Comparative analysis of three Australian finger lime (Citrus australasica) cultivars: Identification of unique citrus chemotypes and new volatile molecules

DOI: 10.1016/j.phytochem.2014.10.023

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Phytochemistry p. 111 - 124 (2015)

Update date:2022-08-04

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Authors:

Delort, Estelle

Jaquier, Alain

Decorzant, Erik

Chapuis, Christian

Casilli, Alessandro

Frérot, Eric

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Article abstract of DOI:10.1016/j.phytochem.2014.10.023

The volatile constituents of the peel of three cultivars of Australian finger lime (Citrus australasica) were investigated: Alstonville, Judy's Everbearing and Durham's Emerald. Both qualitative and quantitative GC-MS analyses were performed on their peel solvent extract. The results showed that the unique phenotypes of finger lime are also correlated to unique molecular compositions. Each cultivar revealed a different chemotype: limonene/sabinene for cv. Alstonville, limonene/citronellal/isomenthone for cv. Judy's Everbearing, and limonene/citronellal/ citronellol for cv. Durham's Emerald. To the best of our knowledge, these chemotypes have never been reported in any other citrus species. Furthermore, the amounts of some volatile constituents (γ-terpinene, α-pinene, β-pinene, citral), which are generally the major constituents besides limonene in lime species, were surprisingly low in the three cultivars. Comparative GC-MS analysis also showed that some volatile molecules tended to be specific to one cultivar and could therefore be considered as markers. Moreover six molecules were reported for the first time in a citrus extract and confirmed by synthesis. Heart-cutting enantioselective two-dimensional GC-MS was performed to determine the enantiomeric distribution of the major chiral constituents. The combined data on three finger lime cultivars gives evidence of their divergence from other citrus species.

Full text of DOI:10.1016/j.phytochem.2014.10.023

Phytochemistry 109 (2015) 111–124

Contents lists available at ScienceDirect

Phytochemistry

journal homepage: w ww.elsevier.com/locate/phytochem

Comparative analysis of three Australian ﬁnger lime (Citrus australasica)

cultivars: Identiﬁcation of unique citrus chemotypes and new volatile

molecules

⇑

Estelle Delort , Alain Jaquier, Erik Decorzant, Christian Chapuis, Alessandro Casilli , Eric Frérot

Firmenich SA, Corporate R&D Division, Geneva, Switzerland

a r t i c l e i n f o

a b s t r a c t

Article history:

Received 4 June 2014

Received in revised form 15 October 2014

Available online 11 November 2014

The volatile constituents of the peel of three cultivars of Australian ﬁnger lime (Citrus australasica) were

investigated: Alstonville, Judy’s Everbearing and Durham’s Emerald. Both qualitative and quantitative

GC–MS analyses were performed on their peel solvent extract. The results showed that the unique phe-

notypes of ﬁnger lime are also correlated to unique molecular compositions. Each cultivar revealed a dif-

ferent chemotype: limonene/sabinene for cv. Alstonville, limonene/citronellal/isomenthone for cv. Judy’s

Everbearing, and limonene/citronellal/ citronellol for cv. Durham’s Emerald. To the best of our knowl-

edge, these chemotypes have never been reported in any other citrus species. Furthermore, the amounts

Keywords:

Alstonville

Judy’s Everbearing

Durham’s Emerald

of some volatile constituents (c-terpinene, a-pinene, b-pinene, citral), which are generally the major con-

stituents besides limonene in lime species, were surprisingly low in the three cultivars. Comparative GC–

MS analysis also showed that some volatile molecules tended to be speciﬁc to one cultivar and could

therefore be considered as markers. Moreover six molecules were reported for the ﬁrst time in a citrus

extract and conﬁrmed by synthesis. Heart-cutting enantioselective two-dimensional GC–MS was per-

formed to determine the enantiomeric distribution of the major chiral constituents. The combined data

on three ﬁnger lime cultivars gives evidence of their divergence from other citrus species.

6-Methyl-1-octylacetate

Isoascaridole

Citronellyl citronellate

1,2:5,6-Diepoxy-p-menthane

2,3-Epoxy-p-menthan-6-one

p-Menth-1-en-3-ol-6-one

1,2-Epoxy-p-methan-5-one

4-Hydroxy-6-isopropyl-3-methylcyclohex-

2-enone

1. Introduction

Durham’s Emerald, Judy’s Everbearing, Pink Ice, Byron Sunrise

and Jali Red. In our previous study (Delort and Jaquier, 2009), the

The Australian ﬁnger lime Citrus australasica is one of ﬁve native

citrus species endemic to Australia. It is native to the rainforests of

South East Queensland and Northern New South Wales. Finger

limes are genetically diverse in shape, peel and pulp color, size

and taste. All have in common the caviar-like pulp, which makes

this fruit unique in the genus Citrus. Because of the increasing

demand for ﬁnger limes, initially from the restaurant trade, some

ﬁnger lime cultivars were selected from the wild and are currently

commercially grown in Australia. For the time being, one ﬁnger

lime variety has been registered with Plant Breeders’ Rights (C. aus-

tralasica var. sanguinea, also called ‘‘Rainforest Pearl’’), and seven

ﬁnger lime cultivars have been registered with the Australian Cul-

tivar Registration Authority: Alstonville, Blunobia Pink Crystal,

volatile composition of the peel extract of Australian ﬁnger lime

of unknown cultivar was investigated by gas chromatography–

mass spectrometry (GC–MS). The work highlighted a unique com-

position (limonene/isomenthone/citronellal). Six new terpenyl

esters were identiﬁed and their structures conﬁrmed by chemical

synthesis. In the present study, three ﬁnger lime cultivars, namely

Alstonville, Judy’s Everbearing and Durham’s Emerald, were inves-

tigated for the ﬁrst time with the aim of comparing, both qualita-

tively and quantitatively, their volatile composition and identifying

the molecular markers of the cultivars.

2. Results and discussion

2.1. Comparison of phenotypic and organoleptic characters of fresh

fruits

⇑

Corresponding author at: Firmenich SA, Corporate R&D Division, Route des

Jeunes 1, CH-1211 Geneva 8, Switzerland. Tel.: +41 22 780 35 32; fax: +41 22 780

33 34.

As shown in Fig. 1, the different cultivars investigated in the

present study were relatively close in terms of shape and size,

but differed in their peel and pulp color. The cultivar Alstonville

E-mail address: estelle.delort@ﬁrmenich.com (E. Delort).

Present address: Universidade Federal do Rio de Janeiro, Instituto de Química, Ilha

do Fundão, Rio de Janeiro, RJ 21941-909, Brazil.

http://dx.doi.org/10.1016/j.phytochem.2014.10.023

112

E. Delort et al. / Phytochemistry 109 (2015) 111–124

Fig. 1. Finger lime fruits investigated in the present study. (a, b) cv. Alstonville; (c,d) cv. Judy’s Everbearing; (e,f) cv. Durham’s Emerald (Photos: A. Jaquier, Firmenich S.A.).

was characterized by a pale to dark green peel and a pale green

pulp. Judy’s Everbearing fruits have a reddish to green and dark

green peel with green to yellow rosy pulp. Durham’s Emerald cit-

rus has a green to dark green peel with green pulp, relatively close

to the cultivar Alstonville, but with a size and shape closer to Judy’s

Everbearing.

green and citronellal-like for Judy’s Everbearing, and citronellal-

like with turpentine and vegetable notes for Durham’s Emerald.

2.2. GC–MS comparative analysis of peel solvent extracts

The volatile composition of the peel extracts was analyzed by

GC–MS. To perform a comparative analysis, the solvent extraction

was repeated under strictly identical conditions for the three culti-

vars and the reported analytical procedure herein was followed for

each peel extract. GC–MS ﬁles were ﬁrst processed manually by

using the MSD Chemstation software (Agilent). The deconvolution

software AMDIS was then used as a support tool for the identiﬁca-

tion of additional trace compounds that were not easily detected

manually because of coelutions. The additional identiﬁcations

given by AMDIS, as well as the absence of molecules in some cul-

tivars, were conﬁrmed or rejected by using manual interpretation

and extract ion mode. Then the percentages of the volatile compo-

nents were determined by manual integration of GC-ﬂame ioniza-

tion detector (FID) areas with the use of an internal standard and

the application of correction factors as reported earlier (de Saint

Laumer et al., 2010). This meticulous analytical process allowed

us to obtain a qualitative and quantitative comparative analysis

of the peel extract of the three cultivars, as given in Table 1.

The peel aroma and the juice taste of fresh fruits were evaluated

by perfumers and ﬂavorists, all experts in citrus. All of these

experts noticed the organoleptic uniqueness of the peel. Compared

to common lime varieties, namely Key lime (Citrus aurantifolia) and

Tahiti lime (Citrus latifolia), the three cultivars were lime-like, but

greener and less ﬂoral. Interestingly, the odor nuances of the peel

were quite different from one cultivar to another. The peel of

Alstonville was greener, more terpenic (myrcene-like, sabinene-

like) and slightly minty. A clear citronellal-like character reminis-

cent of citronella was found in the peel of Judy’s Everbearing. Its

proﬁle was also terpenic, but fresher, more minty-like, and associ-

ated with some spicy and fruity notes. The citronellal-like charac-

ter was also perceived in Durham’s Emerald, but it was distinctive

for its woody lime character with phenolic (p-vinylguaiacol-like)

and slightly smoky notes. The juice of these cultivars was less

unique, being acidic and in-between lemon and lime juice with

the following nuances: bitter and slightly resinous for Alstonville,

E. Delort et al. / Phytochemistry 109 (2015) 111–124

113

Table 1

Volatile composition of ﬁnger lime solvent peel extracts.

LRI

Name

A % FID

JE % FID

DE % FID

Ident.

SPB-1

617

627

636

654

657

668

673

716

725

746

749

753

773

775

819

826

837

(E)-2-Butenal

tr^⁄

–

tr^⁄

–

tr^⁄

–

LRI, MS

3-Methylbutanal

2-Methylbutanal

1-Penten-3-one

1-Penten-3-ol

tr^⁄

3-Pentanone

3-Hydroxy-2-butanone

Tentative: (Z)-2-pentenal

(E)-2-pentenal

1-Pentanol

(Z)-2-Penten-1-ol

3-methyl-2-butenal

(Z)-3-Hexenal

Hexanal

(Z)-2-Hexenal

(E)-2-Hexenal

(Z)-3-Hexen-1-ol

(Z)-2-Hexen-1-ol

1-Hexanol

2-Methylbutyl acetate

Heptanal

tr^⁄

–

tr^⁄

–

847

850

861

879

880

903

tr^⁄

–

tr^⁄

–

tr^⁄

–

(E,E)-2,4-Hexadienal

tr^⁄

Unknown^b

–

927

933

936

948

950

971

977

983

-Thujene

0.12

0.55

0.01

tr^⁄

0.98

LRI, MS

Benzaldehyde

tr^⁄

0.31

a-Pinene

a-Fenchene

Camphene

Sabinene

b-Pinene

Myrcene

(Z)-3-Hexenyl acetate

Hexyl acetate

2-Carene

0.16

1.52

–

20.55

0.32

1.50

tr^⁄

0.58

0.03

1.54

–

986

993

995

tr^⁄

–

1001

1009

1012

1013

1016

1026

1028

1031

1036

1039

1053

1056

1059

1062

1073

1076

1077

1084

1085

1088

1092

1099

1103

1108

1113

1117

1119

1122

1126

1129

1135

1136

1143

1144

1148

1149

1151

a-Phellandrene

0.01

0.09

0.25

0.19

2.00

0.36

tr^⁄

0.32

4.74^a

–

66.27

0.73

0.19

0.01

d-3-Carene

Phenylacetaldehyde

a-Terpinene

tr^⁄

0.16

1.79^a

–

p-Cymene

b-Phellandrene

Eucalyptol

0.18^a

tr^a

61.66

0.15

–

Limonene

64.38

0.35

0.08

0.01

–

(Z)-b-Ocimene

2,6-Dimethyl-5-heptenal

(E)-b-Ocimene

0.21

0.08

-Terpinene

6-methyloctanal

trans-Sabinene hydrate

cis-Linalyl oxide (furanoid)

Methyl benzoate

trans-Linalyl oxide (furanoid)

p-Cymenene

–

tr^⁄

–

tr^⁄

0.10

0.21

–

tr^⁄

0.03

1.94

tr^⁄

–

a-Terpinolene

0.02

0.03

–

Linalool

cis-Sabinene hydrate

Heptyl acetate

–

cis-Rose oxide

tr^⁄

–

Tentative: 1,3,8-p-menthatriene

2,6-Dimethyl-5-hepten-1-ol

trans-2,8-p-Menthadien-1-ol

trans-p-Menth-2-en-1-ol

trans-Rose oxide

(4E,6Z)-Alloocimene

cis-1,2-Epoxy-8-p-menthene

trans-1,2-Epoxy-8-p-menthene

cis-p-Menth-2-en-1-ol

Citronellal

Isopulegol

Neoisopulegol

Menthone

4-Isopropenyl-2-cyclohexen-1-one

4-Methylnonanal

tr^⁄

–

9.26

tr^⁄

tr^a

tr^⁄

–

tr^⁄

–

9.04

tr^⁄

–

tr^⁄

–

Ethyl benzoate

Isomenthone

tr^⁄

–

7.29

–

0.03

(continued on next page)

114

E. Delort et al. / Phytochemistry 109 (2015) 111–124

Table 1 (continued)

LRI

Name

A % FID

JE % FID

DE % FID

Ident.

SPB-1

1152

1165

1159

1164

1165

1169

1174

1176

1178

1179

1182

1185

1187

1191

1195

1199

1202

1206

1207

1210

1211

1216

1218

1222

1223

1228

1235

1239

1240

1243

1245

1247

1255

1259

1260

1263

1265

1276

1277

1278

1283

1291

1297

1301

1311

1313

1321

1324

1331

1334

1338

1341

1345

1358

1361

1362

1371

1376

1380

1386

1389

Phenylacetaldehyde (E)-O-methyl oxime

Phenylacetaldehyde (Z)-O-methyl oxime

trans-Isopulegone

Tentative: thujenol

Tentative: 4-isopropyl-2-cyclohexen-1-one

p-Cymen-8-ol

4-Terpineol

trans-1(7),8-p-Menthadien-2-ol

Methyl salicylate

Neoisomenthol

Isomenthol

tr^⁄

–

0.03

–

0.03

–

LRI, MS

tr^⁄

0.16

tr^⁄

–

0.72^a

tr^a

0.21

–

a-Terpineol

tr^⁄

–

8-p-Menthen-2-ol

Decanal

cis-Piperitol

Octyl acetate

trans-Piperitol

4-Isopropylphenol

trans-Carveol

–

0.10

–

tr^⁄

–

tr^⁄

–

tr^⁄

–

tr^⁄

–

cis-1(7)-8-p-Menthadien-2-ol

Unknown^c

2.00

–

0.96

tr^⁄

–

Citronellol

5.18

–

LRI, MS

trans-Sabinene acetate

(Z)-3-Hexenyl 2-methylbutyrate

neral

4-Isopropylbenzaldehyde (cuminaldehyde)

Carvone

Methyl thymol ether

Piperitone

(E)-2-Decenal

Linalyl acetate

cis-Sabinene acetate

geranial

Isopiperitenone

Perillic aldehyde

1,2-Epoxy-p-menthan-5-one 7

8-Methyldecanal

6-Methyloctyl acetate 1

Thymol

Carvacrol

Bornyl acetate

0.43

tr^⁄

–

0.25

–

tr^⁄

–

tr^⁄

–

tr^⁄

–

p-Vinyl guaiacol

Citronellic acid

–

Terpinen-4-ol acetate

–

Unknown^d

–

cis-isoascaridole cis-3a

Tentative: 2,6-dimethyl-7-octen-2,6-diol (2-hydroxydihydrolinalool)

2,3-Epoxy-p-menthan-6-one 5a

Tentative: 3,7-dimethyl-5-octen-1,7-diol

Tentative: 3-oxo-p-menthen-1-en-7-al

2,3-Epoxy-p-menthan-6-one 5b

Tentative: p-menthane-3,8-diol isomer 1 (8-hydroxymenthol)

8-p-Menthene-1,2-diol isomer 1

Trans-piperitol acetate

Citronellyl acetate

–

tr^⁄

–

LRI, MS

LRI_lit, MS

LRI, MS

0.19^a

0.02^a

–

0.40^a

0.04^a

0.19

0.15

0.21

tr^⁄

–

0.04^a

0.20^a

0.01^a

–

0.04^a

0.20^a

–

1,2:5,6-Diepoxy-p-menthane 4a

Unknown^e

Tentative: p-menthane-3,8-diol isomer 2

Tentative: 2-phenyl-2-hydroxypropanal

1,2:5,6-Diepoxy-p-menthane 4b

Tentative: p-menthane-3,8-diol

Eugenol

–

LRI, MS

0.15^a

–

0.58

–

0.15^a

0.05^a

–

LRI, MS

a-Terpenyl acetate

0.66

tr^⁄

0.61

tr^⁄

–

tr^⁄

–

Neryl acetate

d-Elemene

Geranyl acetate

(Z)-3-Hexenyl hexanoate

Vanillin

0.16

–

tr^⁄

Methyleugenol

cis-p-Menth-1-en-3-ol-6-one 6a

trans-p-Menth-1-en-3-ol-6-one 6b

(E)-7-Tetradecene

Decyl acetate

Dodecanal

4-Hydroxypiperitone

–

tr^⁄

–

tr^⁄

–

tr^⁄

–

tr^⁄

–

E. Delort et al. / Phytochemistry 109 (2015) 111–124

115

Table 1 (continued)

LRI

Name

A % FID

JE % FID

DE % FID

Ident.

SPB-1

1398

1413

1424

1435

1438

1441

1446

1448

1450

1463

1468

1492

1497

1498

1508

1514

1517

1526

1530

1538

1552

1559

1560

1563

1571

1582

1586

1594

1609

1627

1647

1655

1676

1747

1837

1879

1906

1909

1917

1934

1936

2022

2057

2101

2294

2118

2426

2493

2643

b-Elemene

Perillic acetate

Citronellyl propanoate

(E)-Isoeugenol

trans-b-caryophyllene

–

0.38

–

0.60

0.37

–

0.84

–

0.13

–

LRI, MS

tr^⁄

-Elemene

tr^⁄

–

trans- -Bergamotene

(E)-2-Dodecenal

b-Farnesene

Geranyl propanoate

Methyl isoeugenol

–

0.09

0.20

–

3.09

–

tr^⁄

–

tr^⁄

0.90

tr^⁄

0.74

1.80

–

-Humulene

Germacrene-D

(E,E)- -Farnesene

cis- -Bisabolene

Bicyclogermacrene

Bornyl 3-methylbutanoate

Elemicin

d-Cadinene

(Z)-9-Dodecen-12-olide (yuzu lactone)

–

tr^⁄

trans-

a-Bisabolene

(Z)-3-Hexenyl benzoate

Hexyl benzoate

Citronellyl 2-methylbutanoate

Citronellyl 3-methylbutanoate

Germacrene-B

Spathulenol

geranyl 3-methylbutanoate

Globulol

tr^⁄

–

0.36

–

0.12

0.20

–

Benzophenone

–

tr^⁄

–

tr^⁄

Tentative: germacrol

Unknown^f

0.05^a

–

0.32

0.22

–

Tentative: guaia-10(14),6-dien-4-ol

Tentative: valenca-1,9-dien-11-ol

Citronellyl 3-methyl-2-butenoate

LRI, MS

–

0.53

tr^⁄

–

0.21

–

5.67

–

tr^⁄

–

a-Cadinol

-Bisabolol

tr^⁄

Unknown^g

Neophytadiene

Tentative: neophytadiene isomer

Tentative: 8,13-epoxy-15,16-dinorlabd-12-ene

Citronellyl benzoate

6,7-Dimethoxy-2H-chromen-2-one

Hexadecanoic acid

0.23

tr^⁄

LRI, MS

Sinapyl alcohol

–

Citronellyl citronellate 2

Unknown^h

trans-Phytol

tr^⁄

–

LRI, MS

Citronellyl decanoate

Unknownⁱ

0.19

0.16

–

Oxypeucedanin

Tentative: citronellyl dodecanoate

oxypeucedanin hydrate

–

LRI, MS

tr^⁄

–

A: cv. Alstonville; JE: cv. Judy’s Everbearing; DE: cv. Durham’s Emerald. FID: ﬂame ionization detector; LRI: linear retention index; tr: trace (<0.01%). tr^⁄: Trace compound

identiﬁed with AMDIS. LRI_lit: LRI obtained from the literature.

Quantiﬁed on polar phase.

m/z (%) = 140(19),125(10),112(9),111(67), 97(29), 82(51),81(20),79(12), 70(19),69(62),68(9), 67(89),59(23),55(16),53(13),43(100),41(36).

m/z

(%)=151(5),150(30),135(32),122(23),108(63),107(76),

95(20),94(14),93(54),91(17),82(21),81(47),80(33),79(100),77(18),

69(27),68(24),67(55),66(11),58(13),

55(21),54(36),53(64),52(8),51(13),43(10),41(65).

m/z(%) = 137(36),134(34),123(11),121(42),119(30),110(14),109(69),107(11),105 (26),95(33),94(76),93(28),92(16),91(86),84(16),82(10),81(36),79(100),78(13),77(39),71

(13),69(20),67(23),65(13),55(17),53(16),51(12),43(50),41(34).

m/z(%) = 168(7),117(12),97(10),82(100),79(13),71(71),68(10),67(46),58(14),55(12),50(10),45(11),44(28),43(75),41(18).

m/z

(%) = 202(37),187(26),162(22),159(87),149(25),147(31),145(32),135(21),134(19),133(62),

131(55),121(29),120(31),119(36),117(39),109(55),107(52),106(22),

105(77),95(33),93(57),91(100), 83(20),82(38),81(54),79(65),77(45),69(50),67(74),65(23),59(33),55(61),53(36),44(22),43(47), 41(91).

m/z (%) = 202(25),159(56),147(21),145(31),133(62), 131(46), 123(26),121(24),120(23),119(34), 117(35),109(25), 107(43),105(81), 95(51),93(52),91(100), 82(32),81(45),

79(65), 77(51),69(36), 67(61),65(23),55(48),53(33),43(34),41(79).

m/z(%) = 253(11),252(77),209(22),194(43),193(20),192(55),177(21),161(40),154(14),149(100),133(27),131(31),121(26),119(21),118(23),106(43),105(30),103(25),

91(53),89(25),78(42),77(48),65(22),60(36),51(21),45(49),43(94).

m/z(%) = 231(10),230(82),216(16),215(100),213(12),201(15),187(78),176(11),175(90),

147(19),144(18),141(15),131(22),128(14),115(26),100(13),91(15),77(20),69(13),65(13),51(16),41(19).

As shown in Fig. 2, the volatile compositions of the three culti-

vars were very different. Each cultivar revealed a unique chemo-

type: limonene/sabinene for Alstonville, limonene/citronellal/

isomenthone for Judy’s Everbearing and limonene/citronellal/citro-

nellol for Durham’s Emerald. Recently, Lota et al. (2002) compared

the chemical composition of peel and leaf oil of 43 lemon and lime

116

E. Delort et al. / Phytochemistry 109 (2015) 111–124

Abundance

Alstonville

Judy’s Everbearing

Durham’s Emerald

Time

10.00

20.00

30.00

40.00

50.00

60.00

Fig. 2. GC proﬁle of the peel solvent extract of the three cultivars on a non-polar column.

Table 2

Enantiomeric distribution obtained by enantioselective heart-cut 2D-GC–MS.

Alstonville

Judy’s Everbearing

Durham’s Emerald

Limonene (R)-(+)/(S)-(À)

Citronellal (R)-(+)/(S)-(À)

Citronellol (R)-(+)/(S)-(À)

Sabinene (R)-(+)/(S)-(À)

99.4/0.6

5.3/94.7

98.9/1.1

96.7/3.3

97.2/2.8

96.7/3.3

95.6/4.4

95.8/4.2

-Phellandrene (R)-(À)/(S)-(+)

0/100

nd: Not determined due to their low abundance in the extract.

species/cultivars, all cultivated in similar environmental condi-

tions. Results showed the existence of three chemotypes for lime

peel extracts: limonene, limonene/b-pinene, and limonene/b-

one, may contribute to the minty odor of the peel, while the higher

amount of monoterpenes (e.g. sabinene, b-pinene, a-thujene) may

explain the terpenic and green character.

pinene/

-terpinene. In that study, C. australasica (cultivar not men-

Judy’s Everbearing was characterized by an unusually high

amount of citronellal and isomenthone. Many derivatives of these

molecules were also identiﬁed: citronellyl acetate, citronellyl pro-

panoate, citronellyl 2-methylbutanoate, citronellyl 3-methylbut-

anoate, citronellyl benzoate, citronellyl citronellate, citronellyl

decanoate, neoisomenthol, isomenthol and menthone. Such a high

amount of menthone derivatives is common in mint but not in cit-

rus. They are probably responsible for the fresh minty odor per-

ceived in the peel, while citronellal and citronellyl esters provide

the citronella character and the fruity notes, respectively. Com-

pared to those in the other cultivars, linalool and piperitone were

also found in higher amounts. The spicy and slightly smoky notes

of the peel may be explained by the high number of phenols in this

cultivar: eugenol, carvacrol, 4-isopropylphenol, p-vinyl guaiacol

and methyl eugenol.

tioned) showed a unique pattern, with sabinene as the second

major volatile constituent after limonene. To the best of our knowl-

edge, the chemotypes of the three ﬁnger lime cultivars of the pres-

ent study are not only unique in lime species, but also in the genus

Citrus. Moreover, the three peel extracts had in common a surpris-

ingly low amount of

c-terpinene (0.10%), a-pinene (<1.00%), b-

pinene (<0.40%), neral (<0.01%) and geranial (<0.01%), which are

generally the major volatile constituents of lime peel extracts

besides limonene (Lota et al., 2002).

Comparing the three cultivars qualitatively and quantitatively

highlighted the similarities and differences between them. Alston-

ville was characterized by the absence of citronellal and citronellol,

both found in high amounts in the two other cultivars. On the other

hand, its peel extract contained trace amounts of several aliphatic

(heptyl acetate, octyl acetate) and terpenic acetates (linalyl acetate,

trans- and cis-sabinene acetate, bornyl acetate), which were not

detected in the two other cultivars. Such high amounts of various

acetates, which has been reported in kumquat peel (Choi, 2000),

is generally not common in lime. The use of AMDIS and extract

ion mode on polar stationary phase allowed us to identify unam-

biguously eucalyptol (1,8-cineole) in this cultivar, which was

absent in the other two cultivars. Eucalyptol, together with carv-

Durham’s Emerald had a proﬁle that was closer to Judy’s Ever-

bearing than to Alstonville, with a high amount of citronellal and

citronellol. However, both cultivars differed by the amount of some

constituents like isomenthone, found in only trace amount in Dur-

ham’s Emerald, and

a-phellandrene, found in higher amount in

Durham’s Emerald. Several molecules were found only in this cul-

tivar, in particular some aldehydes (4-methylnonanal, 4-isopropyl-

benzaldehyde, (E)-2-dodecenal), phenols (methyl isoeugenol,

E. Delort et al. / Phytochemistry 109 (2015) 111–124

117

Fig. 3. Molecules newly identiﬁed in citrus. ^aReported for the ﬁrst time in a natural product; ^breported for the ﬁrst time in citrus.

(E)-isoeugenol), and sesquiterpenes (trans-

a-bergamotene, cis-and

late 2 (LRI 2022) had never been reported in citrus, but was iden-

trans- -bisabolene). These sesquiterpenes are typical in lime and

may provide the woody lime character perceived in the fresh peel

of this cultivar.

tiﬁed in natural products that are rich in citronellal, such as

citronella (Glichitch, 1926) and Eucalyptus citriodora (Vernin

et al., 2004). It was identiﬁed in Judy’s Everbearing and Durham’s

Emerald extracts, which indeed both contain a signiﬁcant amount

of citronellal. Isoascaridole of undetermined stereochemistry was

recently reported for the ﬁrst time in orange and bergamot essen-

tial oil (Tranchida et al., 2013). In the present study, both the cis

and trans isomers were synthesized, and the presence of the cis-

isoascaridole 3a (LRI 1291) in ﬁnger lime could be conﬁrmed.

1,2:5,6-Diepoxy-p-menthane 4 (2 isomers, LRI 1324 and 1331)

and 2,3-epoxy-p-menthan-6-one 5 (2 isomers, LRI 1297 and

1301) are reported for the ﬁrst time in a natural product. Cis and

trans-p-menth-1-en-3-ol-6-one, cis-6a (LRI 1376) and trans-6b

(LRI 1380), were identiﬁed in the peel extract of the cultivars Judy’s

Everbearing and Durham’s Emerald. The MS of both isomers of 4

and 6 were detected in our previous analysis, but could not be

identiﬁed at that time (Delort and Jaquier, 2009). To the best of

our knowledge, 6 was reported in some natural products but never

in citrus. 1,2-Epoxy-p-Methan-5-one 7 (LRI 1259) is reported here

for the ﬁrst time in a natural extract.

2.3. Chirality of major components

The enantiomeric distribution of the major compounds (limo-

nene, sabinene, citronellol and citronellal) was determined by

using heart-cutting enantioselective two-dimensional GC–MS.

The principle was to transfer the compound of interest during its

elution in the ﬁrst dimension, a non-polar phase, into a second chi-

ral column. Compared to the monodimensional approach, this

multidimensional approach has the advantage of reducing coelu-

tions that may lead to wrong enantiomeric ratios. The results are

given in Table 2. The chirality of limonene was the same as that

already reported in citrus oils (Mondello et al., 2011), with (S)-

(À) as the major isomer. In Alstonville, the major enantiomer of

sabinene was (S)-(À), as already reported in Persian lime and Key

lime by the same authors. However, for citronellal, the (R)-(+)

was the major isomer, which is not common in lime. Indeed, the

major enantiomer of citronellal was reported to be (S)-(À) in Per-

sian lime and Key lime, as well as in other citrus in which it occurs

at a signiﬁcant level, such as bergamot, kafﬁr lime and lemon

(Mondello et al., 2011). The (R)-(+) is generally the major isomer

in citronella oil (Nhu-Trang et al., 2006), as well as in tangerine,

sweet orange, grapefruit and mandarin (Mondello et al., 2011).

2.5. Conﬁrmation by synthesis

All molecules mentioned above were conﬁrmed after compari-

son of their MS spectra and LRIs with those of freshly prepared

standards. 6-Methyloctyl acetate 1 and citronellyl citronellate 2

were synthesized via standard esteriﬁcation. Cis-isoascaridole 3a

was obtained from ascaridole via thermal rearrangement. The mol-

2.4. Identiﬁcation of new molecules

As reported in Table 1, several peaks detected in trace amounts

remained unknown after GC–MS analysis. Although identiﬁcation

of the structure of unknowns based solely on mass spectra inter-

pretation is challenging, seven unknowns could be elucidated dur-

ing this work via high resolution mass spectra interpretation. Their

structures were conﬁrmed after comparison of their MS and LRIs

with those of the seven corresponding freshly prepared standards

(Fig. 3). To the best of our knowledge, six of them had never been

found in a citrus extract, and four of them are reported for the ﬁrst

time in a natural product.

6-Methyloctyl acetate 1 (LRI 1263) was detected in the peel

extract of the cultivar Alstonville and identiﬁed here for the ﬁrst

time in a natural product. The occurrence in citrus was not surpris-

ing, as its corresponding aldehyde was also found in trace amounts

in the same extract and was already known in yuzu (Tajima et al.,

1990) and orange peel oil (Widder et al., 2003). Citronellyl citronel-

ecules 4, 5, 6 and 7 were prepared from

in Table 3. The syntheses were carried out on the only commer-

cially available enantiomer, (R)-(À)- -phellandrene 9, although

its chirality in ﬁnger lime was determined as pure (S)-(+) (Table 2).

Following the work of Schenck et al. (1953), (R)-(À)- -phelland-

a-phellandrene, as shown

rene 9 was photo-oxygenated to produce a 57:43 mixture of

(1S,4R,7R)-(+)-10a/(1R,4S,7R)-(+)-10b. Chromatographic puriﬁca-

tion allowed us to isolate an 83:17 enriched quality of

(1S,4R,7R)-(+)-10a, as well as diastereoisomerically pure

(1R,4S,7R)-(+)-10b. A small sample of pure (1R,4S,7R)-(+)-10b

was treated with a catalytic amount of [(Ph₃P)₃RuCl₂] in CH₂Cl₂

at 20 °C following methods of Suzuki et al. (1989) to afford the dia-

stereoisomerically pure diepoxide (1S,2R,4R,5R,7S)-(À)-4b (Turner

and Herz, 1977) in 80% yield, which proved to be thermally stable

after 4 h in reﬂuxing toluene. (1R,2S,4S,5R,7R)-(À)-4a was isolated

after puriﬁcation of the crude material obtained under the same

118

E. Delort et al. / Phytochemistry 109 (2015) 111–124

Table 3

Syntheses of new

-phellandrene derivatives. In table: 3a: cis-isoascaridole; 4a: (1R,2S,4S,5R,7R)-(À)-4a; 4b: (1S,2R,4R,5R,7S)-(À)-4b; 5a: (1R,2S,5R,6S)-5a; 5b: (1S,2R,5R,6R)-5b;

6a: (4S,5R)-(À)-6a; 6b: (4R,5R)-(+)-6b; 7b: (1S,4S,6R)-7b; 8: (4S,6R)-(À)-8; 10a: (1S,4R,7R)-(+)-10a; 10b: (1R,4S,7R)-(+)-10b. Conditions: i) a) O₂, hv, iPrOH, methylene blue, 40 h,

20 °C; ii) cat [(Ph₃P)₃RuCl₂], CH₂Cl₂, 20 °C, 4 h; iii) toluene, 110 °C, 4 h; iv) toluene, 110 °C, 18 h; v) GC–MS injection on SPB-1 non-polar column (injector temperature 250 °C).

Reagent

Cond.

10a

10b

(R)-(À)-9

10b

10a/10b 83:17

10b

57%

43%

iii

80%

22%

25%

56%

48%

12%

17%

38%

32%

22%

18%

4%^a

15%

11%

11%^a

27%

15%

10b

Tentative identiﬁcation.

conditions starting from the 83:17 mixture of (1S,4R,7R)-(+)-10a/

(1R,4S,7R)-(+)-10b.

the

thermodynamically

stable

diastereoisomers

(1R,2R,5R,6S)-5c (0.00 kcal/mol) and (1S,2S,5R,6R)-5d (0.95 kcal/

mol), respectively. For these reasons, the stereochemistry of 5a

was further investigated for conﬁrmation via successive reduction,

separation and oxidation, as described in Supplementary informa-

tion. (1R,2S,5R,6S)-5a was isolated from the above mixture by ﬂash

chromatography and obtained only in analytical quantity, as it

degraded during puriﬁcation on silica gel into the hydroxy-enone

(4S,5R)-(À)-6a. Similarly, thermal treatment in reﬂuxing toluene

(18 h, 110 °C) of (1R,4S,7R)-(+)-10b afforded a mixture containing

(1S,2R,5R,6R)-5b (11%), from which it could be characterized.

(4R,5R)-(+)-6b was obtained in analytical quantity during the

attempted chromatographic puriﬁcation (cyclohexane/AcOEt 9:1)

of this mixture.

The keto-epoxides 5a and 5b were obtained after thermal treat-

ment of the endoperoxides 10a and 10b. Indeed, the heating of the

83:17 mixture of (1S,4R,7R)-(+)-10a/(1R,4S,7R)-(+)-10b in reﬂuxing

toluene for 4 h afforded, according to ¹³C NMR analysis, a mixture

of (1R,2S,4S,5R,7R)-4a (32%)/(1S,2R,4R,5R,7S)-(À)-4b (25%), cis-

isoascaridole 3a (17%), the major keto-epoxide (1R,2S,5R,6S)-5a

(18%), and two other very minor keto-epoxides tentatively

assigned to (1S,2R,5R,6R)-5b (4%) and (1S,4S,6R)-7b (4%). The pres-

ence of isoascaridole cis-3a may come from thermal rearrangement

of ascaridole found in the 83:17 mixture (1S,4R,7R)-(+)-10a/

(1R,4S,7R)-(+)-10b obtained after photo-oxidation. As shown in

Table 3, epoxy-cyclohexanone 5 may exist as four diastereoiso-

mers. According to STO 6-31G^⁄⁄calculations (Frisch et al., 1998;

Luft et al., 2007), both (1R,2S,5R,6S)-5a (0.41 kcal/mol) and

(1S,2R,5R,6R)-5b (1.10 kcal/mol) may eventually epimerize into

Finally, taking into account the pure (S)-(+) enantiomer form in

ﬁnger lime, as well as the LRIs (both on non-polar and polar

columns) of the above standards, we suggest the following

E. Delort et al. / Phytochemistry 109 (2015) 111–124

119

conﬁguration for the

extracts: (1S,2R,4R,5S,7S)-4a, (1R,2S,4S,5S,7R)-4b, (1S,2R,5S,6R)-5a,

(1R,2S,5S,6S)-5b, (4R,5S)-6a and (4S,5S)-6b.

-phellandrene derivatives in the ﬁnger lime

internal standard. Standard gradient-selected COSY, HSQC and

HMBC experiments were carried out on a Bruker Avance 500 or

on a Bruker Avance 600 spectrometer, and signal assignment was

achieved by using Bruker NMR software TopSpin 2.0.

However, the natural occurrence of di-epoxides 4, keto-epox-

ides 5 and hydroxy-enone 6 in ﬁnger lime should be considered

with the utmost circumspection, in view of the photo and thermal

lability of the endoperoxides 10, readily affording mixtures of 4

and 5, as well as the chromatographic instability of 5 leading to

6. The thermal lability of endoperoxide 10 was conﬁrmed, as the

injection of pure (1R,4S,7R)-(+)-10b into a GC–MS revealed its deg-

radation into the di-epoxide (1S,2R,4R,5R,7S)-(À)-4b and the keto-

epoxides (1S,2R,5R,6R)-5b and (1S,4S,6R)-7b, which may occur in

the injector (Table 3). Consequently, these molecules may come

from the thermal rearrangement during GC–MS of 10, in analogy

to the reported conversion of ascaridole into its corresponding

diepoxide iso-ascaridole (Nitz et al., 1989). For these reasons, the

Infra-red measurements were done on a Perkin Elmer Spectrum

One FT-IR,

m .

in cm^À1

Optical rotations were measured on a Perkin-Elmer 241 polar-

imeter at 20 °C, k_Na= 589 nm; k_Hg= 578, 546, 436, 365 nm; conc.

in g/100 mL.

Oxypeucedanin was purchased from ChromaDex^Ò(Irvin, CA,

USA). For the syntheses, the chemicals were supplied as follows:

dichloromethane (Atrasol, SDS, France); 6-methyloctanoic acid

(SynInnova, Edmonton, Canada); lithium aluminum hydride

(Acros, Gent, Belgium); diethylether (Carlo Erba, Val de Rueil,

France); methyl octanoate, acetyl chloride, diisopropylethylamine

(DIEA) and 4-dimethylaminopyridine (DMAP) (Fluka, Buchs, Swit-

presence of new oxygen derivatives from

a-phellandrene in two

zerland); (R)-(-)-a-phellandrene, methylene blue solution (Sigma

cultivars, not yet reported in any citrus, may rather indicate the

presence of the corresponding endoperoxide as an intermediate

metabolite during the biosynthesis of volatile molecules. The endo-

peroxide 10 had already been reported in the essential oil or

extract of some plants, such as Chenopodium multiﬁdum (De

Pascual-Teresa et al., 1981) and Alpinia densibracteata (Sy and

Brown, 1997), but never in citrus. Its presence in the cultivars

Judy’s Everbearing and Durham’s Emerald could be explained by

Aldrich, Steinheim, Germany); [(Ph₃P)₃RuCl₂] (Strem Chemicals,

Bischeim, France); dicyclohexylcarbamide (Alfa Aesar, Karlsruhe

Germany); ascaridole (City Chemical, West Haven, CT, USA); citro-

nellic acid and citronellol (available in-house).

For the chirality study, (R)-(+)-limonene and (S)-(À)-limonene

were available in-house; (R)-(+)-citronellal, (S)-(À)-citronellal,

(R)-(+)-citronellol, (S)-(À)-citronellol and (R)-(À)-

a-phellandrene

were from Sigma Aldrich; (S)-(+)-a-phellandrene was found in

the signiﬁcant amount of

may undergo a 1,4-cycloaddition of singlet oxygen, as reported to

explain the generation of ascaridole from -terpinene or the degra-

-phellandrene in these cultivars, which

rosemary oil (available in-house); (R)-(+)-sabinene was found in

nutmeg oil (SAFC, Sigma Aldrich); and (S)-(À)-sabinene was found

in bergamot oil (available in-house).

dation of 1,3,8-p-menthatriene in parsley (Nitz et al., 1989).

4.2. Plant material

3. Conclusions

Three cultivars of fresh ﬁnger lime fruits, Alstonville, Judy’s

Everbearing and Durham’s Emerald, were purchased from Global

Fresh Marketing PTY LTD (Hawthorn East, Australia) and delivered

by express air freight courier. They arrived in very good shape and

their peel extracts were immediately prepared. These three culti-

vars have been previously registered by the Australian Cultivar

Registration Authority with the following registration numbers:

ACC1123, ACC1122 and ACC1156.

Australian native ﬁnger limes (C. australasica) are known to be

unique for their caviar-like pulp and diverse shape, peel and pulp

color, and ﬂavor. This study on three cultivars shows that these dis-

tinctive observed characteristics are also observed on the molecu-

lar level. Indeed, the analytical data obtained during this work

showed that the three cultivars possess unique chemical composi-

tions with unusual ratios of major volatile metabolites: limonene/

sabinene in cv. Alstonville, limonene/citronellal/isomenthone in cv.

Judy’s Everbearing and limonene/citronellal/citronellol in cv. Dur-

ham’s Emerald. Finger limes also differ from common lime species

4.3. Preparation of the ﬁnger lime peel extracts

with their low amounts of c-terpinene, a- and b-pinene, and citral,

The peel of 2.4 kg of ﬁnger limes (cv. Judy’s Everbearing) was

removed using a grater (Micropane, Betty Bossy) to give 77.5 g of

peel, which was immediately covered with dichloromethane

(400 mL) and left at room temperature for 24 h. The mixture was

ﬁltered through cotton wool. The peel was covered again with

dichloromethane (300 mL), left at room temperature for 24 h and

then ﬁltered as before. The combined extracts were dried over

MgSO₄and concentrated using a Vigreux column at atmospheric

pressure to give 7.4 g of peel extract. The same procedure was

repeated with 4.5 kg of ﬁnger limes (cv. Alstonville) and 4.8 kg of

ﬁnger limes (cv. Durham’s Emerald) to give peel extracts of

15.6 g and 15.3 g, respectively.

as well as their opposite enantiomeric distribution of citronellal.

Variations in volatile compositions between cultivars were also

observed. Some constituents seem to be speciﬁc to one cultivar

(e.g. aliphatic and terpenyl acetates in Alstonville; citronellyl esters

in Judy’s Everbearing; 4-methylnonanal, cumin aldehyde and

methyl thymol ether in Durham’s Emerald). The combined data

assess the divergence of ﬁnger limes from other Citrus genotypes.

Considering the difﬁculty in tracing the origin of citrus species, fur-

ther chemotaxonomic should be carried out to understand the ori-

gin of these native species and their divergence from other citrus

species.

4. Experimental

4.4. Manual identiﬁcation by GC–MS

4.1. General experimental procedures

GC–MS analyses were performed on a GC–MS 7890A/5975C

(Agilent, Santa Clara, USA) operating in electron ionization (EI)

mode equipped with a split/splitless injector (250 °C); transfer line

temperature 280 °C; split ratio 1:50; equipped with a non-polar

column (SPB-1 capillary column, 30 m Â 0.25 mm, ﬁlm thickness

Abbreviations: cv., cultivar; DIEA, diisopropylethylamine;

DMAP, 4-dimethylaminopyridine; DS, Deans Switch; EI, electron

ionization; EPC, electronic pneumatic control; LTM, low thermal

mass; MSD; mass selective detector; LRI, linear retention index.

¹H NMR and ¹³C NMR spectra were recorded in CDCl₃on a Bru-

ker DPX 400 spectrometer at 25 °C with tetramethylsilane as the

1 lm, Supelco); oven temperature program: 60 °C for 5 min,

increased to 250 °C at a rate of 5 °C/min; carrier gas: helium

(He), constant ﬂow rate 1 mL/min; injection volume: 0.5 L.

120

E. Delort et al. / Phytochemistry 109 (2015) 111–124

GC–MS analyses were also carried out on a GC–MS 6890 N/5973

(Agilent) operating in the same conﬁguration but equipped with a

stant pressure channel was connected to the three-way splitter.

The three-way splitter was connected to the MS using deactivated

fused silica (0.90 m Â 0.10 mm, Agilent) and to an olfactometry

port using deactivated fused silica (0.90 m Â 0.25 mm, Agilent).

The ﬁrst dimension (1D) was a non-polar column (DB1-MS,

polar column (SUPELCOWAX 10 capillary column, 30 m Â 0.25 mm,

ﬁlm thickness 0.25 lm, Supelco); oven temperature program:

50 °C for 5 min, increased to 240 °C at a rate of 5 °C/min; carrier

gas: helium (He), constant ﬂow rate 1 mL/min; injection volume:

20 m Â 0.18 mm, ﬁlm thickness 0.18

lm, J&W) connected to the

0.5

L. Total ion current (TIC) GC–MS proﬁles and mass spectra

DS, and deactivated fused silica (0.45 m Â 0.1 mm, Agilent) was

installed to connect the DS to the three-way splitter. The second

chiral dimension (2D) was installed as an LTM II module between

the DS and the three-way splitter. For limonene and sabinene, the

1D temperature program was as follows: 50 °C to 250 °C at 10 °C/

min, 100 min isotherm; carrier gas: helium (He), ﬂow program:

from 1.0 mL/min (for 30 min) to 0.2 mL/min (for 90 min) at

100 mL/min; split ratio 1:50. The second dimension (2D) was HP-

CHIRAL (20% b-cyclodextrin in (35%-phenyl)-methylpolysiloxane,

were obtained with Data Analysis MSD ChemStation software

(Agilent). Mass spectra were generated at 70 eV from m/z: 27 to

350 with a scan rate of 2.29 scan/s. Linear retention indices (LRIs)

were determined after injection of a series of n-alkanes under

identical conditions.

The compounds were identiﬁed by comparison of their mass

spectra and LRIs obtained from a proprietary database. This data-

base is composed of analytical data obtained from synthesized or

commercially available compounds that were all unequivocally

characterized by NMR spectroscopy. The NIST05 MS database

(National Institute of Standards and Technology, USA) and Mass-

Finder3 (Dr. Hochmut, Scientiﬁc Consulting, Germany) were also

used when necessary.

30 m Â 0.25 mm, ﬁlm thickness 0.25

lm, Agilent). 2D temperature

program: 30 °C, 30 min isotherm, then 3 °C/min to 220 °C, 26.6 min

isotherm; carrier gas: helium, ﬂow program: from 2.5 mL/min (for

30 min) to 1.2 mL/min (for 90 min) at 100 mL/min. For citronellol

and citronellal, the 1D temperature program was as follows:

50 °C to 250 °C at 10 °C/min, 86.6 min isotherm; carrier gas:

helium (He), ﬂow program: from 0.8 mL/min (for 30 min) to

0.2 mL/min (for 76.6 min) at 100 mL/min; split ratio 1:10. The sec-

ond dimension (2D) was MEGA-DEX-DAC (diacetyl tertbutylsilyl-

4.5. Automated mass spectral deconvolution and identiﬁcation system

(AMDIS)

GC–MS ﬁles were processed with the free version 2.69 of AMDIS

software (available from http://chemdata.nist.gov/mass-spc/

amdis/downloads/). To increase the accuracy of our identiﬁcations,

we used both mass spectra and LRIs in the settings/algorithm of

the software (Norli et al., 2010), with the following parameters:

minimum match factor, 60; use retention index data; RI window,

10; match factor penalty, very strong; maximum penalty, 20; no

RI in library, 20.

b-cyclodextrin, 30 m Â 0.25 mm, ﬁlm thickness 0.25

lm, MEGA

S.N.C., Legnano, Italy). 2D temperature program: 30 °C, 30 min iso-

therm, then 3 °C/min to 220 °C, 13.3 min isotherm; carrier gas:

helium, ﬂow program: from 2.5 mL/min (for 30 min) to 1.2 mL/

min (for 76.6 min) at 100 mL/min.

4.7.2. Conﬁguration 2 (for a-phellandrene)

The 2D-GC–MS system consisted of a GC–MS 7890A/5973

(Agilent) retroﬁtted with one DS (Agilent), one three-way splitter

(Agilent), and one LTM II module (Agilent). The GC was equipped

with a split/splitless injector and two additional pneumatic control

module (PCM) devices. The constant ﬂow channel of each PCM was

connected to the DS and to the three-way splitter, respectively. The

second chiral dimension (2D) was installed as an LTM II module

between the DS and the three-way splitter. The three-way splitter

was connected to the MS using deactivated fused silica

(0.90 m Â 0.10 mm, Agilent) and to an olfactometry port using

deactivated fused silica (0.90 m Â 0.25 mm, Agilent).

4.6. Quantitation by GC-FID

The ﬁnger lime extracts were injected into a GC 6890 (Agilent)

equipped with a double injector and two DB-1 capillary columns

(60 m Â 0.25 mm, ﬁlm thickness 0.25

lm, J&W). One column was

connected to an MS 5973 N (Agilent) for identiﬁcation, the other

to an FID for quantitation. The temperature program was as fol-

lows: 50 °C for 5 min, increased to 120 °C at a rate of 3 °C/min, then

increased to 250 °C at a rate of 5 °C/min, 5 min isothermal, then

increased to 300 °C at a rate of 15 °C/min, and then 20 min isother-

The ﬁrst dimension (1D) was a non-polar column (SPB1,

mal; split ratio, 1:50; injection volume, 0.2

L; injector and detec-

30 m Â 0.25 mm, ﬁlm thickness 1

lm, Supelco) connected to the

tor temperature, both 250 °C; carrier gas helium at a constant ﬂow

rate of 1.8 mL/min (FID) and 2.1 mL/min (MS). For the quantitative

data given in Table 1, peel extract (452.2 mg) of ﬁnger lime Alston-

ville was diluted 10 times in dichloromethane with methyl octano-

ate (15.5 mg) as an internal standard. The percentages of the

volatile constituents were obtained from the FID area corrected

with the use of the response factors as described by Delort and

Jaquier (2009). The same experiment was done with Judy’s Ever-

bearing peel extract (448.9 mg) with an internal standard of

15.4 mg, and Durham’s Emerald peel extract (433.5 mg) with an

internal standard of 14.5 mg.

DS, and deactivated fused silica (0.60 m Â 0.1 mm, Agilent) was

installed to connect the DS to the three-way splitter. 1D tempera-

ture program: 60 °C, 5 min isotherm, then 10 °C/min to 250 °C,

72.33 min isotherm; carrier gas: helium (He), ﬂow program: from

1.5 mL/min (for 30 min) to 0.1 mL/min (for 66.33 min) at 100 mL/

min; split ratio 1:5. The second dimension (2D) was MEGA-DEX

DMP Beta (dimethyl pentyl-b-cyclodextrin, 30 m Â 0.25 mm, ﬁlm

thickness 0.25 lm, MEGA S.N.C.). 2D temperature program: 40 °C,

30 min isotherm, then 3 °C/min to 230 °C, 3 min isotherm; ﬂow

program: from 3.0 mL/min (for 30 min) to 1.2 mL/min (for

66.33 min) at 100 mL/min.

For each compound, the chiral phase was ﬁrst tested with stan-

dard compounds to ensure good separation of both enantiomers.

4.7. Enantioselective heart-cut 2D-GC–MS

The injection volume was 1.0 lL of neat citrus sample; injector

4.7.1. Conﬁguration 1 (for limonene, sabinene, citronellol and

citronellal)

temperature 250 °C; MS parameters: mass range, m/z from 29 to

350; ionization, 70 eV; transfer line temperature, 250 °C; source

temperature, 230 °C; quadrupole temperature, 150 °C.

The 2D-GC–MS system consisted of a GC–MS 7890A/5973 (Agi-

lent) retroﬁtted with one Deans Switch (DS) (Agilent), one 3-way

splitter (Agilent), and one low thermal mass (LTM) II module (Agi-

lent). The GC was equipped with a split/splitless injector, with an

additional pneumatic control module (PCM). The PCM constant

ﬂow channel was connected to the Deans Switch and the PCM con-

4.8. High-resolution GC-TOF-MS

Time-of-ﬂight GC–MS analyses were performed on

GCT Premier (Waters, Milford, USA) with a SPB-1 column

E. Delort et al. / Phytochemistry 109 (2015) 111–124

121

(30 m Â 0.25 mm, ﬁlm thickness 1.0

60 °C, 5 min; 5 °C/min to 250 °C; constant helium ﬂow, 1.0 mL/

min; injection volume, 0.1 L; injector temperature, 250 °C; split

ratio, 1:50. The acquisition time was set to 0.49 s with an interscan

delay of 0.01 s over a mass range of 1–300 Da. Spectra were

recorded using the following parameters: electron energy, 70 eV;

m, Supelco). Oven program:

(5); 70 (9); 69 (100); 68 (8); 67 (20); 57 (9); 56 (6); 55 (26); 53

(5); 43 (7); 41 (32). HR-GC-TOF-MS: 308.2713 (C20H36O,

À0.6 ppm). LRI (SPB-1) 2025; LRI (SWax) 2360. In cv. Judy’s Ever-

bearing and Durham’s Emerald extracts: LRI (SPB-1) 2022; LRI

(SWax) 2352.

emission current, 594.6

A: trap current, 200

lA; source tempera-

4.9.3. Isoascarisole 3

ture, 200 °C. Calibration was performed by using heptacosa (perﬂu-

orotributylamine, Mass Spec Std, Alfa Aesar, Karlsruhe, Germany).

Calibration data were collected for 1 min in centroid mode. A total

of 60 spectra were summed to generate a 23-point calibration

curve from m/z 69 to 502 Da. The curve was ﬁtted to a second-

order polynomial such that the standard deviation of the residuals

was 0.001 amu or lower. Heptacosa was continuously introduced

into the ion source and the ion m/z 218.9856 was used as a lock

mass. Mass spectra and molecular formula were obtained by using

MassLynx software (Waters). The difference between the exact

4.9.3.1. Cis-isoascaridole cis-3a. Ascaridole (100.2 mg, 0.596 mmol)

in xylene (15 mL) was heated at reﬂux for 18 h. The cold reaction

mixture was concentrated and puriﬁed by ﬂash chromatography

(cyclohexane/AcOEt 99:1 to 6:4) to afford cis-3a in 85% yield. IR:

2961, 2935, 2874, 1465, 1443, 1419, 1380, 1366, 1310, 1264,

1226, 1208, 1747, 1097, 1065, 1039, 1014, 996, 928, 901, 877,

850, 804, 778, 718, 693, 656, 616. ¹H NMR (600 MHz, CDCl₃):

3.13 (d, J = 3.0, 1H); 3.10 (d, J = 3.0, 1H); 1.86–1.81 (m, 1H); 1.79–

1.73 (m, 1H); 1.67–1.58 (m, 1H); 1.53 (hept, J = 6.9, 1H); 1.36 (s,

3H); 0.98 (d, J = 6.8, 3H); 0.93 (d, J = 6.8, 3H). ¹³C NMR (150 MHz,

CDCl₃): 59.9 (C); 56.0 (C); 54.6 (CH); 54.2 (CH); 34.3 (CH); 27.7

(CH₂); 22.0 (CH₃); 21.1 (CH₂); 17.9 (CH₃); 17.4 (CH₃).

mass calculated (M_calc

) from the measured mass (M_meas) is

expressed in ppm ((M_meas.À M_calc.)/M_calc.Â 10⁶).

MS (EI, 70 eV), m/z (%): 168 (7, M⁺), 150 (10); 140 (12); 139

(29); 135 (25); 126 (11); 125 (52); 119 (32); 110 (14); 109 (13);

107 (39); 105 (10); 99 (11); 98 (15); 97 (97); 95 (34); 93 (11);

91 (29); 85 (22); 83 (22); 82 (29); 81 (24); 79 (36); 77 (17); 71

(33); 70 (13); 69 (66); 67 (26); 65 (10); 60 (12); 57 (10); 55

(48); 53 (19); 43 (100); 42 (10); 41 (68); 39 (29); 29 (11); 27

(17). HR-GC-TOF-MS: 168.1153 (C₁₀H₁₆O₂, +1.8 ppm). LRI (SPB-1)

1287; LRI (SWax) 1867. In cv. Alstonville and Durham’s Emerald

extracts: LRI (SPB-1) 1291; LRI (SWax) 1869.

4.9. Synthesis

4.9.1. 6-Methyloctyl acetate 1

6-Methyloctanoic acid (10 g, 63 mmol) was reduced by lithium

aluminum hydride (2.4 g, 1.0 eq.) in dry diethylether (150 mL) at

0 °C and then left at room temperature overnight to yield after

work-up the corresponding alcohol (7.4 g, GC purity 97%). Acetyla-

tion of 6-methyloctanol (2.4 g, 16.6 mmol) was performed by using

acetyl chloride (1.57 g, 1.2 eq.) and DIEA (4.3 g, 2.0 equiv.) in ethyl

acetate (50 mL). After the usual work-up, the crude product was

distilled under vacuum (ꢀ84 °C, ꢀ5 mmHg) to yield 6-methyloctyl

acetate (2.2 g, 72%, GC purity 98.7%).

4.9.3.2. Trans-isoascaridole trans-3b. Isolated in analytical amount

from commercially available ascaridole by ﬂash chromatography

(cyclohexane/AcOEt 99:1 to 6:4). ¹H NMR (600 MHz, CDCl₃): 3.17

(s, 2H); 1.85–1.71 (m, 4H); 1.50 (hept, J = 7.0, 1H); 1.29 (s, 3H);

0.96 (d, J = 7.0, 3H); 0.93 (d, J = 7.0, 3H). ¹³C NMR (150 MHz, CDCl₃):

64.0 (C); 58.6 (C); 57.2 (CH); 56.2 (CH); 34.1 (CH); 25.3 (CH₂); 22.7

(CH₃); 19.4 (CH₂); 18.0 (CH₃); 17.6 (CH₃). MS (EI, 70 eV), m/z (%):

168 (1, M⁺), 125 (15), 110 (10), 107 (18), 97 (94), 95 (27), 85

(17), 82 (24), 79 (28), 71 (31), 69 (28), 67 (17), 60 (10), 55 (31),

53 (12), 43 (100), 41 (51), 39 (21), 27 (15). LRI (SPB-1) 1291; LRI

(SWax) 1888. Not detected in ﬁnger lime extracts.

¹H NMR (400 MHz, CDCl₃): 4.05 (t, J = 6.7, 2H); 2.05 (s, 3H);

1.59–1.66 (m, 2H); 1.25–1.38 (m, 7H); 1.08–1.16 (m, 2H); 0.86 (t,

J = 7.1, 3H); 0.85 (d, J = 6.5, 3H). ¹³C NMR (100 MHz, CDCl₃): d

171.2 (C); 64.7 (CH₂); 36.5 (CH₂); 34.3 (CH); 29.5 (CH₂); 28.7

(CH₂); 26.7 (CH2); 26.3 (CH₂); 21.0 (CH₃); 19.2 (CH₃); 11.4 (CH₃).

MS (EI, 70 eV), m/z (%): 186 (0, M⁺); 98 (22); 97 (100); 83 (17);

71 (15); 70 (54); 69 (39); 68 (11); 61 (41); 57 (27); 56 (31); 55

(75); 43 (72); 42 (10); 41 (29); 29 (10). LRI (SPB-1) 1264; LRI

(SWax) 1544. In cv. Alstonville extract, LRI (SPB-1) 1263; LRI

(SWax) 1543.

4.9.4. Phellandrene endoperoxyde 10

4.9.4.1. (+)-(1S,4R,7R)-7-Isopropyl-5-methyl-2,3-dioxabicyclo[2.2.2]

4.9.2. Citronellyl citronellate 2

oct-5-ene ((1S,4R,7R)-(+)-10a). A solution of (R)-(-)-a-phelland-

Dicyclohexylcarbamide (2.67 g, 12.92 mmol, 1.1 eq.) was added

to a solution of citronellic acid (2.00 g, 11.75 mmol, 1 eq.), citronel-

lol (1.84 g, 11.75 mmol, 1 eq.) and DMAP (0.15 g, 1.18 mmol) in

dichloromethane (20 mL) and stirred at room temperature for

6 h. The mixture was poured on water and extracted with diethyl

ether. The combined organic phase was washed with brine and

dried over MgSO₄. The solvent was removed to give 3.91 g of crude

product. The residue was puriﬁed by ﬂash-chromatography using

2% diethyl ether in pentane, yielding pure product (2.63 g, 73%,

GC–MS purity 97%) after bulk-to-bulk distillation (160 °C,

0.056 mbar).

rene 9 ([

²⁰= À170, conc. = 4.1, CHCl₃; 16.6 g, 122 mmol) in

iPrOH (90 mL) was irradiated at 20 °C for 40 h (125 W equivalent

daylight-balanced; color temperature 6500 °K; LED camera light)

in the presence of a few drops of methylene blue solution, while

air was bubbled through a glass frit into the mixture at a rate of

480 mL/min. Every 8 h, both the level of solvent and the blue color

were readjusted. The reaction mixture of 57:43 (1S,4R,7R)-(+)-10a/

(1R,4S,7R)-(+)-10b was then concentrated under vacuum and puri-

ﬁed by ﬂash chromatography (cyclohexane/AcOEt 97:3 to 95:5) to

afford a 70:30 mixture of (1S,4R,7R)-(+)-10a/(1R,4S,7R)-(+)-10b in

23% yield (4.71 g). During an additional puriﬁcation by ﬂash chro-

matography, an enriched 83:17 (1S,4R,7R)-(+)-10a/(1R,4S,7R)-(+)-

10b fraction (800 mg, 3.9% yield) was obtained and used for analyt-

¹H NMR (400 MHz, CDCl3): d 5.09 (br t, J = 7.05, 2 H); 4.04–4.17

(m, 2 H); 2.30 (dd, J = 5.9, 14.5, 1 H); 2.10 (dd, J = 8.2, 14.5, 1H);

1.98 (m, 1.89–2.07, 5 H); 1.68 (s, 6 H); 1.60 (s, 6 H); 1.49–1.72

(m, 2 H); 1.13–1.48 (m, 5 H); 0.94 (d, J = 6.5, 3 H); 0.91 (d, J = 6.5,

3 H). 13C NMR (100 MHz, CDCl3): d 173.3 (C), 131.5 (C), 131.3

(C), 124.6 (CH), 124.3 (CH), 62.7 (CH2), 41.9 (CH2), 37.0 (CH2),

36.8 (CH2), 35.6 (CH2), 30.1 (CH), 29.5 (CH), 25.7 (2CH3), 25.4

(CH2), 25.4 (CH2), 19.6 (CH3), 19.4 (CH3), 17.6 (2 CH3). MS (EI,

70 eV), m/z (%): 308 (2, M+); 170 (2); 153 (10); 152 (8); 139 (5);

138 (42); 136 (5); 124 (5); 123 (45); 110 (19); 109 (42); 97 (7);

96 (11); 95 (49); 94 (12); 83 (26); 82 (33); 81 (54); 80 (6); 71

ical and synthetic purposes. [a]

²⁰= + 43.0, conc. = 4.0, CHCl₃. IR:

3047, 2958, 2871, 1661, 1470, 1443, 1385, 1369, 1206, 1121,

1065, 1006, 961, 943, 909, 795, 726, 676. ¹H NMR (500 MHz,

CDCl₃): 6.33 (dq, J = 1.7, 6.5, 1H); 4.57 (dt, J = 1.5, 6.5, 1H); 4.38

(dq, J = 1.8, 3.6, 1H); 2.35 (ddd, J = 4.3, 8.6, 13.1, 1H); 1.94 (s, 3H);

1.82 (dt, J = 4.2, 13.2, 1H); 1.70 (ddd, J = 2.5, 11.0, 13.2, 1H); 1.12–

1.07 (m, 1H); 0.99 (d, J = 6.6, 3H); 0.98 (d, J = 6.6, 3H). ¹³C NMR

(125 MHz, CDCl₃): 141.4 (C); 126.0 (CH); 76.2 (CH); 73.1 (CH);

41.6 (CH); 30.5 (CH); 27.8 (CH₂); 21.3 (CH₃); 20.8 (CH₃); 18.6

122

E. Delort et al. / Phytochemistry 109 (2015) 111–124

(CH₃). Because of its thermal instability, the MS and LRI analyses

could not be measured and its presence in the extracts could not

be studied.

(22); 85 (33); 83 (29); 82 (13); 81 (25); 79 (24); 77 (14); 71

(15); 70 (13); 69 (35); 67 (16); 57 (12); 55 (46); 53 (16); 43

(100); 41 (47); 39 (28); 29 (12); 27 (15). (1S,2R,4R,5R,7S)-(À)-4b:

LRI (SPB-1) 1333; LRI (SWax) 1963. HR-GC-TOF-MS: 168.1157

(C₁₀H₁₆O₂, +4.2 ppm). In Judy’s Everbearing and Durham’s Emerald

extracts, 4b: LRI (SPB-1) 1331; LRI (SWax) 1957.

4.9.4.2. (+)-(1R,4S,7R)-7-Isopropyl-5-methyl-2,3-dioxabicyclo[2.2.2]

oct-5-ene ((+)-(1R,4S,7R)-10b).. During puriﬁcation by ﬂash

chromatography described above, an enriched 4:96 (1S,4R,7R)-

(+)-10a/(1R,4S,7R)-(+)-10b fraction (100 mg, 0.5% yield) was

obtained and used for analytical and synthetic purposes.

4.9.6. 2,3-Epoxy-p-menthan-6-one 5

4.9.6.1. (1R,2S,5R,6S)-5-Isopropyl-2-methyl-7-oxabicyclo[4.1.0]hep-

tan-3-one ((1R,2S,5R,6S)-5a). Thermal treatment in reﬂuxing tolu-

ene (4 h, 110 °C) of the 83:17 mixture of (1S,4R,7R)-(+)-10a/

(1R,4S,7R)-(+)-10b afforded, according to 13C NMR analysis, a mix-

ture of (1R,2S,4S,5R,7R)-4a(32%)/(1S,2R,4R,5R,7S)-(À)-4b(25%),

together with cis-isoascaridole 3a (17%), the major keto-epoxide

(1R,2S,5R,6S)-5a (18%), and two other very minor keto-epoxides

tentatively assigned, from spectroscopic data, to (1S,2R,5R,6R)-5b

(4%) and (1S,4S,6R)-7b (4%). (1R,2S,5R,6S)-5a could only be isolated

in analytical quantity, as its puriﬁcation by ﬂash chromatography

(cyclohexane/AcOEt 9:1) resulted in its transformation into the

hydroxy-enone (4S,5R)-(À)-6a.

[a]

²⁰= + 62.8, conc. = 4.1, CHCl₃. IR: 2958, 2938, 2871, 1470,

1443, 1385, 1368, 1207, 1065, 1006, 982, 960, 942, 905, 795,

726, 676. ¹H NMR (600 MHz, CDCl₃): 6.19 (dq, J = 2.0, 6.1, 1H);

4.60 (ddd, J = 1.5, 3.5, 5.7, 1H); 4.43 (dq, J = 1.7, 3.6, 1H); 2.35

(ddd, J = 4.3, 8.7, 13.0, 1H); 1.94 (s, 3H); 1.91 (ddt, J = 4.0, 5.7, 9.5,

1H); 1.09 (ddd, J = 1.7, 4.7, 13.0, 1H); 1.06–1.01 (m, 1H); 0.90 (d,

J = 6.5, 3H); 0.85 (d, J = 6.5, 3H). ¹³C NMR (150 MHz, CDCl₃):

142.2 (C); 122.8 (CH); 75.6 (CH); 73.9 (CH); 41.7 (CH); 32.6 (CH);

28.5 (CH₂); 20.4 (CH₃); 19.8 (CH₃); 18.6 (CH₃). Because of its ther-

mal instability, the MS and LRI analyses could not be measured and

its presence in the extracts could not be studied.

IR: 2960, 2930, 2874, 1711, 1463, 1378, 1370, 1247, 1204, 1177,

4.9.5. 1,2:5,6-Diepoxy-p-menthane 4

1111, 1096, 1064, 1015, 921, 893, 858, 833, 778, 730, 699, 654. ¹

4.9.5.1.

(À)-(1R,2S,4S,5R,7R)-5-Isopropyl-1-methyl-3,8-dioxatricy-

NMR (500 MHz, CDCl₃): 3.37 (d, J = 4.2, 1H); 3.20 (dd, J = 2.0, 4.2,

1H); 2.59 (ddq, J = 0.8, 1.9, 7.2, 1H); 2.27, (dd, J = 13.0, 16.5, 1H);

2.17–2.15 (m, 1H); 2.15–2.12 (m, 1H); 1.87 (dsept, J = 5.1, 6.8,

1H); 1.31 (d, J = 7.2, 3H); 1.03 (d, J = 6.8, 3H); 1.01 (d, J = 6.8, 3H).

¹³C NMR (125 MHz, CDCl₃): 210.7 (C); 54.7 (CH); 54.4 (CH); 41.7

(CH); 39.7 (CH); 37.4 (CH₂); 31.3 (CH); 19.6 (CH₃); 19.4 (CH₃);

14.1 (CH₃). MS (EI, 70 eV), m/z (%): 168 (7, M⁺); 153 (16); 135

(10); 126 (49); 125 (30); 111 (22); 98 (37); 97 (40); 95 (11); 83

(14); 81 (15); 79 (12); 71 (62); 70 (100); 69 (43); 68 (10); 67

(11); 57 (18); 56 (10); 55 (71); 53 (11); 43 (24); 42 (19); 41

(46); 39 (28); 29 (10); 27 (12). HR-GC-TOF-MS: 168.1149

(C₁₀H₁₆O₂, -0.6 ppm). (1R,2S,5R,6S)-5a: LRI (SPB-1) 1299; LRI

(SWax): degrades on polar phase. In Judy’s Everbearing and Dur-

ham’s Emerald extracts, 5a: LRI (SPB-1) 1297; LRI (SWax) not

detected.

clo[5.1.0.02,4]octane ((1R,2S,4S,5R,7R)-(-)-4a). A 83:17 mixture of

(1S,4R,7R)-(+)-10a/(1R,4S,7R)-(+)-10b (1500 mg, 8.93 mmol) was

treated with [(Ph₃P)₃RuCl₂] (13.8 mg) in CH₂Cl₂(2 mL) at 20 °C

for 4 h to obtain, according to 13C NMR analysis, a mixture of

(1R,2S,4S,5R,7R)-(À)-4a (38%), (1S,2R,4R,5R,7S)-(À)-4b (22%),

keto-epoxides (1R,2S,5R,6S)-5a (22%), hydroxy-enone (4S,5R)-(-)-

6a (6%), and cis-isoascaridole 3a (12%), from which

(1R,2S,4S,5R,7R)-(À)-4a could be isolated in analytical amount by

ﬂash chromatography (cyclohexane/AcOEt 9:1).

[

²⁰= À13.9,

conc. = 0.4, CHCl₃. IR: 2959, 2931, 2873, 1464, 1445, 1416, 1369,

1220, 1109, 1096, 1062, 920, 905, 891, 859, 838, 810, 776, 718,

701, 628. ¹H NMR (500 MHz, CDCl₃): 3.13 (d, J = 4.1, 1H); 3.11

(dd, J = 0.6, 4.2, 1H); 2.88 (dd, J = 2.9, 7.0, 1H); 1.82–1.74 (m, 4H);

1.47 (s, 3H); 0.98 (d, J = 6.6, 3H); 0.975 (d, J = 6.6, 3H). ¹³C NMR

(125 MHz, CDCl₃): 54.7 (CH); 53.7 (C); 53.5 (CH); 50.5 (CH); 39.0

(CH); 30.9 (CH); 22.6 (CH₂); 20.7 (CH₃); 20.0 (CH₃); 19.7 (CH₃).

MS (EI, 70 eV), m/z (%): 168 (1, M⁺); 153 (8); 135 (10); 126 (15);

125 (20); 119 (15); 111 (13); 110 (10); 109 (20); 107 (29); 98

(25); 97 (100); 95 (25); 93 (22); 91 (19); 85 (18); 83 (28); 82

(12); 81 (24); 79 (21); 77 (13); 71 (26); 70 (35); 69 (37); 67

(18); 57 (14); 55 (60); 53 (18); 43 (90); 42 (11); 41 (53); 39

(35); 29 (14); 27 (17). (1R,2S,4S,5R,7R)-(À)-4a: LRI (SPB-1) 1326;

LRI (SWax) 1949. HR-GC-TOF-MS: 168.1151 (C₁₀H₁₆O₂,

+0.6 ppm). In Judy’s Everbearing and Durham’s Emerald extracts,

4a: LRI (SPB-1) 1324; LRI (SWax) 1945.

4.9.6.2. (1S,2R,5R,6R)-5-Isopropyl-2-methyl-7-oxabicyclo[4.1.0]hep-

tan-3-one

((1S,2R,5R,6R)-5b). When

pure

endoperoxide

(1R,4S,7R)-(+)-10b was heated in reﬂuxing toluene for 18 h, a mix-

ture containing, according to 13C NMR analysis, (1S,2R,4R,5R,7S)-

(À)-4b

(56%)/(1R,4R,6S)-7b

(15%)/(À)-(4S,6R)-8

(13%)/

(1S,2R,5R,6R)-5b (or eventually the thermodynamically more sta-

ble (1S,2S,5R,6R)-5d) (11%)/(4R,5R)-(+)-6b (5%)) was obtained.

The spectroscopic data given for (1S,2R,5R,6R)-5b were deduced

from the mixture. These are tentative attributions, as they could

refer eventually to the thermodynamically more stable stereoiso-

mer (1S,2S,5R,6R)-5d. ¹H NMR (500 MHz, CDCl₃): 3.31–3.30 (m,

1H); 3.25 (d, J = 3.3, 1H); 2.67 (q, J = 7.1, 1H); 2.40–1.60 (m, 4H);

1.29 (d, J = 7.1, 3H); 1.03 (d, J = 6.7, 3H); 0.98 (d, J = 6.7, 3H). ¹³C

NMR (125 MHz, CDCl₃): 210.8 (C); 57.8 (CH); 55.6 (CH); 42.6

(CH); 41.4 (CH); 38.2 (CH₂); 30.7 (CH); 20.3 (CH₃); 19.8 (CH₃);

13.6 (CH₃). MS (EI, 70 eV), m/z (%): 168 (5, M⁺); 153 (19); 126

(49); 125 (37); 111 (24); 98 (33); 97 (42); 95 (10); 83 (18); 81

(11); 79 (11); 71 (62); 70 (100); 69 (58); 67 (12); 57 (17); 56

(11); 55 (84); 53 (11); 43 (26); 42 (21); 41 (51); 39 (27); 29

(12); 27 (12). HR-GC-TOF-MS: 168.1157 (C₁₀H₁₆O₂,+4.2 ppm).

(1S,2R,5R,6R)-5b: LRI (SPB-1) 1304; LRI (SWax): degrades on polar

phase. In Judy’s Everbearing and Durham’s Emerald extracts, 5b:

LRI (SPB-1) 1301, LRI (SWax) not detected.

4.9.5.2.

clo[5.1.0.02,4]octane

(67 mg, 0.07 mmol) was added to a solution of pure stereoisomer

(1R,4S,7R)-(+)-10b (309 mg, 1.84 mmol) in CH₂Cl₂(10 mL). After

4 h at 20 °C, the reaction mixture was concentrated, and then puri-

ﬁed by ﬂash chromatography (cyclohexane/AcOEt 9:1) to afford

(À)-(1S,2R,4R,5R,7S)-5-Isopropyl-1-methyl-3,8-dioxatricy-

((1S,2R,4R,5R,7S)-(-)-4b). [(Ph₃P)₃RuCl₂]

pure (1S,2R,4R,5R,7S)-(À)-4b in 80% yield.

[a]

²⁰= À41.8,

conc. = 1.5, CHCl₃. IR: 2959, 2932, 2874, 1465, 1448, 1416, 1379,

1369, 1298, 1264, 1252, 1203, 1093, 1075, 1015, 931, 912, 892,

868, 808, 775, 696, 665. ¹H NMR (500 MHz, CDCl₃): 3.13 (d,

J = 4.0, 1H); 2.91 (t, J = 3.3, 1H); 2.88 (dd, J = 2.2, 4.0, 1H); 1.88

(dt, J = 3.1, 12.5, 1H); 1.70–1.67 (m, 3H); 1.53 (s, 3H); 1.00 (d,

J = 6.5, 3H); 0.96 (d, J = 6.5, 3H). ¹³C NMR (125 MHz, CDCl₃): 56.3

(CH); 53.1 (C); 52.0 (CH); 51.9 (CH); 37.0 (CH); 30.3 (CH); 24.3

(CH₂); 21.0 (CH₃); 20.4 (CH₃); 20.1 (CH₃). MS (EI, 70 eV), m/z (%):

168 (1, M⁺); 153 (9); 135 (13); 125 (19); 119 (30); 111 (13); 110

(10); 109 (20); 107 (30); 98 (19); 97 (91); 95 (25); 93 (20); 91

4.9.7. p-Menth-1-en-3-ol-6-one 6

4.9.7.1.

(À)-(4S,5R)-4-Hydroxy-5-isopropyl-2-methylcyclohex-2-

enone ((4S,5R)-(-)-6a). (4S,5R)-(À)-6a was obtained in 8% yield

during the attempted chromatographic puriﬁcation of

E. Delort et al. / Phytochemistry 109 (2015) 111–124

123

(1R,2S,5R,6S)-5a from the mixture obtained after thermal treat-

ment (4 h, 110 °C in toluene) of (1S,4R,7R)-(+)-10a/(1R,4S,7R)-(+)-

4.9.8.2.

(À)-(4S,6R)-4-Hydroxy-6-isopropyl-3-methylcyclohex-2-

enone ((4S,6R)-(À)-8). (À)-(4S,6R)-8 constitutes 13% of the mixture

10b (reported above) and could be characterized. [

]

²⁰= À108.0,

obtained after thermal treatment of (1R,4S,7R)-(+)-10b (18 h,

conc. = 2.1, CHCl₃. IR: 3426, 2959, 2923, 2871, 1660, 1473, 1448,

1418, 1384, 1366, 1245, 1183, 1139, 1113, 1088, 1042, 1021,

947, 925, 887, 834, 721, 707, 654. ¹H NMR (600 MHz, CDCl₃):

6.78 (dq, J = 1.5, 6.0, 1H); 4.42 (br s, 1H); 2.56 (dd, J = 4.0, 16.6,

1H); 2.45 (dd, J = 13.0, 16.6, 1H); 2.04 (br s, 1 OH); 1.81 (br s,

3H); 1.80–1.77 (m, 1H); 1.67–1.62 (m, 1H); 1.03 (d, J = 6.6, 3H);

0.96 (d, J = 6.6, 3H). ¹³C NMR (150 MHz, CDCl₃): 200.1 (C); 142.8

(CH); 137.3 (C); 64.3 (CH); 45.9 (CH); 36.9 (CH₂); 28.5 (CH); 20.4

(CH₃); 20.2 (CH₃); 15.6 (CH₃). MS (EI, 70 eV), m/z (%): 168 (24,

M⁺); 135 (19); 126 (49); 125 (14); 111 (38); 107 (10); 98 (100);

97 (25); 82 (13); 79 (15); 77 (12); 70 (47); 69 (35); 55 (14); 43

(15); 42 (10); 41 (24); 39 (14). HR-GC-TOF-MS: 168.1153

(C₁₀H₁₆O₂, +1.8 ppm). (4S,5R)-(À)-6a: LRI (SPB-1) 1378; LRI (SWax)

2339. In Judy’s Everbearing and Durham’s Emerald extracts, 6a: LRI

(SPB-1) 1376; LRI (SWax) 2340.

110 °C in toluene). It was obtained pure in 9% yield after ﬂash chro-

matography (cyclohexane/AcOEt 99:1 to 6:4).

[

]

₅₈₉= À34.5,

conc. = 3.1, CHCl₃.

[

]

₅₈₉= À58.8,

]

₅₇₈= À62.0, [

]

₅₄₆= À72.0,

]

₄₃₆= À145.7,

[a]

₃₆₅= À147.9, conc. = 1.8, MeOH. IR: 3408,

2958, 2872, 1651, 1464, 1439, 1370, 1309, 1248, 1208, 1072,

1034, 1009, 988, 976, 906, 879, 732, 685, 673. ¹H NMR

(600 MHz, CDCl₃): 5.81 (br d, J = 1.0, 1H); 4.36 (t, J = 4.8, 1H);

2.37 (ddd, J = 4.7, 5.9, 9.0, 1H); 2.26 (dq, J = 6.8, 7.0, 1H); 2.15

(ddd, J = 4.2, 9.0, 13.3, 1H); 2.05 (dt, J = 9.0, 5.3, 1H); 2.03 (d,

J = 1.0, 3H); 1.96 (br s, 1 OH); 0.94 (d, J = 7.0, 3H); 0.89 (d, J = 6.8,

3H). ¹³C NMR (150 MHz, CDCl₃): 201.3 (C); 159.9 (C); 127.1 (CH);

67.1 (CH); 48.5 (CH); 32.3 (CH₂); 26.4 (CH); 21.0 (CH₃); 20.7

(CH₃); 19.1 (CH₃). MS (EI, 70 eV), m/z (%): 168 (12, M⁺); 135 (26);

126 (53); 125 (32); 124 (34); 111 (34); 109 (20); 98 (100); 97

(13); 83 (10); 70 (31); 69 (34); 55 (13); 43 (11); 42 (13); 41

(22); 39 (13). LRI (SPB-1) 1390; LRI (SWax) 2444. Not detected in

ﬁnger lime extracts.

4.9.7.2.

(+)-(4R,5R)-4-Hydroxy-5-isopropyl-2-methylcyclohex-2-

enone ((4R,5R)-(+)-6b). (4R,5R)-(+)-6b was obtained in analytical

quantity during the chromatographic puriﬁcation (cyclohexane/

AcOEt 9:1) of the mixture obtained after thermal treatment

(18 h, 110 °C in toluene) of (1R,4S,7R)-(+)-10b, which contained,

according to 13C NMR analysis, (1S,2R,4R,5R,7S)-(À)-4b (56%)/

(1R,4R,6S)-7b (15%)/(À)-(4S,6R)-8 (13%)/(1S,2R,5R,6R)-5b (or even-

tually the thermodynamically more stable (1S,2S,5R,6R)-5d) (11%)/

Acknowledgements

We are indebted to Alain Bagnoud, Claude-Alain Richard and

Tim Vernet for their skillful experimental work; Fred Durham (Glo-

bal Fresh Marketing) for supplying the ﬁnger limes; Florence Fou-

illet, Helen Claussen Jaquier, Josef Limacher and Matthis Van de

Waal for the organoleptic evaluations of fruits and extracts; Dr.

Jean-Yves de Saint Laumer for theoretical calculations; Robert Bra-

uchli for NMR stereochemical elucidations; Anna Castelli for the

bibliography; Giovanni Cipolla for helpful discussions on deconvo-

lution software; Prof. Carlo Bicchi and Stefano Galli for their valu-

able advices on enantioselective GC analysis.

(4R,5R)-(+)-6b (5%)). [a] a]

²⁰= +67.0, conc. = 0.9, CHCl₃. [ ²⁰= +92.2,

conc. = 0.6, EtOH. IR: 3418, 2958, 2928, 2873, 1661, 1466, 1448,

1421, 1386, 1369, 1356, 1256, 1143, 1117, 1090, 1048, 1007,

984, 927, 893, 754, 728. ¹H NMR (600 MHz, CDCl₃): 6.65 (quint,

J = 1.6, 1H); 4.35 (br d, J = 8.0, 1H); 2.46 (dd, J = 3.8, 16.0, 1H);

2.18 (dhept, J = 3.3, 7.0, 1H); 2.12 (dd, J = 13.2, 16.0, 1H); 1.96

(ddt, J = 3.3, 9.4, 13.2, 1H); 1.78 (t, J = 1.6, 3H); 1.55 (br s, 1OH);

0.97 (d, J = 7.0, 3H); 0.90 (d, J = 7.0, 3H). ¹³C NMR (150 MHz, CDCl₃):

199.8 (C); 148.3 (CH); 135.3 (C); 69.2 (CH); 50.1 (CH); 36.3 (CH₂);

26.4 (CH); 20.5 (CH₃); 16.6 (CH₃); 15.3 (CH₃). MS (EI, 70 eV), m/z

(%): 168 (31, M⁺); 150 (11); 135 (36); 126 (50); 125 (17); 111

(50); 107 (11); 98 (100); 97 (34); 95 (12); 91 (10); 79 (16); 77

(12); 70 (48); 69 (42); 55 (15); 43 (18); 42 (11); 41 (27); 39 (15).

HR-GC-TOF-MS: 168.1150 (C₁₀H₁₆O₂, +0.0 ppm). (4R,5R)-(+)-6b:

LRI (SPB-1) 1381; LRI (SWax) 2375. In Judy’s Everbearing and Dur-

ham’s Emrald extracts, 6b: LRI (SPB-1) 1380; LRI (SWax) 2376.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in

the online version, at http://dx.doi.org/10.1016/j.phytochem.2014.

10.023.

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