first chemical synthesis of three natural depsides involved in flavonol catabolism and related to...

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First Chemical Synthesis of Three NaturalDepsides Involved in Flavonol Catabolism andRelated to Quercetinase CatalysisSylvain Tranchimand a , Thierry Tron a , Christian Gaudin a & Gilles Iacazio aa Laboratoire de Bioinorganique Structurale, Faculté des Sciences etTechniques, Université Paul Cézanne Aix‐Marseille III, Marseille Cedex,FrancePublished online: 16 Aug 2006.

To cite this article: Sylvain Tranchimand , Thierry Tron , Christian Gaudin & Gilles Iacazio (2006) FirstChemical Synthesis of Three Natural Depsides Involved in Flavonol Catabolism and Related to QuercetinaseCatalysis, Synthetic Communications: An International Journal for Rapid Communication of Synthetic OrganicChemistry, 36:5, 587-597

To link to this article: http://dx.doi.org/10.1080/00397910500406534

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First Chemical Synthesis of Three NaturalDepsides Involved in Flavonol Catabolismand Related to Quercetinase Catalysis

Sylvain Tranchimand, Thierry Tron, Christian Gaudin,

and Gilles Iacazio

Laboratoire de Bioinorganique Structurale, Faculte des Sciences et

Techniques, Universite Paul Cezanne Aix-Marseille III,

Marseille Cedex, France

Abstract: We report here the first chemical synthesis of three depsides related to

quercetinase-catalyzed degradation of kaempferol, quercetin, and myricetin. The three

depsides were constructed through the coupling of suitably protected phloroglucinol

carboxylic acid and hydroxy-perbenzylated, derivatives of gallic, protocatechuic, and

4-hydroxy benzoic acids. The three synthesized target compounds proved to be

identical to their natural counterparts, arising from quercetinase action on correspond-

ing flavonols.

Keywords: Depsides, kaempferol, myricetin, quercetin, quercetin 2,3-dioxygenase,

quercetinase

INTRODUCTION

Quercetinases (quercetin 2,3-dioxygenase, E.C. 1.13.11.24) are copper-

containing dioxygenases produced by various filamentous fungi. They act

on flavonols and generate a depside and carbon monoxide (Scheme 1).[1 – 4]

Received in Poland July 28, 2005

Address correspondence to Gilles Iacazio, Laboratoire de Bioinorganique

Structurale (CBRL UMR CNRS 6517), Case 432, Faculte des Sciences et Techniques,

Universite Paul Cezanne Aix-Marseille III, avenue Escadrille Normandie-Nieman,

13397 Marseille Cedex 20, France. Tel.: 33 491 282 856; Fax: 33 491 983 208;

E-mail: [email protected]

Synthetic Communicationsw, 36: 587–597, 2006

Copyright # Taylor & Francis Group, LLC

ISSN 0039-7911 print/1532-2432 online

DOI: 10.1080/00397910500406534

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Very recently an iron-containing quercetinase has been found in Bacillus

subtilis,[5,6] and a quercetinase activity has been detected in pirin originating

both from Escherichia coli and humans.[7] Pirin is implicated in transcrip-

tional activation and apoptosis and is highly conserved both in prokaryotes

and eukaryotes. Quercetinase activity is generally quantified by UV-

spectroscopy following a decrease in absorbance of quercetin at 367 nm.[8]

Detection of the depside was never used in this context, probably because

of the lack of authentic samples. Since the discovery of quercetinase

activity in pirins,[7] it appears to be widely distributed over all kingdoms of

life. We have therefore planned the synthesis of three different depsides

related to quercetinase activity to facilitate quercetinase detection in

complex media such as cellular extracts.

RESULTS AND DISCUSSION

Strategy for the synthesis was based on the coupling of suitably protected

phloroglucinol carboxylic acid with each of the three hydroxy-perbenzylated

benzoic acid derivatives arising from gallic, protocatechuic, and 4-hydroxy

benzoic acids. Benzyl 2,4-bis(benzyloxy)-6-hydroxybenzoate 2 was obtained

following two different synthetic approaches (Scheme 2).

Scheme 1. Reaction catalyzed by quercetinases on flavonols.

Scheme 2. a) K2CO3, BnBr, DMF, rt, 17% yield; b) trifluoroacetic anhydride,

acetone, trifluoroacetic acid, 08C to rt, 5 h, 34% yield; c) K2CO3, BnBr, DMF, rt,

91% yield; d) benzyl alcohol, Na, THF, rt, 15 min, 84% yield.

S. Tranchimand et al.588

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In a single-step experiment, unprotected phloroglucinol carboxylic

acid was benzylated with three equivalents of benzyl bromide in DMF.

The targeted compound 2 was obtained with a yield of 17% after two chroma-

tographic steps, necessary to eliminate various mono-, di-, tri-, and tetra-

benzylated derivatives co-obtained. Considering this poor yield, we set up a

multistep synthesis experiment in which phloroglucinol carboxylic acid

was first protected as 5,7-dihydroxy-2,2-dimethyl-4H-1,3-benzodioxin-4-one

according to Dushin and Danishefsky with a yield of 34%.[9] The two

remaining free phenolic functionalities were then benzylated (benzyl

bromide, K2CO3, DMF, 91%), and 2 was finally obtained after deprotection

with benzyl alcoholate (benzylic alcohol, Na, 84%) in an overall yield of

26%, slightly higher than for the previous direct synthesis.

The synthesis of the three hydroxy-perbenzylated benzoic acids was then

set up as described in Scheme 3.

The three starting compounds (methyl gallate and protocatechuic and

p-hydroxy benzoic acids) were first perbenzylated (benzyl bromide, K2CO3,

dimethyl formamide (DMF), 91–92%) and then transformed to free

carboxylic acids (potassium hydroxide (KOH), EtOH/H2O, 80–98%).

The coupling reaction between 2 and the three different hydroxy-

perbenzylated benzoic acids 11, 12, and 13 was performed using N,N0-

dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP) in

Scheme 3. a) K2CO3, BnBr, DMF, rt, 91–92% yield; b) KOH, H2O, EtOH, reflux,

1 h, 80–98% yield.

Scheme 4. a) DCC, DMAP, CH2Cl2, reflux, 85–95% yield; b) H2, Pd/C, THF, rt,

79–94% yield.

Synthesis of Three Natural Depsides 589

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anhydrous CH2Cl2 (85–95%). A final deprotection step (Pd/C, H2, 79–94%)

afforded the three targeted depsides 17, 18, and 19 (Scheme 4).

Finally, the chemically neosynthesised depsides were then compared

to their enzymatically generated counterparts. Pure quercetinase was

added to a solution containing the three flavonols (myricetin, quercetin, and

kaempferol), and the products of the reaction were analysed by reverse-

phase high performance liquid chromatography (HPLC). Coinjection

of enzymatically generated and chemically synthesized depsides finally

established the identity of these compounds (Figure 1).

In conclusion we describe here the first chemical synthesis of three

naturally occuring depsides related to quercetinase activity and established

their identity to their enzymatically generated counterparts. On the basis of

this work, the analysis of quercetinase activity in complex media where

UV-spectroscopy is inapplicable is now possible. In this context with

chemical standards available, HPLC, alone or in conjunction with mass

spectroscopy, could be an extremely valuable tool both for biochemical and

biomimetic[10] studies on quercetinase activity.

Figure 1. Reverse phase (C18) HPLC chromatogramms set for the comparison

between enzymatically generated depsides 17, 18, and 19 (bottom) and coinjection

of the former with the same depsides, but chemically synthesized, added to get

chromatographic peaks of comparable intensity (top).


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EXPERIMENTAL

Melting points were determined on an Electrothermal 9300 capillary melting-

point apparatus and are uncorrected. IR spectra were measured on KBr plates

on a Bruker IFS25 spectrophotometer. NMR spectra were recorded on Bruker

Avance DPX-300 (1H, 300 MHz; 13C, 75.5 MHz); chemicals shifts (d) are

reported in ppm relative to the NMR solvent. Microanalyses were

performed on a Thermo Finnigan EA 1112. Flash column chromatography

was carried out with silica gel 60 (Merck, particle size 230–400 mesh).

All reactions and chromatographic separations were monitored by TLC.

All reagents and solvents were commercially available (Acros or Aldrich).

N,N-dimethylformamide and THF were distilled under anhydrous conditions

before use; dichloromethane was purified on activated silica gel.

HPLC Conditions

HPLC analysis were conducted using a LiChrospher100 RP-18 (5mm)

column from Agilent Technologies and an HP 1100 series system (DAD).

Solvent flow: 0.5 mL/min. Solvent gradient: 100% A to 40% A–60% B in

10 min and then to 100% B in 20 min (A: 90/10/0.1: water/acetonitrile/acetic acid; B: 90/10/0.1: acetonitrile/water/acetic acid). The detection

was performed at 280 nm.

Depside Biosynthesis

MES buffer (2 ml, 0,1 M, pH ¼ 6) and 2 ml of a purified quercetinase solution

(0.141 U/mL) in the same buffer were added to a 1-mL solution of the three

flavonols (quercetin, kaempferol, and myricetin) in DMSO (at respectively

0.16, 0.03, and 0.34 mg/mL). The mixture was stirred for 1 h and then

analyzed by reverse-phase HPLC.

Benzyl 2,4-bis(Benzyloxy)-6-hydroxybenzoate (2)

Dry K2CO3 (16.23 g, 117.6 mmol) and benzyl bromide (14 ml, 117.7 mmol)

were added to a stirred solution of 2,4,6-trihydroxybenzoic acid monohydrate

(9 g, 47.9 mmol) 1 in DMF (250 ml) under argon. The mixture was stirred

overnight at room temperature; then a 0.5% HCl solution (750 ml) was

added and the mixture extracted with Et2O (6 � 250 ml). The combined

organic layers were washed with a 1.5% HCl solution (2 � 100 ml),

dried (Na2SO4), filtered, and concentrated in vacuo. Two successive flash

chromatographies (dichloromethane/n-pentane: 1/1 then dichloromethane)

afforded 2.937 g (17%) of 2 as a white solid. Mp: 123–1248C, Rf ¼ 0.36


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(dichloromethane/n-pentane, 1:1). IR (KBr, cm21): 3031, 2967, 1648, 1312,

1296, 1213, 1199, 1165, 819, 697. 1H NMR (CDCl3) d: 4.99 (s, 2H), 5.02

(s, 2H), 5.34 (s, 2H), 6.12 (d, J ¼ 2.5 Hz, 1H), 6.20 (d, J ¼ 2.5 Hz, 1H),

7.19–7.43 (m, 15H), 1.04 (s, 1H); 13C NMR (CDCl3) d: 171.1, 166.0,

164.4, 161.2, 136.1, 135.9, 135.6, 128.6, 128.5, 128.4, 128.2, 128.1, 128.0,

127.8, 127.6, 127.3, 97.1, 94.6, 93.3, 70.7, 70.1, 66.8. Anal. calc. for

C28H24O5: C, 76.36; H, 5.45. Found: C, 76.42; H, 5.65.

5,7-Dihydroxy-2,2-dimethyl-4H-1,3-benzodioxin-4-one (3)

At 08C, anhydrous acetone (1 ml, 13.6 mmol) and trifluoroacetic anhydride

(5 ml, 36.0 mmol) were added to a stirred suspension of 2,4,6-trihydroxy-

benzoic acid (741.6 mg, 4.36 mmol) 1 in trifluoroacetic acid (8 ml,

107.7 mmol) under argon. The mixture was warmed slowly to room tempera-

ture and stirred for 5 h. The slightly yellow homogeneous mixture was then

concentrated in vacuo, poured into a saturated solution of NaHCO3

(100 ml), and extracted with ethyl acetate (2 � 100 ml). The combined

organic layers were washed with water (50 ml), dried (Na2SO4), filtered,

and concentrated in vacuo. Flash chromatography (n-pentane/ethyl acetate:

3/1) afforded 315 mg (34%) of 3 as a white solid. Mp: decomposition at

1958C, Rf ¼ 0.36 (n-pentane/ethyl acetate: 3/1). IR (KBr, cm21): 3200,

1645, 1505, 1271, 1172, 1094, 815, 843. 1H NMR (CD3OD) d: 1.70

(s, 6H), 5.91 (d, J ¼ 2.2 Hz, 1H), 6.00 (d, J ¼ 2.2 Hz, 1H); 13C NMR

(CD3OD) d: 168.3, 166.6, 164.4, 158.7, 108.0, 98.3, 96.5, 93.1, 25.7. Anal.

calc. for C10H10O5: C, 57.14; H, 4.80. Found: C, 57.19; H, 4.92.

5,7-bis(Benzyloxy)-2,2-dimethyl-4H-1,3-benzodioxin-4-one (4)

Dry K2CO3 (500 mg, 3.6 mmol) and benzyl bromide (0.25 ml, 2.1 mmol) were

added to a stirred solution of 3 (185 mg, 0.88 mmol) in N,N-dimethylforma-

mide (10 mL) under argon. The mixture was stirred overnight, then poured

in water (100 ml) and extracted with diethylether (3 � 100 ml). The

combined organic layers were washed with water (50 ml), dried (Na2SO4),

filtered, and concentrated in vacuo. Flash chromatography (dichloromethane)

afforded 313 mg (91%) of 4 as a white solid. Mp: 119–1208C, Rf ¼ 0.20

(dichloromethane). IR (KBr, cm21): 3062, 3027, 2999, 2990, 2944, 1728,

1618, 1579, 1271, 1169, 1119, 1033, 735, 718. 1H NMR (CDCl3) d: 1.70

(s, 6H), 5.02 (s, 2H), 5.19 (s, 2H), 6.14 (d, J ¼ 2.3 Hz, 1H), 6.26

(d, J ¼ 2.3 Hz, 1H), 7.25–7.55 (m, 10H); 13C NMR (CDCl3) d: 165.3,

161.7, 159.4, 157.9, 136.1, 135.5, 128.7, 128.6, 128.4, 127.8, 127.6, 126.6,

105.0, 97.4, 95.9, 94.7, 70.6, 70.4, 25.6. Anal. calc. for C24H22O5: C, 73.85;

H, 5.64. Found: C, 74.30; H, 5.85.


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Benzyl 2,4-bis(Benzyloxy)-6-hydroxybenzoate (2)

Na (360 mg) was added to a stirred solution of benzyl alcohol (1.87 g,

17.3 mmol) in THF (50 ml, 08C). The suspension was stirred until Na disap-

peared; then 4 (1.411 g, 3.6 mmol) was added and the mixture was stirred

15 min at rt. The mixture was then concentrated in vacuo, poured into a

1.5% HCl solution (100 ml), extracted with diethylether (3 � 100 ml) and

ethyl acetate (100 ml), dried (Na2SO4), filtered, and concentrated in vacuo.

Flash chromatography (dichloromethane/n-pentane: 1/1) afforded 1.34 g

(84%) of 2 (same characteristics as before).

General Procedure for the Synthesis of Hydroxy-perbenzylated

Benzoic Esters (6, 8, 10)

Dry K2CO3 (3 g, 21.7 mmol) and benzyl bromide (2.6 ml, 21.9 mmol) were

added to a stirred solution of 3,4-dihydroxybenzoic acid (1 g, 6.5 mmol) 5

in N,N-dimethylformamide (20 ml) under argon. The mixture was stirred

overnight, then poured into water (100 ml) and extracted with diethyl ether

(3 � 100 ml). The combined organic layers were washed with water

(50 ml), dried (Na2SO4), filtered, and concentrated in vacuo. Flash chromato-

graphy (dichloromethane) afforded 2.5 g (91%) of 6 as a white solid. Mp:

66–678C, Rf ¼ 0.76 (dichloromethane). IR (KBr, cm21): 3068, 3037, 3029,

2855, 1693, 1516, 1432, 1290, 1216, 1140, 1024, 763, 741, 696. 1H NMR

(CDCl3) d: 5.18 (s, 2H), 5.20 (s, 2H), 5.31 (s, 2H), 6.91 (d, J ¼ 8.8 Hz, 1H),

7.24–7.49 (m, 15H), 7.66 (dd, J1 ¼ 8.8 Hz, J2 ¼ 2 Hz, 1H), 7.67 (d, J ¼ 2 Hz,

1H); 13C NMR (CDCl3) d: 166.0, 152.9, 148.3, 136.8, 136.5, 136.2, 128.6,

128.5, 128.4, 128.1, 128.0, 127.9, 127.8, 127.4, 127.1, 124.1, 123.0, 115.6,

113.2, 71.2, 70.8, 66.5. Anal. calc. for C28H24O4: C, 79.24; H, 5.66. Found: C,

79.33; H, 5.77.

Following the same procedure as described previously, from 7 (502 mg,

3.6 mmol), N,N-dimethylformamide (20 ml), K2CO3 (1.1 g, 8 mmol), and

benzyl bromide (0.87 ml, 7.3 mmol), 1.063 g of 8 (92%) were obtained as a

white solid. Mp: 117–1188C, Rf ¼ 0.74 (dichloromethane). IR (KBr,

cm21): 2970, 2952, 2891, 2876, 1698, 1604, 1509, 1275, 1243, 1170, 1109,

1003, 756, 704. 1H NMR (CDCl3) d: 5.11 (s, 2H), 5.33 (s, 2H), 6.98

(d, J ¼ 9 Hz, 2H), 7.28–7.52 (m, 10H), 8.03 (d, J ¼ 9 Hz, 2H); 13C NMR

(CDCl3) d: 166.1, 162.6, 136.3, 136.2, 131.7, 128.7, 128.5, 128.2, 128.1,

128.0, 127.4, 122.8, 114.5, 70.1, 66.4. Anal. calc. for C21H18O3: C, 79.24;

H, 5.66. Found: C, 79.62; H, 5.85.

Following the same procedure as described previously, from 9 (2.43 g,

13.2 mmol), N,N-dimethylformamide (50 ml), K2CO3 (12 g, 87 mmol), and

benzyl bromide (8 ml, 67.3 mmol), 5.38 g of 10 (92%) were obtained after

flash chromatography (dichloromethane/n-pentane: 1/1 then dichloromethane)

as a white solid. Mp: 97–988C, Rf ¼ 0.46 (dichloromethane/n-pentane: 1/1).


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IR (KBr, cm21): 3033, 2949, 1717, 1589, 1428, 1335, 1216, 1112, 1003,

756. 1H NMR (CDCl3) d: 3.88 (s, 3H), 5.11 (s, 2H), 5.13 (s, 4H), 7.20–7.47

(m, 17H); 13C NMR (CDCl3) d: 166.6, 152.5, 142.4, 137.4, 136.6, 128.5,

128.4, 128.1, 128.0, 127.9, 127.5, 125.2, 109.1, 75.1, 71.2, 52.2. Anal. calc.

for C29H26O5: C, 76.65; H, 5.73. Found: C, 76.88; H, 5.81.

General Procedure for the Synthesis of Hydroxy-perbenzylated

Benzoic Acids (11, 12, 13)

KOH (1.5 g, 27 mmol) was added to a stirred solution of 6 (2.3 g, 5.4 mmol) in

water (20 ml) and ethanol (80 ml). The mixture was refluxed for 1 h until it

became homogeneous and then concentrated in vacuo. The residue was

poured into water (100 ml) and washed with diethyl ether (2 � 50 ml). The

organic phases were discarded, and the aqueous phase acidified with concen-

trated H2SO4 until the formation of a white solid in suspension. The mixture

was extracted with diethyl ether (3 � 100 ml) and ethyl acetate (2 x 100 ml).

The combined organic layers were washed with a solution of 1.5% HCl

(100 ml), dried (Na2SO4), filtered, and concentrated in vacuo to give 1.777 g

(98%) of 11 as a white solid. Mp: 187–1888C. IR (KBr, cm21): 3091,

3064, 3031, 2906, 2865, 1677, 1601, 1521, 1441, 1306, 1277, 1226, 1135,

1025, 761, 729, 693. 1H NMR (DMSO-D6) d: 5.18 (s, 2H), 5.22 (s, 2H),

7.15 (d, J ¼ 8.9 Hz, 1H), 7.28–7.50 (m, 10H), 7.56 (dd, J1 ¼ 8.9 Hz,

J2 ¼ 2 Hz, 1H), 7.57 (d, J ¼ 2 Hz, 1H), 12.7 (brs, H); 13C NMR (DMSO-D)

d: 166.9, 152.0, 147.5, 136.9, 136.6, 128.4, 128.3, 127.8, 127.7, 127.5,

127.4, 123.4, 123.2, 114.5, 113.0, 69.9, 69.8. Anal. calc. for C21H18O4: C,

75.45; H, 5.39. Found: C, 75.26; H, 5.53.


3.3 mmol), water (10 ml), ethanol (30 ml), and KOH (5 g, 89 mmol), 598 mg

of 12 (80%) were obtained after flash chromatography (diethyl ether/n-

pentane/acetic acid: 50/50/0.2, then dichloromethane/methanol: 7/3) as a

white solid. Mp: 191–1928C. IR (KBr, cm21): 3063, 3038, 2667, 2558,

1685, 1608, 1258, 1169, 772, 738, 693, 656. 1H NMR (DMSO-D6) d: 5.18

(s, 2H), 7.10 (d, J ¼ 8.8 Hz, 2H), 7.30–7.50 (m, 5H), 7.90 (d, J ¼ 8.8 Hz,

2H), 12.45 (brs, 1H); 13C NMR (DMSO-D6) d: 166.9, 161.8, 136.4, 131.3,

128.4, 127.9, 127.7, 123.1, 114.5, 69.4. Anal. calc. for C14H12O3: C, 73.68;

H, 5.26. Found: C, 73.92; H, 5.50.


11.9 mmol), water (100 ml), ethanol (300 ml), and KOH (15 g, 268 mmol),

4.15 g of 13 (80%) were obtained after recrystallization from dichloromethane

as a white solid. Mp: 195–1968C. IR (KBr, cm21): 3089, 3065, 3030, 2867,

1686, 1595, 1431, 1338, 1129, 735, 694. 1H NMR (DMSO-D6) d: 5.05

(s, 2H), 5.18 (s, 4H), 7.24–7.52 (m, 17H), 12.96 (brs, 1H); 13C NMR

(DMSO-D6) d: 166.8, 151.9, 140.9, 137.3, 136.8, 128.3, 128.1, 128.0,


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127.8, 127.5, 125.9, 108.1, 74.1, 70.1. Anal. calc. for C28H24O5: C, 76.36; H,

5.45. Found: C, 76.40; H, 5.55.

General Procedure for the Synthesis of Perbenzylated

Depsides (14–16)

To a stirred solution of 11 (1.04 g, 3.1 mmol) in dichloromethane (50 ml)

was added 2 (864 mg, 1.96 mmol), N,N-dimethylaminopyridine (248 mg,

2.03 mmol) and N,N0-dicyclohexylcarbodiimide (740 mg, 3.6 mmol). The

mixture was refluxed overnight. Then ethanol (1 ml) and acetic acid (1 ml)

were added; the mixture was refluxed for 2 h and concentrated in vacuo.

Flash chromatography (dichloromethane) afforded 1.329 g (88%) of 14 as a

white solid. Mp: 97–988C, Rf ¼ 0.56 (dichloromethane). IR (KBr, cm21):

3090, 3064, 3031, 2932, 2881, 1726, 1616, 1275, 1187, 1175, 1127, 1096,

749, 730, 699. 1H NMR (CDCl3) d: 4.98 (s, 2H), 5.06 (s, 2H), 5.10 (s, 2H),

5.13 (s, 2H), 5.23 (s, 2H), 6.46 (d, J ¼ 2.1 Hz, 1H), 6.49 (d, J ¼ 2.1 Hz,

1H), 6.90 (d, J ¼ 9 Hz, 1H), 7.16 (m, 5H), 7.24–7.52 (m, 20H), 7.61 (dd,

J1 ¼ 9 Hz, J2 ¼ 2 Hz, 1H), 7.62 (d, J ¼ 2 Hz, 1H); 13C NMR (CDCl3) d:

164.3, 164.1, 161.3, 158.3, 153.3, 150.9, 148.3, 136.7, 136.4, 136.1, 135.9,

135.4, 128.6, 128.5, 128.2, 128.1, 128.0, 127.9, 127.8, 127.7, 127.5, 127.4,

127.04, 127.03, 124.8, 121.6, 115.5, 113.1, 110.2, 101.4, 98.9, 70.9, 70.71,

70.68, 70.3, 66.9. Anal. calc. for C49H40O8: C, 77.78; H, 5.29. Found: C,

77.42; H, 5.50.

Following the same procedure as described previously, 145 mg of 15

(95%) were obtained as a white solid from 12 (78 mg, 0.34 mmol),

dichloromethane (10 ml), N,N-dimethylaminopyridine (30 mg, 0.24 mmol),

N,N0-dicyclohexylcarbodiimide (78.2 mg, 0.38 mmol), and 2 (100.7 mg,

0.23 mmol). Mp: 97–988C, Rf ¼ 0.66 (dichloromethane). IR (KBr, cm21):

3090, 3064, 3032, 1735, 1614, 1259, 1167, 1089, 738, 697. 1H NMR

(CDCl3) d: 4.98 (s, 2H), 5.04 (s, 2H), 5.11 (s, 2H), 5.15 (s, 2H), 6.49

(s, 2H), 6.95 (d, J ¼ 8.9 Hz, 2H), 7.05–7.46 (m, 20H), 7.98 (d, J ¼ 8.9 Hz,

2H); 13C NMR (CDCl3) d: 164.4, 164.1, 162.9, 161.2, 158.3, 150.9, 136.2,

136.1, 135.9, 135.5, 132.3, 128.64, 128.59, 128.5, 128.2, 127.83, 127.80,

127.5, 127.4, 127.1, 121.5, 114.6, 110.3, 101.4, 98.8, 70.7, 70.3, 70.1, 66.9.

Anal. calc. for C42H34O7: C, 77.54; H, 5.23. Found: C, 77.75; H, 5.58.


(85%) were obtained as a white solid from 13 (150 mg, 0.34 mmol),

dichloromethane (10 ml), N,N-dimethylaminopyridine (33.6 mg, 0.28 mmol),

N,N0-dicyclohexylcarbodiimide (75.2 mg, 0.37 mmol), and 2 (100.7 mg,

0.23 mmol). Mp: 132–1338C, Rf ¼ 0.52 (dichloromethane). IR (KBr,

cm21): 3089, 3065, 3032, 1732, 1614, 1427, 1262, 1187, 1161, 1119, 753,

733, 698. 1H NMR (CDCl3) d: 5.02 (s, 2H), 5.09 (s, 2H), 5.16 (s, 2H), 6.46

(d, J ¼ 2.1 Hz, 1H), 6.52 (d, J ¼ 2.1 Hz, 1H), 7.05–7.49 (m, 32H);13C NMR (CDCl3) d: 164.3, 164.1, 161.4, 158.6, 152.5, 150.9, 143.0, 137.4,


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136.6, 136.2, 135.9, 135.4, 128.7, 128.6, 128.5, 128.3, 128.21, 128.16, 128.04,

127.98, 127.93, 127.85, 127.6, 127.1, 123.9, 110.2, 109.5, 101.4, 99.1, 75.1,

71.1, 70.9, 70.4, 67.0. Anal. calc. for C56H42O9: C, 77.96; H, 4.87. Found:

C, 77.87; H, 5.42.

General Procedure for Depsides Deprotection (17, 18, 19)

In a Schlenk tube, 14 (1.18 g, 1.6 mmol), THF (10 ml), and Pd/C 3% (1 g)

were mixed. The reaction vessel was capped with a septum, vacuum was

applied, and the Schlenck tube was filled with hydrogen. The suspension

was stirred overnight. Pd/C was then filtered on Whatman paper and THF

evaporated in vacuo to afford 447 mg of 17 (94%) as a white solid. Mp:

decomposition at 1658C. IR (KBr, cm21): 3441, 1719, 1696, 1652, 1606,

1455, 1313, 1272, 1224, 1198, 1106, 748. 1H NMR (CD3OD) d: 6.12

(d, J ¼ 2.4 Hz, 1H), 6.26 (d, J ¼ 2.4 Hz, 1H), 6.85 (d, J ¼ 8.9 Hz, 1H), 7.56

(dd, J1 ¼ 2.1 Hz, J2 ¼ 8.9 Hz, 1H), 7.55 (d, J ¼ 2.1 Hz, 1H); 13C NMR

(CD3OD) d: 172.5, 167.2, 166.6, 164.8, 154.9, 152.2, 146.3, 124.5, 122.3,

118.0, 116.0, 104.6, 101.7, 100.6.

Following the same procedure as described previously, 80 mg of 18 (79%)

were obtained as a white solid from 15 (229 mg, 0.35 mmol), THF (5 ml), and

Pd/C 3% (200 mg). Mp: decomposition at 1618C. IR (KBr, cm21): 3364,

3232, 1696, 1637, 1607, 1446, 1276, 1192, 1095, 1028, 839, 625. 1H NMR

(CD3OD) d: 6.13 (d, J ¼ 2.4 Hz, 1H), 6.26 (d, J ¼ 2.4 Hz, 1H), 6.87

(d, J ¼ 8.8 Hz, 2H), 7.99 (d, J ¼ 8.8 Hz, 2H); 13C NMR (CD3OD) d: 172.5,

167.1, 166.6, 164.8, 164.0, 154.9, 133.5, 122.0, 116.3, 104.6, 101.7, 100.7.


(87%) were obtained as a white solid from 16 (218.3 mg, 0.25 mmol), THF

(5 ml), and Pd/C 3% (200 mg). Mp: decomposition at 1658C. IR (KBr,

cm21): 3428, 2541, 1721, 1696, 1653, 1446, 1282, 1046, 749. 1H NMR

(CD3OD) d: 6.13 (d, J ¼ 2.4 Hz, 1H), 6.27 (d, J ¼ 2.4 Hz, 1H), 7.19

(s, 2H); 13C NMR (CD3OD) d: 172.4, 167.4, 166.5, 164.8, 154.9, 146.5,

140.2, 121.1, 110.8, 104.5, 101.7, 100.6.

ACKNOWLEDGMENT

This work was supported by a grant (No. 10659) to S. T. The Ministere

Delegue a l’Enseignement Superieur et a la Recherche is gratefully

acknowledged.

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8. Hund, H.-K.; Breuer, J.; Lingens, F.; Huttermann, J.; Kappl, R.; Fetzner, S.Flavonol 2,4-dioxygenase from Aspergillus niger DSM 821, a type 2 CuII-containing glycoprotein. Eur. J. Biochem. 1999, 263, 871–878.

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first chemical synthesis of three natural depsides involved in flavonol catabolism and related to...

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