Convenient synthesis of 2,4-diacetylphloroglucinol a natural antibiotic involved in the control of take-all disease of wheat

Article abstract of DOI:10.1021/jf9907135

2,4-Diacetylphloroglucinol (DAPG) is an antibiotic with broad-spectrum antibacterial and antifungal activities. It is a major determinant in the biological control of several plant diseases. DAPG is produced by Pseudomonas fluorescens both in vitro and in the rhizosphere of wheat. It is involved in the natural suppression of take-all disease known as take-all decline, which develops in soils following extended monoculture of wheat or barley. A one- step synthesis of DAPG from the commercially available 2- acetytlphloroglucinol is described. This reaction involves the direct alkylation of 2-acetylphloroglucinol using acetic anhydride as the acetylation reagent, with boron trifluoride-etherate as the catalyst. This synthesis is simple and produces higher yields of DAPG (90%) as compared with previously described procedures. As ecological concerns are gaining equal status with agricultural concerns, the demand for natural biocontrol measures is increasing. There is tremendous pressure from society on agriculture to reduce the use of pesticides. A discussion is given on the agricultural and ecological importance of this natural antibiotic and its application as an alternative to reduce the use of synthetic pesticides.

Full text of DOI:10.1021/jf9907135

1882
J. Agric. Food Chem. 2000, 48, 1882−1887
Con ven ien t Syn th esis of 2,4-Dia cetylp h lor oglu cin ol, a Na tu r a l
An tibiotic In volved in th e Con tr ol of Ta k e-All Disea se of Wh ea t
Patrice A. Marchand,†,‡ David M. Weller, and Robert F. Bonsall*
§
,#
Institute of Biological Chemistry and Department of Plant Pathology, Washington State University, and
Root Disease and Biological Control Research Unit, Agricultural Research Service,
U.S. Department of Agriculture, Pullman, Washington 99164
2
,4-Diacetylphloroglucinol (DAPG) is an antibiotic with broad-spectrum antibacterial and antifungal
activities. It is a major determinant in the biological control of several plant diseases. DAPG is
produced by Pseudomonas fluorescens both in vitro and in the rhizosphere of wheat. It is involved
in the natural suppression of take-all disease known as take-all decline, which develops in soils
following extended monoculture of wheat or barley. A one-step synthesis of DAPG from the
commercially available 2-acetylphloroglucinol is described. This reaction involves the direct alkylation
of 2-acetylphloroglucinol using acetic anhydride as the acetylation reagent, with boron trifluoride-
etherate as the catalyst. This synthesis is simple and produces higher yields of DAPG (90%) as
compared with previously described procedures. As ecological concerns are gaining equal status
with agricultural concerns, the demand for natural biocontrol measures is increasing. There is
tremendous pressure from society on agriculture to reduce the use of pesticides. A discussion is
given on the agricultural and ecological importance of this natural antibiotic and its application as
an alternative to reduce the use of synthetic pesticides.
Keyw or d s: O-Acetylated; total intensity chromatogram (TIC); photodiodide array UV spectroscopy;
high-pressure liquid chromotograpy (HPLC); mass spectrum (MS); retention time (RT); melting point
(
mp); phloroglucinol (Phl); 2-acetylphloroglucinol (MAPG); 2,4-diacetylphloroglucinol (DAPG); boron
trifluoride-etherate; Pseudomonas fluorescens
INTRODUCTION
1992). Mazzola et al. (1995) reported that DAPG-
producing strain Q2-87 suppressed take-all caused by
three DAPG-sensitive isolates of G. graminis var. tritici
but failed to suppress take-all caused by two isolates of
the pathogen that were insensitive to DAPG at 3.0 µg/
mL. Furthermore, DAPG-producing fluorescent Pseudo-
monas spp. have been shown to be responsible for take-
all decline, a natural biological control of take-all which
develops in soils following extended monoculture of
wheat or barley (Raaijmakers et al., 1997; Raaijmakers
and Weller, 1998). The positive role for DAPG in the
biological control of plant diseases demonstrated by the
use of genetic approaches was confirmed by direct
isolation of the antibiotic from the rhizosphere of wheat.
When seeds of wheat were coated with P. fluorescens
Q2-87 and then sown in raw Shano silt loam and
American agriculture is under increasing pressure to
reduce the use of synthetic chemical pesticides for the
control of plant diseases, insect pests, and weeds
because of concerns about the adverse impact of pesti-
cides on public health and the environment. Thus, there
is considerable interest in finding and developing natu-
ral products with pesticidal activity. Phloroglucinol (Phl)
antibiotics are phenolic metabolites produced by bacte-
ria and plants with broad-spectrum antibacterial, anti-
fungal, antiviral, anthelmintic, and phytotoxic proper-
ties (Thomashow and Weller, 1996). Incontrovertible
evidence has accumulated that the polyketide antibiotic
2
,4-diacetylphloroglucinol (DAPG) is a major determi-
nant in the biological control of several plant diseases
by certain strains of Pseudomonas fluorescens. A genetic
approach was used to demonstrate the role of DAPG in
the suppression of black root rot of tobacco (Thielavi-
opsis basicola) and take-all of wheat (Gaeumannomyces
graminis var. tritici) by P. fluorescens CHA0 (Keel et
al., 1990, 1992), take-all of wheat by P. fluorescens Q2-
7
Ritzville silt loam, 0.16 and 0.33 µg of DAPG/10 colony-
forming units (CFU) of Q2-87/g of root plus rhizosphere
soil, respectively, were isolated (Bonsall et al., 1997).
The amount of DAPG produced in the rhizosphere
recently was shown to be directly related to the popula-
tion size of DAPG producers present (Raaijmakers et
al., 1999).
8
7 (Harrison et al., 1993; Vincent et al., 1991), and
damping-off of sugar beet (Pythium ultimum) by P.
fluorescens F113 (Fenton et al., 1992; Shanahan et al.,
DAPG can be readily isolated in milligram quantities
from cultures of fluorescent Pseudomonas spp. contain-
ing the DAPG biosynthetic locus strains (Keel et al.,
*
Author to whom correspondence should be addressed [fax
1
996), and production of DAPG can be enhanced when
(
509) 335-7674; e-mail bonsall@wsu.edu].
plant roots are added to the culture (Aino et al., 1995).
The DAPG biosynthetic locus has been cloned (Bangera
and Thomashow, 1996, 1999) and sequenced (Genbank
accession no. U41818). However, no efficient chemical
synthesis for DAPG has been described. The purpose
†
Institute of Biological Chemistry.
‡
Present address: Max Planck Institute f u¨ r Chemische
O¨ kologie, Tatzend Promenade 1A, D-07745 J ena, Germany.
§
U.S. Department of Agriculture.
Department of Plant Pathology.
#
1
0.1021/jf9907135 CCC: $19.00 © 2000 American Chemical Society
Published on Web 04/22/2000

Synthesis of 2,4-Diacetylphloroglucinol
J. Agric. Food Chem., Vol. 48, No. 5, 2000 1883
of this study was to develop a simple, convenient, and
affordable synthesis of DAPG to facilitate studies of this
natural product for plant disease control. 2-Acetylphloro-
glucinol (MAPG) was used as the starting material
because it is commercially available and inexpensive.
Ta ble 1. P h lor oglu cin ol a n d C- or O-Acetyla ted
Der iva tives
MW
R1a R2 R3 R4 R5 R6 (m/z)
b
RTc
compound
Phl
H
H
H
H
Ac
Ac
H
H
H
H
H
H
H
Ac
Ac
H
H
H
H
H
H
H
H
H
H
H
Ac
H
Ac
H
H
H
H
126
168 12.35
210 18.19
3.59
MAPG
DAPG
TAPG
Ac
Ac
Ac
H
H
H
MATERIALS AND METHODS
Ac 252 25.02
1
1
1
1
5
3
-O-acetyl-Phl
,3-di-O-acetyl-Phl
,3,5-tri-O-acetyl-Phl Ac
-O-2-diacetyl-Phl
-O-2-diacetyl-Phl
-O-2,4-triacetyl-Phl
H
H
H
H
H
H
H
168
9.87
Gen er a l Exp er im en ta l P r oced u r es. Solvents were of
either HPLC grade (CH CN, MeOH) or ACS grade (EtOAc,
3
Ac
Ac
H
210 13.79
252 16.56
210 14.43
210 15.66
252
hexanes) from J . T. Baker. Column and analytical thin layer
chromatographic separations (Al Si G/UV 254) were performed
using silica gel 60 (230-400 mesh, Whatman). IR spectra were
recorded on a Perkin-Elmer (Spectrum 2000) FTIR spectrom-
eter, and NMR spectra were obtained using a Bruker ARX500
Ac Ac
H
H
Ac
H
Ac Ac Ac
a
Substituent groups on the phloroglucinol ring as shown in
Figure 1. b Molecular weight based on the parent molecular weight
ion as determined by mass spectrometry. RT, retention time.
(500 MHz) spectrometer. All NMR spectra were recorded in
c
acetone-d using TMS as internal reference, with chemical
6
shifts (δ) expressed in parts per million.
Electron ionization mass spectrometry (EI/MS) analyses
were carried out with an HPLC/MS (Waters, Integrity). The
Waters Integrity system consisted of an Alliance 2690 separa-
tion module with a 996 photodiode array detector and an
electron ionization Thermabeam mass detector (TMD). The
separation was achieved using reversed phase column chro-
matography (Waters, Symmetry C-18; 150 × 3.0 mm). Solvent
conditions included a flow rate of 0.35 mL/min with a 2 min
initialization at 10% CH
followed by a 20 min linear gradient to 100% CH
acetic acid). MS analyses of total intensity chromatograms
3
CN in water with 2% v/v acetic acid,
3
CN (2% v/v
F igu r e 1. Direct acetylation of Phl using acetic anhydride
(
TIC) were performed from m/z 70 to 355 at a rate of 1 scan
(Ac
2
O) and Et
2
3
O‚BF as catalyst.
-
1
s
. The nebulizer, ion source, and expansion region temper-
atures were 84 , 220, and 80 °C, respectively. These temper-
atures were optimized using commercial MAPG (Aldrich) and
natural DAPG as references. High-resolution mass spectrom-
etry (HRMS) analyses employed a VG 7070 EHF mass
spectrometer at 70 eV. Melting points are uncorrected and
were recorded on a B u¨ chi apparatus B-540. Elemental analysis
was conducted by Desert Analytics (Tucson, AZ).
P r oced u r e for Syn th esis of DAP G. Acetic anhydride (10
mL, 106.5 mmol, 1.8 equiv) was added dropwise at room
temperature to MAPG (Aldrich; 10 g, 59.5 mmol), dried at 80
°
C under vacuum for 12 h in a 250-mL round-bottom flask.
Boron trifluoride-etherate (Et O‚BF ) as a catalyst (50 mL,
9.7 mmol) was slowly added with a syringe to the stirring
2
3
3
mixture, followed by continuous stirring for 24 h at room
temperature. Water (30 mL) was then slowly added, maintain-
ing vigorous stirring, followed by 60 mL of water/methanol v/v
(
1:1). Potassium carbonate (K
2 3
CO ; 40 g, 290 mmol) was then
F igu r e 2. TIC of the synthesis of DAPG from Phl. TIC was
performed from m/z 70 to 355 at a rate of 1 scan/s. Numbers
shown are retention times. Peaks at retention times of 14.43
and 15.66 are either 1-O-2-diacetyl-Phl or 5-O-2-diacetyl-Phl
because both peaks exhibit similar mass spectra.
carefully added followed by the addition of water/methanol 1:1
(
∼20 mL) to maintain stirring of the solution. The basic
solution was stirred overnight, followed by neutralization/
acidification (pH 5-6) with 10% HCl. Methanol was evapo-
rated from the mixture by rotor evaporation under vacuum
(40 °C). Distilled water (500 mL) was added to the moist
RESULTS
residue followed by stirring for 5 min and then filtration
through a B u¨ chner funnel apparatus. This operation was
repeated twice to remove salts from the crude extract. The
residue was dried overnight in a desiccator under vacuum at
room temperature. The residue was dissolved in a minimum
amount of acetone, subjected to silica column chromatography
Syn th esis of DAP G fr om P h l. Dean and Robertson
1953) reported a synthesis of DAPG involving the
(
acetylation of Phl (Table 1; Figure 1). As a first step in
our study, we repeated their procedures. LC/MS analy-
sis revealed the formation of many different side
products due to random O-acetylation of Phl and MAPG
together with the formation of MAPG and DAPG (Table
1; Figure 1) production. However, from the total inten-
sity chromatogram (TIC), the peak that corresponded
(400 × 40 mm), and eluted with hexanes/EtOAc v/v (4:1);
DAPG was eluted first (monitored by TLC; UV ) 254 nm).
The elutant was rotor-evaporated under vacuum (50 °C
maximum) to dryness, followed by recrystallization from
benzene/petroleum ether v/v 2:1 (15 mL), suction filtered
through a B u¨ chner funnel, and dried overnight in a desiccator
under vacuum. To recover the remainder of DAPG, the
recrystallization filtrate was rotor-evaporated to dryness under
vacuum (50 °C maximum) and purified by silica gel column
chromatography (300 × 20 mm) as described above. The white
solid DAPG fractions (obtained from recrystallization and the
second silica gel purification) were combined to give a total
+
to DAPG (M at m/z ) 210) was a minor peak when
compared to that of the O-acetylated compounds and
MAPG produced (Figure 2).
O-Acetylation of Phl. 1-O-acetyl-Phl (Table 1; Figure
+
1
1
; RT ) 9.87, Figure 2) has a molecular ion M at m/z
+
68 (which is the expected M mass for MAPG) but
mass of 11.2 g (53.3 mmol) at a 90% yield. TLC: R
hexanes/EtOAc v/v [1:1]).
f
) 0.55
exhibits a TIC RT (Figure 2) and a mass spectrum
(Figure 3D) different from those of MAPG (Figure 3B).
(

1884 J. Agric. Food Chem., Vol. 48, No. 5, 2000
Marchand et al.
F igu r e 3. Mass spectra of Phl, MAPG, DAPG, and O-acetylated forms of Phl and MAPG: (A) Phl [m/z (relative intensity) 126
+
+
+
+
(
(
1
100, [M] ), 111 (8, [M - CH
3
] )]; (B) MAPG [m/z (relative intensity) 168 (45, [M] ), 153 (100, [M - CH
3
] )]; (C) DAPG [m/z
+
+
+
+
relative intensity) 210 (90, [M] ), 195 (100, [M - CH
3
] ), 177 (75, [M - H
2
O - CH
3
] ); 149 (10, [M - H
2
O - COCH
CO] ), 97 (6)]; (E) 1,3-di-O-acetyl-
CO)] ), 111 (5), 97 (10)]; (F) 1,3,5-tri-
CO)] ), 126 (100, [M - 3(CH
3
] )]; (D)
+
+
+
-O-acetyl-Phl [m/z (relative intensity) 168 (16, [M] ), 126 (100, [M - CH
3
] ), 111 (4, [M - CH
3
+
+
+
Phl [m/z (relative intensity) 210 (25, [M] ), 168 (40, [M - CH
3
CO] ), 126 (100, [M - 2(CH
3
+
+
+
+
O-acetyl-Phl [m/z (relative intensity) 252 (6, [M] ), 210 (12, [M - CH
3
CO] ), 168 (37, [M - 2(CH
3
3
CO)] ),
+
+
9
(
-
7 (5)]; (G, H) either 1-O-2-diacetyl-Phl or 5-O-2-diacetyl-Phl [m/z (relative intensity) 210 (10, [M] ), 168 (55, [M - CH
3
CO] ), 153
+
+
+
+
100, [M - CH
3
CO - CH
3
] ), 126 (20, [M - 2(CH
3
CO)] ), 97 (3) or 210 (22, [M] ), 168 (48, [M - CH
3
3
CO] ), 153 (100, [M - CH CO
+
+
CH
3
] ), 126 (36, [M - 2(CH
3
CO)] ), 97 (3)].

Synthesis of 2,4-Diacetylphloroglucinol
J. Agric. Food Chem., Vol. 48, No. 5, 2000 1885
This peak was identified as 1-O-acetyl-Phl because the
molecular ion M+ of m/z 168 (Figure 3D) was followed
by a mass spectrum pattern identical to that of Phl
+
(Figure 3A), with a neutral fragment loss of C2H2O (M
-
42 at m/z 126).
1
,3-Di-O-acetyl-Phl (Table 1; Figure 1; RT ) 13.79,
Figure 2) exhibited a mass spectrum with a molecular
ion M at m/z 210, which is the expected mass for
+
DAPG. Mass spectra analysis (Figure 3E) revealed that
+
the molecular ion M at m/z 210 was followed by a mass
spectrum pattern identical to that obtained for 1-O-
acetyl-Phl (Figure 3D) and Phl (Figure 3A), with a
+
neutral fragment loss of C2H2O (M - 42 at m/z 168).
1
,3,5-Tri-O-acetyl-Phl (Table 1; Figure 1; RT ) 16.56,
Figure 2) exhibited a mass spectrum with a molecular
ion M at m/z 252, which is the expected mass for 2,4,6-
+
triacetylphloroglucinol (TAPG; Table 1; Figure 1). The
mass spectrum (Figure 3F) of this peak showed a
+
neutral fragment loss of C2H2O (M - 42 at m/z 210)
similar to those of 1,3-di-O-acetyl-Phl (Figure 3E) and
1
-O-acetyl-Phl (Figure 3D).
O-Acetylation of MAPG. 1-O-2-Diacetyl-Phl (Table 1;
Figures 1 and 2) and 5-O-2-diacetyl-Phl (Table 1;
Figures 1 and 2) also exhibit a mass spectrum with a
+
molecular ion M at m/z 210, which is the expected mass
for DAPG. Because the MS spectra (Figure 3G,H) of
both peaks at RT ) 14.43 and 15.66 (Figure 2) are
similar, it was impossible to assign which peak belonged
to what product. However, these two peaks are related
to the O-acetylation of MAPG, due to the fact that the
molecular ion at m/z 210 (Figure 3G,H) is followed by a
mass spectrum pattern identical to that of MAPG
+
(Figure 3B) with a neutral fragment loss of C2H2O (M
-
42 at m/z 168). Therefore, the only two possibilities
that can exist are 1-O-2-diacetyl-Phl (Table 1; Figure
) and 5-O-2-diacetyl-Phl (Table 1; Figure 1) for the two
F igu r e 4. Photodiode array UV spectra of (A) MAPG and (B)
DAPG.
1
peaks found at RT ) 14.43 and 15.66 (Figure 2).
Production of MAPG and DAPG from Acetylation of
Phl. The peaks at RT ) 12.35 (MAPG) and 18.19
(
DAPG) on the LC/MS profile (Figure 2) gave UV and
mass spectra that matched our referenced MAPG
Figures 4A and 3B) and DAPG (Figures 4B and 3C).
(
Again, the amount of DAPG formed was a very minor
fraction (3%) when compared to the total amount of
O-acetylated products and MAPG formed (Figure 2).
Syn th esis of DAP G fr om MAP G. Due to the
problems with the O-acetylated compounds that were
encountered by the synthesis of DAPG from Phl, a
small-scale conversion was set up using MAPG as the
starting material that could be later scaled up (Figure
F igu r e 5. Conversion of MAPG to DAPG using acetic
anhydride (Ac
2
O) and Et
2
3
O‚BF as catalyst.
chromatography (silica gel). The purity of DAPG after
silica gel column chromatography was demonstrated by
HPLC/MS using TIC (Figure 6).
5
). It should be mentioned that extending the reaction
The proton NMR spectra, 13C NMR spectra, EI mass
spectra, high-resolution mass spectra (HRMS), Fourier
transform infrared spectra (FTIR), photodiode array UV
spectra, elemental analysis, and melting point (mp) data
time (48 h) of the conversion gives O-acetylation of
DAPG yielding 3-O-2,4-triacetyl-Phl (Table 1; Figure 1).
This conclusion was made on the basis of MS analysis
that exhibited a molecular ion M+ of m/z 252 followed
by an MS spectrum pattern similar to that of DAPG,
1
for the properties of DAPG are as follows: H NMR
(CD3COCD3) δ 5.87 (m, 1H CHAr), 3.90-3.30 (br s, 3H,
+
13
with a neutral fragment loss of C2H2O (M - 42 at m/z
OH), 2.61 (s, 3H, CH3); C NMR (CD3COCD3) δ 205.1
2
10) (data not shown). In addition, this was confirmed
(CdO), 173.4 (s, Cq-OH: C-1 and C-5), 170.8 (s, Cq-
OH: C-3), 105.3 (s, Cq-CdO), 96.3 (d, CH) 33.5 (q, CH3);
1
3
by C NMR, which exhibited a symmetrical spectrum
1
3
+
pattern: only one C peak for both C-2 and C-4, as
expected for a substitution position C-3 (data not
shown). Later, it was observed that DAPG could be
recovered from the O-acetylated product by simple basic
treatment with K2CO3 in aqueous methanol. In conclu-
sion, the conversion of MAPG to DAPG with basic
treatment will yield only nonconverted MAPG with
DAPG as products, which can be separated by column
EI/MS, m/z (relative intensity) 210 (90, [M] ), 195 (100,
+
+
[M - CH3] ), 177 (75, [M - H2O - CH3] ), 149 (10, [M
+
- H2O - COCH3] ); HRMS calcd for C10H10O5 210.0528,
-1
found 210.0509; FTIR ν (cm ) 3400-3000 (OH br) 1610,
1562, 1468 (aromatic conjugated CdO with intramo-
lecular hydrogen bonds and aromatic CdC), 1364 (C-
OH), 1283 (C-O); photodiode array analysis (UV spec-
tra) λmax ) 270, 330 nm. Anal. calcd for C10H10O5 C,

1886 J. Agric. Food Chem., Vol. 48, No. 5, 2000
Marchand et al.
would mislead the investigator to believe that they had
pure quantities of MAPG and DAPG (as the major
products) with TAPG (as the minor product) from the
synthetic reaction.
In our study, using MAPG as the starting material,
purity was assessed after recrystallization (on the whole
mass), using both spectroscopic (UV, IR, NMR, MS) and
elemental analysis. In addition, direct alkylation of
MAPG by our method yields pure DAPG with no
O-acetylated products, as determined by mass spectral
analysis on the HPLC/MS profile, using TIC (Figure 6).
Our synthesis of DAPG gave a melting point of 173 °C,
which was identical to that reported by Nowak-
Thompson et al. (1994; 173-174 °C), whose sample was
1
13
F igu r e 6. TIC of the synthesis of DAPG from MAPG. TIC
was performed from m/z 70 to 355 at a rate of 1 scan s-1
analyzed by H and C NMR. In contrast, Dean and
Robertson (1953) and Mani et al. (1991) reported a lower
melting point of 168 °C indicating impure material. Also
questionable is the purity of synthetic DAPG claimed
by Ranjaneyulu et al. (1987) because they obtained
purple crystals in the reaction with Ac O/ZnCl instead
.
5
7.14; H, 4.80. Found: C, 56.30; H, 4.99 (taking into
account 0.18 molecule of water associated with one
molecule of DAPG C, 57.18; H, 4.81). mp ) 173 °C [lit.
2
2
1
73-174 °C (Nowak-Thompson et al., 1994)].
of the white crystals that we obtained. In addition, the
previously reported yields for MAPG and DAPG appear
to be in error. Ranjaneyulu et al. (1987) reported a yield
of MAPG of 50%; however, the actual yield should be
36% because they did not consider that MAPG will
crystallize with one molecule of water (MAPG, H2O;
C8H10O5; commercially available form of MAPG). In
addition, their report of a 25% yield of DAPG should
actually be 20.5% (Ranjaneyulu et al., 1987). Mani et
al. (1991) made the same type of error, such that the
reported yield of 85% should be 51%.
Because Phl and MAPG are commercially available
as the dihydrate and monohydrate (Phl, 2H2O; MAPG,
H2O), respectively, the water content of DAPG was
investigated by elemental analysis and by thermal
gravimetric analysis. This was done to obtain informa-
tion concerning the real molecular weight and water
content of DAPG for an accurate determination of a
percent yield of synthesis from MAPG. Elemental
analysis of synthetic DAPG at room temperature re-
vealed C, 56.30, and H, 4.99. This value was compared
with theoretical values for the molecular formula of
C10H10O5, X ) 0 (C, 57.14; H, 4.80) and C10H12O6, X )
Our chemical synthesis using MAPG as the starting
material with basic (K CO ) treatment and silica column
2
3
1
(C, 52.63; H, 5.30). It was concluded that synthesized
chromatography separation produces only DAPG as the
final product (Figure 6). However, the crystals of this
extract are slightly yellow in color and, after recrystal-
lization (benzene/petroleum ether), gave pure white
DAPG crystals. This reaction is a simple one-step
reaction at low cost yielding high quantities of DAPG
with no O-acetylated side products as contaminants.
Purity was demonstrated by a melting point of 173 °C,
elemental analysis, photodiode array, mass spectrom-
DAPG does not contain any molecules of water associ-
ated with it (maximum hydration + 0.18 molecule of
water). Reconfirmation by thermal gravimetric analysis
on recrystallized DAPG also revealed no degrees of
hydration (<0.5% H2O). Thus, the molecular C10H10O5
formula for DAPG was confirmed with a molecular mass
-
1
of 210 g mol
.
1
13
etry, FTIR, and both H and C NMR. Furthermore,
by elemental and thermal gravimetric analyses it was
determined that there were no degrees of hydration
associated with synthetic DAPG.
DISCUSSION
There have been a limited number of attempts to
chemically synthesize DAPG in which Phl has been the
preferred starting material (Dean and Robertson, 1953;
Mani et al., 1991; Ranjaneyulu et al., 1987). We began
our study by duplicating the synthesis of DAPG from
Phl as described by Dean and Robertson (1953). Their
study involved the C-acetylation of Phl and C-methyl
Phl together with their mono- and dimethyl ethers,
using boron trifluoride-etherate as catalyst, to produce
DAPG, 2,4-diacetyl-6-methylphloroglucinol, and their
corresponding methyl ethers. We demonstrated that the
direct alkylation of Phl resulted in many O-acetylated
Biological production of DAPG (Aino et al., 1995;
Nowak-Thompson et al., 1994) is slow and results in
very low yields (120 mg/L in 2 days with a maximum of
1
.1 g/L in 15 days). Moreover, the need of a multistep
purification process involving extractions and column
chromatography with further repetitive separations by
HPLC makes obtaining even small quantities of DAPG
tedious. Compared to the biological production of DAPG,
the chemical synthesis described in this paper is more
efficient, quicker, and less costly with a 90% yield.
DAPG is an antibiotic known to be active against a
broad spectrum of microorganisms and is involved in
the suppression of many plant diseases. It is particularly
important in the suppression of take-all disease of wheat
(G. graminis var. tritici) by both naturally occurring and
introduced strains of P. fluorescens. Take-all is consid-
ered by agriculturists to be among wheat’s most damag-
ing root diseases, causing millions of dollars in losses
annually in U.S. wheat production alone. In infested
fields, crop yields drop 10-50%, but the diseases can
“take-all” of the crop.
+
molecules, with the same molecular weight (M at m/z
2
10) and with the same theoretical elemental analysis
(C10H10O5) as those of DAPG. As a result, the credibility
of the synthesis described by Dean and Robertson (1953)
was further investigated. In their paper, assessing the
purity of the crude extract involved only elemental
1
13
analysis and no mass spectral or H and C NMR data.
These O-acetylated compounds have the same molecular
weights that would be expected for MAPG, DAPG, and
TAPG. Therefore, straight elemental analysis that is not
1
13
reconfirmed by mass spectrometry and H and C NMR

Synthesis of 2,4-Diacetylphloroglucinol
J. Agric. Food Chem., Vol. 48, No. 5, 2000 1887
Traditionally, take-all has been controlled by a com-
bination of crop rotation and tillage. However, modern
wheat farming practices, including direct seeding and
growing several wheat crops before a break, exacerbate
take-all. There are no varieties of wheat with resistance
to take-all, and methods of chemical control are inef-
fective. A natural suppression of take-all, known as
take-all decline, will develop in the soil following
extended wheat or barley monoculture; however, few
growers have been able to make use of this biocontrol
measure because they cannot sustain the severe disease
losses that occur until decline develops. In nature, wheat
roots in take-all suppressive soils become colonized by
DAPG-producing bacteria, which produce small amounts
of the antibiotic in the rhizosphere and inhibit the
pathogen.
This paper provides a means of synthesizing large
quantities of DAPG quickly with scale-up possibilities.
The chemical synthesis avoids the long and tedious
extractions and purifications that are associated with
biologically produced samples. The availability of pure,
synthetic DAPG provides a standard reference for
positive identification of DAPG from microorganisms
and for calculating the amounts of the antibiotic natu-
rally present in the rhizosphere of plants. It also
facilitates studies concerning the stability of the anti-
biotic in soils from soil-borne pathogens and in research
on DAPG analogues concerning biosynthetic pathway
elucidation. Perhaps most important, the availability of
synthetic DAPG makes it possible to assess the activity
of this natural product as seed and foliar treatments
against plant diseases in the field.
Keel, C.; Wirthner, P. H.; Oberh a¨ nsli, T. H.; Voisard, C.;
Burger, U.; Hass, D.; D e´ fago, G. Pseudomonas as antago-
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Microbe Interact. 1992, 5, 4-13.
Keel, C.; Weller, D. M.; Natsch, A.; D e´ fago, G.; Cook, R. J .;
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Mazzola, M.; Fujimoto, D. K.; Thomashow, L. S.; Cook, R. J .
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2
1
Raaijmakers, J . M.; Weller, D. M.; Thomashow, L. S. Fre-
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Received for review J une 29, 1999. Revised manuscript
received February 15, 2000. Accepted March 7, 2000.
J F9907135