Chemoenzymatic Synthesis of the Microbial Elicitor (-)-Syringolide via a Fructose 1,6-Diphosphate Aldolase-Catalyzed Condensation Reaction

Article abstract of DOI:10.1021/jo991989e

Syringolide 2, an elicitor of the bacterial plant pathogen Pseudomonas syringae pv. tomato which triggers a hypersensitive defense response in resistant soybean plants, has been synthesized in five steps via a fructose 1,6-diphosphate aldolase reaction.

Full text of DOI:10.1021/jo991989e

J . Org. Chem. 2000, 65, 4529-4531
4529
Ch em oen zym a tic Syn th esis of th e Micr obia l Elicitor
(-)-Syr in golid e via a F r u ctose 1,6-Dip h osp h a te Ald ola se-Ca ta lyzed
Con d en sa tion Rea ction
Robert Cheˆnevert* and Mohammed Dasser
De´partement de chimie, Faculte´ des sciences et de ge´nie, Universite´ Laval,
Que´bec (Que´bec), Canada G1K 7P4
robert.chenevert@chm.ulaval.ca
Received December 28, 1999
Syringolide 2, an elicitor of the bacterial plant pathogen Pseudomonas syringae pv. tomato which
triggers a hypersensitive defense response in resistant soybean plants, has been synthesized in
five steps via a fructose 1,6-diphosphate aldolase reaction.
Sch em e 1a
In tr od u ction
Some plant pathogens produce signal molecules (elici-
tors) which are recognized specifically by resistant plants
and enable the plants to initiate active defense responses
against these pathogens.1 In 1993, Sims et al.2 isolated
novel nonprotein elicitors syringolides 1 and 2 from
Pseudomonas syringae pv. tomato. These compounds
elicit a hypersensitive response in resistant cultivars of
soybeans. This defense reaction involves a rapid, localized
cell death and accumulation of phytoalexins (antimicro-
bial compounds) around the infection site. Syringolides
attract considerable interest since they have features in
common with antigens that are recognized by the im-
mune systems of vertebrates. Recently, several enanti-
oselective synthesis of syringolides have been reported.3
Syringolides have been synthesized from chiral pool
precursors such as xylose,3a-c tartaric acid,3d,e glycer-
aldehyde,3f or via the Sharpless catalytic asymmetric
dihydroxylation of butenolides.3g,h
are rare examples of this strategy. We report here a
chemoenzymatic synthesis of (-)-syringolide 2 via a
fructose 1,6-diphosphate aldolase (FDP aldolase) reac-
tion.
Resu lts a n d Discu ssion
Aldehyde 16 was first prepared in two steps by alkyl-
ation of allyl alcohol with p-methoxyphenylmethyl chlo-
ride and ozonolysis of the corresponding ether, followed
by reductive workup with dimethyl sulfide.
The use of aldolases in the synthesis of carbohydrates
and close analogues (azasugars, cyclitols) has been amply
demonstrated.4 However, the application of aldolases for
the synthesis of natural products other than sugars has
received very little attention. The syntheses of brevi-
comin,5a aspicilin5b (C3-C9 fragment), pentamycin5c (C11-
C16 fragment), and amphotericin5d (C12-C20 fragment)
The reaction of aldehyde 1 with dihydroxyacetone
phosphate 2 (DHAP)7 in the presence of fructose 1,6-
diphosphate aldolase in water/DMF (10/1), followed by
hydrolysis in situ of the intermediate phosphate ester
with acid phosphatase, afforded ketotriol 3 in 65% yield
(Scheme 1). FDP aldolase catalyzes the formation of C-C
bonds having the D-threo (3S,4R) configuration;8 com-
pound 3 is presumed to have the 3S,4R absolute config-
uration. Compound 3 was treated with acetone under
acid catalysis to give acetonide 4. Acylation of 4 with
octanoyl Meldrum’s derivative9 5 in refluxing THF led
to the corresponding ester which upon standing or
treatment with silica gel gave keto-ester 6 via an in-
tramolecular Knoevenagel reaction (Scheme 2). Removal
of the MPM protecting group was accomplished with
(1) Schro¨der, F. Angew. Chem., Int. Ed. 1998, 37, 1213.
(2) Midland, S. L.; Keen, N. T.; Sims, J . J .; Midland, M. M.; Stayton,
M. M.; Burton, V.; Smith, M. J .; Mazzola, E. P.; Graham, K. J .; Clardy,
J . J . Org. Chem. 1993, 58, 2940.
(3) (a) Henschke, J . P.; Rickards, R. W. Tetrahedron Lett. 1996, 37,
3557. (b) Zeng, C. M.; Midland, S. L.; Keen, N. T.; Sims, J . J . J . Org.
Chem. 1997, 62, 4780. (c) Yoda, H.; Kawauchi, M.; Takabe, K.; Hosoya,
K. Heterocycles 1997, 45, 1895. (d) Kuwahara, S.; Moriguchi, M.;
Miyagawa, K.; Konno, M.; Kodama, O. Tetrahedron 1995, 51, 8809.
(e) Wood, J . L.; J eong, S.; Salcedo, A.; J enkins, J . J . Org. Chem. 1995,
60, 286. (f) Yu, P.; Wang, Q. G.; Mak, T. C. W.; Wong, H. N. C.
Tetrahedron 1998, 54, 1783. (g) Ishihara, J .; Sugimoto, T.; Murai, A.
Tetrahedron 1997, 53, 16029. (h) Honda, T.; Mizutani, H.; Kanai, K.
J . Org. Chem. 1996, 61, 9374.
(4) (a) Schoevaart, R.; van Rantwijk, F.; Sheldon, R. A. Tetrahe-
dron: Asymmetry 1999, 10, 705. (b) Fessner, W. D. Current Opin.
Chem. Biol. 1998, 2, 85. (c) Takayama, S.; McGarney, G. J .; Wong, C.
H. Chem. Soc. Rev. 1997, 26, 407.
(5) (a) Schultz, M.; Waldmann, H.; Vogt, W.; Kunz, H. Tetrahedron
Lett. 1990, 31, 867. (b) Cheˆnevert, R.; Lavoie, M.; Dasser, M.; Can. J .
Chem. 1997, 75, 68. (e) Shimagaki, M.; Muneshima, H.; Kubota, M.;
Oishi, T. Chem. Pharm. Bull. 1993, 41, 282. (d) Malleron, A.; David,
S. New J . Chem. 1996, 20, 153.
(6) (a) England, P.; Chun, K. H.; Moran, E. J .; Armstrong, R. W.
Tetrahedron Lett. 1990, 31, 2669. (b) Kwart, H.; Sarner, S. F.; Slutsky,
J . J . Org. Chem. 1973, 38, 5234.
(7) J ung, S. H.; J eong, J . H.; Miller, P.; Wong, C. H. J . Org. Chem.
1994, 59, 7182.
(8) (a) Wong, C. H.; Whitesides, G. M. Enzymes in Synthetic Organic
Chemistry; Pergamon: Oxford, 1994; Chapter 4, pp 195-251. (b) Kaber,
K.; Biotransformations in Organic Chemistry; Springer-Verlag: Berlin,
1995; Chapter 2, pp 219-235.
(9) Oikawa, Y.; Sugano, K.; Yonemitsu, O. J . Org. Chem. 1978, 43,
2087.
10.1021/jo991989e CCC: $19.00 © 2000 American Chemical Society
Published on Web 06/30/2000

4530 J . Org. Chem., Vol. 65, No. 15, 2000
Cheˆnevert and Dasser
Sch em e 2a
solvent was evaporated. The crude product was purified by
flash chromatography (hexane/EtOAc, 9/1) to give aldehyde
16(647 mg, 3.59 mmol) as a colorless oil. IR (neat) 3100-3090,
1760, 1300-1100 cm-1; 1H NMR (CDCl3) 9.68 (s, 1H), 7.28 (d,
J ) 8.6 Hz, 2H), 6.88 (d, J ) 8.6 Hz, 2H), 4.55 (s, 2H), 4.05 (s,
2H), 3.79 (s, 3H); 13C NMR (CDCl3) 200.48, 159.52, 129.62,
128.77, 113.87, 74.89, 73.20, 55.15; HRMS (EI, 70 eV) calcd
for C10H12O3 (M+) 180.0786, found 180.0792 ( 0.0005.
(3S,4R)-5-p-Meth oxyben zyloxy-1,3,4-tr ih yd r oxy-2-p en -
ta n on e (3). Aldehyde 1 (600 mg, 3.33 mmol) was added to a
solution of DHAP 2 (680 mg, 4.0 mmol) in 25 mL of water/
DMF (9/1) under nitrogen atmosphere, and the pH was
adjusted to 6.8 with 1 N NaOH. Aldolase was added (36 mg,
360 U), and the mixture was shaken at room temperature.
After 24 h and again after 48 h, the pH was readjusted to 6.8,
and additional aldolase (33 mg, 330 U) and DHAP (374 mg,
2.2 mmol) were added. After 72 h, the pH was set at 4.8 with
1 N HCl, the acid phosphatase (143 mg, 57 U) was added, and
the mixture was stirred at room temperature for 12 h.
Additional phosphatase (57 U) was added, and the mixture
was stirred for 12 h. The pH was then raised to 7 with 1 N
NaOH. The solution was freeze-dried, the residue was taken
up in ethyl acetate, and the solution was filtered and evapo-
rated. The crude product was purified by flash chromatography
(CH2Cl2/MeOH, 19/1) to afford 3 as an oil (588 mg, 2.18 mmol,
DDQ. Treatment of the resultant compound 7 with an
excess of p-toluenesulfonic acid in acetone-water pro-
vided syringolide 2 (8) in 77%. Recrystallization from
pentane/ether (2/1) gave analytically pure syringolide 2
in 54% yield, [[R]25 -75.0 (c 0.06, CHCl3); lit.2 [R]24
-75.91 (c 0.22, CHCl3)]. The spectroscopic and physical
data of synthetic syringolide 2 (8) were identical to those
reported in the literature.2,3 The synthesis of the natural
enantiomer of syringolide 2 proved that the aldolase-
catalyzed reaction provided an addition product (3) with
the 3S,4R configuration.
In conclusion, we have completed the enantioselective
synthesis of the natural enantiomer of syringolide 2 in
five steps and 14% overall yield via an aldolase-catalyzed
condensation. The present work demonstrates that the
aldolase-catalyzed reactions are efficient processes in the
asymmetric synthesis of natural products.
D
D
65%). [R]25 -1.8 (c 2.0, CHCl3); IR (neat) 3640-3000, 1730,
D
1300-1100 cm-1; H NMR (CDCl3) 7.24 (d, J ) 8.5 Hz, 2H),
1
6.89 (d, J ) 8.5 Hz, 2H), 4.57 (d, J ) 19.8 Hz, 1H), 4.47 (s,
2H), 4.42 (d, J ) 19.8 Hz, 1H), 4.33 (d, J ) 2.3 Hz, 1H), 4.12
(m, 1H), 3.81 (s, 3H), 3.64 (dd, J 1 ) 4.4 Hz, J 2 ) 1.2 Hz, 2H),
2.2 (br s, 3H); 13C NMR (CDCl3) 211.05, 159.83, 129.46, 129.23,
113.84, 76.12, 73.22, 70.58, 66.65, 55.15; HRMS (CI, NH3) calcd
for C13H18O6 (M+) 270.1103, found 270.1108 ( 0.0008.
(3S,4R)-5-p-Meth oxyben zyloxy-3,4-isopr opyliden edioxy-
1-h yd r oxy-2-p en ta n on e (4). A solution of 3 (541 mg, 2.0
mmol) and p-toluenesulfonic acid (45 mg) in freshly distilled
acetone (10 mL) was stirred for 5 h in the presence of molecular
sieves (4 Å, 40 mg). The mixture was filtered, neutralized with
triethylamine (2 mL), and concentrated. The residue was
dissolved in CH2Cl2 and washed with water. The organic layer
was dried and evaporated. The crude product was purified by
flash chromatography (ether/hexane, 2/3) to give 4 (416 mg,
Exp er im en ta l Section
Gen er a l. Aldolase (D-fructose-1,6-bisphosphate-D-glyceral-
dehyde-3-phosphate-lyase; EC 4.1.2.13, from rabbit muscle)
and acid phosphatase (from wheat germ, EC 3.1.3.2) were
purchased from Sigma Chem. Co. Melting points are uncor-
rected. NMR spectra were recorded at 300 MHz (1H) and 75.44
MHz (13C).
1.34 mmol, 67%) as a colorless oil. [R]25 -7.7 (c 1.45, CHCl3);
D
IR (neat) 3600-3100, 1730, 1380, 1370 cm-1; 1H NMR (CDCl3)
7.18 (d, J ) 8.6 Hz, 2H), 6.80 (d, J ) 8.6 Hz, 2H), 4.47 (s, 2H),
4.44 (m, 2H), 4.31 (d, J ) 8.0 Hz, 1H), 4.11 (m, 1H), 3.72 (s,
3H), 3.67 (dd, J 1 ) 10.7 Hz, J 2 ) 3.1 Hz, 1H), 3.55 (dd, J 1
)
10.7 Hz, J 2 ) 5.2 Hz, 1H), 2.88 (t, J ) 5.0 Hz, 1H), 1.39 (s,
3H), 1.34 (s, 3H); 13C NMR (CDCl3) 209.10, 159.21, 129.67,
129.22, 113.72, 111.36, 79.98, 76.90, 73.21, 69.23, 66.12, 55.13,
26.65, 26.01; HRMS (CI, NH3) calcd for C16H22O6 (M+) 310.1416,
found 310.1421 ( 0.0009.
p-Meth oxyben zyloxya ceta ld eh yd e (1). A solution of allyl
alcohol (0.340 mL, 5 mmol) in THF (100 mL) was added
dropwise to a suspension of NaH (180 mg, 7.5 mmol) in THF
(24 mL) at 0 °C under dry atmosphere. The reaction mixture
was stirred at 0 °C for 45 min. Tetrabutylammonium iodide
(25 mg, 0.067 mmol) and p-methoxybenzyl chloride (0.88 mL,
6.5 mmol) were added, and reaction mixture was stirred at 0
°C for 30 min and then at room temperature for 12 h. The
reaction mixture was filtered, and the solvent was evaporated.
The residue was dissolved in ether, and the organic layer was
washed with brine, dried (MgSO4), and evaporated. The crude
product was purified by flash chromatography (petroleum
ether /diethyl ether, 9/1) to give allyl p-methoxybenzyl ether6b
(850 mg, 95%) as a colorless oil. IR (neat) 3100-3000, 1650,
(1′R,2′R)-3-[1′,2′-(Isop r op ylid en ed ioxy)-3′-(p-m eth oxy-
ben zyloxy)p r op yl]-2-octa n oyl-4-olid e (6). A solution of 4
(190 mg, 0.612 mmol) and octanoyl Meldrum’s derivative 5
(prepared by stirring equimolar amounts of 2,2-dimethyl-1,3-
dioxane-4,6-dione and n-octanoyl chloride in CH2Cl2 at 0 °C
for 8 h) in THF was stirred at reflux temperature for 3 h. The
solvent was evaporated, and the residue was dissolved in
hexanes-ethyl acetate 9/1 (15 mL) in the presence of silica
gel (1 g). The mixture was stirred at room temperature for 18
h. Silica gel was filtered, and the solvents were evaporated.
Flash chromatography (EtOAc/hexane, 3/7) gave 6 (202 mg,
1
1300-1100 cm-1; H NMR (CDCl3) 7.27 (d, J ) 8.6 Hz, 2H),
6.89 (d, J ) 8.6 Hz, 2H), 5.87 (m, 1H), 5.31 (ddd, J 1 ) 16.1
Hz, J 2 ) 3.2 Hz, J 3 ) 1.4 Hz, 1H), 5.22 (ddd, J 1 ) 10.0 Hz, J 2
) 2.5 Hz, J 3 ) 1.4 Hz, 1H), 4.46 (s, 2H), 4.05 (m, 2H), 3.80 (s,
3H); 13C NMR (CDCl3) 159.10, 134.79, 130.30, 129.23, 116.86,
113.67, 71.67, 70.75, 55.12; MS (E1, 70 eV) 178 (M+). A solution
of allyl p-methoxybenzyl ether (800 mg, 4.49 mmol) in CH2-
Cl2 (30 mL) was cooled to - 78 °C, and a stream of O2/O3 was
passed through until the persistence of a blue color. Nitrogen
was bubbled through the solution to remove the excess ozone.
Dimethyl sulfide (496 mg, 6.75 mmol) was added dropwise,
and the temperature was raised to room temperature. The
solution was stirred at room temperature for 3 h, and the
71%) as a colorless oil. [R]25 -23.6 (c 1.2, CHCl3); IR (neat)
D
1780, 1700, 1640, 1370, 1360 cm-1; H NMR (CDCl3) 7.23 (d,
1
J ) 8.6 Hz, 2H), 6.84 (d, J ) 8.6 Hz, 2H), 5.43 (d, J ) 8.2 Hz,
1H), 5.03 (d, J ) 19.8 Hz, 1H), 4.83 (d, J ) 19.8 Hz, 1H), 4.55
(d, J ) 11.6 Hz, 1H), 4.45 (d, J ) 11.6 Hz, 1H), 3.97 (m, 1H),
3.78 (s, 3H), 3.72 (d, J ) 4.8 Hz, 2H), 2.91 (t, J ) 7.0 Hz, 2H),
1.57 (m, 2H), 1.43 (s, 6H), 1.27 (m, 8H), 0.86 (t, J ) 5.7 Hz,
3H); 13C NMR (CDCl3) 196.56, 172.47, 170.08, 159.16, 129.69,
129.31, 126.71, 113.61, 110.96, 80.69, 73.76, 73.15, 69.78,
68.88, 55.10, 41.84, 31.53, 28.95, 28.87, 26.64, 23.03, 22.45,
13.92; HRMS (CI, isobutane) calcd for C26H36O7 (M+) 460.2461,
found 460.2464 ( 0.0014.

Synthesis of the Microbial Elicitor (-)-Syringolide
J . Org. Chem., Vol. 65, No. 15, 2000 4531
(1′R,2′R)-3-[3′-(H yd r oxy)-1′,2′-(isop r op ylid en ed ioxy)-
p r op yl]-2-octa n oyl-2-bu ten -4-olid e (7). Compound 6 (175
mg, 0.38 mmol) was dissolved in a mixture of CH2Cl2 (10 mL)
and water (0.5 mL). DDQ (130 mg, 1.5 equiv) was added, and
the mixture was stirred at room temperature. The reaction
was monitored by TLC and quenched after 3 h. The reaction
mixture was diluted with CH2Cl2, filtered, and washed with
saturated aqueous NaHCO3 and brine. The organic layer was
dried (Na2SO4) and evaporated, and the crude product was
purified by flash chromatography (EtOAc/hexane, gradient 1/4
solution was filtered on silica gel (2 g). The solution was
evaporated, and the solid residue was washed with hexane to
give syringolide 2 (49 mg, 77%) as a pale yellow solid.
Recrystallization from pentane/ether (2/1) gave pure syrin-
golide 2 (34 mg, 54%) as a colorless solid. Mp 121-123 °C;
[R]25D -75.0 (c 0.06, CHCl3); lit.2 mp 123-124 °C, [R]24D -75.91
(c 0.22, CHCl3); lit.3a mp 120-122 °C, [R]20 -74.7 (c 0.10,
D
CHCl3); IR (KBr) 3600-3100, 1700 cm-1 1H NMR (CD3-
;
COCD3) 5.38 (d, J ) 1.9 Hz, 1H), 4.68 (d, J ) 10.2 Hz, 1H),
4.49 (br s, 1H), 4.33 (d, J ) 10.2 Hz, 1H), 4.30 (d, J ) 4.2 Hz,
1H), 4.13 (m, 1H), 3.94 (dd, J 1 ) 10.1 Hz, J 2 ) 1.0 Hz, 1H),
3.83 (dd, J 1 ) 10.1 Hz, J 2 ) 2.8 Hz, 1H), 3.10 (s, 1H), 1.89 (m,
2H), 1.70-1.40 (m, 2H), 1.30 (m, 8H), 0.87 (t, J ) 7.0 Hz, 3H);
13C NMR (CDCl3) 172.70, 108.72, 99.00, 92.16, 75.50, 75.32,
74.81, 59.63, 39.36, 32.40, 30.45, 29.40, 24.27, 23.15, 14.18.
HRMS (CI, NH3) calcd for C15H25O6 (MH+) 301.1651, found
301.1657 ( 0.0009.
to 2/3) to yield 7 (107 mg, 83%) as a colorless oil. [R]25 -60.0
D
(c 1.6, CHCl3); IR (neat) 3600-3100, 1790, 1710, 1620, 1370,
1360 cm-1; 1H NMR (CDCl3) 5.39 (d, J ) 8.0 Hz, 1H), 5.10 (d,
J ) 19.8 Hz, 1H), 4.85 (d, J ) 19.8 Hz, 1H), 3.96-3.80 (m,
3H), 3.09 (m, 1H), 2.92 (m, 1H), 1.58 (m, 2H), 1.45 (s, 6H),
1.27 (m, 8H), 0.97 (t, J ) 7.0 Hz, 3H); 13C NMR (CDCl3) 198.75,
173.33, 169.74, 126.75, 110.54, 81.44, 73.82, 69.02, 61.42,
41.92, 31.49, 28.91, 28.84, 26.65, 23.04, 22.46, 13.93; HRMS
(CI, NH3) calcd for C18H29O6 (MH+) 341.1964, found 341.1956
( 0.0010.
(-)-Syr in golid e 2 (8). A solution of 7 (70 mg, 0.21 mmol)
and 10 equiv of TsOH (399 mg, 2.1 mmol) in water/acetone
(1.2/1, 10 mL) was stirred for 56 h at room temperature. The
solution was neutralized with saturated aqueous NaHCO3 (2
mL), and the solvents were concentrated in vacuo. The residue
was taken up in EtOAc (20 mL) and washed with brine (3 ×
5 mL). The organic layer was dried (Na2SO4) and evaporated.
The solid product was redissolved in EtOAc (3 mL), and the
Ack n ow led gm en t. The authors thank the Natural
Sciences and Engineering Research Council of Canada
(NSERC) for financial support.
Su p p or tin g In for m a tion Ava ila ble: Spectrometric in-
formation (1H and 13C NMR) for new compounds. This material
is available free of charge via the Internet at http://pubs.acs.org.
J O991989E