Trans-4-aminoproline, a phytotoxic metabolite with herbicidal activity produced by Ascochyta caulina

Article abstract of DOI:10.1016/S0031-9422(99)00507-5

A phytotoxic metabolite, characterized through NMR techniques and synthetic methods as trans-4-aminoproline, was isolated from the culture filtrates of Ascochyta caulina, a promising mycoherbicide for biological control of Chenopodium album. The metabolite, which shows interesting phytotoxic properties, together with ascaulitoxin (recently characterized as N²-β-D-glucoside of the unusual bis-amino acid 2,4,7-triamino-5- hydroxyoctandioc acid) and another unidentified compound, compose an active fraction of A. caulina culture filtrates with promising herbicidal properties. When assayed on leaves of host and non host dicots, including wild and cultivated plants, the trans-4-aminoproline showed a wide range of toxicity, with leaves of C. album being the most sensitive. Other interesting aspects were its inefficacy on several monocots, both cultivated and wild, and its lack of antifungal, antibiotic and zootoxic activities. This is the first report on trans-4-aminoproline as naturally occurring compound and phytotoxic metabolite produced by A. caulina. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0031-9422(99)00507-5

Phytochemistry 53 (2000) 231±237
www.elsevier.com/locate/phytochem
Trans-4-aminoproline, a phytotoxic metabolite with herbicidal
activity produced by Ascochyta caulina
Antonio Evidentea,*, Anna Andol®a, Maurizio Vurrob, Maria Chiara Zonnob,
Andrea Mottac
a
Â
Â
Dipartimento di Scienze Chimico-Agrarie, Universita di Napoli Federico II, Via Universita 100, I-80055 Portici, Italy
bIstituto Tossine e Micotossine da Parassiti Vegetali, CNR, Viale L. Einaudi 51, I-70125 Bari, Italy
cIstituto per la Chimica di Molecole di Interesse Biologico, CNR, Via Toiano 6, I-80072 Arco Felice, Italy
Received 8 July 1999; received in revised form 14 September 1999; accepted 15 September 1999
Abstract
A phytotoxic metabolite, characterized through NMR techniques and synthetic methods as trans-4-aminoproline, was isolated
from the culture ®ltrates of Ascochyta caulina, a promising mycoherbicide for biological control of Chenopodium album. The
metabolite, which shows interesting phytotoxic properties, together with ascaulitoxin (recently characterized as N 2-b-D-glucoside
of the unusual bis-amino acid 2,4,7-triamino-5-hydroxyoctandioc acid) and another unidenti®ed compound, compose an active
fraction of A. caulina culture ®ltrates with promising herbicidal properties. When assayed on leaves of host and non host dicots,
including wild and cultivated plants, the trans-4-aminoproline showed a wide range of toxicity, with leaves of C. album being the
most sensitive. Other interesting aspects were its inecacy on several monocots, both cultivated and wild, and its lack of
antifungal, antibiotic and zootoxic activities. This is the ®rst report on trans-4-aminoproline as naturally occurring compound
and phytotoxic metabolite produced by A. caulina. # 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Chenopodium album; Ascochyta caulina; Phytotoxins; Nonproteigenic amino acids; Trans-4-aminoproline; Weed biocontrol
1. Introduction
produced in vitro phytotoxic hydrophilic low molecu-
lar weight metabolites (Scheepens, Kempenaar,
Adreanas, Eggers, Netland & Vurro, 1997), which
could be relevant for an alternative, or in addition, to
the use of pathogens in weed biocontrol (Strobel,
The perthotrophic fungal species Ascochyta caulina
(P. Karst.) v.d. Aa and v. Kest. has been proposed as
a
mycoherbicide against Chenopodium album (L.)
(Kempenaar, 1995). This is known as common lambs-
quarter or fat hen and is a common world-wide weed
of many arable crops as sugar beet and maize (Holm,
Pluckett, Pancho & Herberger, 1977). The application
of pycnidiospores of the fungus to C. album plants
causes the appearance of large necrosis of leaves and
stems, and depending on the amount of necrosis devel-
oped, plants show retarded growth or death.
Preliminary experiments had showed that A. caulina
Kien®eld, Bunkers, Sugawara
&
Clardy, 1991).
Recently, the main phytotoxin, named ascaulitoxin,
has been isolated and characterized as N 2-b-D-gluco-
side of the unusual bis-amino acid 2,4,7-triamino-5-
hydroxyoctandioc acid (Evidente et al., 1998).
Considering that the other toxic metabolites also
exhibited an amino acidic nature, the culture ®ltrates
were preliminarily fractionated by cationic-exchange
chromatography obtaining a mixture of phytotoxins.
The puri®cation of this mixture by gel ®ltration chro-
matography allowed isolation of a further phytotoxin
which, together with ascaulitoxin and other still
unknown metabolites, is responsible for the high phy-
totoxicity of the fungal culture ®ltrate.
* Corresponding author. Tel.: +39-081-7885224; fax: +39-081-
7755130.
E-mail address: evidente@unina.it (A. Evidente).
0031-9422/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(99)00507-5

232
A. Evidente et al. / Phytochemistry 53 (2000) 231±237
Table 1
1H- and 13C-NMR D2O data of trans-4-aminoproline (1) and trans-4-hydroxy-L-proline (3)
C
1a
HMBC
3
d
m
dH
m
J (Hz)
d
m
dH
m
J
2
3
58.4
28.1
d
t
4.71
2.82
2.44
4.55
3.83
3.79
dd
9.8, 8.4
12.3, 9.8, 9.8
12.3, 9.8, 8.4
55.6
40.1
d
t
4.35
2.43
2.17
4.67
3.49
3.37
±
dd
10.2, 7.9
14.1, 7.9
14.1,10.2,4.4
ddd
ddd
m
4.71, 4.55, 3.83, 3.79
2.82, 2.44
brdd
ddd
m
4
5
60.0
63.6
d
t
72.7
62.5
d
t
dd
dd
13.0, 3.7
13.0, 5.1
dd
12.7, 3.8, 1.8
12.7, 1.48
±
brd
±
COOH
177.1
s
4.71, 2.82, 2.44
176.9
s
a 2D 1H, 1H (COSY) and 2D 13C, 1H (HMQC) NMR experiments delineated correlations of all protons and corresponding carbons.
This paper reports on the isolation and chemical
and biological characterization of this new phytotoxin.
ied ranging from very wide necrosis (poppy, annual
mercury, cucumber, wild cucumber), to medium (tree
of Heaven, tomato, common sowthistle), to small
(black nightshade). An interesting aspect is the lack of
toxicity when 1 was assayed on several monocots, both
2. Results and discussion
cultivated (wheat, oat, barley) as well as wild (canary-
5
The culture ®ltrate of A.caulina, showing high phy-
tototoxicity on leaves and cuttings of both host and
non-host plants, was fractionated by cation-exchange
chromatography, which allowed separation of the mix-
ture of phytotoxic metabolites from other metabolites
and the large amount of saccharose used as carbon
source in the culture medium. The mixture of toxic
metabolites was successively puri®ed by a Bio-Gel P-2
column, as detailed in Section 3, yielding the second
fraction group containing ascaulitoxin (Evidente et al.,
1998) and the third one corresponding to a homo-
geneous compound characterized as trans-4-aminopro-
line (1, 150 mg l 1), as described below.
Assayed at 1 mg/ml on punctured leaves, the toxin
had a drastic e€ect on the host, causing the fast
appearance of large necroses (6±7 mm diameter) sur-
rounding the puncture. At concentration ®ve times
lower, the toxin was still active, causing smaller
necroses. On other dicot leaves the phytotoxicity var-
grass, slender foxtail, wild oat). Tested up to 10
M
on cut young fat-hen seedlings the toxin caused wide
necrosis and dryness of cotyledons, while no e€ects
could be seen on stems. The same symptoms on leaves
but not on stems, could be observed on cut host
4
plants, only at higher concentrations (10 M).
The toxin (1) proved to be a non-aromatic amino
acid considering the typical reactivity of its TLC with
ninhydrin associated to the lack of the characteristic
UV absorption. It showed a molecular weight of 130
as deduced by mass spectrometry. In fact, the ions
generated from the molecular ion by loss of H2O and
by cluster formation with both K+ and Na+ were
observed in the FAB and ES mass spectra at m/z 112
1
and 169 and 153, respectively. The H-NMR spectrum
(see Table 1) showed signal systems very similar to
those present in the spectrum of the proteigenic trans-
4-hydroxy-L-proline (3) recorded in the same con-
ditions. In particular, the double doublet of the a-
Table 2
1H-NMR data of N1,N4-ditosyl methyl ester derivatives of trans-4-aminoproline and cis-4-amino-L-proline (2 and 9)
H
2
9
d
m
J (Hz)
d
m
J (Hz)
2
4.39
2.44
dd
ddd
ddd
m
9.5, 7.6
11.8, 9.5, 6.4
11.8, 7.6, 5.6
4.21
2.14
dd
ddd
10.1, 2.3
14.6, 10.1, 6.30
14.6, 2.3, 2.3, 1.2
3
3'
4
2.26
4.23
1.82
3.99
br ddd
m
dd
ddd
s
5
4.21
4.18
dd
dd
s
10.8, 4.6
10.8, 5.3
3.25
3.18
10.3, 4.9
10.3, 2.4, 1.2
5'
OMe
3.67
3.76
Ph
Me±Ph
Me±Ph
7.80±7.35
2.47
2.46
m
7.72±7.32
2.45
2.43
m
s
s
s
s

A. Evidente et al. / Phytochemistry 53 (2000) 231±237
233
Fig. 1. Synthesis of N 1,N4-ditosyl-cis-4-amino-L-proline methyl ester.
amino acidic proton (H-2) at d 4.71, coupled, in the
COSY spectrum (Bax & Freeman, 1981), with both
protons of the adjacent methylene group (H2C-3),
which appeared as two double doublets at d 2.82 and
2.44. Both of them correlated with the H-4 proton
resonating as a complex multiplet at d 4.55, a chemical
shift characteristic of a secondary carbon bearing an
amino group (Pretsch, Clerc, Seibl & Simon, 1983).
Finally, the latter coupled with the protons of a
further methylene group (H2C-5), appearing as two
double doublets at d 3.83 and 3.79, which are typical
of the methylene bonded to the nitrogen of proline de-
rivatives (Pretsch et al., 1983). Taken together, these
data suggest the structure of 4-aminoproline for this
new toxin. The 13C-NMR data were consistent with
those of 3 (Table 1) but with an amino group and a
carboxylic groups at C-4 and C-2, respectively resonat-
ing at the typical chemical shift values of d 60.0 and
58.4, together with the signi®cant singlet of the car-
boxylic group observed at d 177.1 (Pretsch et al., 1983;
Breitmaier & Voelter, 1987). Furthermore, the struc-
ture 1 was corroborated by the correlations recorded
in the HMQC spectrum (Table 1) (Bax, Ikura, Kay,
Torchia & Tschudin, 1990).
The structure of 4-aminoproline was con®rmed by
preparing the corresponding N 1,N 4-ditosylmethyl ester
derivative (2). Its 1H-NMR (see Table 2), besides the
signals of the two tosyl (Ts) groups and the singlet of
the ester methoxy group at d 3.67, showed a signal
pattern very similar to that of 1 except for the down-
®eld shift ꢀDd 0.38 and 0.39, respectively) of both H-5
and H-5', observed as two double doublets at d 4.21
and 4.18. The EIMS did not show the molecular ion
at m/z 452 but the ion at m/z 394, generated from it
by loss of the a-lactone (2-etanolide), which is a frag-
mentation mechanism typical of proline derivatives
(Porter, 1985). Similarly, the parent ion, by successive
loss of CO2Me and Ts residues, generated the ion at
m/z 222. By an alternative fragmentation mechanism,
the molecular ion, losing in succession two TsNH, or
TsNH2 residues, produced the ions at m/z 282, 92 and
281, respectively. The base peak, corresponding to the
expected tropylium ion, was recorded at m/z 91.
Considering that 4-aminoproline can exist as four
stereoisomers and that biological activity of pyrrolidine
derivatives depend on their substitution pattern, func-
tionalization and absolute con®guration (Massiot &
Delaude, 1986; Numata & Ibuka, 1987; Tanaka,
Suzuki
&
Sawanishi, 1996; Thorbek, Hjeds
&
Schaumburg, 1981), a study was undertaken to deter-
mine the stereochemistry of the toxin. Unfortunately, 1
and crystals of derivative 2 proved to be unsuitable for
X-ray analysis. Therefore, the determination of the
relative stereochemistry of 4-aminoproline was carried
out by chemical methods and comparing the physical
and the spectroscopic properties of the N 1,N 4-ditosyl
methyl ester derivative, obtained from 1, and from the
commercially available trans-4-hydroxy-L-proline (3),
whose absolute con®guration is 2S, 4R.
The well known synthetic procedure (Andreatta,
Nair, Robertson, & Simpson, 1967; Portoghese &
Mikhail, 1966) adopted to convert 3 into the corre-
sponding N 1,N 4-ditosyl methyl ester derivative, is
depicted in Fig. 1. The proteigenic amino acid (3) was
®rst transformed into the N-tosyl derivative (4), which
in turn was esteri®ed with ethereal diazomethane and
then the resulting methyl ester (5) converted into the
corresponding N,O-ditosyl derivative (6). The latter,

234
A. Evidente et al. / Phytochemistry 53 (2000) 231±237
by nucleophilic substitution, was transformed into the
corresponding azido derivative (7) with inversion of
the con®guration at C-4. This was ®rst reduced by cat-
alytic hydrogenation and the resulting very unstable
amino derivative converted into N 1,N 4-ditosyl-cis-4-
amino-L-proline methyl ester (9). Derivative 9 proved
to be di€erent from the N 1,N 4-ditosyl methyl ester (2)
prepared starting from 1. In fact, 2 and 9 showed
di€erent optical rotations and TLC behaviours in
three di€erent systems (see Section 3). Moreover, their
1H-NMR spectra (Table 2), recorded in the same con-
ditions, appeared signi®cantly di€erent in terms of
chemical shifts and coupling constants. In particular,
comparing 2 and 9, the H-3', H-4 and both H-5 and
H-5' appeared low®eld shifted ꢀDd 0.44, 0.24, 0.96 and
1.00, respectively) at d 2.26, 4.23, 4.21 and 4.18, re-
spectively, while the coupling constants between H-3'
with both H-2 and H-4 and H-4 with H-5' were mark-
edly di€erent. On these bases the two ditosyl methyl
ester derivatives (2 and 9) appeared to be diastereo-
mers and therefore the relative stereostructure of trans-
4-aminopyrrolidine-2-carboxylic acid can be assigned
to 4-aminoproline (1). Furthermore, the speci®c optical
very interesting and are in progress.
3. Experimental
3.1. General
Mp were taken on a Reichert Thermovar and are
uncorrected; optical rotations were measured in CHCl3
on a JASCO DIP 370 Digital polarimeter, unless
otherwise noted; IR spectra were recorded (neat) on a
Perkin-Elmer FT-IR 1720X spectrometer. UV spectra
were taken on a Perkin-Elmer Lamba 3B UV/Vis spec-
trophotometer in MeCN, unless otherwise noted. 1H-
and l3C-NMR: were recorded in CDCl3 (unless other-
wise noted) at 500 or 250 and at 125 or 62.5 MHz, re-
spectively, on DRX 500 and AM 250 Bruker
spectrometers. The same solvent used as internal stan-
dard. For spectra recorded in D2O, TPS (sodium-3-tri-
methylsylilpropionate-2,2,3,3-d4) was used as internal
standard. Carbon multiplicities were determined by
DEPT spectra (Breitmaier & Voelter, 1987). DEPT,
COSY-45, HMQC and HMBC (Bax & Summers,
1986) NMR experiments were performed using Bruker
microprograms. EI and Electrospray MS were
recorded at 70 eV on a Fisons Trio-2000 and a Perkin-
Elmer API-100 spectrometer, respectively. FAB MS
were recorded in glycerol/thioglycerol using Cs as
bombarding atoms on a VG ZAB 2SE. Analytical and
preparative TLC were performed on silica gel (Merck,
Kieselgel, 60 F254, 0.25 and 0.50 mm, respectively) or
on reverse phase (Whatman, KC18 F254, 0.20 mm)
plates; the spots werc visualized by exposure to UV
radiation and/or by spraying with 0.5% ninhydrin in
Me2CO followed by heating at 1108C for 10 min. CC:
Dowex-50 and Bio-Gel P-2. Solvent systems: (A)
BuOH±HOAc±H2O (3:1:1); (B) iso-PrOH±H2O (7:3);
(C) CHCl3-iso-PrOH (49:1); (D) CHCl3-iso-PrOH
(97:3), (E) CHCl3-iso-PrOH (9:1), (F) CHCl3-iso-PrOH
(19:1); (G) EtOAc-n-hexane (1:1); (H) EtOH±H2O
(1.5:1).
rotation value ꢀ‰aŠ2D1+47.2) measured for 1 and that
21
D
reported for the trans-4-amino-L-proline ꢀ‰aŠ
57.8)
shared their enantiomeric relation, therefore suggesting
the absolute trans-4-amino-D-proline stereostructure
for 1. Further attempts are in progress to con®rm this
relative and possibly absolute stereochemistry by X-
ray analysis of a suitable derivative.
Chiral non racemic pyrrolydines are common struc-
tural subunits found in many natural and synthetic
products with biological activity (Massiot & Delaude,
1986; Numata & Ibuka, 1987). Biological activities of
pyrrolidines are a€ected by both the functionalization
and stereochemistry of the pyrrolidine ring (Massiot &
Delaude, 1986; Numata & Ibuka, 1987; Tanaka et al.,
1996; Thorbek et al., 1981). Among these, cucurbitine
(Tanaka et al., 1996; Fang, Li, Niu & Tseng, 1961;
Shiao, Shao, Ho, Yang & Mao, 1962), containing (S )-
a-amino acid function at 3-position in the pyrrolidine
ring and cis-3-amino-L-proline (Hatanaka, 1969) are
the naturally occurring non proteigenic bioactive
amino acids more related to trans-4-aminoproline (1).
Trans-4-aminoproline is already known as synthetic
product (Andreatta et al., 1967; Mauger & Witkop,
1966), but this is its ®rst report as naturally occurring
compound and as phytotoxin produced by A. caulina
with potential bioherbicidal activity.
Considering the high phytotoxic activity of the com-
pound on fat-hen, the range of activity for other
dicots, and its inecacy against monocots associated
to the lack of antifungal, antibiotic and zootoxic ac-
tivity in the used assays, investigations on the practical
use of the toxin as natural herbicide, as alternative or
in addition to the use of the producing pathogen are

A. Evidente et al. / Phytochemistry 53 (2000) 231±237
235
3.2. Production and bioassays of trans-4-aminoproline
(1)
remove ammonia. The residual lyophilized solution
gave a residue consisting of a mixture of phytotoxic
amino acids (243 mg). An aliquot of this (11.0 mg)
was puri®ed by a Bio-Gel P-2 column (3 Â 57 cm)
equilibrated and eluted with ultrapure Milli-Q water,
as in detail previously described (Evidente et al., 1998);
®ve groups of homogeneous fractions were obtained.
Only residues of groups 2 (2.9 mg), 3 (1.8 mg) and 5
(3.9 mg) showed phytotoxic activity. The residue
obtained from group 2 resulted to be ascaulitoxin
(Evidente et al., 1998), while that of group 3 proved to
be another homogeneous compound (150 mg/l of cul-
ture ®ltrate) as showed by TLC analysis on silica gel
and reverse phase (Rf 0.21 and 0.75, using eluents A
and B, respectively). It was identi®ed as trans-4-amino-
proline (1): ‰aŠ2D1: +47.2 (c 0.4, H2O) UV (H2O) lmax
nm ꢀlog e† <220; 1H- and 13C-NMR: Table 1; FAB
(+) m/z (rel. int.): 112 [M-H2O]+ (100); ES m/z: 169
[M + K]+, 153 [M + Na]+.
A strain of A. caulina (P. Karst) v.d. Aa and v. Kest
freshly isolated from diseased leaf of C. album was
kindly supplied by Dr P.C. Scheepens, AB-DLO,
Wageningen, The Netherlands, and stored as single
spore culture in the Collection of Istituto Tossine e
Micotossine da Parassiti Vegetali, CNNR, Bari, Italy
(ITEM 1058). The method used for the preparation of
culture ®ltrates of A. caulina is that presented in a
recent paper (Evidente et al., 1998). Culture ®ltrates,
chromatographic frs and trans-4-aminoproline were
assayed on host plants using the leaf-puncture assay.
The pure toxin was assayed, using the same assay, at 1
mg/m1 also on monocots, both cultivated (oat: Avena
sativa, barley: Hordeum vulgare; durum wheat:
Triticum durum ) and weedy (canarygrass: Phalaris
canariensis; slender foxtail: Alopecurus myosuroides;
wild oat: Avena fatua), as well as on dicots, cultivated
(tomato: Lycopersicon esculentum; cucumber: Cucumis
sativus ) or weeds (poppy: Papaver rhoeas; annual mer-
cury: Mercurialis annua; wild cucumber: Ecballium ela-
terium, black nightshade: Solanum nigrum; tree of
Heaven: Ailanthus glandulosa; common sowthistle:
Sonchus oleraceus )
3.4. N 1,N 4-Ditosyl-trans-4-aminoproline methyl ester
(2)
The trans-4-aminoproline(1, 11, 0 mg) was dissolved
in 2 N NaOH (2 ml) and shaken for 1 day with a TsCl
Et2O solution (75 mg/2 ml). The aqueous layer was
separated, acidi®ed with 6 N HCl and refrigerated
overnight. The yielded crude product (15.2 mg) was
separated by ®ltration and its cold solution in MeOH
(2 ml) was treated with a ethereal CH2N2, until a pale
yellow color persisted. The solvent was removed in
vacuo and the white residue puri®ed by prep. TLC
(silica gel, eluent C) to give 2 as a homogeneous com-
pound [15.0 mg; Rf 0.69 and 0.47 (silica gel eluent F
and G, respectively) and 0.41 (reverse phase, eluent
H)]: ‰aŠ2D5: +19.8 (c 0.3); UV lmax, nm ꢀlog e† 225 (4.3);
Trans-4-aminoproline was also assayed on young cut
fat-hen seedlings (having only cotyledons) up to
6
5 Â 10 M as well as on cut host plants (at the 4-
5
true-leaf-stage, up to 8 Â 10 M. Plants were cut and
immersed in the toxin solution (or in water as control)
for 2 days under continuous light, then were trans-
ferred in water. Symptoms were observed daily up to 5
days.
The antifungal activity of the toxins was checked on
Geotrichum candidum, while the antibiotic one was
assayed on Pseudomonas syringae subsp. syringae and
on Escherichia coli, as already described in detail, up
to 50 mg/disk (Evidente et al., 1998). The zootoxic ac-
tivity was tested on Artemia salina brine shrimps up to
40 mg/ml of sea solution (Bottalico, Logrieco &
Visconti, 1989).
1
1
IR nmax cm : 1731, 1597; H-NMR: Table 2; EIMS,
m/z (rel. int.): 394 [M-COOCH2 (a-lactone)]+ (9), 282
[M-TsNH]+ (6), 281 [M-TsNH2]+ (44), 268 [M-CHO-
Ts]+ (72), 240 [M-HCO-CO-Ts]+ (21), 222 [M-
CO2Me-Ts]+ (77), 155 [Ts]+ (98), 92 [M-2 Â TsNH]+
(33), 91 [C7H7]+ (100). ES: m/z, 491 [M+K]+, 475
[M+Na]+, 453 [M+H]+.
3.3. Puri®cation and characterization of trans-4-
aminoproline (1)
3.5. Synthesis of N 1,N 4-ditosyl-cis-4-amino-L-proline
methyl ester (9)
The lyophilized culture ®ltrate of A. caulina (10 g,
corresponding to 275 ml) was dissolved in 1 N
HCOOH (3 ml) and adsorbed on a Dowex-50 H+
form resin packed in a chromatographic column
(2 Â 30 cm). The column was washed with ultrapure
water (50 ml), which permitted to remove both the
great amount of saccharose used as carbon source in
the culture medium, and other not basic substances.
Than, the column was eluted with 1 N NH4OH (300
ml) and the eluate was ¯uxed by a N2 stream to
Trans-4-hydroxy-L-proline (3, 500 mg) was con-
verted, according to described procedures (Andreatta
et al., 1967; Portoghese & Mikhail, 1966) and as
depicted in Fig. 1, into the corresponding N-tosyl de-
rivative (4, 500 mg). The latter was esteri®ed with
CH2N2 and the resulting methyl ester (5, 450 mg) con-
verted into the corresponding N,O-ditosyl derivative
(6, 400 mg), which in turn, by nucleophilic substitution
with NaN3 was transformed in the expected N-tosyl-

236
A. Evidente et al. / Phytochemistry 53 (2000) 231±237
cis-4-azido-L-proline (7, 200 mg). The intermediates 4±
7 were characterized by the following physical and
spectroscopic properties compared with the very few
spectroscopic data available from literature (Andreatta
et al., 1967; Portoghese & Mikhail, 1966 ).
UV lmax nm ꢀlog e† 227 (4.1); IR nmax cm 1: 2108,
1757, 1598 [lit. 15: nmax (CHCl3) 2108, 1750]; 1H-
NMR, d: 7.82 (2H, Ph), 7.34 (2H, Ph), 4.53 (1H, dd,
0
J2, 3 ˆ 9:0 and J2, 3 ˆ 4:1 Hz, H-2), 4.07 (1H, m, H-4),
3.75 (3H, s, OMe), 3.65 (1H, dd, J4, 5 ˆ 6:0 and
0
0
J5, 5 ˆ 10:9 Hz, H-5), 3.33 (1H, ddd, J3, 5 ˆ 1:0,
0
0
3.6. N-Tosyl-trans-4-hydroxy-L-proline (4)
J4, 5 ˆ 4:0 and J5, 5 ˆ 10:9 Hz, H-5'), 2.46 (3H, s,
0
MePh), 2.36 (1H, dddd, J2, 3 ˆ 9:0, J3, 3 ˆ 13:4, J3, 4
ˆ
4 had: mp 148±1508C; ‰aŠ2D5: 101.3 (c 0.6, EtOH);
4:5 and J3, 5 ˆ 1:0 Hz, H-3), 2.26 (1H, dt, J2; 3 ˆ J3 ,
0
0
0
23
D
0
[Portoghese & Mikhail, 1966: mp 153±1558C.; ‰aŠ
4 ˆ 4:5 and J3, 3 ˆ 13:4 Hz, H-3'); EIMS, m/z (rel.
105.48 (c 2% EtOH)]; UV (MeOH) lmax nm ꢀlog e†
228 (4.0); IR nmax cm 1: 3395, 1736, 1598; 1H-NMR
data (CD3OD), d: 7.77 (2H, Ph), 7.41 (2H, Ph), 4.35
int.): 265 [M-CO2Me]+ (55), 237 [M-CO2Me-N2]+
(27), 155 [Ts]+ (88), 91 [C7H7]+ (100).
(1H, m, H-4), 4.25 (1H, t, J2; 3 ˆ J2; 3 ˆ 7:9 Hz, H-2),
3.10. N 1,N4-Ditosyl-cis-4-amino-L-proline methyl ester
(9)
0
0
3.59 (1H, dd, J4, 5 ˆ 4:2 and J5, 5 ˆ 10:9 Hz, H-5),
0
0
0
3.28 (1H, ddd, J3, 5 = 1.5, J4, 5 ˆ 2:6 and J5, 5 ˆ 10:9
Hz, H-5'), 2.44 (3H, s, MePh), 2.11 (2H, m, H2-3); EI
MS, m/z (rel. int.): 240 [M-COOH]+ (43), 155 [Ts]+
(29), 91 [C7H7]+ (81), 86 [M-Co2-Ts]+ (30), 58 (100)
N-Tosyl-cis-4-azido-L-proline methyl ester (7, 24 mg)
in MeOH (1 ml) was hydrogenated in the presence of
10% Pd on charcoal (100 mg) at room temperature
and 1 atmospheric pressure. After 4 h the catalyst was
®ltrated o€ and the solvent was removed under vac-
uum. The reaction mixture (28 mg) was puri®ed by
TLC prep. on silica gel (eluent E) giving the very un-
stable N-tosyl-cis-4-amino-L-proline (8, 7.0 mg) as
homogeneous compound, which was immediately con-
verted into the corresponding ditosylderivative as fol-
lows: 8 (7 mg) was dissolved in dry pyridine (100 ml)
and the solution was chilled in an ice-bath. A cold sol-
ution of TsCl (28 mg) in dry pyridine (100 ml) was
added and the reaction mixture was left at 08 for 3
days. The oily residue left by the reaction work-up was
puri®ed by prep. TLC (silica gel, eluent F) giving
N 1,N 4-ditosyl-cis-4-amino-L-proline methyl ester (9) as
3.7. N-Tosyl-trans-4-hydroxy-L-proline methyl ester (5)
25
D
5 had: mp 98±1008C; ‰aŠ
102.1 (c 0.5); [Andreatta
20
et al., 1967: mp 103±1048C; ‰aŠ
98.548 (c 1%
D
CHCl3)]; UV lmax ꢀlog e† 225 (4.3); IR nmax cm 1: 3515,
1
1741, 1598; H-NMR: d: 7.79 (2H, Ph ), 7.32 (2H, Ph),
0
4.46 (1H, m, H-4), 4.43 (1H, t, J2; 3 ˆJ2; 3 ˆ8:1 Hz, H-
2), 3.75 (3H, s, OMe), 3.62 (1H, dd, J4, 5 ˆ 4:1 and
0
0
J5, 5 ˆ 11:4 Hz, H-5), 3.40 (1H, dt, J4; 5 ˆ J3; 5 ˆ 1:8
0
and J5, 5 ˆ 11:4 Hz, H-5'), 2.44 (3H, s, MePh), 2.20
(1H, m, H-3), 2.13 (1H, m, H-3'); EIMS, m/z (rel.
int.): 240 [M-CO2Me]+ (49), 155 [Ts]+ (40), 91
[C7H7]+ (100), 58 (55).
homogeneous compound (9.5 mg): ‰aŠ2D5:
10.4 (c
1
3.8. N,O-Ditosyl-trans-4-hydroxy-L-proline methyl ester
(6)
0.12); UV lmax nm ꢀlog e† 226 (4.3); IR nmax cm
:
1738, 1598; 1H-NMR: Table 2. EIMS, m/z (rel. int.):
394 [M-COOCH2 (a-lactone)]+ (2), 393 [M-CO2Me]+
(10), 282 [M-TsNH]+ (4), 281 [M-TsNH2]+ (28), 268
[M-CHO-Ts]+ (9), 222 [M-CO2Me-Ts]+ (100), 155
[Ts]+ (81), 92 [M-2 Â TsNH]+ (29), 91 [C7H7]+ (86)
6 had: mp 92±948C; ‰aŠD25: 49.5 (c 0.5); [Andreatta
20
D
et al., 1967: mp 958C; ‰aŠ
54.88 (c 2% CHCl3)]; UV
lmax nm ꢀlog e† 225 (4.3); IR nmax cm 1: 1757, 1598;
1H-NMR, d: 7.72 (2H, Ph), 7.63 (2H, Ph), 7.34 (2H,
Ph), 7.29 (2H, Ph), 5.00 (1H, m, H-4), 4.29 (1H, t, J2; 3
0
ˆ J2; 3 ˆ 7:8 Hz, H-2), 3.75 (3H, s, OMe), 3.69 (1H,
Acknowledgements
0
dd, J4, 5 ˆ 4:4 and J5, 5 ˆ 12:3Hz, H-5), 3.58 (1H, dt,
0
0
0
J3; 5 ˆJ4; 5 ˆ1:9 and J5, 5 ˆ 12:3 Hz, H-5'), 2.47 (3H,
This investigation was supported in part by grants
to A.E. from the European Project entitled
``Optimizing biological control of a dominant weed in
major crops'' (FAIR5-PL97-3525) and in part by
grants from the Italian Ministry of University and
Scienti®c and Technological Research. The authors
thank the `Centro Interdipartimentale di Metodologie
Chimico-Fisiche, Universita di Napoli Federico II,
Napoli', Italy for NMR spectra and Mr. C. Iodice
(ICMIB-CNR, Arco Felice) for technical assistance.
Mass spectral data were provided by `Servizio di
Spettrometria di Massa del CNR e dell'Universita di
s, MePh), 2.45 (3H, MePh), 2.35 (1H, br ddd,
0
0
J2, 3 ˆ 7:8, J3, 3 ˆ 12:3 and J3, 5 ˆ 1:9 Hz, H-3), 2.20
0
0
0
(1H, ddd, J2, 3 =7.8, J3, 3 ˆ 12:3 and J3 , 4 ˆ 5:0 Hz,
H-3'); EIMS, m/z (rel. int.): 394 [M-CO2Me]+ (27),
240 [M-CO2Me-Ts]+ (50), 222 [M-CO2Me-Ts-H2O]+
(98), 155 [Ts]+ (96), 91 [C7H7]+ (100).
3.9. N-Tosyl-cis-4-azido-L-proline methyl ester (7)
7 had: mp 65±688C; ‰aŠD25: 44.3 (c 0.5); [Andreatta
20
D
et al., 1967: mp 69±708C; ‰aŠ
47.88 (c 2% CHCl3)];

A. Evidente et al. / Phytochemistry 53 (2000) 231±237
237
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