Identification of thiazolidines in guava: stereochemical studies

FLAVOUR AND FRAGRANCE JOURNALFlavour Fragr. J. 2001; 16: 274–280DOI: 10.1002/ffj.996

Identification of thiazolidines in guava: stereochemicalstudies

Xavier Fernandez,1 Elisabet Dunach,1∗ Roland Fellous,1 Louisette Lizzani-Cuvelier,1 Michel Loiseau,1

Vincent Dompe,2 Francis Cozzolino,2 Gerard George,2 Sophie Rochard2 and Christine Schippa2

1 Laboratoire Aromes, Syntheses, Interactions, Faculte des Sciences de Nice Sophia-Antipolis, Parc Valrose, 06108 Nice cedex 2,France2SKW Biosystems, Business Unit Flavours and Fruit Systems, Le Plan B.P. 82067 06131, Grasse cedex, France

Received 25 November 2000Revised 12 February 2001Accepted 19 February 2001

ABSTRACT: Volatile compounds from guava (Psidium guajava L.) were extracted using dichloromethane.Among these volatile compounds, methyl 2-methyl-thiazolidine-4-(R)-carboxylate (cis and trans) and the ethyl2-methyl-thiazolidine-4-(R)-carboxylate (cis and trans) could be identified for the first time in fruits. The chemicalsynthesis of the reference compounds and other analogues allowed the further comparison with the guava extractedthiazolidines. The absolute configuration of the cis/trans isomers was determined by nuclear magnetic resonance(1H-NMR) and by nuclear overhauser enhancement (2D NMR NOESY). Copyright 2001 John Wiley & Sons,Ltd.

KEY WORDS: Psidium guajava L.; GC–MS; GC–IR; thiazolidine; epimerization; NMR; NOESY

Introduction

There has been an increasing consumption of exoticfruits during the last few years. This has resulted inseveral recent studies aimed at obtaining more insightinto the chemical composition of these fruits.1 Amongexotic fruits, the guava, Psidium guajava L., of themyrtle family (Myrtaceae) is well known.2 Raw guavasare eaten unprepared but are preferred seeded and servedsliced as dessert or in salads. More commonly, uponcooking the fruit, its strong odour is eliminated. Thehigh concentration of ascorbic acid (40 mg/100 g pulp)3

and the various medicinal properties4 may explain theincreasing interest of guava fruit. A leaf decoction istaken as a remedy for coughs, throat and chest pain.

Idstein and Schreir (1985)5 were able to identify 154volatile compounds in guava. Among these, the authorsdescribed for the first time six new sulphur-containingcompounds (2-pentanethiol, diethyl disulphur, dimethyltrisulphur, 2-ethylthiophene, 2-methylthiophene and3-methylthiophene), three pyrazine derivatives (2,3,5-trimethylpyrazine, methyl and acetylpyrazine) and threethiazole structures (5-ethoxythiazole, 2-methylthiobenzo-thiazole and benzothiazole). Ortega and Pino (1986)

*Correspondence to: E. Dunach, Laboratoire Aromes, Syntheses,Interactions, Faculte des Sciences de Nice Sophia-Antipolis, Parc Val-rose, 06108 Nice cedex 2, France. E-mail: [email protected]/grant sponsor: Ministry of Education, France.

were able to identify up to 320 compounds in guava,among all the different varieties of this fruit.3

Our recent studies on yellow-flesh guava led to theidentification of 20 novel compounds of this fruit,such as mercaptoalcohols, thioesters and oxathianes.6

We have been particularly interested in sulphur- andnitrogen-containing heterocyclic compounds possessingthiazolidine structures. Four thiazolidines are identi-fied for the first time in guava: methyl 2-(R or S)-methyl-thiazolidine-4-(R)-carboxylate and ethyl 2-(R orS)-methyl-thiazolidine-4-(R)-carboxylate. To the best ofour knowledge, these compounds have not been yet iden-tified in any other fruit or vegetable. In order to confirmthe structures and allow stereochemical assignments,these heterocyclic compounds (and related analogues)were synthesized by condensing methyl or ethyl estersof L-cysteine with several aldehydes.

Experimental

Fruit material

Yellow-flesh guava was imported from Jakarta (Indone-sia). The fruits (1800 g) were sliced into small cubes andintroduced into a 2 l flask with 800 g dichloromethane.Volatile compounds were extracted under ultrasonictreatment (Branson 3200, 47 kHz, 25 °C, 95 min). Afterfiltration, the organic layer was dried over MgSO4 andconcentrated to 1.5 ml by careful solvent distillation.

Copyright 2001 John Wiley & Sons, Ltd.

THIAZOLIDINE IDENTIFICATION IN GUAVA 275

Analytical GC

GC analysis was carried out using a Hewlett-Packard5890 gas chromatograph, under the following operationconditions: vector gas, He; injector and detector temper-atures, 250 °C; injected volume, 1 µl; splitless; HP1 col-umn, polydimethylsiloxane (50 m ð 0.32 mm i.d., filmthickness 0.52 µm; pressure 100 kPa); temperature pro-gramme 40–130 °C at 2 °C/min, 130–250 °C at 4 °C/minand 250 °C for 25 min; column HP20m, polyethyleneg-lycol (50 m ð 0.2 mm i.d., film thickness 0.1 µm; pres-sure 165 kPa); temperature programme 60–130 °C at2 °C/min, 130–220 °C at 4 °C/min and 220 °C for 25 min.Retention indices were with C5 –C28 alkane standards asthe reference.

Specific Detectors

The sulphur-specific detector used was a flame photo-metric detector (Hewlett-Packard 19256A) under the fol-lowing operation conditions: temperature, 250 °C; flowrates: air, 100 ml/min; H2, 75 ml/min. The nitrogen-specific detector used was a thermoionic detector (Hew-lett-Packard 19234B/C); temperature, 250 °C; flow rates:air, 100 ml/min; H2 3 ml/min; He, 20 ml/min.

GC–MS

GC–MS analysis was accomplished by using a HP 5890gas chromatograph coupled to a 5970A mass selectivedetector. Mass spectra were obtained by electron ioniza-tion at 70 eV and a source temperature of 250 °C (onlythe most abundant ions are given); and a magnetic spec-trometer Fisons-VG-Prospec for high-resolution massspectrometry.

GC–IR

Spectral acquisitions of 700–4000/cm were performedusing a Hewlett-Packard 5965A; vector gas, He; pres-sure, 140 kPa; auxiliary gas pressures, AUX A�N2� D65 kPa, AUX B �N2� D 65 kPa.

1H and 13C NMR

1H and 13C NMR spectra were recorded on a Bruker AC200 FT spectrometer at room temperature, with 50 mgproduct in 0.5 ml CDCl3, with TMS as internal standard.1H 2D NMR spectra were recorded on a Bruker AMX400 spectrometer. Acquisition and processing parametersfor NOESY experiment: data points in F1, 256; and inF2, 1024; mixing time, 1–2 s; zero filling, 1024.

Synthesis

Methyl or ethyl thiazolidine-4(R)-carboxylates were syn-thesized by condensing methyl or ethyl esters of (L)-cysteine with aldehydes.7

To a stirred solution of (L)-cysteine ethyl ester hydro-chloride (10 mmol) and potassium acetate (0.98 g/10 mmol) in ethanol (30 ml), a solution of freshly dis-tilled acetaldehyde in 10 ml ethanol was added at 0 °C.After stirring for 4 h at 0 °C, the reaction mixture wasfiltered off and the solvent evaporated under reducedpressure. The crude product was washed with a satu-rated solution of aqueous NaCl (15 ml) and extractedthree times with CH2Cl2 (3 ð 15 ml). The organic phasewas dried over MgSO4 and the solvent evaporated. Acolourless oil (1.5 g) was obtained in a yield of 93%.

In the case of the A/B diastereomers, the major isomerA (cis isomer) was eluted first by GC analysis. For C/Ddiastereomers also, the cis isomer, C, was the first eluted.

Ethyl 2-methyl-thiazolidine-4(R)-carboxylates, Aand B

1H NMR: 1.3(t, 3H, CH3CH2Otrans, J D 7.2 Hz),1.3(t, 3H, CHCH2Ocis, J D 7, 2 Hz) 1.5(dd, 3H,CH3trans , J D 1 and 6.5 Hz), 1.6(dd, 3H, CH3cis , J D1 and 6 Hz), 2.9(td, 1H, H-5cis, J D 1 and 9.5 Hz),3.1(dd, 1H, H-5trans, J D 6 and 10.5 Hz), 3.3(m, 2H,H-5’cis and trans), 3.8(t, 1H, H-4cis, J D 7 Hz), 4.2(m,8H, OCH2cis and trans , N-Hcis and trans), 4.55(qd, 1H, H-2cis,J D 1 and 6 Hz), 4.8(q, 1H, H-2trans, J D 6.5 Hz).

13C-NMR: 14.26 and 14.27�CH3CH2O�, 20.73 and23.75�CH3�, 38.48 and 39.33�OCH2�, 61.60 and61.67(C-5), 64.34 and 65.01(C-4), 64.79 and 65.97(C-2),171.38 and 171.45�C D O�. GC–MS : 175(MC, 11.6),160(9.3), 102(100), 100(18.1), 86(25), 85(50), 75(54),59(25.8), 55(26.3), 44(23.5).

IR: 1765 (S), 1233 (S).The same procedure was applied with methyl (or

ethyl) ester derivatives of (L)-cysteine and other alde-hydes, in methanol or ethanol solutions, and the spectraldata of the products obtained are described hereafter.

Reaction of (L)-cysteine methyl ester hydrochlorideand acetaldehyde; yield, 90%.

Methyl 2-methyl-thiazolidine-4(R)-carboxylates. Cand D

1H-NMR: 1.5(d, 3H, CH3trans , J D 6.5 Hz), 1.6(d,3H, CH3cis , J D 6 Hz), 2.2(s, 2H, N-H), 2.9(td, 1H,H-5cis, J D 1 and 9.5 Hz), 3.1(dd, 1H, H-5trans, J D6 and 10.5 Hz), 3.3(m, 2H, H-50

cis and trans), 3.71(s,3H, OCH3trans ), 3.73(s, 3H, OCH3cis), 3.85(1H, H-4cis),4.2(t, 1H, H-4trans, J D 6.5 Hz), 4.5(q, 1H, H-2cis, J D5.5 Hz), 4.75(q, 1H, H-2trans, J D 6.5 Hz).

Copyright 2001 John Wiley & Sons, Ltd. Flavour Fragr. J. 2001; 16: 274–280

276 X. FERNANDEZ ET AL.

13C-NMR: 20.73 and 23.75�CH3�, 38.41 and 39.30(C-5), 52.59 and 52.59�OCH3�, 64.31 and 64.98(C-4), 65.64and 65.93(C-2), 171.82 and 172.23�C D O�.

GC–MS : 161(MC, 15.6), 146(29), 115(14.6), 102(100),87(17), 86(41), 85(56), 75(74.7), 59(64.3), 55(52.2),44(19.6).

IR: 1768 (S), 1230 (S).Reaction of (L)-cysteine ethyl ester hydrochloride and

propionaldehyde; yield, 90%.

Ethyl 2-ethyl-thiazolidine-4(R)-carboxylates

1H-NMR: 0.95(t, 3H, CH3CH2trans , J D 7.5 Hz), 1(t,3H, CH3CH2cis , J D 7.5 Hz), 1.25(6H, 2t, OCH2CH3),1.8(2m, 2H, CH3CH2), 2.6(m, 2H, N-Hcis and trans),2.8(dd, 1H, H-5cis, J D 1 and 9.5 Hz), 2.95(dd, 1H,H-5trans), 3.2(m, 2H, H-50

cis and trans), 3.75(1H, dd, H-4cis,J D 7 and 9.5 Hz), 4.05(t, 1H, H-4trans, J D 6.5 Hz),4.15(m, 4H, OCH2CH3), 4.4(1H, dd, H-2cis, J D 5.5 and6.5 Hz), 4.55(dd, 1H, H-2trans, J D 6 and 8 Hz).

13C-NMR: 11.95 and 12.17�CH3CH2�, 14.3 and14.31�CH3CH2O�, 28.81 and 31.38�CH3CH2�, 37.63and 37.97(C-5), 61.64 and 61.7�OCH2CH3�, 64.24 and65.6(C-4), 71.88 and 72.84(C-2), 171.46 and171.51�C O�.

GC–MS : 189(MC, 8.9), 160(100), 132(8.6), 116(71.7),114(13.2), 99(21.5), 89(27.5), 86(51.9), 69(13.5),59(19.7).

Reaction of (L)-cysteine ethyl ester hydrochloride andbutyraldehyde; yield, 92%.

Ethyl 2-propyl-thiazolidine-4(R)-carboxylates

1H-NMR: 0.9(t, 6H, CH3�CH2�2, J D 7 Hz), 1.2(2t, 6H,OCH2CH3, J D 7 Hz), 1.4 and 1.6(2m, 2H, CH2CH2

CH3), 1.7 and 1.9(2m, 2H, CH2CH2CH3), 2.2(s, 2H, N-H), 2.7(t, 1H, H-5cis, J D 10 Hz), 2.9(dd, 1H, H-5trans,J D 6.5 and 10.5 Hz), 3.2(m, 2H, H-50

cis and trans),3.7(dd, 1H, H-4cis, J D 7 and 9.5 Hz), 4(t, 1H, H-4trans,J D 7 Hz), 4.15(m, 4H, OCH2CH3), 4.4(dd, 1H, H-2cis,J D 6 and 7 Hz), 4.6(t, 1H, H-2trans, J D 7 Hz).

13C-NMR: 13.9 and 14.08�CH3�CH2�2�, 14.3 and14.3�CH3CH2O�, 20.95 and 21.32�CH2CH2CH3�, 37.60and 37.90�CH2CH2CH3�, 38 and 38.09(C-5), 61.66and 61.72�OCH2H3�, 64.49 and 65.56(C-4), 70.01 and71.11(C-2), 171.44 and 171.48�C O�.

GC–MS : 203(MC, 5.1), 160(100), 130(50), 131(8.9),113(8.7), 103(18.6), 87(10), 86(44.3), 82(9.2), 59(15.7),55(11.3), 53(9.7).

Reaction of (L)-cysteine methyl ester hydrochlorideand isobutyraldehyde; yield, 94%.

Methyl 2-isopropyl-thiazolidine-4(R)-carboxylates

1H-NMR: 0.9, 0.95, 1 and 1.05(4d, 12H, CH�CH3�2,J D 6 Hz, 6.5 Hz, 6.5 Hz and 6.5 Hz), 1.75(heptuplet,

1H, CH�CH3�2trans , J D 7 Hz), 1.9(heptuplet, 1H,CH�CH3�2cis , J D 7 Hz), 2.2(s, 2H, N-H), 2.7(t, 1H,H-5cis, J D 10 Hz), 2.95(dd, 1H, H-5trans, J D 7 and10.5 Hz), 3.1(dd, 1H, H-50

trans, J D 6.5 and 10.5 Hz),3.2(dd, 1H, H-50

cis, J D 7 and 10.5 Hz), 3.7(2s, 3H,OCH3trans), 3.73(2s, 3H, OCH3cis ), 3.75(dd, 1H, H-4cis),4.05(t, 1H, H-4trans, J D 6.5 Hz), 4.3(d, 1H, H-2cis,J D 7 Hz), 4.45(d, 1H, H-2trans, J D 7.5 Hz).

13C-NMR: 19.81, 20.44, 20.6 and 20.80�CH�OH3�2�,33.70 and 35.17�CH�CH3�2�, 37.37 and 37.6(C-5), 52.59and 52.59(OCH3), 64.5 and 65.56(C-4), 77.87 and78.14(C-2), 171.3 and 171.89�C O�.

GC–MS : 189(MC, 2.1), 147(7.3), 146(100), 130(8.3),87(9.3), 86(34.1), 74(6.6), 59(10.3), 55(9.6), 41(6.7).

Reaction of (L)-cysteine methyl ester hydrochlorideand benzaldehyde; yield, 90%.

Ethyl 2-phenyl-thiazolidine-4(R)-carboxylates

1H-NMR: 1.2(t, 6H, OCH2CH3, J D 7 Hz), 2.2(s, 2H,N-H), 3.05(dd, 1H, H-5cis, J D 9 and 10 Hz), 3.12(dd,1H, H-5trans, J D 6 and 3 Hz), 3.35(dd, 1H, H-50

trans,J D 7 and 10.5 Hz), 3.45(dd, 1H, H-50

cis, J D 7.1 and10.2 Hz) 3.91(dd, 1H, H-4cis, J D 7.2 and 8.2 Hz),4.1(dd, 1H, H-4trans, J D 6.5 and 7 Hz), 4.2(m, 4H,OCH3CH3), 5.55(s, 1H, H-2cis), 5.8(s, 1H, H-2trans),7.31(m, 10H, H-Ph).

13C-NMR: 14.05 and 14.06�CH3CH2O�, 38.05 and39.09(C-5), 61.36 and 61.50�OCH2CH3�, 84.24 and84.44(C-4), 70.67 and 72(C-2), 126.81, 127.35, 127.76,128.31, 128.59, 128.55(C-Ph), 171 and 171.81�C O�.

GC–MS : 237(MC, 4.1), 164(96.5), 137(100),121(32.9), 118(28.5), 117(79.9), 106(23.4), 104(24.5),86(23.8), 77(23.8), 59(20.9).

Results and Discussion

Identification

Within our interest in identifying new sulphur and/ornitrogen compounds in yellow-flesh guava, mercaptoal-cohols, thioesters and oxathianes were found in this fruitafter analysis of the extracts using specific sulphur andnitrogen GC detectors.6

Two new compound, A and B, not previously iden-tified, presented retention indices of 1275 and 1280 onan apolar column, and of 1895 and 1905 on a polarcolumn, with a ratio A : B of 63 : 37. In the GC–MSspectrum, both compounds showed the same fragmenta-tion that did not correspond to the compounds alreadyidentified (Figure 1). By high resolution mass spectrom-etry, the molecular weight of both of these compounds Aand B was found to be 175.066916, corresponding to themolecular formula C7H13O2NS. These compounds con-tain two unsaturations. For both A and B the base peak



Figure 1. Mass spectrum of ethyl 2-methyl-thiazolidine-4(R)-carboxylate A/B

Figure 2. Mass spectrum of methyl 2-methyl-thiazolidine-4(R)-carboxylate C/D

m/z D 102 had a mass of 102.037782, corresponding tothe formula C4H8NS (Figures 1 and 2).

Infrared absorption at 1765 cm�1 and 1233 cm�1

indicated the presence of an ester group. By mass spec-trometry, the loss of a fragment with m/z D 73 shouldtherefore correspond to an ethyl ester group, CO2Et. Aloss of a methyl group was also observed in the massspectrum. The peak at m/z D 102 is compatible with athiazolidine structure. This peak is characteristic of theN,S ring in our mass spectra database.

With retention indices of 1210 and 1220 on apolar andof 1854 and 1881 on polar GC columns, we could alsofind two other S-containing compounds, C and D, in arelative C : D ratio of 62 : 38. For both C and D the basepeak m/z D 102 gave an identical high resolution mass of102.037782, corresponding to same formula, C4H8NS.All these compounds present the same heterocyclic ringsystem. According to the mass spectra, the molecularweight for both C and D was 161, which corresponds to

a difference of 14 (CH2 group) with respect to that of Aand B. Infrared absorption at 1765 cm�1 and 1233 cm�1

indicated the presence of an ester group. The combinedIR and mass spectra are compatible with a methyl estergroup linked to a thiazolidine ring. The concentrationrange of these four compounds A–D is 10–100 ppm inthe guava.

The biosynthesis of the thiazolidine ring present innatural products is generally issued from (L)-cysteinederivatives after their reaction with aldehydes. In thecase of A–D, the reaction with acetaldehyde was there-fore considered (Eq. 1).

�1�

The experimental data collected led us to propose,for A and B, the two diastereoismeric forms in ratio of



Figure 3. New thiazolidines identified in guava

ethyl 2-methyl-thiazolidine-4-(R)-carboxylates, in theircis and trans forms. For compounds C and D we proposethe structures of the corresponding methyl esters, methyl2-methyl-thiazolidine-4-(R)-carboxylate, also in the formof two cis/trans diastereoisomers (Figure 3).

We then could establish the structure and the con-figuration of these different derivatives by effecting thedirect chemical synthesis of the proposed molecules, asdescribed hereafter. Thiazolidines have not been oftenidentified in natural products. Lee et al.8 were able toidentify 2-methyl-thiazolidine and 2-propyl-thiazolidinein heated butter aroma. These products were proposed tobe produced during cooking by the condensation reactionof cysteamine (2-aminoethanethiol) and acetaldehyde orbutyraldehyde via the Maillard reaction.9

These volatile thiazolidine derivatives are used asflavouring agents in various foods such as soups, saucesand beverages.10,11 Thiazolidines may produce a spec-trum of olfactory notes ranging from fruity, green andvegetable-like (onion, radish, bean, asparagus) to nutty,butter-like, fatty and caramel to smoky, spicy and meator fish-like, depending on the type of substitution in theheterocyclic ring.12 They are introduced in low concen-tration, generally in the range 0.1–10 ppm, alone or withother flavouring compounds.10

Thiazolidine structures are also used for the determi-nation of trace aldehydes in food and beverages,13 asthey are quantitatively formed in the presence of 1,2-aminothiols. The method is based on the reaction ofvolatile carbonyl compounds with cysteamine, and thestable thiazolidines were subsequently analysed by GC,using sulphur or nitrogen detectors.

The biosynthesis of these compounds involves thereaction of cysteine or one of its derivatives with alde-hydes (Eq. 1).9 In the case of reaction with (L)-cysteineesters, the cyclization gives rise to a new chiral centreat the C-2 position of the heterocyclic ring. A mix-ture of two cis- and trans-diastereomers can thereforebe obtained (Figure 3).

We then independently prepared compounds A–Dand completed these synthesis with a series of dif-ferently substituted thiazolidine derivatives from (L)-cysteine methyl or ethyl esters and six aldehydes (seeEq. 1 and Table 1). The thiazolidines were obtained inyields of 90–94% (see Experimental). We establishedthat the chemically synthetized derivatives A–D pre-sented the same mass spectral data, IR absorptions andGC retention indices as the extracted compounds. For

Table 1. Yields of thiazo-lidines obtained followingEq. 1

R1 R2 Yield (%)

Et Me 93Me Me 90Et Et 90Et n-Pr 92Me i-Pr 94Et Ph 90

each prepared thiazolidine, the separation of the cis andtrans isomers by distillation, liquid chromatography (sil-ica gel) and preparative chromatography was attempted.However, all the separation trials led to a mixture of thetwo cis : trans diastereomers in the same initial ratio.This ratio, determined according to NMR data, is shownin Table 2. Previous literature data reported the possibil-ity of an epimerization reaction involving carbon 2, viaan imine-thiol intermediate14,15 (Eq. 2).

�2�

The H NMR of the mixture of the two isomers A : Band C : D was studied in detail. NMR proved to be thebest method of analysis; the spectra of the two isomericcompounds showed distinct signals for the two cis : transisomers, and the protons on the C(2) positions wereobtained with a good resolution in all the examples weexamined.

To determine the absolute configuration of the C(2)centre, we performed 2D NOESY spectra in CDCl3.1H–1H nuclear overhauser effect has been widely usedto determine molecular structures in solution.16 In thecase of thiazolidines, the NOE experiments showed anH(4) to H(2) correlation which was adequate for deter-mining the cis : trans configuration (Figure 4). Thus,the 2D NOESY spectrum of the methyl 2-methyl-thiazolidine-4-(R)-carboxylates C : D (Figure 4) showsthe interaction between H(2) and H(4) for the majorisomer, C, while the H(4) proton is only correlatedwith H(5) in the minor isomer, D. The space-proximityof H(2) and H(4) protons in C gives evidence for acis configuration of the ester and the R2 groups inthe thiazolidine ring (Figure 5). The absolute configu-ration of C(4) being determined by the starting mate-rial as (R), we can conclude that the absolute config-



Figure 4. Diastereomers obtained by condensing (L)-cysteine derivatives with aldehydes

H-4cis

H-2cis

ppm

ppm

4

3

2

4 3 2

Figure 5. 2D NOESY spectrum of methyl 2-methyl-thiazolidine-(R)-carboxylates (C and D) in CDCl3 at 20 °C



Table 2. Ratio between thetwo thiazolidine diastereo-mers determined by 1H-NMRin CDCl3 at 20 °C

R1 R2 cis trans

Me Me 61 39Et Me 71 29Et Et 67 33Et Pr 69 31Me i-Pr 70 30Et Ph 66 34

Table 3. Variation of the cis:trans ratio of ethyl 2-ethyl-thiazolidine-4 R -carboxvalates as a function of time indifferent solvents at 20 °C

CDCl3 CD3OD DMSO-d6t (% cis/trans) (% cis/trans) (% cis/trans)

20 min 63/37 54/46 59/412 h 68/32 52/48 53/475 h 67/33 52/48 50/508 h 67/33 52/48 46/54

24 h 67/33 52/48 46/54

uration of C(2) is also (R), for the major isomer ofmethyl (2R, 4R)-2-methyl-thiazolidine-4-carboxylate, C.NOESY experiments were also performed with severalother thiazolidines (in CDCl3). In all the cases exam-ined, the cis isomers were the major compounds (seeTable 2).

The cis:trans ratios were strongly dependent on thenature of the solvent (Table 3). The equilibration timeof ethyl 2-ethyl-thiazolidine-4(R)-carboxylate occurredbetween 20 min and 8 h. In solvents such as CDCl3 orCD3OD, the equilibration was faster, while in DMSO-d6,our data indicated a slower equilibration process.

In CDCl3 and CD3OD, the major isomer was thecompound cis. However, in DMSO-d6, the cis isomerwas the major compound after 20 min, but the proportionof the trans isomer rose with time. In this solvent, themajor diastereomer after complete equilibration was thetrans, with a reverse ratio compared with that obtainedin CDCl3 or CD3OD.

On the other hand, primary olfactory tests revealedthat ethyl 2-methyl-thiazolidine-4(R)-carboxylate A : Bpresented a leek-like note. Methyl 2-methyl-thiazolidine-4(R)-carboxylate C : D was less powerful from an olfac-tory point of view, with notes of leek, broad bean

and fennel. Moreover, ethyl 2-ethyl-thiazolidine-4(R)-carboxylate and ethyl 2-propyl-thiazolidine-4(R)-car-boxylate presented a leek-like note, methyl 2-isopropyl-thiazolidine-4(R)-carboxylate presented a leguminousnote, interesting for potential food applications.

Conclusions

In conclusion, four new thiazolidine structures derivedfrom (L)-cysteine have been identified in guava fruit.These four compounds correspond to the ethyl andmethyl esters of 2-methyl-thiazolidine-4-(R)-carboxylate,in their cis or trans stereochemistry, respectively, aswas confirmed by their independent chemical synthesis.The chemical preparation of the different thiazolidinederivatives showed that each pair of diastereomers wasin equilibrium and that a mixture of the cis:trans isomerswas isolated in each case. The association of analyticaland synthetic work enabled the assignment of the abso-lute configurations of the compounds identified in guavafruits.

Acknowledgements—We are grateful to Dr R. Faure, Faculte des Sci-ences St. Jerome, Marseille, France, for his contribution in recording2D NMR spectra. We also thank the French Ministry of Education fora doctoral scholarship to X.F.

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Identification of thiazolidines in guava: stereochemical studies

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