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First Biosynthetic Studies on Trail Pheromones in Ants.

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[4] M. Schmittel. U.Baumann, Angex' Chem. 1990,102,571-572, Angew. Chem.
nization of the colonies in which they live. The division of labor
I n t . Ed. Engl. 1990,29, 541 -543; b) M. Schmittel, M. Rock, Chem. Ber. 1992,
of the thousands of individuals living in an ant-state can only
125, 1611-1620.c)M.Schmittel, Top.Curr.Chem.1994,169,1X3-230;d)M
work with the aid of a communication system. Communication
Schmittel. A. Langels, Liehigs Ann Chem. 1996.999-1004.
in the ant-state largely depends on chemical signals. When, for
[5] The synthesis ofenol E was straightforward. Dimesitylketene, prepared readily
from dimesitylacetic acid, was allowed to react with 4-dimethylaminophenylexample, a new food source is discovered, the successful scout
lithium to yield enol E after aqueous workup. The pure compound was ohmarks the way back to the nest with a trail pheromone. Nesttained after column chromatography and recrystallization from ethanol. M.p.
205- 208'C;IR(KBr):a = 3494cm~'(s,0H);'HNMR(CDCI3,200MHz): mates are stimulated to follow this odor trail to the newly discovered resource, which leads finally to the well-known ant6 = 1.90.2.20, and 2.26(3 s, coalescence, lXH, Mes-CH,). 2.91 (s,6H.NCH3),
5.05 (s, 1 H. OH), 6.49 (d. 2 H , J = 9.0 Hz, Ar-H), 6.67 (s, 2 H , Mes-H). 6.87
streets. Only for a few ant species (about 20-25) is the chemical
(s.~H,M~~-H),~.~O(~,~H,J=~.OHZ,A~-H).
structure of these trail pheromones known.".
Biosynthetic
[6] All potentials in this paper are referenced against the ferroceneiferrocenium
on
trail
pheromones
in
insects
generally
and ants in
studies
couple (Fc Fc') and were recorded at a scan rate of 100 mVs-' unless noted
particular have not been reported so far.[31
otherwise ( E , = 0.39 V vs SCE).
[7] The oxidation reaction was quenched after one minute by addition of a soluRecently we identified in the rectal bladder of several ant
tion of sodium bicarbonare. B: ' H N M R (CDCI,, 200 MHz): 6 = 1.85 (s, 3H,
species of the subfamily Formicinae two classes of compounds
p-CH,-Mes).2.02 (s,6H,o-CH3-Mes), 2.35 and 2.37 (2s, 6 H , 4 - and 6-CH3that function as trail pheromones, namely 3,4-dihydroisocouBf), 2.50 (5. 3 H . 7-CH3-Bf), 2.93 ( s , 6 H , NCH,), 6.60 (d. 2 H , J = 9.2 Hz,
marins and 3,5-dimethyl-6-alkyl-tetrahydro-2H-pyran-2-ones
Ar-H). 6 71 (s, 1 H. 5-Bf-H). 6.96 (s, 2 H , Mes-H), 7.39 (d, 2 H , J = 9.2 Hz,
Ar-HI. Bf = benzofuran.
(6-lactones) .I4] In addition to the biologically active compo[8] K. Izutsu. Acid- Base Dissociation Constants in Dipolar Aproric Solvents.
nents the rectal bladder often contains other derivatives of the
Blackwell. Oxford, 1990.
same class of compounds, but no behavioral function could be
[9] In structurally related ammonium ions hindered rotation about the C-N
attributed to them. Table 1 shows the species and their respecbond was reported: U Pindur, Arch. Pharm. 1980,3f3, 301-306.
[lo] M. J. S . Dewar. E. G. Zoebisch, E. F. Healy, J J P. Stewart, J: Am. Chem. SOC.
tive trail pheromone examined. We investigated whether the
1985. 107,3902-3909.
3,4-dihydroisocoumarins and S-lactones derive from related
' using
[ l l ] Unfortunatel). attempts to measure the kinetics of the cyclization of C
biosynthetic
pathways.
fast scan cyclic voltammetry failed due to rapid electrode coating. The occurAs can be seen in Table 1, the rectal bladders of many
rence of an irreversible wave at li = 500 mVs- ' for R --t C' only indicates that
the half-life of cation C' is less than I s. For comparison, kinetic investigations
species contain 3,4-dihydro-8-hydroxy-3-methylisocoumarin
(1,
on the cyclization of the related p-anisoyl-dimesitylmethyl cation provided
mellein). This compound is fairly ubiquitous in n a t ~ r e . [ ~For
1
k = 1.4 x 10's I : M. Schmittel, M. Rock, unpublished results.
instance, it has been found in the species Aspergillus and in
[12] Notably. in all multisweep experiments the current of the reduction wave at
various insects like the Oriental fruit moth, Grapholita moEpL= 0 69 V increased with the number of cycles.
[13] Cyclic voltammetric investigation of pure B revealed two reversible waves at
lesta Busck,[61in the wing glands of the bumble-bee wax moth,
E , = 0.23 V and at 0.72 V, the first of which was assigned to B B + and the
Aphomia sociella L.,[71in the gaster of Rhytidoponera rnetallilatter to B * + EL+
ca,['I
and in the mandibular gland secretion of some carpenter
I141 Wave 111 reflects two oxidation processes, namely the oxidation of X + t o X"+
ant species, for example Camponotus pennsylvanicus,
and of B + to Bz*.thus explaining the high peak intensity of wave 111.
[15) The almost complete disappearance of wave 11 in the multisweep experiment
C. herculeanus, and C. noveboracensi~,~~~
to which we can add
starting with E - H + is in full agreement with the proposed mechanism. AcC. rufbes and C . silvicola according to our own investigacordingly, the depletion of E-H' in the diffusion layer is expected as a result
ti~ns.[~'.gl
It was therefore tempting to assume that the ants take
of the redox process between E-H' and E". In addition, the wave of
up mellein (for example with food), methylate it during diges6 + B" is no longer reversible in the presence of acid because the enderyonic disproportionation 2 B + + B 2 + + B is followed by quantitative prototion at various positions on the aromatic ring and then store the
nation of B.
methylated derivatives in the rectal bladder as trail pheromones.
[16] The deprotonation of E'+ is a fast chemical reaction ( k z lo4 sCi) since even
To test this hypothesis we synthesized (R,S)-[D,]mellein (D5-l]
at ti = 15000 V b - ' no sign of reversibility was found in the CV experiment.
(Figure 1) by a method adapted from the literature['*I and fed
[I71 M. F Marcus, M. D. Hawley. J Electroanal. Chem. 1968, 18, 175-183.
I181 a) B. Speiser, A Rieker, Electrochim. Acra 1978,23.983-989; b) B. Speiser, A.
the labeled compound to C. rufipes, C. silvicola, and L. niger,
Rieker. J. Elecrrriunrrl Chem. 1979, fO2, 373-395.
--f
A
First Biosynthetic Studies on
Trail Pheromones in Ants**
1
Hans Jurgen Bestmann,* Elke Ubler, a n d
Bert Holldobler
2
3
Qq-q&
Ants are amongst the most important animals in almost all
terrestrial ecologic systems. There are about 9500 known species
of ants, and their ecological success is based on the social orga-
OH 0
O H 0
OH0
[*] Prof. Dr. H. J. Bestmann. Dr. E. Ubler
Instttut fur Organische Chemie der Universitat Erlangen-Niirnberg
Henkestrasse 42, D-91054 Erlangen (Germany)
Fax: Int. code +(9131)85-6864
Prof. Dr. B Holldobler
Theodor-Boveri-Institut, Lehrstuhl fur Verhaltensphysioiogie und Soziobiologie der Untversitat Wiirzburg (Germany)
[**I Pheromones. Part 107. This work was supported by the Deutsche Forschungsgemeinschaft. We thank Dr. Kunesch for stimulating advice. Part 106:
E. Janssen. H. J Bestmann. B. Holldobler, F. Kern, K. Tsuji, J. Chem. Ecol.,
in press
Angew. Chem. In1 Ed. Engf.
1997. 36. No. 4
5
4
4T
a
0
OH 0
0 VCH VerlagsgesellschafrmhH, 0-69451
7
Weinheim, 1997
8
6
qo
+
*
9
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395
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DS-I
11
D3C-CK2--COOH
4q
D,
OH
12
7
OH
0
3
0
Ds-5
4-4
D3
OH
OH
0
Ds-6
0
Dg-7
Figure 1. Deuterium-labeled precursors and labeled 3,4-dihydroisocoumarins obtained after feeding with CD,CH,COOH.
which all use “methylated” melleins as trail pheromone. After a
feeding period of 14 days we excised the hindguts of the freshly
killed worker ants and analyzed the chemical composition of the
rectal bladders by gas chromatography coupled with mass spectrometry (GC-MS), using a solid-sampling injection technique.[”] Since in all cases we found large quantities of the
labeled substance D,-1 among the other well known volatile
constituents, we were certain that the labeled compound had
been taken up; however, none of the trail pheromones (see
Table 1 ) had incorporated the deuterium label.
In subsequent experiments we fed the ants [D,]methionine
( l l ) , a compound known to transfer methyl groups during
biosynthesis, but again no evidence was obtained to support the
incorporation of labeled methyl groups into trail pheromones.
According to these results the modification of mellein by methylation as a possible biosynthetic pathway to the trail
pheromones of C. rufipes, C. silvicola, and L. niger could be
excluded.
Biosynthetic studies on Aspergillus mellusr‘21and Rhytidoponeru chalybaea“31 with labeled acetate demonstrated that
mellein can arise by the polyketide pathway. To establish a
de novo biosynthesis for all identified 3,4-dihydroisocoumarins listed in Table 1, and for the pheromones 4 and 6 especially,
we fed C. rujipes, C. silvicola and L. niger with [D,]acetate over
14 days. In all cases deuterium was weakly incorporated into
the pheromones, as indicated directly by the MS data of the
GC- MS analysis, which clearly revealed a molecular ion with
increased mass M + + X (X = 1-6). Because of the low concentration of deuterium-enriched 3,4-dihydroisocoumarins we
could not determine with certainty the defined distribution of
the label.
The methyl groups at the aromatic ring are unlikely to be
derived from acetate. To check whether they originate from
incorporation of propionate we synthesized P-[D,]propionic
acid (12) and fed an aqueous solution of its sodium salt to the
species named above. After an incorporation time of 14 days we
focused on 6 and the homologue 7 (occurrence in C. silvicola
and L. niger), and on 4 and 5 (C. rujpes). The resulting GCanalysis exhibited new peaks in front of the expected and known
signals, which were unambiguously assigned to the deuterated
compounds. The molecular ion of 4 increased by three, those of
5 and 6 by six, and those of 7 by nine mass units. The fragmentation pattern definitly demonstrated the incorporation of CD,
groups into the aromatic ring and also into the 3-ethyl group of
5 and 7. In Table2 the MS data of natural and labeled
pheromones 4 and 6 are compared.
Table 2. MS data (EI, 70 eV) recorded from unlabeled and labeled 3,4-dihydroisocoumarins 4 and 6 after feeding with CD,CH,COOH.
Fragment
Mi
M i -H20
M i - C,H,
M + - H,O - CH, [a]
M i - H,O - CD, [b]
Table 1. 3,4-Dihydroisocoumanns 1-7 and b-lactones 8-10 (+ + =compound serves as M‘ - CH3CH0
trail pheromone; + = compound merely detected) found in the rectal bladder of ants of M’ - H,O - CO
the subfamily Formicinae.
M + - H,O - CH, - CO [a]
M + - H,O - CD, - CO [b]
Art
1
2
3
4
5
6
7
8
9
10
M + - CH,CHO - CO
C. silvicola [4c,g]
+
C . rufpes [4c, g]
+
+
+
++ +
C . inequalis [4h]
C. herculeanus[4b]
C. ligniperdus [4f]
++ +
+
++
+
+
C. pennsylvanicus [4fI
C. socius [4 f]
++
L./iu/iginosus[4e]
++
E rufu (4 a]
++
++
++
195(100)
177(33)
166(18)
162(13)
159(1)
151(39)
149(9)
134(2)
131(1)
123(15)
192(100)
174(42)
163(22)
159(26)
212(100)
194(20)
183(23)
179(10)
176(6)
168120)
166(6)
151(7)
148(5)
14019)
206(100)
188( 17)
177(24)
173(25)
-
148(42)
146(12)
131(13)
-
120(15)
-
162(19)
160(6)
145(15)
-
134(12)
++ ++ ++
These results clearly illustrate that in ants of the subfamily
Formicinae the 3,4-dihydroioscoumarin pheromones are biosynthesized from acetate as well as from propionate, presumably by a polyketide pathway as illustrated schematically in
Figure 2. Thus the construction of 4 would require one acetyl-
t
propionate
acetate
+ -
+
E sanguinea [4 a]
396
6(%)
+
L. niger [4a]
Efusca [4al
D6-6(Yo)
++
C. atriceps [4d]
C.floridunus[4d]
4(%)
[a] 3-methyl-fragmentation and for 6 and 4 in addition 7-methyl-fragmentation
(ortho-effect) [4a]. [b] 7-CD3-fragmentation (ortho-effect) [4a].
++
++ +
++
c. vugus [4 fI
D,-4(%)
+
0 VCH Verlagsgesellschuft
o
R
c
R
0
+
+
mbH, 0-69451 Weinhelm, 1997
- SCoA
J $
0
-
pR
\
OH
0
Figure 2. Model for the formation of 3,4-dihydroisocoumarins via a pentaketide
precursor biosynthesized from acetate (R = H) and propionate (R = CH,).
OS70-0833/97/3604-0396S 15.00+ -2510
Angew. Chem. Int. Ed. Engl. 1997, 36, No. 4
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CoA, three malonyl-CoA, and one methylmalonyl-CoA unit,
of 6 one acetyl-CoA, two malonyl-CoA, and two methylmalonyl-CoA units, of 5 one propionyl-CoA, three malonylCoA, and one methylmalonyl-CoA unit, and of 7 one propionyl-CoA, two malonyl-CoA, and two methylmalonyl-CoA
units. For 6 the GC-MS analysis also showed a compound that
had incorporated only one CD, group because one natural and
one deuterated propionate molecule was used in the biosynthesis. Significant amounts of labeled compounds biosynthesized
during the feeding experiment were detected by GC-MS
(30- 40 yo).
These results prompted us to investigate whether the second
class of trail pheromones occurring in the rectal bladders of
formicine ants, the &lactones, are biosynthesized in a related
way by lipid metabolism. We fed labeled propionate 12 to
C. ligniperdus and C . herculeanus, which both utilize lactone 8
and 10 (and for C. lignzperdus also 9) as trail pheromones. Subsequently. after a feeding period of two weeks, the hindgut contents of the ants were analyzed by GC-MS. For both species the
gas chromatogram showed in front of the peaks for 8 and 10
new signals assigned to the compounds with incorporated CD,
groups. Only for 9 this was not so distinct because of its very low
concentration in the rectal bladder. The molecular ion of 8
showed an increase of six, those of 9 of nine, and those of 10 of
twelve mass units. The fragmentation pattern definitly pointed
to the structures D,-8, D,-9 and D,,-10 (Table 3).
1 Acety-CoA
3 Methylmalonyl-CoA
1 Propionyl-CoA
3 Methylmalonyl-CoA
0
09-9
Ds-8
8(%)
D9-9(%) 9(%)
Mi
148(1)
j 84(2)
210(1)
-
142(2)
lOO(4)
193(<1)
M' - C,H6
M t - C,D,H,
M i - 6-alkyl
M + - 6-alkyl - CO
M' - CH,CHO
M + - CH,CHO - CO
M + - C3H6 - C 0 2
M + - C3D3H3- COz
CH,CHCO
CD,CHCO
-
142(17)
-
103(2)
133(1)
105(1)
104(20)
76(12)
-
-
127(2)
9%1)
98(23)
70(14)
56(100)
3
D11-10
D&%)
Fragment
D12-10(%) lo(%)
165(12)
148(8)
133(100) 127(100) 133(100)
105(21)
99(23) 105(27)
-
-
-
-
-
98(15)
198(2)
156(19)
-
127( 100)
99128)
-
-
-
112(2)
-
104(13)
-
121(2)
-
561100)
-
56(37)
-
56(42)
59(100)
-
59(20)
-
59(32)
-
59(100)
These results establish the incorporation of labeled acetate
and particulary propionate into the biologically active &lactones found in the rectal bladders of formicine ants and are
consistent with the biosynthetic pathway shown in Figure 3.
The amount of labeled pheromones produced is considerable
(up to 85 Yo).
While the 3,4-dihydroisocoumarins are biosynthesized from
pentaketide chains by the use of at least one acetyl unit and a
variable number of propionyl units, the &lactones require three
or four carboxylic acid equivalents, among which the propionyl
precursor predominates. Acetate is not essential: pheromone 10
is biosynthesized by the exclusive use of propionate.
Many of these ant species are richly endowed with endosymbionts living in the gut system. Whether these micro-organisms
play a role in the synthesis of the trail pheromones is not yet
Angen. Chem. Inr. Ed Engl. 1997,36, No. 4
S
&
&
A
A
-
-
lo
Received: July 8. 1996 [Z9304IE]
German version: Angew Chem. 1997, 109.391 -394
3 a - , .
a
G
S
known.['] Preliminary investigations by feeding experiments
showed that the biosynthesis of the trail pheromones in the ants
is not considerably impaired by antimycotics and antibiotics.
a;
CD,
-
q
Figure 3. Model of the biosynthesis of 3.5-dimethyl-6-alkyI-tetrahydro-2H-pyran2-ones (6-lactones) .
Table 3. MS data (EI, 70 eV) recorded from unlabeled and labeled &lactones after feeding
with CD,CH,COOH.
D7-J;
-
Keywords: biosynthesis * lipids * pheromones
[l] B. Holldobler, E. 0. Wilson, The Ants, Springer, Berlin, 1990.
[2] B. D. Jackson, E. D. Morgan, Chemoecoiogy 1993, 4, 125- 1 4 4
[3] Only a few studies on the biosynthesis of defense compounds exist: G. M.
Happ, J. Meinwald, J. Am. Chem. SOC.1965,87, 2507-2508; A. Hefetz, M. S .
Blum, Science 1978,201,454-455; J. H. Kim, R F. Toia, J. .Waf.Prod. 1989,
52, 63-66; B. Renson, P. Merlin, D. Daloze, J. C. Breakman, Y. Roisin, J. M.
Pasteels, Can. J: Chem. 1994, 72, 105-109.
141 a) H. J. Bestmann, F. Kern, D. Schafer, M. C. Witschel, Angew,. Chem. 1992,
104, 757-758; Angew. Chem. Int. Ed. Engl. 1992,31, 795-796; b) H. J. Bestmann, U. Haak, F Kern, B. Holldobler, Naturwissenschafen 1995, 82, 142144; c) E. ubler, F. Kern, H. J. Bestmann, B. Holldobler, A. B. Attygalle, ibid.
1995,82.523-525; d)U. Haak, B. Holldobler, H. J. Bestmann, F. Kern, Chem.
Okoi., in press; e) F. Kern, R. W. Klein, E. Janssen, H. J. Bestmann, D. Schafer,
U. Maschwitz, A. B. Attygalle, J. Chem. Ecoi., in press; f) U Haak, Dissertation, Universitat Erlangen-Niirnberg, 1995;g) E. Ubler, Dissertation, Universitat Erlangen-Niirnberg, 19%; h) J Tenschert, Diplomarbeit, Universitat
Erlangen-Niirnberg, 1995
[5] R. A. Hill in Fortschritfe der Chemie Organischer Natursfoffe. Vol. 49 (Eds.: W.
Herz, H. Griesebach, G. W. Kirby, C. Tamm), Springer, New York, 1986, pp.
1-78.
161 T. C. Baker, R. Nishida, W. L. Roelofs, Science 1981,214, 1359-1361.
[7J G Kunesch, P. Zagatti, A. Pouvreau, R. Cassini, Z Nuturforsch. C 1987, 42,
657-659.
[8] J. J. Brophy, G . W. K. Cavill, W. D. Plant, Insect Biochem. 1981. f f , 307-310;
ihid. 1984, 14, 738.
[9] T. H. Jones, H. M. Fales, Tefrahedron L e f t . 1983, 24, 5439-5440.
[lo] a) R. S . Mali, P. G. Jagtap, S G. Tilve, Synth. Commun. 1990,20,2641-2652;
b) R. S . Mali, P. G. Jagtap, S . R. Patil, P N. Pawar, J Chem. SOC.Chem.
Commun. 1992,883-884.
Ill] a) E. D. Morgan, L. J. Wadhams, J. Chromafogr. Sci. 1972, 10, 528-529;
b) A. B. Attygalle, M. Herrig, 0. Vostrowsky, H. J. Bestmann, J Chem. Ecoi
1987, f3, 1299-1311.
1121 a) J. S . E. Holker, T. F. Simpson, J Chem. SOC.Perkin f 1981, 1397-1400,
b) C. Abell, M. J. Garson, F. J. Lepper, J Staunton, J. Chem. Soc Chem.
Commun. 1982. 1011-1013;c)C. Abell, D. M. Doddrell, M J. Garson,E. D.
Laue, J. Staunton, ibid. 1983, 694-696.
[13] C M. Sun, R. F. Toia, J. Nut. Prod. 1993, 56, 953-956.
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