N5-(4-methoxyphenyl)methyl-L-glutamine in xylem sap from squash root

Article abstract of DOI:10.1016/S0031-9422(99)00055-2

A new amino acid derivative, N⁵-(4-methoxyphenyl)methyl-L-glutamine was found in the xylem sap from squash root. The structure was deduced from spectroscopic data and identified by chemical synthesis.

Full text of DOI:10.1016/S0031-9422(99)00055-2

Phytochemistry 51 (1999) 425±428
N 5-(4-Methoxyphenyl)methyl-L-glutamine in xylem sap
from squash root
Yoshinobu Inouyea,*, Takashi Wakahoia, Shinobu Satohb
aDepartment of Chemistry, University of Tsukuba, Tsukuba, Ibaraki 305-8571, Japan
bInstitute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan
Received 30 July 1998; accepted 6 October 1998
Abstract
A new amino acid derivative, N 5-(4-methoxyphenyl)methyl-L-glutamine was found in the xylem sap from squash root. The
structure was deduced from spectroscopic data and identi®ed by chemical synthesis. # 1999 Elsevier Science Ltd. All rights
reserved.
Keywords: Cucurbita maxima DuchesneÂC. moschata Duchesne; Cucurbitaceae; Xylem sap from squash root; Amino acid derivative; N 5-(4-
Methoxyphenyl)methyl-L-glutamine
1. Introduction
region was con®rmed by the WEFT technique. The
major signals are marked with `Ã', which show relative
intensities in integer multiples and several other minor
signals are also present. The presence of a (4-methoxy-
phenyl)methyl group, probably attached to a hetero
atom, is indicated from the NMR signals of d 6.91,
7.20, 3.75 and 4.24, the UV absorptions (222 and 274
nm) and the fragment ion m/z 121 formed from m/z
267 in the linked-scan ESI+ mass spectrum. The other
signals (d 2.06, 2.35, 2.39 and 3.67) in the 1H NMR
spectrum indicate the presence of a partial structure,
Z±CH2±CH2±CH±Y, where Y is an electron-with-
drawing group and Z must be an unsaturated group
because the geminal coupling constant (J=14.8 Hz) is
slightly larger than the typical methylene group.
The water soluble nature of 1 and the facile for-
mation of (M+H)+ and (M±H) ion in the ESI mass
spectra suggested that 1 could be a glutamic acid or
glutamine (or isoglutamine) derivative, as represented
by Z±CH2±CH2±CH±Y. The amino acid analysis of 1
(hydrolysis in 6 M HCl at 1108C) showed that only
glutamic acid was detected. The high resolution mass
spectrum gave (M+H)+ indicative of a glutamine (or
isoglutamine) derivative of the molecular formula
C13H18N2O4. The presence of a carboxylic acid was
Various organic compounds produced in the root
cells of the higher plants transport through xylem
vessels to the aerial parts and many important activi-
ties of the aerial organs rely on the supply of inorganic
and organic compounds from the xylem sap.
Previously, one of us (Satoh) found proteins and
carbohydrates in xylem sap from squash root (Satoh,
Iizuka, Kikuchi, Nakamura,
&
Fujii, 1992).
Subsequently, we isolated a new amino acid derivative
1 based on its biological activity towards adventitious
root formation of cucumber hypocotyls in shoot cut-
tings cultures. In the present work, we describe the
structure of the amino acid derivative as N 5-(4-meth-
oxyphenyl)methyl-L-glutamine (1a).
2. Results and discussion
The 1H NMR spectrum of 1 in D2O is shown in
Fig. 1(A). The absence of signals underneath the water
* Corresponding author.
0031-9422/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(99)00055-2

426
Y. Inouye et al. / Phytochemistry 51 (1999) 425±428
N 2-(t-Butoxycarbonyl)-L-glutamic acid 1-t-butyl
ester (2) was prepared from L-glutamic acid 5-benzyl
ester according to the literature (Olsen, Ramasamy, &
Emery, 1984). The carboxyl group in 2 was condensed
with 4-methoxybenzylamine in the presence of the
water soluble carbodiimide (WSCIÁHCl) (Sheehan,
Preston, & Cruickshank, 1965) to give an N 5-(4-meth-
oxyphenyl)methyl-N 2-(t-butoxycarbonyl)-L-glutamine
1-t-butyl ester (3). Removal of both protecting groups
with tri¯uoroacetic acid followed by neutralization
gave N 5-(4-methoxyphenyl)methyl-L-glutamine (1a) as
1
a white solid. The H NMR spectrum of 1a (Fig. 1B)
was completely coincident with that of the major com-
ponent of 1. The UV spectrum of 1a di€ered from 1 in
the absence of 358 nm (weak and broad); this absorp-
tion is probably due to minor component(s) present in
1.
The synthetic 1a contained 5.1% of D-isomer as
revealed by GC±MS analysis. By comparison with 1a,
the absolute con®guration of N 5-(4-methoxyphenyl)-
methylglutamine in 1 was determined as L.
The analogous compound N 5-(4-hydoxyphenyl)-
methyl-L-glutamine has been isolated from Fagopyrum
esculentum Moench (Koyama, Tsujizaka, & Sakumura,
1973), Sinapsis alba L. (Larsen, Olsen, Pedersen,
Sorensen, 1964) and Sinapsis arvensis L. (Larsen et al.,
1964), but N 5-(4-methoxyphenyl)methyl-L-glutamine is
a new compound. Synthetic 1a inhibited both adventi-
tious root formation from hypocotyl of cucumber and
development of 1st leaves of cucumber in the culture
of shoot cuttings (5 mM in water) but showed no
e€ects at a 0.5 mM concentration. Thus, the major in-
hibitory factor in the n-butanol fraction may be due to
the minor component(s) present in 1.
Fig. 1. 1H NMR spectra of 1 (A) and 1a (B) in D2O.
further indicated by the facts that 1 was adsorbed on
the anion-exchange resin and could be eluted out by
hydrochloric acid. These results established 1 as the N-
(4-methoxyphenyl)methyl derivative of glutamine (1a)
or isoglutamine (1b). The structure was ®nally con-
®rmed by chemical synthesis as shown in Scheme 1.
The role of N 5-(4-methoxyphenyl)methyl-L-gluta-
mine in nature and the structure of the minor com-
ponent(s) are now under investigation. The details on
their biological activity will be reported elsewhere.
Scheme 1. Synthesis of 1a.

Y. Inouye et al. / Phytochemistry 51 (1999) 425±428
427
3. Experimental
Bu), 1.90 (m, 1H, H-4a), 2.15 (m, 1H, H-4b), 2.42 (m,
2H, H-3), 4.20 (m, 1H, H-2), 5.15 (m, 1H, H-N).
3.1. General
3.4. N-(4-Methoxyphenyl)methyl-N 2-(t-
butoxycarbonyl)-L-glutamine 1-t-butyl ester (3)
M.p.'s uncorrected. HPLC: ODS-80Tm (4.6 Â 150
mm, Tosoh, Tokyo) and ODS-120T (4.6 Â 250 mm,
Tosoh) columns with acetonitrile±H2O gradient (0 to
60%). GC±MS: Chirasil-L-Val column (0.25 mm  25
m, Chrompak), helium (1.5 ml min 1) as the carrier
gas, programmed from 608C (1 min), up to 1208C
(108C min 1) and then to 1808C (58C min 1).
To a cold soln of 2 (850 mg, 3 mmol), HOBt (1-
hydroxybenzotriazole, 405 mg, 3 mmol) and 4-methox-
ybenzylamine (0.39 ml, 3 mmol) in CH2Cl2 (5 ml), was
added
a
suspension of WSCIÁHCl (1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride, 580
mg, 3 mmol) in CH2Cl2 (3 ml). The mixture was stir-
red at room temp. for 1 h EtOAc (50 ml) and H2O (20
ml) were added and the organic layer was separated.
The organic layer was washed successively with H2O,
0.5 M HCl, a satd NaHCO3 soln, H2O (2 times) and
saline. After drying over Na2SO4, the solvent was
evaporated to give almost pure crystalline 3 (1.20 g,
95%).
3.2. Plant material and isolation
Seeds of Cucurbita maxima DuchesneÂC. moschata
Duchesne cv. Shintosa-ichigou and seeds of Cucumis
sativus cv. Shimoshirazu-jibai were obtained from
Sakata Seed Co. (Kanagawa, Japan). The squash
plants were grown in the ®eld for 2±3 months (May to
August).
Xylem sap (2 l) collected from squash root was
mixed with four volumes of EtOH and the ppt was
removed. The ®ltrate was concentrated and partitioned
between water and n-butanol. The ppt and water frac-
tion accelerated the adventitious root formation of
cucumber in the culture of shoot cuttings, but the n-
butanol fraction inhibited this growth. After concen-
trating the n-butanol fraction, the residues were puri-
3: m.p. 98±1008C (triturated with pentane); [a]D22
1
218 (MeOH; c 0.10); H NMR (CDCl3, 200 MHz): d
1.41 (s, 9H, t-Bu), 1.44 (s, 9H, t-Bu), 1.90 (m, 1H, H-
4a), 2.15 (m, 1H, H-4b), 2.23 (m, 2H, H-3), 3.78 (s,
3H, OCH3), 4.15 (m, 1H, H-2), 4.35 (d, 2H, J=6 Hz,
CH2±N), 5.20 (m, 1H, H±N), 6.37 (m, 1H, H±N), 7.05
(d, 2H, J=9 Hz, H±Ar), 7.20 (d, 2H, J=9 Hz, H±Ar).
Found: C, 62.45; H, 8.25; N, 6.59%. Calcd. for
C22H34N2O6: C, 62.54; H, 8.11; N, 6.63%.
®ed by HPLC to give 0.2 mg of 1 as a white solid.
H2O
max
3.5. N 5-(4-Methoxyphenyl)methyl-L-glutamine (1a)
1: UV l
nm: 222 (strong), 274 (medium), 358
1
(weak and broad). H NMR (D2O, 500 MHz; only the
signals designated with `Ã'): d 2.06 (dt, 2H, J=6.2, 7.9
Hz, H-3), 2.35 (dt, 1H, J=14.8, 7.9 Hz, H-4a), 2.39
(dt, 1H, J=14.8, 7.9 Hz, H-4b), 3.67 (t, 1H, J=6.2
Hz, H-2), 3.75 (s, 3H, OCH3), 4.24 (s, 2H, CH2±N),
6.91 (d, 2H, J=8.7 Hz, H±Ar), 7.20 (d, 2H, J=8.7
Hz, H±Ar). ESI+ (electrospray ionization) mass: m/z
267 (M+H)+, 289 (M+Na)+; m/z 121 by linked scan
from m/z 267. ESI mass: m/z 265 (M±H) .
TFA (0.5 ml) was added to 3 (200 mg) and the mix-
ture was stirred at room temp for 1 h. After evaporat-
ing the excess TFA under vacuum, residues were
dissolved in H2O (1 ml). A 0.5 M NaOH soln was
added until pH 7, when ppt appeared. The ppt was
collected by ®ltration and washed with cold H2O fol-
lowed by MeOH and dried in a desiccator to give 1a
(80 mg, 63%) as a white solid.
High resolution ESI+ mass: found: m/z 267.138.
calcd for C13H19N2O4 (M+H): m/z 267.142.
1a: m.p. 214±2158C (dec); [a]2D2 +238 (1 M HCl; c
H2O
max
0.10); l
nm (log e): 224 (4.1), 273 (3.2).
Found: C, 58.52; H, 6.96; N, 10.37%. Calcd for
C13H18N2O4: C, 58.63; H, 6.81; N, 10.52%.
3.3. N 2-(t-Butoxycarbonyl)-L-glutamic acid 1-t-butyl
ester (2)
3.6. GC±MS analysis
N 2-(t-Butoxycarbonyl)-L-glutamic acid 1-t-butyl
ester (2) was prepared from L-glutamic acid 5-benzyl
ester according to the known procedure (Olsen et al.,
1984) in 55% overall yield.
1a (ca. 0.2 mg) was stirred in 5% HCl/MeOH (0.4 ml)
at room temp. for 3 h. After evaporating the reagents
under vacuum the residue was treated with tri¯uoro-
acetic anhydride (0.2 ml) for 30 min. Evaporating the re-
agents gave an analytical sample, which was dissolved in
acetone (1 ml). TIC (total ion chromatogram: m/z=100
to 400) of the solution was measured. The two peaks (31
and 32 min) showed a similar mass spectrum and were
2: m.p. 112±1148C (from pentane-ether); [a ]2D2 298
(MeOH; c 0.11) (lit. Olsen et al., 1984: m.p. 102±
1058C; [a]2D5 26.58 (MeOH; c 1); lit. Tomasz, 1971:
m.p. 110±1148C; [a ]2D5 30.28 (MeOH; c 1)); H NMR
1
(CDCl3, 200 MHz) d 1.43 (s, 9H, t-Bu), 1.45 (s, 9H, t-

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Y. Inouye et al. / Phytochemistry 51 (1999) 425±428
identi®ed as D- and L-isomers, respectively. The ratio of
D- and L-isomer in 1a was calculated from the area of m/z
245 (M±OMe) to be 5.1:94.9.
References
Koyama, M., Tsujizaka, Y., & Sakumura, S. (1973). Agric. Biol.
Chem., 37, 2749.
Larsen, L. M., Olsen, O., Pedersen, L. H., & Sorensen, H. S. (1964).
Phytochemistry, 23, 896.
Acknowledgements
Olsen, R. K., Ramasamy, K., & Emery, T. (1984). J. Org. Chem.,
49, 3527.
The authors are grateful to Dr. Kenji Matsuura
(JEOL) for the measurements of ESI (electrospray ion-
ization) mass spectra and also to Dr. Hajime Mita
(University of Tsukuba) for his generous assistance in
GC±MS analyses.
Satoh, S., Iizuka, C., Kikuchi, A., Nakamura, N., & Fujii, T. (1992).
Plant Cell Physiol., 33, 841.
Sheehan, J. C., Preston, J., & Cruickshank, P. A. (1965). J. Am.
Chem. Soc., 87, 2492.
Tomasz, J. (1971). J. Acta Chim. Acad. Sci. Hung., 70, 255.