13C NMR (the dihydrate 2) (D2O, MeOH at 49.5 ppm, 50.3 MHz) d 37.4 (C-4), 64.2
(C-6), 70.9 (C-1), 76.4 (C-5), 92.9 (C-3), 93.9 (C-2). The product was used without
further purification.
1,5-Anhydro-D-glycero-hex-1-en-3-ulose (1). Compound 9 (1.04 g, 7.2 mmol)
was dissolved in dry pyridine (100 mL) and 4A molecular sieves (10.8 g) added. The
mixture was heated at 120°C in an atmosphere of N2 for 1 h and concentrated in vacuo
to give a syrup, which was dissolved in H2O (50 mL) and acidified with 1 M HCl until
pH 4–5. The aqueous phase was extracted with EtOAc (5 Â 100 mL) and the combined
organic phase was dried (MgSO4) and concentrated to a brown syrup. Upon addition of
EtOAc-hexane, 1 crystallised (0.190 g, 18%): mp 90–95°C; [a]D +139.5 (c 1.0, H2O)

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ASCOPYRONE P
575
[Lit.[1] mp 98.5–99°C, [a]D + 155 (c 1.1, H2O)]. The mother liquer was purified by
chromatography (20 g silica, eluted with EtOAc, then CHCl3-MeOH, 4:1) to afford 9
(0.57 g) and 1 (0.0494 g). Total yield of 1: 23% (51% when subtracting recoved
starting material). 1H NMR (D2O, MeOH at 3.34 ppm, 300 MHz) d 2.53 (ddd,
J
4,6 = 1.0, J4,5 =3.5, J4,4’ = 17.5, 1H, H-4), 2.87 (ddd, J4’,6’ =1.0, J4’,5 =14.5, 1H, H-4’),
3.79 (ddd, J5,6 =5.5, J6,6’ = 12.5, 1H, H-6), 3.88 (ddd, J5,6’ =3.0, 1H, H-6’), 4.57 (m, 1H,
H-5), 7.53 (s, 1H, H-1). 13C NMR (D2O, MeOH at 49.5 ppm, 75.5 MHz) d 37.7 (C-4),
63.7 (C-6), 81.0 (C-5), 136.1 (C-2), 152.3 (C-1), 192.9 (C-3).
Anal. Calcd for C10H12O6: C, 50.00; H, 5.59. Found: C, 50.86; H, 5.98.
1,5-Anhydro-4-deoxy-D-glycero-hexo-2,3-diulose 2,4-dinitrophenylosazone
(10a).[14,18] 2,3,4,6-Tetra-O-acetyl-1,5-anhydro-D-arabino-hex-1-enitol (5)[15,16] (3.0
g, 9.1 mmol) was dissolved in dry MeOH (20 mL) and kept at 0°C. MeOH saturated
with ammonia (10 mL) was added and the reaction kept at 0°C overnight. The mixture
was concentrated in vacuo at ambient temperature and co-concentrated with dry MeOH
(2 Â 10 mL). Finally, the mixture was dissolved in dry EtOH (9 mL) and by addition of
dry Et2O (44 mL) an oily product was obtained (1.8 g). To a filtered solution of 2,4-
dinitrophenylhydrazine (6.0 g, 30.3 mmol) in 2 M HCl (1.0 L) the oily product was
added and the mixture was stirred overnight at ambient temperature. The precipitate
was filtered off and washed with H2O (2 Â 20 mL) and dry EtOH (2 Â 20 mL)
affording the osazone 10a (3.05 g, 67% yield from 5, mp 239–242°C). Recrystallisa-
tion in EtOH-acetone afforded an analytical sample: mp 246°C. The sample was too
1
coloured to obtain an optical rotation. [Lit.[14] mp 237°C, [a]D +176 (pyridine)]. H
NMR (DMSO-d6 at 2.50, 300 MHz) d 2.74 (dd, J4,5 =10.5, J4,4’ =17.5, 1H, H-4), 3.00
(dd, J4’,5 =4.0, 1H, H-4’), 3.56 (t, J=4.5, 2H, H-6 and H-6’, ABX-system), 3.86 (dddd,
J= 4.5, 4.5, 4.5, 10.5, 1H, H-5, ABX-system), 4.51 (d, J1,1’ = 15.0, 1H, H-1), 4.64 (d,
1H, H-1’), 5.03 (t, JOH,6 = JOH,6’ =4.5, 1H, -OH), 8.0–8.9 (6H, 2,4-dinitrophenyl), 11.07
(broad s, 1H, NH), 13.80 (broad s, 1H, NH). 13C NMR (DMSO-d6 at 39.5, 50.3 MHz)
d 28.8 (C-4), 63.3 (C-6), 70.2 (C-1), 75.1 (C-5), 116.8, 117.4, 122.5, 129.6, 130.0,
130.9, 131.3, 138.6, 138.9 (2,4-dinitrophenyl), 140.0 (C-3), 142.7, 144.6 (2,4-
dinitrophenyl), 148.7 (C-2).
Anal. Calcd for C18H16N8O10: C, 42.86; H, 3.20; N, 22.22. Found: C, 43.11; H,
3.18; N, 22.02.
6-O-Acetyl-1,5-anhydro-4-deoxy-D-glycero-hexo-2,3-diulose phenylosazone
(12).[13] To a solution of 2,3,4,6-tetra-O-acetyl-1,5-anhydro-D-arabino-hex-1-enitol
(5)[15,16] (2.37 g, 7.16 mmol) in 50% AcOH (36 mL), PhNHNH2 (2.9 mL, 29.4 mmol)
was added. The reaction mixture was refluxed for a few minutes, until the product
started precipitating, and then stirred at rt for 2 h. The mixture was poured onto ice
(150 g) and the precipitated product 12 was filtered off (1.90 g, 73%, mp 176–178°C).
Two recrystallisations from EtOH afforded an analytical sample: mp 203–204°C; [a]D
1
À 120.1 (c 1.0, CHCl3) [Lit.[13] mp 205°C, [a]D À 98.6 (pyridine)]. H NMR (CDCl3 at
7.27, 300 MHz) d 2.16 (s, 3H, OCOCH3), 2.44 (dd, J4,5 =11.0, J4,4’ =16.5, 1H, H-4),
2.63 (dd, J4’,5 =4.5, 1H, H-4’), 3.97 (dddd, J5,6’ =4.0, J5,6 =6.0, 1H, H-5), 4.22 (dd,
J
6,6’ = 12.0, 1H, H-6), 4.32 (dd, 1H, H-6’), 4.46 (d, J1,1’ =15.0, 1H, H-1), 4.70 (d, 1H, H-
1’), 6.87–7.43 (10H, phenyl), 7.45 (broad s, 1H, NH), 12.65 (broad s, 1H, NH). 13C
NMR (CDCl3 at 77.0, 75.5 MHz) d 20.9 (OCOCH3), 27.2 (C-4), 66.0 (C-6), 71.1 (C-1),

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576
ANDERSEN, JENSEN, AND YU
72.4 (C-5), 113.4, 120.8, 121.7 (phenyl), 127.9 (C-3), 129.3, 129.6 (phenyl), 137.9 (C-2),
143.6, 144.4 (phenyl), 170.9 (OCOCH3).
Anal. Calcd for C20H22N4O3: C, 65.56; H, 6.05; N, 15.29. Found: C, 65.30; H,
6.24; N, 15.06.
1,5-Anhydro-4-deoxy-D-glycero-hexo-2,3-diulose phenylosazone (10b).[13] To
the phenylosazone 12 (1.03 g, 2.81 mmol) was added 0.5 M NaOMe/MeOH (30 mL) at
1
0°C and the mixture was stirred for /2 h at 0°C. The reaction mixture was acidified
(pH< 6) with AcOH and concentrated to a syrup, from which 10b crystallised upon
addition of 10% AcOH (150 mL). The precipitate was washed with H2O (2 Â 20 mL)
to afford 10b (0.74 g, 82%, mp 162–164°C). Two recrystallisations from EtOH-H2O
afforded an analytical sample: mp 180–182°C; [a]D À 141.6 (c 1.0, pyridine) [Lit.[13]
1
mp 183°C, [a]D-159.8 (pyridine)]. H NMR (DMSO-d6 at 2.50, 300 MHz) d 2.42 (dd,
J
4,5 =11.0, J4,4’ = 17.5, 1H, H-4), 2.77 (dd, J4’,5 =4.0, 1H, H-4’), 3.55 (t, J= 5.5, 2H, H-6
and H-6’, ABX-system), 3.75 (dddd, J= 4.5, 4.5, 4.5, 11.0, 1H, H-5, ABX-system), 4.36
(d, J1,1’ =14.5, 1H, H-1), 4.47 (d, 1H, H-1’), 4.93 (t, JOH,6 =JOH,6’ =5.5, 1H, -OH), 6.79–
7.41 (10H, phenyl), 9.67 (broad s, 1H, NH), 12.75 (broad s, 1H, NH). 13C NMR
(DMSO-d6 at 39.5, 50.3 MHz) d 28.4 (C-4), 63.9 (C-6), 70.0 (C-1), 75.2 (C-5), 112.6,
113.0, 120.2, 120.4, 129.5 (phenyl), 130.4 (C-3), 139.3 (C-2), 144.4, 144.6 (phenyl).
Anal. Calcd for C18H20N4O2: C, 66.65; H, 6.21; N, 17.27. Found: C, 66.40; H,
6.16; N, 17.39.
Assay of Antioxidant Activity of Ascopyrone P (1). Ascopyrone P was eva-
luated for possible antioxidative activity by measuring its inhibition on the formation of
oxidation products from the polyunsaturated fatty acid, linolenic acid. The oxidation
products formed, lipid hydroperoxide, malonadehyde (MDA) and 4-hydroxyalkenals (4-
HNE), were measured using the assay kit of Bioxytech1 LPO-560 and Bioxytech1
LPO-586 from Oxis International, Inc. (Portland, USA). The lipid hydroperoxide assay
is based on its oxidation of Fe2 + to Fe3 + ; the formed Fe3 + gives colour with xylenol
orange having a maximum absorbance at 560 nm. Linolenic acid (20 mL) was dissolved
in oxygenated water (50 mL) containing Tween 40 (0.2 mL) (Solution 1). To solution 1
(0.96 mL) was added 0.1 M EDTA (20 mL) and water (20 mL) (control) or ascopyrone
P (20 mL) (experimental) with a final concentration of 160 ppm. The reaction mixture
was incubated at 24°C in the dark. At the 6th day, samples (0.1 mL) were taken and
mixed with working solution from the assay kit containing Fe2 + xylenol orange (0.9
mL). Absorbance at 560 nm was measured after incubation at 24°C in the dark for 60
min against reagent blank, which had the same composition as the control and
experimental groups, but not incubated. With no antioxidant added the absorbance was
0.748. In the presence of 160 ppm ascopyrone P the absorbance was 0.397. The assay
for MDA and 4-HNE is based on their reaction in acid with the chromogenic reagent
1-methyl-2-phenylindole at 45°C to form a stable chromophore that has maximum
absorbance at 586 nm. At the 6th day samples (40 mL) were taken and after mixing
with 0.5 M BHT (5 mL), freshly diluted N-methyl-2-phenylindole (7.7 mM, 130 mL)
and methanesulfonic acid (15.4 M, 30 mL) were added and mixed. The reaction
mixture was incubated at 45°C for 45 min and cooled to ambient temperature and
the absorbance at 590 nm was measured using a micro plate reader within 2 hours.

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ASCOPYRONE P
577
Without antioxidant the absorbance was 0.280. In the presence of 160 ppm ascopyrone
P the absorbance was 0.116.
ACKNOWLEDGMENTS
The authors wish to acknowledge Jytte G. Rasmussen, Department of Organic
Chemistry, Technical University of Denmark and Merete K. Hansen, Danisco for their
technical assistance, Robert K. Hansen, Danisco for recording NMR spectra and
Michael Thorsen, Danisco for LC-MS results.
REFERENCES
1. Shafizadeh, F.; Furneaux, R.H.; Stevenson, T.T.; Cochran, T.G. 1,5-Anhydro-4-
deoxy-D-glycero-hex-1-en-3-ulose and other pyrolysis products of cellulose.
Carbohydr. Res. 1978, 67, 433–447.
2. Stevenson, T.T.; Stenkamp, R.E.; Jensen, L.H.; Cochran, T.G.; Shafizadeh, F.;
Furneaux, R.H. The crystal structure of 1,5-anhydro-4-deoxy-D-glycero-hex-1-en-3-
ulose. Carbohydr. Res. 1981, 90, 319–325.
3. Baute, M.-A.; Deffieux, G.; Vercauteren, J.; Baute, R.; Badoc, A. Enzymatic
activity degrading 1,4-a-D-glucans to ascopyrones P and T in pezzizales and
tuberales. Phytochemistry 1993, 33 (1), 41–45.
4. Yu, S.; Bojsen, K.; Kragh, K.M.; Bojko, M.; Nielsen, J.E.; Marcussen, J. a-1,4-
Glucan Lyase From a Fungus Infected Algae, Its Purification, Gene Cloning and
Expression in Microorganisms. US Patent 6013504, 2000.
5. Bojsen, K.; Yu, S.; Kragh, K.M.; Christensen, T.M.I.E.; Marcussen, J. a-1,4-Glucan
Lyase From a Fungus, Its Purification, Gene Cloning and Expression in Micro-
organisms. US Patent 5908760, 1999.
6. Andersen, S.M.; Isak, T;. Jensen, H.M.; Marcussen, J.; Yu, S. Anti-oxidant. PCT
Int. Patent WO 0056838, 2000.
7. Andersen, S.M.; Lundt, I.; Marcussen, J.; Søtofte, I.; Yu, S. Structure of 1,5-
anhydro-D-fructose: X-ray analysis of crystalline acetylated dimeric forms. J.
Carbohydr. Chem. 1998, 17 (7), 1027–1035.
8. Freimund, S.; Ko¨pper, S. Dimeric structures of 1,5-anhydro-D-fructose. Carbohydr.
Res. 1998, 308, 195–200.
9. Brehm, M.; Dauben, W.G.; Ko¨hler, P.; Lichtenthaler, F.W. Enantiomerically pure
building blocks from sugars. 5. Proof of the (S,S)-configuration of (–)-Bissetone by
synthesis from D-glucose. Angew. Chem. 1987, 99 (12), 1318–1319.
10. Lichtenthaler, F.W.; Lo¨he, A.; Cuny, E. Sugar enolones. XIX. An approach to
actinospectose-type 1,2-diketo sugars.—Synthetic proof for the R configuration of
‘‘Herzgift-Methylreduktionsaure’’. Liebigs Ann. Chem. 1983, 11, 1973–1985.
11. Jarglis, P.; Lichtenthaler, F.W. Sugar enolones. Part 15. A stereospecific synthesis
of (S,S)-Palythazine from D-glucose. Angew. Chem. 1982, 94 (2), 140–141.
12. Kametani, S.; Akanuma, H. Oxide of 1,5-Anhydroglucitol and Method for
Measuring 1,5-Anhydoglucitol Using the Same. Jpn. Patent 8217773, 1996.

MARCEL DEKKER, INC. • 270 MADISON AVENUE • NEW YORK, NY 10016
©2002 Marcel Dekker, Inc. All rights reserved. This material may not be used or reproduced in any form without the express written permission of Marcel Dekker, Inc.
578
ANDERSEN, JENSEN, AND YU
13. Bergmann, M.; Zervas, L. Unsaturated reduction products of the sugars. XVI.
New dismutation products of the sugars. Ber. Dtsch. Chem. Ges. 1931, 64, 1434–
1438.
14. Go¨ckel, V.H. 1,5-Anhydro-D-Ketosen Aus 2-Hydroxy-D-Glycalestern: Darstellung
und Konformationsanalyse. Ph.D. Dissertation; Technischen Hochschule: Darm-
stadt, 1984.
15. Lemieux, R.U. Tetra-O-acetyl-a-D-glucopyranosyl bromide. Methods Carbohydr.
Chem. 1963, 2, 221–222.
16. Blair, M.G. 2-hydroxy-D-glucal. Acyl and alkyl derivatives. Methods Carbohydr.
Chem. 1963, 2, 411–414.
17. Lichtenthaler, F.W.; Jarglis, P. Selective deacylation of enol esters with hydro-
xylamine. Tetrahedron Lett. 1980, 21, 1425–1428.
18. Corbett, W.M. Reaction of phenyl- and 2,4-dinitrophenylhydrazine with some un-
saturated D-glucose derivatives. J. Chem. Soc., Sec. C. 1959, 3213–3216.
19. Bayne, S. Aldosuloses (osones). From phenylosazones. Methods Carbohydr. Chem.
1963, 2, 421–423.
20. Maynez, S.R.; Pelavin, L.; Erker, G. Regeneration of ketones from tosylhydrazones,
arylhydrazones, and oximes by exchange with acetone. J. Org. Chem. 1975, 40
(22), 3302–3303.
21. McMurry, J.E.; Silvestri, M. A method for cleaving 2,4-dinitrophenylhydrazones to
ketones. J. Org. Chem. 1975, 40 (10), 1502–1504.
22. Gottlieb, H.E.; Kotlyar, V.; Nudelman, A. NMR chemical shifts of common
laboratory solvents as trace impurities. J. Org. Chem. 1997, 62 (21), 7512–7515.
¨
23. Jarglis, P. Synthese und Reaktionen Pyranoider 2-Ketozucker und Ihre Uberfu¨hrung
in Chirale Synthone. Ph.D. Dissertation; Technischen Hochschule: Darmstadt,
1980.
Received May 29, 2002
Accepted August 30, 2002