Microbial Secondary Metabolites as a Source of Agrochemicals

Chapter 3

Microbial Secondary Metabolites as a Source of Agrochemicals Downloaded by UNIV OF CALIFORNIA SAN DIEGO on April 15, 2016 | http://pubs.acs.org Publication Date: December 20, 1993 | doi: 10.1021/bk-1994-0551.ch003

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Richard J. Stonard , Stephen W. Ayer , John J. Kotyk , Leo J. Letendre , Carolyn I. McGary , Thomas E. Nickson , Ngo LeVan , and Paul B. Lavrik 3

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1

Agricultural Group, Monsanto Company, 700 Chesterfield Parkway, Chesterfield, MO 63198 Institute for Marine Biosciences, National Research Council of Canada, Halifax, Nova Scotia B3H 3Z1, Canada Bristol Myers-Squibb, Evansville, IN 47721 Sandoz Agro Ltd., Crop Protection Research, CH-4002 Basel, Switzerland 2

3

4

The isolation and structure elucidation of four novel microbial secondary metabolites are described. Diabroticin A (C H N O ) and B (C H N O ) are Bacillus derived pyrazines possessing potent activity against the southern corn rootworm. Arginidiene (C H N O ) and phytophthoradiene (C H N O ) are weakly fungicidal dipeptides incorporating an unusual cyclohexadiene moiety. 16

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6

6

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13

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3

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6

5

5

5

6

Several of the top 20 pharmaceuticals in gross sales in 1992 were natural products or close derivatives thereof (eg. Mevacor, Ceclor). Of the top 20 crop chemicals in this same period, only one class of compounds, the pyrethroids, are based on a natural substance, the plant metabolite pyrethrin. Three active areas of chemistry have the potential to impact this situation within the next ten years: the methoxyacrylate family of fungicides based upon the fungal metabolite, strobilurin; Basta, a postemergent broad-spectrum herbicide based upon the Streptomyces viridochromogenes metabolite phosphinothrieylalanylalanine; and avermectin, and its semi-synthetic derivatives, insecticides originating from Streptomyces avermitilis. Several reasons are cited for the success of natural products in health care as compared to agriculture. The most prevalent of these are; their frequent lack of amenability to cost effective total synthesis, or that they are too structurally complex to serve as models for analogue synthesis programs; the absence of environmental stability; insufficient efficacy (typically too specific/ too narrow a spectrum of activity); and the high costof-goods of the natural substance per se. The latter is a particularly significant distinction between crop chemicals and pharmaceuticals. For example, the average costs per treatment in 1992 for some of the more common agrochemicals were: cotton caterpillar control, $10/acre; wheat foliar disease control, $17/acre; and broad and narrow leaf weed control in 0097-6156/94/0551-0025$06.00/0 © 1994 American Chemical Society

Hedin et al.; Natural and Engineered Pest Management Agents ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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NATURAL AND ENGINEERED PEST MANAGEMENT AGENTS

corn, $17/acre. For comparative purposes, perhaps the least expensive fermentation product, penicillin at $50/lb, as a hypothetical agrochemical would have to be used in the range of 40g/acre or less in order to be economically viable. This use rate is within the range of only a few modern crop chemicals. While significant hurdles to commercialization exist, natural products offer several potential advantages including: target specificity; novel mechanisms of action; and unique structures upon which to base synthesis efforts. Consequently, several major agrochemical companies have been or are presently engaged in natural product screening programs. In this chapter we report on the discovery in our laboratories of a new class of natural insecticides derived from micro-organisms, the diabroticins, and two structurally related microbial secondary metabolites, arginidiene and phytophthoradiene, possessing antifungal activity. DjabrQtiçing Microbial secondary metabolites have proven to be a modestly significant source of insecticidally active substances. Avermectin, a metabolite of Streptomyces avermitilis, and its derivatives have found utility as commercial pest control agents (2). Others such as pyrrolnitrin have served as the basis for the development of novel synthetic insecticides (2). As part of an investigation of antiinsectan metabolites produced by microorganisms, we identified a new family of water soluble pyrazine-amino acid conjugates produced by Bacillus subtilis having potent activity against the southern corn rootworm, Diabrotica undecimpunctata. Herein, we describe the isolation, characterization and biological activity of the most abundant members of this family, diabroticin A (1) and diabroticin Β (2). The diabroticins were isolated from fermentation broths of B. subtilis (isolate ALB 102) obtained from a soil sample collected in an oat field in South Dakota. Bioassay guided purification versus D. undecim punctata afforded 1, 13 mg/L, and 2, 3 mg/L, as colorless oils. The 0.45μπι filtered broth was purified by open column, gradient cation

1 2 3 4 5 6 11 12

Κ,Κχ,Η^Η^^ΟΗ R,Ri,R2,Ra =H R , R i = Ac,R2 = H , R = OAc R, Ri = Ac-cfe, Ra = H, R = OAc-cfe R = H, Ri, R2 = =CHN(CH ) , R = OH R, Ri = H, R2 = 2,4-dinitrophenyl, R = OH R , R i = Ac, R 2 , R » H R , R = H, Ri, R2 = =CHN(CH ) 3

3

3

2

3

3

3

3

3

2


3. STONARDETAL.

Microbial Secondary Metabolites

27

+

exchange chromatography (IRC 50 NH4 form; 0-5N NH4OH) followed by repeated cation exchange HPLC (Whatman SCX). Crude broths showed significant activity against Colorado potato beetle, boll weevil, mosquito larvae, Staphylococcus aureus and Micrococcus lutea, but were inactive against European corn borer, Escherichia coli, B. subtilis and Pseudomonas aeruginosa. Diabroticin A was also isolated (7 mg/L from 1.8L of filtrate) from fermentation broths of B. cereus (isolate B739) isolated from a soil sample taken from a cantaloupefieldin Georgia. Mass spectral analysis (EI, CI, +/- FAB) of diabroticin A (1, [OC]D (C 2.1 in MeOH) -6.7) failed to detect a molecular ion or characteristic mass fragments. The molecular formula of 1, Cie^eNôOe (MW 400 Da, 6 sites of unsaturation), was deduced on the basis of mass spectral analysis of acetylated, dimethylamidine, and hydrogenated derivatives (Table I). A UV maximum at 276 nm in conjunction with a downfield singlet resonance at 58.67 in the H NMR spectrum (Table II) and a C NMR spectrum (Table III) displaying resonances at 5145.0 and 5157.2 suggested the presence of a heteroaromatic moiety. Absorption bands at 1660 and 1560 cm in the FTIR spectrum in combination with a C NMR APT resonance at 5173.52 (qC) indicated the presence of a secondary amide functionality. The Ή NMR and C NMR spectra of 1 in D2O contained resonances for 9 protons and 8 carbons, respectively, denoting a high degree of molecular symmetry. Acetylation of 1 afforded hexa-acetyldiabroticin A (3, Table I). Derivatization with Ac20-dg (4) and analysis by FABMS and *H NMR confirmed the synthetic origin of all six acetyl groups. The H NMR spectrum of 3 in CDCI3 (Table II) exhibited two acetyl methyl singlet resonances at 52.19 (12H) and 52.10 (6H) and two D2U-exchangeable two proton doublet resonances at 57.06 (J=10.0 Hz) and 56.97 (J=9.0 Hz). In conjunction with the molecular formula and IR data, this indicated the presence of four hydroxyl and two primary amine groups in 1. The presence of the two primary amine groups was confirmed by the reaction of 1 with iV,iV-dimethylformamide dimethyl acetal (DMF/DMA) to give the bisdimethylamidine derivative 5 (see Table I). In addition, treatment of 1 with Sanger's reagent led to the formation of 6 with incorporation of two 2,4-dinitrophenyl units. The proton decoupled N NMR (40.5 MHz, 5 in ppm upfield of internal nitromethane at 0 ppm) spectrum of 1 contained three signals at 5 -61.7, 5 -263.9 and 5 -341.9 indicating, when taken in conjunction with the molecular formula, the presence of two aromatic, two amide and two amine nitrogen atoms, respectively. Hydrogénation of 1 resulted in the uptake of three moles of hydrogen (Table I). Subsequent acetylation of the hydrogénation product, 7, gave an octaacetyl derivative, 8, having a protonated molecular ion at ml ζ 743 Da (+FABMS; confirmed by a parallel Ac20-c?e acetylation experiment), thereby confirming that the heteroaromatic ring was six membered and contained two nitrogen atoms.


23

X

13

1

13

13

1

1 5


28


Table I. HRFAB Mass Spectral Data for Derivatives of Diabroticin A Derivative Hexa-acetyl (3) Bisdimethylamidine (5) Hexahydro (7) b


ΔΜ mmu 2.4 3.6 0.5

Formula C 8H40N Ol2 C 2H38N 06 0ΐ6Η34Νβθ6

a

a

2

6

2

8

b

Ac 0/4-pyrroli(Iinopyridine, rt, 24 hr H2/Pt02/TPA rt, 1 atm, 4 hrs, 3 moles absorbed. 2

Table Π. *H NMR Data for Diabroticin A (1), Β (2) and Hexa acetyldiabroticin A (3) diabroticin position A (IV 3 6 1'

b

e

2' 3'

8.67 (2H, s)

8.46 (2H, s)

8.58 8.45 (2H, d, 10.5) 3.13 2.86 (2H, dddd, 10.5,10,3,3) 4.24 (2H, dd, 12, 3) 3.73 (2H, dd, 12, 3) 3.88 (2H, d, 10.) 3.54 (2H, dq, 9, 7) (2H, d, 9) (6H, d, 7) 1.31 4.75 4.16 3.63 3.58 1.34 (12H, s) (6H, s)

4.97 (2H, d, 7)

5.64

e

4.28 (2H, brq, 7) 4.76 3.76 (4H, d, 7) 4.61 4.17 7.06 3.94 (2H q, 7) 4.50 6.97 1.44 (6H, d, 7) 1.24

4' 6' r 8' 1" 2" 3" 6" 8" OAc NAc

diabroticin B(2V

hexacetyldiabroticmA(3)

2.19 2.10

a

(1H, d,1.3) (1H, d,1.3) (1H, dd, 14,4) (1H, dd,14,10) (1H, m) (1H, dd, 11,1.5) (1H, dd, 11, 6) d

(1H, q, 7)

(3H, d, 7) (1H, d, 8.5) (1H, m) (2H, m) (1H, q, 7) (3H, d, 7)

b

d

Ή NMR spectra were recorded at 300 MHz (D 0) or at 400 MHz in CDCI3 or at 500 MHz in CD3OD. assignments of 6' and 6" may be interchanged. Assignments were aided by DQCOSY (3) experiments. J values are in hertz, and chemical shifts, δ in ppm relative to HOD at δ 4.63 ppm. Signals correspond to position 3 and 6. 2

c

e


3. STONARDETAL.


29

Compound 7 showed a UV maximum at 222 nm and disappearance of the aromatic protons observed at δ 8.67 in 1. Thus, the twelve heteroatoms present in 1 could be accounted for by 4 -OH, 2 -HNCO-, 2 -NH and 2ArN and the six sites of unsaturation could be ascribed to two amide groups and one six membered aromatic ring containing two nitrogen atoms. 2


ο

OR

OR

7 R=H 8 R = Ac The structure of diabroticin A (1) was established by a series of NMR experiments. The COSY spectra of 1 and of hexa-acetyldiabroticin A (3) showed essentially the same proton-proton correlations, fully consistent with two isolated spin systems as illustrated in partial structures A and Β for hexacetyldiabroticin A (3). The two amide proton doublet resonances at δ7.06 (Hb) and δ6.97 (H ) in 3 showed strong correlations with a methine resonance at δ4.76 (H , dddd) and a methine resonance at δ4.50 (H , dq), respectively. In addition, observation of a correlation between the aromatic proton resonance at δ8.67 in 1 and the methine doublet resonance at δ4.97 (Ha , not resolved in ID - Ή NMR) established the connection of the heteroaryl group as shown in A. c

e

g

Determination of the side chain structure was facilitated by two acid hydrolyses and subsequent derivatizations of the reaction products. Treatment of 1 with 2N-HC1 in the presence of trace amounts of methanol at 85°C for 3 hr followed by acetylation with


30

NATURAL AND ENGINEERED PEST MANAGEMENT AGENTS 1 3

Table ΠΙ. C NMR Data for Diabroticin A (1), Β (2) and Hexaacetyldiabroticin A (3) hexa-acetylcarbon diabroticin diabroticin diabroticin no. Β (2) A(3) LUT a


b

2 5 3,6 r 1" 2', 2" 3,3" 5', 5" 7', 7" 8', 8" CH3C=0 £H C=0

157.2 C 145.0 73.8

CH CH

58.5 62.5 173.5 51.9 19.4

CH CH C C CH

d

3

151.4

164.5 C 155.4 C 144.2 144.0 , 146.0 CH 70.9 38.0 CH 73.6 CH 54.3 ,58.4 CH 49.7 65.5 , 62.0 C H 63.2 173.5, 173.5 C 171.6 47.8 52.0,52.0 C 21.0 26.0 , 19.8 C H 171.5,170.6,169.5,169.4 15.0, 23.3 C

C

d

2

2

C

C

e

C

2

e

3

e

3

a 13

b

c

C NMR were recorded at 75 MHz in D 0 or, in CDC1 . Assignments of carbons 3/6, 272", 373", 878" may be interchanged. Assignments for 1 and 2 were aided by APT (4) experiments. Chemical shifts, δ in ppm downfieldfromTMS. Signals represent positions Γ and 1". 2

3

d

Ac 0/4-PP resulted in the formation of compounds 9 and 10. Compound 9 exhibited a protonated molecular ion ai m I ζ 511 Da (+FABMS). The Ή NMR spectrum of 9 contained an aromatic proton singlet resonance at δ8.62 and three acetyl methyl resonances (δ2.17, δ2.07 and δΐ.92), consistent with the formation of a symmetrical degradation product of 1 lacking two alanine residues. More vigorous acid hydrolysis (6N HCl/llO'C/24 hr) of 1 resulted in the formation of two mole equivalents of alanine as determined by quantitative PTH and ΟΡΑ amino acid analysis. As the IR spectrum of diabroticin A (1) contained only amide carbonyl bands and since reaction with DMF/DMA and Sanger's reagent gave bis and not tetra derivatives, it was concluded that A and Β were connected as shown in partial structure C for diabroticin A (1). The nature of the six-membered aromatic heterocyclic ring was revealed by comparison of spectral data between diabroticin A (1) and several model compounds (see Table IV). As mentioned above, NMR analysis of compound 1 showed one aromatic nitrogen resonance at δ 61.8, two aromatic carbon resonances both adjacent to nitrogen (δ144.96, δ157.15) and one aromatic proton resonance at δ8.67, consistent with a 2,5-disubstituted pyrazine. The 2,6-disubstituted pyrazine possibility was ruled out on the basis of the equivalence of the 2 new acetyl methyl signals 2


3. STONARD ET AL.


31

OAc


9

10

f

Table TV. NMR and UV Comparison of 1 and Related Model Compounds UV Xmax

15

240 nm

Pyrimidine

NNMR

13

a

CNMR

b

e

122, 160,157 7.36,8.78, 9.26 7.54, 9.24 128, 153 145 d, 153 s 8.3 s

-89»

d

Pyridazine* 247 nm 2,6-Dimethylpyrazine 275 nm

+17« +53 (t), +56 (s ) -52 ND> -61

g

c

h

br

g

2,5-Dimethylpyrazine 274 nm Fructosazine (5) 276 nm Diabroticin A (1) 275 nm

h

145 d, 153 s 8.3 s 145 d, 158 s 8.7 s 145 d, 157 s 8.67 s

40.5 MHz, nitromethane as external reference at Oppm in D2O. See Table 2 and 3 for detail. H-4, H-3, resp., J ,4=4.7 Hz, J =9.0 Hz. H-2, H-4, H-5, resp., J ,5=5.0 Hz. C-5, C-2, C-4,6 resp. 'In D 0 unless indicated. Aromatic resonances only. CDCl3. CD30D. *Not determined. a

b

c

d

3

4 j 5

e

4

2

g

h

observed in Ή NMR spectra of 8, resultingfromthe acetylation of the hydrogénation product (7) of 1. Furthermore, 2,6-disubstituted pyrazines show two discrete aromatic nitrogen signals in the proton decoupled N NMR spectrum (Table IV). Consequently, the structure of diabroticin A is concluded to be as shown in 1. Confirmation of this regiochemical assignment was provided by the structure determination of diabroticin Β as discussed below. As was the case with diabroticin A ( 1), diabroticin Β (2) failed to yield a molecular ion or useful mass fragments by either EI, CI or +/- FABMS. The molecular formula of 2, C i H 8 N 0 5 (MW 384 Da, 6 sites of unsaturation), was deduced by mass spectral analysis (+FABHRMS) of bisdimethyl-amidine ( 12) and pentacetyl derivatives (11). Comparison of 15

6

2

6


32

NATURAL AND ENGINEERED PEST MANAGEMENT AGENTS 1

1 3

the molecular formulae, UV, H NMR and C NMR spectra clearly indicated that 1 and 2 differed only by the presence of a single hydroxyl group on one of the two side chains. This was confirmed by treatment with acetic anhydride resulting in the incorporation of five acetyl units (CIMS, ml ζ 594 Da; Ac20-ds, CIMS, m I ζ 609 Da) versus six as in 1. The observation of resonances attributable to a benzylic methylene group at 53.13 (dd, J = 14, 4 Hz) and 52.86 (dd, J = 14,10 Hz) in the Ή NMR spectrum and at 838.0 in the C NMR APT spectrum of 2, assigned the missing hydroxyl in 2 to one of the two benzylic positions (i.e. C-l'). Consequently, the structure of diabroticin Β was concluded to be as shown in 2, above. Further evidence for the presence of two intact alanine residues in diabroticin Β (2) was provided by acid hydrolysis (6N HCl/110°C/24 hr) followed by quantitative PTH and ΟΡΑ amino acid analysis. Treatment of 2 with DMF-DMA resulted in formation of a bisdimethylamidine adduct (FABMS, m I ζ 494 Da) as predicted. Confirmation of the 2,5-pyrazine substitution pattern in diabroticin A (1) and Β (2) was facilitated by the unsymmetric nature of 2. Two chemically nonequivalent aromatic proton resonances were observed in the H NMR spectrum at 58.58 and 58.45 as 1.3 Hz doublets consistent with J3.6 in mono-substituted pyrazines (£). 2Methylpyrazine and other mono-substituted pyrazines show long range coupling constants of J3-6 = 1.33-1.54 Hz, J3-5 = 0.01-0.35 Hz and Js-6=2.42.9 Hz. Isolated diabroticin A ( 1) was active against first instar larvae of Ζλ undecimpunctata at 2-4 ppm in an in vitro diet incorporation assay(7). Similarly, diabroticin Β (2) was active at 25-50 ppm. No effect was observed on egg hatch with either compound, and 1 did not deter feeding of D. undecimpunctata first instar larvae in a choice assay (S). The toxicity of the diabroticins to mammals has not been determined. Several metabolites related to 1 and 2 have been isolated from fermentations of B. subtilis (isolate ALB 102) but have not been fully characterized. NMR analysis suggests that they differ from 1 in their amino acid substitutions.


1 3

X

Argjr4dierie and Phytophthoradiene Several microbial metabolites have found utility in crop agriculture as control measures for plant pathogenic fungi, mainly in Japan (eg. blasticidin, polyoxin and validamycin). Significant patent activity has occurred during the past few years surrounding a new class of methoxyacrylate fungicides based upon the fungal metabolite, strobilurin (9). These compounds have not been commercialized to date. During the course of screening for new fungicides from micro organisms we encountered two closely related dipeptides with modest activity principally against Phytophthora spp. Herein we describe the isolation and structure elucidation of a novel fungicidal dipeptide,



3. STONARDETAL.


33

arginidiene (13), and briefly the related compound, phytophthoradiene ( 14). Both molecules contain a previously unreported amino diacid moiety and yet interestingly, arginidiene was isolatedfromBacillus megaterium while phytophthora-diene was isolated from a Streptomyces sp. Arginidiene was isolated from the cell-free filtrate of a 10 liter fermentation of B. megaterium (isolate Β18252), obtained from a sand and soil sample collected in San Diego, California. The cell free fermentation broth was loaded onto a 600 X 100 mm column of AG50WX8 cation exchange resin and eluted with 2L of 10% (ν/ν) NH4OH. The active fraction was concentrated in vacuo and chromatographed on a 150 X 30 mm RP flash column eluting with 250 ml each of water, 1:1 methanoliwater and methanol. The active water fractions were combined, concentrated in vacuo and further purified on an LH-20 column (900 X 25 mm) using 25% methanol in water. The active fractions (ca. 5 grams of hydroscopic material) were again combined and purified on an LH-20 column (500 X 25 mm) using a water mobile phase. The bioactive fractions were then combined, lyophilized and dissolved in 8 mL of 0.25M ammonium formate, pH 9.1, and further purified on a QAE-Sephadex column (750 X 20 mm) which had been previously equilibrated in the starting buffer. The column was washed with 150 mL of 0.25M ammonium formate, pH 9.1, followed by a gradient made from 200 mL each of pH 9.1 and pH 6.3 ammonium formate. The active fractions were combined and crystallized from water to yield 18 mg of 1. The sterile fermentation broth of B. megaterium Β18252 was initially observed to be active against Phytophthora megasperma, the causative agent of soybean root rot, in an agar-based in vitro assay. Subsequent testing of purified arginidiene demonstrated in vitro activity against Botrytis cinerea, the causative agent of eggplant gray mold. High resolution FABMS analysis of arginidiene (13) provided a protonated molecular ion of ml ζ 340.1656 Da corresponding to a molecular formula of C14H21N5O5 (ΔΜ 3.5 mmu, 6 sites of unsaturation). Treatment of 13 with 6N HC1 for 12 hours produced arginine and isophthalic acid, accounting for all of the carbon atoms present in the molecule. Ή NMR and C NMR spectra of 13 (Table V) contained several resonances which could be readily assigned, except for the amide carbon, by comparison with spectra for arginine. An APT spectrum clearly indicated that the remaining carbons could be accounted for by the presence of a cyclohexadiene ring containing one aliphatic and two olefinic methines, one aliphatic methylene and two quaternary olefinic carbons, rather than the fully aromatic isophthalic acid obtained as a product of hydrolysis. The geminal coupling constant of 23 Hz observed for the two aliphatic methylene protons (83.19, 83.28 ppm) is consistent with the characteristically large couplings observed in 1,4-cyclohexadiene rings (see partial structure D). Additional support for this assignment was provided by the observation of large (6.8 and 8.3 Hz) homo-allylic coupling constants between the methylene protons and the aliphatic methine proton at 85.4 ppm (HI, see D). The allylic juxtaposition of the olefinic methine protons (86.52, 86.54 ppm) and the aliphatic methylene protons was further suggested by the observed coupling constant (1.9 Hz, see D). 13


34

NATURAL AND ENGINEERED PEST MANAGEMENT AGENTS 13

Table V. Ή and C NMR Data for Arginidiene (13) and Phytophthoradiene (14) CO,H

C0 H Downloaded by UNIV OF CALIFORNIA SAN DIEGO on April 15, 2016 | http://pubs.acs.org Publication Date: December 20, 1993 | doi: 10.1021/bk-1994-0551.ch003

2

14

13 R = CH NHC(=NH)NH 2

Carbon/

Proton

13

C NMR δ ppm (mult)

a

R = CONH

2

Ή NMR δ ppm" (mult)

13

CNMR δ ppm a

2

ΉΝΜ δ ppm'

5.35m 44.1 CH 5.40 ddd, «7=3,4,8.3,6.8 48.5 130.2, 130.3 6.68m 125.3, 125.4 CH 6.52, 6.54 m 138.9, 139.0 135.0, 135.2 C 25.3 3.12m 3.19 ddt, «7=23,8.3,1.9 29.5 CH 3.18m 3.28 ddt, «7=23,6.8,1.9 170.9 Acid 173.4 C Amide 166.8 161.9 C 4.03t, 51.4 55.5 α CH 4.121, «7=6.5 «7=7 β 26.4 2.10 m 28. CH 9 2.23 q*7=7 2.51t, 32.8 21.9 1.84 m CH γ «7=7 δ 38.7 3.41 td, «7=6.4,1.7 170.9 CH Guanidine 155.2 C 1 2,6 3,5 4

2

b

2

2

2

Proton and C NMR spectra were recorded in D2O at 300 and 75 MHz respectively. Carbon multiplicities were determined using the APT (4) experiment. Assigments were made based upon a series of HMBC and HMQC experiments. a

13

b

Assignment of the protonated carbons was facilitated by a protondetected heteronuclear one bond correlation (HMQC) experiment. Assignment of key carbon resonances and the determination of the point of attachment of the arginine and cyclohexadiene groups was accomplished by the analysis of three proton-carbon HMBC experiments which differed in their MCH values (see partial structure E). The arginine carbonyl carbon (δ 166.8 ppm) was assigned based upon its correlation with the arginine α-methine (δ 4.12 ppm) and β-methylene (δ 2.10 ppm) protons. The cyclohexadiene carbonyl carbons were assigned based upon their correlations to the olefinic protons.


3. STONARD ET AL.


Js

J s


35

1.9 Hz

8.3,6.8 Hz

D

Ε

Observation of a correlation between the methine proton (HI, δ 5.4ppm) and the amide carbonyl carbon (δ 166.8 ppm) in an HMBC spectrum obtained with J c H = 4 Hz demonstrated that the cyclohexadiene moiety was connected to the arginine residue as shown in 13 (see E). This was confirmed by a COSY experiment performed in pH 4.5 10% D2O in which HI was coupled to a previously unobserved proton resonance at δ 8.9 ppm which was assigned to the amide proton (see E). Further evidence for this attachment was provided by a Ή - ^ Ν HMBC experiment in which HI correlated to an amide nitrogen (δ 216 ppm, see E). n

F The structure proposed for arginidiene ( 13), based upon chemical and spectroscopic evidence, was subsequently confirmed by single crystal Xray analysis (see F). Shortly after the elucidation of arginidiene we isolated (5-10 mg/L) a closely related molecule from a fungicidally active (in vitro activity was observed versus P. infestans and P. megasperma) fermentation broth of Streptomyces sp. (isolate A16240) isolated from a soil sample collected in a wooded savannah in Boual, Central West Africa. The structure of phytophthoradiene (14) was readily deduced by comparison of spectroscopic information with data gathered on arginidiene (Table V). Hydrolysis of 14, as described for 13 above, afforded isophthalic acid ((Μ Η)- ml ζ 165 Da) and glutamic acid ((M+H) ml ζ 148 Da). The presence +


36


of glutamine as opposed to glutamic acid in the molecule was confirmed by ion spray mass spectral analysis; (M+H) ml ζ 311.8 Da; (M+Na) m/z 333.8 Da. The position of the ring was deduced from a series of HMBC experiments ( JcH = 4 or 8 Hz) wfich clearly demonstrated a correlation between C-Γ and H-l. +

+

n


Conclusion The discovery and subsequent development of microbial secondary metabolites that have direct utility in crop agriculture is rare and costly. In general structural complexity will limit utility unless synthesis and modelling lead to simplified analogues with favorable cost-of-goods per treatment. At Monsanto we have been involved in the search for crop chemicals of natural origin for several years. While numerous active compounds have been characterized, the diabroticins and the recently reported herbicide, herboxidiene ( 10), are by far the most highly active that we have discovered to date. Acknowledgments We would like to thank Nancy Biest, Minhtien Tran, Barb Reich, Mike Prinsen, Jay Pershing, Steve Sims, John Greenplate and Margann MUlerWideman for biological support, and Hideji Fujiwara and Tom Solsten for performing the HRMS analyses. Literature Çited 1. Campbell, W. C.; Burg, R. W.; Fisher, M. H.; Dybas, R. A. In Pesticide Synthesis Through Rational Approaches, ACS Symposium Series No. 255; ACS,Washington, DC, 1984; Chapter 1. 2. Addor, R. W. et al., In Synthesis and Chemistry ofAgrochemicals III, ACS Symposium Series No. 504, ACS, Washington, DC, 1992; Chapter 25. 3. Piantini, U.; Sorenson, O. W.; Ernst, R. R. J. Am. Chem. Soc. 1982, 104, 6800. 4. Patt, S. L.; Shoolery, J. N. J. Magn. Reson. 1982, 46, 535. 5. Tchushida, H.; Komoto, M.; Kato, H.; Fujimaki, M. Agr. Biol. Chem. 1973, 11, 2571. 6. Cox, R. H.; Bothner-By, A. A. J.Phys.Chem. 1968, 5, 1646-1649. 7. Marrone, P.G.; Ferri, F. D.; Mosley, T. R.; Meinke, L. J. J. Econ. Entomol. 1985, 78, 290. 8. Higgans, R. Α.; Pedigo, L. P. J. Econ. Entomol. 1968, 72, 238-244. 9. Beautement, K; Clough, J. M.; de Fraine, P. J.; Godfrey, C. R. A. Pestic. Sci. 1991, 31, 499-519. 10. Isaac, B. G.; Ayer, S. W.; Elliott, R. C.; Stonard, R. J. J. Org. Chem. 1992, 57, 7220. RECEIVED September 21, 1993


Microbial Secondary Metabolites as a Source of Agrochemicals

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