Host marking pheromone (HMP) in the Mexican Fruit Fly Anastrepha ludens

Article abstract of DOI:10.2533/chimia.2010.37

Host marking pheromones (HMPs) are used by insects to mark hosts (usually a fruit) where they have already laid eggs. The compounds serve as a deterrent to conspecifics avoiding over-infestation of hosts (i.e. repeated egg-laying into an already occupied/used host). If these HMPs are sprayed onto commercially valuable fruit they act as deterrents preventing attack by females interested in laying eggs into the valuable commodity. Having no insecticidal or toxic properties, and being natural products (or close derivatives thereof) they could be used as fruit sprays to replace insecticides, or in combination with other products to improve efficacy. This review discusses the isolation, and synthesis of the HMP of the Mexican fruit fly Anastrepha ludens a feared pest of citrus and mangos in Mexico and Central America. This compound is also recognized by females of other pestiferous species in the same genus Anastrepha distributed from the Southern USA to Northern Argentina, including many Caribbean Islands. The synthetic HMP was shown to exhibit strong electrophysiological activity against A. ludens and excellent interspecies cross recognition with other Anastrepha species. Behavioural tests confirmed the HMP deterring effect of the synthetic natural product. Further studies enabled us to drastically simplify the structure of the HMP and obtain a derivative, which we named Anastrephamide, which shows HMP deterring effects very similar to the natural product in laboratory and field tests. The potential use of such HMP derivatives in a crop protection scenario is briefly discussed. Schweizerische Chemische Gesellschaft.

Full text of DOI:10.2533/chimia.2010.37

From ChemiCal researCh to industrial appliCationsꢀ
CHIMIAꢀ2010,ꢀ64,ꢀNo.ꢀ1/2ꢀ 37
doi:10.2533/chimia.2010.37ꢀꢀ
Chimiaꢀ64ꢀ(2010)ꢀ37–42ꢀ ©ꢀSchweizerischeꢀChemischeꢀGesellschaft
Host Marking Pheromone (HMP) in the
Mexican Fruit Fly Anastrepha ludens
AndrewꢀJ.ꢀF.ꢀEdmunds*a,ꢀMartin Alujab,ꢀFransicoꢀDiaz-Fleischercd,ꢀBrunoꢀPatriane,ꢀandꢀ
LeonhardꢀHagmanna
DedicatedꢀtoꢀProfessorꢀDanielꢀBellušꢀonꢀtheꢀoccasionꢀofꢀhisꢀ70thꢀbirthday
Abstract:ꢀHostꢀmarkingꢀpheromonesꢀ(HMPs)ꢀareꢀusedꢀbyꢀinsectsꢀtoꢀmarkꢀhostsꢀ(usuallyꢀaꢀfruit)ꢀwhereꢀtheyꢀhaveꢀ
alreadyꢀlaidꢀeggs.ꢀTheꢀcompoundsꢀserveꢀasꢀaꢀdeterrentꢀtoꢀconspecificsꢀavoidingꢀover-infestationꢀofꢀhostsꢀ(i.e.ꢀ
repeatedꢀegg-layingꢀintoꢀanꢀalreadyꢀoccupied/usedꢀhost).ꢀIfꢀtheseꢀHMPsꢀareꢀsprayedꢀontoꢀcommerciallyꢀvaluableꢀ
fruitꢀtheyꢀactꢀasꢀdeterrentsꢀpreventingꢀattackꢀbyꢀfemalesꢀinterestedꢀinꢀlayingꢀeggsꢀintoꢀtheꢀvaluableꢀcommodity.ꢀ
Havingꢀnoꢀinsecticidalꢀorꢀtoxicꢀproperties,ꢀandꢀbeingꢀnaturalꢀproductsꢀ(orꢀcloseꢀderivativesꢀthereof)ꢀtheyꢀcouldꢀbeꢀ
usedꢀasꢀfruitꢀspraysꢀtoꢀreplaceꢀinsecticides,ꢀorꢀinꢀcombinationꢀwithꢀotherꢀproductsꢀtoꢀimproveꢀefficacy.ꢀThisꢀreviewꢀ
discussesꢀtheꢀisolation,ꢀandꢀsynthesisꢀofꢀtheꢀHMPꢀofꢀtheꢀMexicanꢀfruitꢀflyꢀAnastrepha ludensꢀaꢀfearedꢀpestꢀofꢀcitrusꢀ
andꢀmangosꢀinꢀMexicoꢀandꢀCentralꢀAmerica.ꢀThisꢀcompoundꢀisꢀalsoꢀrecognizedꢀbyꢀfemalesꢀofꢀotherꢀpestiferousꢀ
speciesꢀinꢀtheꢀsameꢀgenusꢀAnastrephaꢀdistributedꢀfromꢀtheꢀSouthernꢀUSAꢀtoꢀNorthernꢀArgentina,ꢀincludingꢀmanyꢀ
CaribbeanꢀIslands.ꢀTheꢀsyntheticꢀHMPꢀwasꢀshownꢀtoꢀexhibitꢀstrongꢀelectrophysiologicalꢀactivityꢀagainstꢀA. ludens
andꢀexcellentꢀinterspeciesꢀcrossꢀrecognitionꢀwithꢀotherꢀAnastrephaꢀspecies.ꢀBehaviouralꢀtestsꢀconfirmedꢀtheꢀHMPꢀ
deterringꢀeffectꢀofꢀtheꢀsyntheticꢀnaturalꢀproduct.ꢀFurtherꢀstudiesꢀenabledꢀusꢀtoꢀdrasticallyꢀsimplifyꢀtheꢀstructureꢀ
ofꢀtheꢀHMPꢀandꢀobtainꢀaꢀderivative,ꢀwhichꢀweꢀnamedꢀAnastrephamide,ꢀwhichꢀshowsꢀHMPꢀdeterringꢀeffectsꢀveryꢀ
similarꢀtoꢀtheꢀnaturalꢀproductꢀinꢀlaboratoryꢀandꢀfieldꢀtests.ꢀTheꢀpotentialꢀuseꢀofꢀsuchꢀHMPꢀderivativesꢀinꢀaꢀcropꢀ
protectionꢀscenarioꢀisꢀbrieflyꢀdiscussed.
Keywords:ꢀAnastrephamide ·ꢀCropꢀprotection ·ꢀFruitꢀflies ·ꢀHostꢀmarkingꢀpheromones ·ꢀNaturalꢀproducts
Fig.ꢀ1.ꢀHMPꢀofꢀtheꢀ
Europeanꢀcherryꢀfruitꢀ
fly,ꢀRhagoletisꢀcerasi.
1. The Importance of Host Marking
Pheromones (HMPs)
Host marking pheromones (HMPs) are
used by certain insects to mark hosts where
theyhavealreadylaideggs.Inthecaseoftrue
fruit flies (family Tephritidae as opposed to
Drosophilidae), this is achieved by drag-
ging its aculeus (tip of ovipositor), over the
surface of the fruit after an egg-laying bout.
The HMPs are also found in the fly faeces.[1]
The compounds serve as a deterrent to con- isolation from methanol extracts of the fly
specifics, thus avoiding over-infestation of faeces, and total synthesis.[3] The natural
hosts (i.e. repeated egg-laying into an al- product ((1), Fig. 1) when applied to cherry
ready occupied/used host).[2] If these HMPs orchards, showed high levels (up to 98%)
are sprayed onto commercially valuable of activity (i.e. reduced fruit infestation).[4]
fruit they act as deterrents preventing attack
So why has this seemingly environ-
by females interested in laying eggs into mentally benign method of pest control
the valuable commodity. Having no insec- not been further pursued? Are HMPs
ticidal or toxic properties, and being natu- confined to the European cherry fruit fly?
ral products (or close derivatives thereof), Clearly one would not expect this to be the
they could be used as fruit sprays to replace case, and HMPs have indeed been found
insecticides, or in combination with other in about 100 species from five insect or-
products to improve efficacy. There have al- ders.[5] In the most cases, the structures of
ready been successful applications of such a the HMPs remain unknown often due to
crop protection strategy reported in the lit- the difficulty of isolation and structure de-
erature. In particular, Ciba Geigy (now Syn- termination, and consequently there has
genta) in collaboration with the Swiss Fed- been very little undertaken to look at these
eral Research Station for Fruit-Growing, HMPs as crop protection agents.[6]
*Correspondence: Dr. A. J. F. Edmundsa
Tel.: +41 61 323 3663
E-Mail: andrew_jf.edmunds@syngenta.com
aSyngenta Crop Protection Muenchwilen AG
Schaffhauserstrasse, CH-4332 Stein
bInstituto de Ecología, A.C
Apartado Postal 63
91000 Xalapa, Veracruz, Mexico
cCampaña Nacional Contra las Moscas de la Fruta
Apartado Postal 368,
30700 Tapachula, Chiapas, Mexico
Viticulture and Horticulture,ꢀ Wädenswil,
In this article we briefly describe our
dCurrent Address:
Switzerland (now Swiss Federal Research findings concerning the HMP of another
Station Agroscope Changins-Wädenswil fruit fly pest, namely the Mexican fruit fly
ACW) were able to identify the HMP of Anastrepha ludens, a feared pest of citrus
the European cherry fruit fly, Rhagoletis and mangos in Mexico and Central Amer-
cerasi by a combination of bioassay-guided ica. A detailed account of isolation, syn-
INBIOTECA
Apartado Postal Universidad Veracruzana
91000 Xalapa, Veracruz, Mexico
eAgroscope Changins-Wädenswil Research Station
ACW
Schloss, CH-8820 Wädenswil

38ꢀ CHIMIAꢀ2010,ꢀ64,ꢀNo.ꢀ1/2ꢀ
From ChemiCal researCh to industrial appliCations
Fig.ꢀ2.ꢀStructureꢀofꢀtheꢀ
HMPꢀofꢀAnastrepha
ludens.
theses, and biological/field testing of com-
pounds described here have appeared in
the patent[7] and mainstream literature,[8,9]
but the work has never been reviewed in
terms of chemistry, biological activity and
structure–activity relationships.
2. Isolation and Structure of
Anastrepha ludens HMP
scribed in detail by Dethier[12] and Stä- Would it be possible to simplify this natu-
dler,[13] the chemosensillum is stimulated ral product and retain activity? We were
and the ensuing spikes (action potentials) very pleasantly surprised, when the struc-
are analyzed. Spike activity varies accord- tural elucidation[7] presented a relatively
ing to the level of response by the insect simple looking compound containing an
and can be used to distinguish between isopalmitc fatty acid chain substituted by
active and inactive HPLC fractions of the methyl at the 2-position and coupled to a
HMP. Using this bioassay, we were even- glutamic acid (2, Fig. 2), apparently as a
tually able to isolate 5 mg of a single com- single diastereomer.[15]
Several species of Anastrepha are im-
portant pests in the Americas. For exam-
ple, commercial crops including guava, or-
anges, grapefruit, mango, tropical plums,
mammee apple, chico zapote are infested
by various Anastrepha species such as A.
fraterculus, A. grandis, A. ludens, A. ob-
liqua and A. serpentina throughout South
America and southern USA.[10]
pound with very high electrophysiologi-
It is immediately apparent that this
cal activity from 167 g of fly faeces. How- molecule is considerably simpler than that
ever, since a positive electrophysiological of the European cherry fruit fly shown
response does not always translate into a in Fig. 1. However, some work had to be
positive behavioural response, additional invested in the first instance to determine
bioassays had to be performed in the lab. the absolute stereochemistry of the natural
When a HMP comes into contact with its product, as it contained two stereocentres
chemosensilla, a female fly responds by and thus could have been one of four pos-
shaking its legs, cleaning them and walk- sible diastereomers: The four possible di-
ing or flying away. Since females mark astereomers can be described as (R)-l, (S)-
the fruit surface with HMP after laying l, (R)-d, (S)-d, in which R or S describe
eggs by dragging its aculeus (tip of ovi- the chirality at the 2-methyl position in the
positor), when the same female or another fatty acid chain, and l or d indicate the chi-
one contacts a marked surface (fruit), it rality of the glutamic acid (Fig. 3).
It has been observed that this fruit fly
species exhibited similar behavioural activ-
ity to the European cherry fruit fly (Rhago-
letis cerasi), also in terms of oviposition.[2]
Thus, it could be demonstrated that metha-
nol extracts obtained from the faeces of one
of the species of these fruit flies (Anastrepha
ludens) showed HMP deterring activity, not
only to Anastrepha ludens, but with com-
plete interspecies cross recognition to A.
obliqua from the fraterculus species group
and A. serpentina.[8] The next step was thus
clear: Isolate the active principle from the
faeces methanol extracts, determine the
structure, test the biological activity, and
simplify the molecule whilst retaining the
HMP deterring properties.
The first part of this story is beyond
the scope of this review, but clearly bio-
assay-guided isolation of the active prin-
ciple from the methanol extract of Anast-
repha ludens faeces was key. In short, the
methanol concentrate was subjected to
HPLC purification and the fractions ana-
lysed using electrophysiology. This test
is based upon the fact that chemorecep-
tor sensilla (chemosensilla) containing a
receptor cell sensitive to the HMP have
been identified on the distal ventrolateral
portions of the second, third and fourth
tarsomeres of each leg (lowermost part
of leg with which the insect contacts the
surfaces it walks over).[11] These sensilla
are known as ‘D’-chemosensilla. Usually,
every tarsi has six of these sensilla, and as
insects have six legs, there are a total of 36
‘D’-chemosensilla potentially responding
to the HMP. D-chemsensilla are thus ex-
tremely useful when trying to determine
the response by the insect to the HMP dur-
ing analysis of HPLC fractions. Using an
electrophysiological test, female flies are
decapitated and mounted ventral side up
to expose the D-sensilla which are humid-
ified continuously with a water-saturated
air stream. Using electrophysiological
techniques known as ‘tip recordings’ de-
walks or flies to another fruit or a leaf.
The configuration of the amino acid was
This behavioral response is measured by, determined by hydrolysing the natural prod-
for example, counting the total number of uct under acidic conditions, and the glutamic
fruit visited by a female per unit time or acid portion derivatised to N-trifluoroacetyl
the amount of time a female spends on top glutamic acid isopropyl ester. This was then
of a fruit. Instead of using fresh fruit as subjected to GC analysis on a chiral column
oviposition substrates, we utilized green (Chirasil-l-Val). Under these conditions,[16]
2.5 cm diameter agar spheres wrapped the l-isomer showed a retention time of 25.6
in parafilm. The flies were continuously min., the d-isomer 24.2 min. based on the
observed, and their landings, oviposition synthetic compounds. The sample of the
attempts or successful ovipositions on derivatised natural product consisted of 21%
treated or untreated spheres, recorded.[4]
d-glutamic acid and 79% l-glutamic acid
With information on successful ovi- which was confirmed by co-injection au-
positions, a discrimination coefficient thentic samples.This result was surprising to
(DC) for the flies to the artificial fruits us as the 1H and 13C NMR indicated the pres-
could be calculated as described by Boller ence of only one diastereomer, and thus we
and Hurter.[14] The DC can vary between suspected that partial epimerisation of the
–100 and +100. A DC of –100 indicates amino acid occurred under the conditions of
that all eggs were laid into treated ovipo- amide hydrolysis. Therefore, we addressed
sition substrates. No difference between the question of the absolute configuration
test substance and control would produce of the natural product by total synthesis of
a DC of zero and hence no deterrent effect all four diastereomers. Comparison of the
was achieved. A DC of +100 indicates that spectral and biological properties would en-
no eggs were laid into treated oviposition able us to assign the absolute configuration
substrates and hence an absolute deterrent of A. ludens HMP (2), and also give us initial
effect was achieved.
results on the importance of stereochemistry
The 5 mg of isolated material showed upon biological activity.
a high deterring effect (90%) in the be-
havioural assay, confirming that the elec-
trophysiological activity was related to a 3. Synthesis and Absolute
HMP effect.
With the pure HMP in hand, structure
elucidation could be undertaken. Would
Configuration of A. ludens HMP
To achieve the synthesis of all four
this be a complicated natural product? diastereomers, we required a stereoselec-

From ChemiCal researCh to industrial appliCationsꢀ
CHIMIAꢀ2010,ꢀ64,ꢀNo.ꢀ1/2ꢀ 39
enolate.[16,17] The synthesis is illustrated
for the (R)-l-(2) in Scheme 1, which also
shows our route for the preparation of iso-
palmitic acid for completeness.
Fig.ꢀ3.ꢀPossibleꢀ
diastereomersꢀofꢀtheꢀ
HMPꢀofꢀA. ludensꢀ(2).
Thus, 9-bromo-1-nonanol (5) was pro-
tected as its THP ether and converted to
the Grignard (6). This was coupled to the
branched chain tosylate (8) in THF in the
presence of lithium chloride-cupric chlo-
ride catalyst,[18] followed by THP-depro-
tection to give the branched chain alcohol
(9). The alcohol (9) was tosylated, convert-
ed to the cyanide, which after basic hydrol-
ysis yielded the isopalmitic acid (10). This
acid was converted to the acid chloride (11)
and then coupled to the sodium salt (S)-
camphor sultam (3) in toluene according
to the method of Oppolzer.[17] Face-selec-
tive alkylation was achieved using n-butyl
lithium in THF:DMPU (4:1) and methyl
iodide at –78 oC.[17] The diasteroselectivity
of the alkylations was 95:5 as determined
by 1H NMR analysis[19] and a single recry-
tallisation gave diastereomerically pure 13.
The (S)-camphor sultam was removed by
peroxide assisted LiOH hydrolysis[20] to
give 2-(R)-14-dimethylpentadecanoic acid
(14). This was coupled to l-glutamic acid
dibenzyl ester yielding 15, which was fi-
nally debenzylated by catalytic hydrogen
transfer reduction using aqueous formic
acid and Pd /C, to give (R)-l-(2).
(R)-D-(2)
(S)-L-(2)
(R)-L-(2)
(S)-D-(2)
As both (S) and (R)-camphor sultams
are commercially available, as well as
the (l)- and (d)-dibenzyl glutamic acids,
this strategy enabled preparation of all
four diastereomers of the A. ludens HMP
(Scheme 2).
(R)-L-(2)
Schemeꢀ1. Reagentsꢀandꢀconditions:ꢀa)ꢀ2,3-Dihydropyrane,ꢀcat.ꢀp-TsOH,ꢀCH2Cl2,ꢀr.t.ꢀb)ꢀMg,ꢀTHF.ꢀ
c)ꢀTsCl,ꢀEt3N,cat.ꢀDMAP,ꢀCH2Cl2,ꢀr.t.ꢀd)ꢀLi2CuCl4ꢀ(0.02ꢀequiv.),ꢀaddꢀ(8),ꢀTHF,ꢀ–78ꢀoCꢀtoꢀr.t.ꢀe)ꢀp-TsOH,ꢀ
MeOH,ꢀr.t.ꢀf)ꢀTsCl,ꢀEt3N,cat.ꢀDMAP,ꢀCH2Cl2,ꢀr.t.ꢀg)ꢀKCN,ꢀDMSO,ꢀ80ꢀoC.ꢀh)ꢀKOH,ꢀEtOH,ꢀreflux.ꢀi)ꢀ
SOCl2,ꢀcat..ꢀDMF,ꢀr.t.ꢀj)ꢀNaH,ꢀToluene,ꢀr.t.ꢀk)ꢀn-BuLi,ꢀTHF:DMPUꢀ(4:1),ꢀ–78ꢀoC,ꢀthenꢀaddꢀMeIꢀ–78ꢀoC
ꢀtoꢀr.t.ꢀl)ꢀLiOH,ꢀH2O2ꢀTHF:H2Oꢀ(9:1).ꢀm)ꢀl-Glutamicꢀacidꢀdibenzylꢀesterꢀ4-toluenesulfonate,ꢀEDC,ꢀ
Et3N,ꢀcat.ꢀDMAP,ꢀCH2Cl2.ꢀn)ꢀHCO2Hꢀ(88%ꢀinꢀH2O):MeOH,ꢀ(4:1),ꢀPd/Cꢀ(10%).
The (R)-l-(2) and (S)-d-(2) enantio-
mers had identical 1H NMR spectral prop-
erties to the isolated natural product while
the diastereomeric (S)-l-(2) and (R)-d-(2)
enantiomers showed minor differences,
particularly for the methyl doublet at C(2)
and the NH signal. More importantly, the
diastereomers could also be distinguished
under specific HPLC conditions (Nucle-
osil-100-7um-Phenyl, 10 × 250 mm, 4 ml/
min, 40/60% acetonitrile/water, 0.1% for-
mic acid, 195 nm UV detection). The re-
tention time for (R)-l-(2) and (S)-d-(2) un-
der these conditions was 46 min. whereas
(S)-l-(2) and (R)-d-(2) eluted after 48 min.
The natural pheromone eluted as a single
peak after 46 min, ruling out the (S)-l-(2)
and (R)-d-(2) diastereomers as the natural
product. Since the chirality of the 2-me-
thyl group of the isopalmitic acid moiety
could be confidently predicted from the
face selective alkylation of 12 (Scheme
1) and the configuration of the amino acid
was secure, we were confident at this stage
that the natural product possessed the (R)-l
configuration. With all the diasteromers of
natural product in hand, biological activ-
ity would deliver the final confirmation.
As the isolated natural product showed
ꢀ(S)-Camphorꢀsultam
ꢀ(R)-D-(2)
ꢀ(R)-L-(2)
ꢀ(S)-L-(2)
ꢀ(S)-D-(2)
ꢀ(R)-Camphorꢀsultam
Schemeꢀ2.ꢀSynthesisꢀofꢀtheꢀdiastereomersꢀofꢀtheꢀHMPꢀofꢀA. ludensꢀ(2).
tive synthesis of the 2-methyl isopalmitic the Oppolzer camphor sultam (3) to isopal-
acid side chain with known absolute con- mitic acid (10), carry out a stereoselective
figuration, coupling of this to a suitably α-amide alkylation, and then remove the
protected form of either l-, or d-glutamic chiral auxiliary. The choice of the camphor
acid, and subsequent deprotection with- sultam (3) for confident prediction of the
out epimerisation of the chiral centres. To stereochemical outcome of the alkylation
prepare the 2-methyl isopalmitic of known is due to attack of the alkyl halide to the
absolute configuration, we chose to attach least hindered face of the lithium chelated

40ꢀ CHIMIAꢀ2010,ꢀ64,ꢀNo.ꢀ1/2ꢀ
From ChemiCal researCh to industrial appliCations
Tableꢀ1.ꢀBiologicalꢀactivityꢀofꢀHMPꢀcompounds.ꢀElectrophysiologyꢀandꢀbehavioralꢀlaboratoryꢀtestsꢀ
(unlessꢀorthewiseꢀstated)ꢀwereꢀperformedꢀwithꢀA. ludensꢀfemales.ꢀTheꢀfieldꢀtestꢀwasꢀcarriedꢀoutꢀ
withꢀA. obliquaꢀ(seeꢀreferencesꢀ[7]ꢀandꢀ[23]ꢀforꢀdetails).
only one peak under the above-mentioned
HPLC conditions, this also confirmed our
suspicion that partial racemisation had oc-
curred in the GC amino acid chirality de-
termination, and that the isolated sample
of natural product was in fact a single di-
astereomer.[21]
En route to the natural product, we
were in a position to prepare compounds
that would determine the importance of the
2-methyl group in isopalmitic moiety, sim-
ply by coupling the isopalmitic acid (10) to
l- and d-dibenzyl glutamic acids, yielding
l-(16) and d-(16), which were deprotected
to yield the 2-desmethyl natural product
analogues l-(17) and d-(18) (Scheme 3).
Biological Activity
Electro-
physiology Laboratoryꢀ
1ꢀppm
Spikes/
msec
Behavioralꢀ
Test
100ꢀppm
Efficacyꢀ%
(Abbott)
Entry
Compound
Test
100ꢀppm
DC
Natural
Product
1
2
78
76
50
16
6
90
----
----
----
----
----
----
----
----
----
64.37
----
----
----
77.1
(R)-L-(2)
(R)-D-(2)
(S)-L-(2)
ꢀ(S)-D-(2)
L-(17)
82.3
23.2
----
4. Biological Activity. Some
Requirements for HMP Deterring
Activity
3
All of the diastereomers of the natural
product (2), the corresponding dibenzyl
esters (15), and 2-desmethyl natural prod-
ucts (17) described in Section 3 were now
subjected to electrophysiology testing. The
results for these evaluations are shown in
Table 1.
4
5
----
These results clearly show that the syn-
thetic (R)-l-(2) has the same activity as the
natural product obtained from bioassay
guided isolation from A. ludens faeces (en-
tries1and2), andthat(R)-l-(2)hadsuperior
activity to the other diasteromers (compare
entries 2 with 3, 4 and 5). This, combined
with the identical spectroscopic and HPLC
chromatographic properties of synthetic
and natural A. ludens HMP, unambiguously
confirms that the natural product has the R
configuration at the 2-methyl position of
the isopalmitic acid, and the l-amino acid
configuration. Some other points can be ex-
tracted from Table 1: The l-amino acid con-
figuration gives compounds of superior ac-
tivity (compare entries 2 with 3, and 4 with
5). The 2-methyl group of the isopamitic
acid moiety is required for activity (com-
pare entry 2 with 6), and the preferred stere-
ochemistry is the R configuration (compare
entry 2 with 4). Although not depicted in
Table 1, we also showed that esters of the
glutamic acid moiety led to compounds (15
and 16) which showed no activity whatso-
ever in the electrophysiology tests.
To confirm that the electrophysiology
results translate to a HMP deterring ef-
fect, compounds (R)-l-(2), (R)-d-(2), and
l-(17) were studied in the behavioural
test with living A. ludens, as described
in Section 1. As shown in Table 1, (R)-
l-(2) was clearly more active than its (R)-
d-(2) diastereomer, and the 2-desmethyl
analogue l-(17). As expected (based on
results with A. ludens faeces methanol
extracts where complete interspecies
cross recognition was observed between
6
25
2
23.7
----
7
D-(17)
8
(R)-L-(2)
(R)-L-(2)
(R)-L-(22)
(R)-D-(22)
(R)-L-(29)
(S)-L-(29)
72a
67b
78
23
----
----
----
----
9
----
10
11
12
13
14
84.8
----
84.3
61.7
78.4
(R/S)-
L-(29)
Anastre-
phamide
aꢀElectrophysiologyꢀwith A. striata. bꢀElectrophysiologyꢀwithꢀA. serpentina

From ChemiCal researCh to industrial appliCationsꢀ
CHIMIAꢀ2010,ꢀ64,ꢀNo.ꢀ1/2ꢀ 41
lent activity (Table 1, entry 10). At this
stage, it will not go unnoticed to the read-
er, that the HMP of Rhagoletis cerasi and
that of Anastrepha ludens have similari-
ties in their structures, i.e. both contain-
ing a long fatty acid residue attached to an
amino acid. In the case of 1, the acid is a
palmitic acid. As palmitic acid is a natu-
rally occurring compound, we envisaged
that a 2-methyl palmitic acid attached to
l-glutamic acid might offer some ben-
efits. Further, at this stage we knew that
diastereomers differing in the configura-
tion of the C(2) methyl of the fatty acid
chain could be separated by HPLC. Thus,
racemic 2-methyl palmitic acid (26) was
prepared very simply according to the
method shown in Scheme 6.[22] This was
converted to the acid chloride (27), which
could be directly coupled to l-glutamic
acid monosodium salt (28) in THF using
anhydrous LiCl to solubilise the mixture.
This gave a 1:1 diastereomeric mixture
of (R/S)-l-(29). The diastereomers could
be separated by preparative HPLC using
the conditions previously described for 2.
The first eluting diastereomer eluted af-
ter 53 min and the second after 56 min.
These were assumed to be (R)-l-(29) and
(S)-l-(29), respectively.[23] All three com-
pounds were tested in the behavioural
assay where it was shown that there was
little difference in the activity between the
distereomeric mixture, and the separated
isomers (Table 1, entries 12, 13 and 14).
L-(17)
L-(16)
D-(16)
D-(17)
Schemeꢀ3.ꢀReagentsꢀandꢀConditions:ꢀa)ꢀl-ꢀorꢀd-Glutamicꢀacidꢀdibenzylꢀesterꢀ4-toluenesulfonate,ꢀ
EDC,ꢀEt3N,ꢀcat.ꢀDMAP,ꢀCH2Cl2.ꢀb)ꢀHCO2Hꢀ(88%ꢀinꢀH2O):MeOH,ꢀ(4:1),ꢀPd/Cꢀ(10%).
Schemeꢀ4. Reagentsꢀ
andꢀConditions:ꢀa)ꢀ
SOCl2,ꢀcat.ꢀDMF,ꢀr.t.ꢀ
b)ꢀNaH,ꢀtoluene,ꢀr.t.ꢀ
c)ꢀn-BuLi,ꢀTHF:DMPUꢀ
S-(3)
(4:1),ꢀ–78ꢀoC,ꢀthenꢀaddꢀ
MeIꢀ–78ꢀoCꢀtoꢀr.t.ꢀd)ꢀ
LiOH,ꢀH2O2,ꢀTHF:H2Oꢀ
(9:1).ꢀ
R-(21)
(R)-(21)
(R)-D-(22)
(R)-L-(22)
Scheme 5.
6. Field Testing
Based upon the results of the behav-
ioural tests, and the clear advantages of
ease of synthesis, we chose to prepare
large amounts of (R)-l-(22), the 14-des-
methyl derivative of A. ludens HMP, and
(R/S)-l-(29), (which we have named Anas-
trephamide) to test in fruit orchards. Tests
in tropical plum (Spondias purpurea) con-
sisted of applying the compounds (100
ppm in water) with a knapsack sprayer to
single fruit-bearing branches of S. purpu-
rea trees visited by Anastrepha obliqua
females. The results reported in Table 1[9]
show that (R)-l-(22), reduced infestation
in plums by 64% and Anastrephamide
((R/S)-l-(22)) by 77%, compared to un-
treated trees.
(R/S)-L-(29)
Scheme 6.
Anastrepha sp. in electrophysiology, be- less impact upon biological activity, was
havioural, and field tests) the synthetic the removal of methyl group at C(14) of the
natural product (R)-l-(2) also showed isopalmitic acid chain. The 14-desmethyl
complete interspecies cross recognition derivatives ((R)-l-(22) and (R)-d-(22))
with A. striata and A. serpentina (Table were easily prepared by routes analogously
1, entries 8 and 9).
to those described for the natural product
from commercially available pentanedeca-
noic acid (Schemes 4 and 5).
7. Conclusion
In conclusion, we have been able to iso-
late the HMP of Anasterpha ludens, dras-
tically been able to simplify its structure
whilst maintaining the biological activity
against fruit infestation by A. ludens and
other flies of this genus such as A. obliqua,
A. striata, and A. serpentina under labora-
5. Simplification of the Natural
Product
The electrophysiology results for (R)-
l-(22) confirmed that the natural product
could be simplified, and that the (R)-l con-
As we had shown that the methyl at figuration was more active than the (R)-d
C(2) of the isopalmitic acid was required configuration (Table 1, entries 10 and 11).
for optimal activity, the next most obvious In the behavioural test, the 14 desmethyl
simplification, perhaps expected to have derivative (R)-l-(22) also showed excel-

42ꢀ CHIMIAꢀ2010,ꢀ64,ꢀNo.ꢀ1/2ꢀ
From ChemiCal researCh to industrial appliCations
2001, 99, 273. It has been found that HMPs
can affect interspecifically the behaviour of
insects, and not just act as ovipostion deterring
pheromones. See ref. [4].
[3] J. Hurter, E. F. Boller, B. Städler, H. Blattmann,
R. Buser, N.U. Bosshard, L. Damm, M.W.
Kozlowski, R. Schöni, F. Raschdorf, R.
Dahinden, E. Schlumpf, H. Fritz, W.J. Richter,
J. Schreiber, Experientia, 1987, 43, 157; B.
Ernst, M. Heneghan, B. Wagner, E. F. Boller, J.
Hurter, E. Staedler, Eur. Pat. Appl. EP 474590,
1992; B. Ernst, B. Wagner, Helv. Chimica Acta
1989. 72, 165. For further relevant references
see; B. Wagner, M. Heneghan, G. Schnabel, B.
Ernst, Synlett. 2003, 9, 1303.
ShiftꢀmethylꢀatꢀC-(14)
toꢀC-(15)
RemovalꢀofꢀmethylꢀatꢀC-(14)
[4] M. Aluja, E. F. Boller, Entomol. Exp. Appl.
1992, 65, 141.
[5] P. Anderson, ‘Chemoecology of Insect Eggs
and Egg Deposition’, 2002, pp 235.
[6] Apart from the European cherry fruit fly, there
has only been one other related study which
involved the use of fly faeces methanol extracts
(R/S)-L-(29)
from the Mediterranean fruit fly, (Ceratitis
capitata) to reduce infestation in coffee bushes.
See; J. Arredondo, F. Diaz-Fleischer, Bull.
Entomol. Res. 2006, 96, 35.
(R)-L-(2)
(R)-L-(22)
(R)-L-(2)
[7] M. Aluja, F. Diaz-Fleischer, A. J. F. Edmunds,
L. Hagmann, US Pat. Appl. US6555120, 2000.
[8] M. Aluja, F. Diaz-Fleischer, J. Chem. Ecol.
2006, 32, 367.
Removeꢀ
stereochemistry
atꢀC-(2)
[9] Full details of the field trials have been be
reported in M. Aluja, F. Diaz-Fleischer, E. F.
Boller, J. Hurter,A. J. F. Edmunds, L. Hagmann,
B. Patrian, J. Reyes, J. Econ. Entomol. 2009,
102, 2268.
[10] M. Aluja, ‘Future trends in fruit fly
management’, in ‘Economic fruit flies: a world
assessment of their biology and management’,
Ed. B. A. McPheron, G. J. Steck, St. Lucie
Press, Delray Beach, FL, 1996, p. 309; M.
Aluja, Ann. Rev. Entomol. 1994, 39, 155.
[11] R. M. Crnjar, R. J. Prokopy, J. Ins. Physiol.
1982, 28, 393.
Fig.ꢀ4.ꢀMethanolꢀextractionꢀofꢀA.ꢀludensꢀfaecesꢀleadsꢀtoꢀtheꢀA.ꢀludensꢀextract.ꢀTheꢀactiveꢀprincipleꢀ
containedꢀinꢀthisꢀextractꢀisꢀtheꢀhostꢀmarkingꢀpheromoneꢀ(HMP)ꢀofꢀA.ꢀludensꢀHMPꢀ((R)-l-(2))ꢀwhichꢀ
hasꢀtheꢀstructureꢀshownꢀinꢀtheꢀFigure.ꢀLeavingꢀoutꢀtheꢀmethylꢀgroupꢀatꢀC(14)ꢀgreatlyꢀsimplifiesꢀ
theꢀsynthesisꢀofꢀtheꢀsecondꢀcompoundꢀ(R)-l-(22)ꢀusedꢀinꢀthisꢀstudy.ꢀForꢀtheꢀthirdꢀcompoundꢀ
discussedꢀhereꢀ(whichꢀweꢀhaveꢀnamedꢀAnastrephamide),ꢀtheꢀmethylꢀgroupꢀwasꢀshiftedꢀtoꢀC(15),ꢀ
andꢀtheꢀstereochemistryꢀatꢀC(2)ꢀwasꢀalsoꢀdiscarded.ꢀ
[12] V. G. Dethier, F. E. Hanson, F. E. Proc. Nat.
Acad. Sci. 1968, 60, 1296.
[13] E. Städler, R. Schöni, R. M. W. Kozlowski,
Physiol. Entomol. 1987, 12, 339; E. Städler, R.
Schöni, R. M. W. Kozlowski, Physiol. Entomol.
1991, 16, 117.
Avendaño, Adán Ochoa, Álvaro Meza, Dina
Orozco, Juan Rull and Jesús Reyes (Campaña
Nacional Contra las Moscas de la Fruta, Metapa
de Domínguez, Chiapas, Mexico) for laboratory
and field assistance and administrative support
(D. Orozco, J. Rull, J. Reyes). This work was fi-
nanciallysupportedbySandozA.G., bytheSwiss
Federal Research Station for Fruit Growing,
Viticulture and Horticulture (Eidgenössische
Forschungsanstalt für Obst-, Wein- und Garten-
bau, Wädenswil, Switzerland), the Mexican
Campaña Nacional Contra Moscas de la Fruta,
the Instituto de Ecología, A.C. and two anony-
mous donors. M. Aluja also acknowledges sup-
port from the Mexican Consejo Nacional de
Ciencia y Tecnología (CONACyT) through a
SabbaticalYear Fellowship (Ref. 79449-2008/9).
tory and field conditions. The work is sum-
marised in Fig. 4.
Further studies will be required to as-
certain if the deterrent effect of synthetic
HMP derivatives will be successful in a
crop protection scenario as such, or wheth-
er this ‘push’ strategy has to be combined
with a ‘pull’ strategy were the females de-
terred from ovipositing are attracted to a
poison trap as flies deterred from treated
fruit and trees are not killed but fly away
in search of clean, untreated fruit. Another
option would be to treat certain rows with
the synthetic HMP and treat a number of
‘pull’ rows with conventional insecticides
to kill the flies moving away from the
HMP-treated trees.
[14] E. F. Boller, J. Hurter, Entomol. Exp. Appl.
1985, 39, 16; E. F. Boller, M. Aluja, J. App,
Entomol. 1992, 113, 113.
[15] Single sets of signals were seen in 1H NMR
and 13C NMR spectra, and HPLC analysis also
showed a single signal.
[16] Temperature program: 70 °C (3 min. isocratic),
2 °C/min. to 190 °C.
[17] W. Oppolzer, R. Moretti, S. Thomi, Tet. Lett.
1989, 30, 5603; W. Oppolzer, R. Moretti, S.
Thomi, Tet. Lett. 1989, 30, 6009.
[18] S. Nunomoto, Y. Kawakami, Y. Yamashita, J.
Org. Chem. 1983, 48, 1912.
[19] 1H-NMR analysis of the crude product (NMR
integration of triplets at 3.893 ppm (J = 6.3
Hz, major isomer) and 3.667 ppm (J = 6.3 Hz,
minor isomer)) showed the diastereoselectivity
of the alkylation to be at least. 95:5. A single
recrytallisation gave diastereomerically pure
13.
[20] W. Oppolzer, J. Blagg, I. Rodriguez, E. Walther,
J. Am. Chem. Soc. 1990, 112, 2767; D. A.
Evans, T. C. Britton, J. A. Ellman, Tet. Lett.
1987, 28, 6141.
[21] If the natural product had been an 80:20 mixture
of l- and d-glutamic acid isomers, two signals
at a ratio of 80:20 would have been observed in
the HPLC at 46 min and 48 min. respectively.
[22] M. L. Hoefle, A. Holmes, B. D. Roth, U.S. Pat.
Appl. US 4716175, 1987.
Received: January 18, 2010
[1] A. L. Averill, R. J. Prokopy, ‘Host marking
pheromones’, in ‘Fruit Flies, their Biology,
Natural Enemies and Control’, Eds. A. S.
Robinson, G. Hooper, Elsevier, Amsterdam,
1989, p. 207.
[2] A. L.Averill, R. J. Prokopy, J. Chem. Ecol. 1987.
13, 167; B. D. Roitberg, R. J. Prokopy. Nature,
1981, 292, 540; A. L. Averill, R. J. Prokopy,
Physiol. Entomol. 1983, 8, 69; M. Aluja, J.
Piñero, I. Jácome, F. Díaz-Fleischer, J. Sivinski,
‘Behaviour of flies in the genus Anastrepha
(Trypetinae: Toxotrypanini)’, in ‘Fruit Flies
(Tephritidae): Phylogeny and Evolution of
Behaviour’, Eds. M. Aluja, A. Norrbom, CRC
Press, Boca Raton, Florida, USA, 2000, p 375;
C. R. Nufio, D. R. Papaj, Entomol. Exp. Appl.
Acknowledgements
We acknowledge Prof. Daniel Belluš for
his support during my early years in indus-
try; the important contribution to this work
by Ernst F. Boller, Jacob Hurter and Erich
Städler (all retired, formerly at Eidgenössische
Forschungsanstalt für Obst-, Wein- und Garten-
bau [FAW], Wädenswil, Switzerland [currently
Agroscope Changins-Wädenswil Research
Station ACW)]), thank Thomas Degen (FAW),
Alejandro Vázquez, Jaime Piñero, Isabel
Jácome, Anita Sánchez, César Ruíz (Instituto de
Ecología, A.C., Xalapa, Veracruz, Mexico) and
José Arredondo, José Luis Márquez, Fernando
[23] The stereochemistry is assumed based upon the
observation that (R)-l-(2) eluted before (R)-d-
(2) under identical HPLC conditions.