Preparation of synthetic auxin-amino acid conjugates

Article abstract of DOI:10.1080/00397911.2019.1605446

Auxin amide conjugates are regulators of the most important auxin, indole-3-acetic acid (IAA), which is considered responsible for many important processes within the plants. Herein, amide conjugates of IAA were synthesized employing a simple and efficient coupling method with WSCI·HCl, a water-soluble condensing reagent, in the presence of 1-hydroxybenzotriazole. IAA conjugates with 10 amino acids along with their corresponding methyl esters were prepared in excellent yields, up to 95%, aiming to facilitate their identification in plant species. Eight IAA-amino acid methyl ester conjugates are characterized here for the first time.

Full text of DOI:10.1080/00397911.2019.1605446

Synthetic Communications
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Preparation of synthetic auxin-amino acid
conjugates
Panagiota-Kyriaki Revelou & Violetta Constantinou-Kokotou
To cite this article: Panagiota-Kyriaki Revelou & Violetta Constantinou-Kokotou (2019):
Preparation of synthetic auxin-amino acid conjugates, Synthetic Communications, DOI:
10.1080/00397911.2019.1605446
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https://doi.org/10.1080/00397911.2019.1605446
Preparation of synthetic auxin-amino acid conjugates
Panagiota-Kyriaki Revelou and Violetta Constantinou-Kokotou
Chemical Laboratories, Department of Food Science and Human Nutrition, Agricultural University of
Athens, Athens, Greece
ABSTRACT
ARTICLE HISTORY
Auxin amide conjugates are regulators of the most important auxin,
indole-3-acetic acid (IAA), which is considered responsible for many
important processes within the plants. Herein, amide conjugates of
IAA were synthesized employing a simple and efficient coupling
method with WSCIÁHCl, a water-soluble condensing reagent, in the
presence of 1-hydroxybenzotriazole. IAA conjugates with 10 amino
acids along with their corresponding methyl esters were prepared in
excellent yields, up to 95%, aiming to facilitate their identification in
plant species. Eight IAA-amino acid methyl ester conjugates are char-
acterized here for the first time.
Received 5 August 2018
KEYWORDS
Amide conjugates;
indole-3-acetic acid;
phytohormones; WSCIÁHCl
GRAPHICAL ABSTRACT
HN
O
OH
HN
HN
O
O
O
O
1. NaOH 2N
MeOH, r.t., 90 min
NH2
R
·
HCl
NH
NH
O
HO
WSCI·HCl, HOBt, Et3N,
CH2Cl2, r.t., o/n
O
2. H
R
R
O
up to 95% yields
up to 99% yields
R = amino acid side chain.
Introduction
Auxins are a class of endogenous phytohormones, mainly represented by indole-3-acetic
[1]
acid (IAA), a plant hormone essential in plant growth and development. A significant
amount of endogenous IAA is conjugated at the carboxyl group with sugars or amino
[2]
acids and peptides via the formation of ester or amide bonds, respectively. Several IAA
amide conjugates with amino acids have been identified in plants, with the carboxylic
group of the amino acid being either free or as methyl ester, although undescribed conju-
[
3–6]
gates are likely to exist.
important processes within plants such as: storage and transport of IAA, IAA catabol-
IAA conjugates with amino acids are responsible for many
[7]
[8]
[9]
[10]
ism, regulation of IAA levels and prevention against enzymatic degradation of IAA.
Synthesis of IAA amide conjugates is crucial for their identification, study of their
potential biological role in plants and exploration of possible interactions with microbes.
Several auxin-amino acid conjugates are not commercially available, hence simple and
CONTACT Violetta Constantinou-Kokotou
vikon@aua.gr
Chemical Laboratories, Department of Food Science and
Human Nutrition, Agricultural University of Athens, Iera odos 75, Athens, 11855, Greece.
Supplemental data for this article is available online at on the publisher’s website.
ß 2019 Taylor & Francis Group, LLC

2
P.-K. REVELOU AND V. CONSTANTINOU-KOKOTOU
efficient methods for their preparation are required. Methods for their synthesis include
[
11]
the activation of the IAA carboxylic group via p-nitrophenyl active esters,
mixed
or the use of N,N’-dicyclohexylcarbodiimide (DCC) as
However, characterization data of the IAA-amino acid conju-
[
12]
[13]
anhydrides,
acid chlorides
[
14–17]
the coupling agent.
gates are often incomplete. The most recent synthetic method utilizes the coupling of
preformed benzotriazolyl esters of IAA with various amino acids leading directly to
[
18]
IAA-amino acids conjugates with free carboxylic group in low to moderate yields.
To clarify the role and the biochemical pathways of the auxins in plants, IAA-amino
acid methyl ester conjugates may also be useful. Herein, we report the synthesis of
IAA-amino acid methyl esters conjugates employing a water-soluble condensing reagent,
1
-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (WSCIÁHCl), in the
presence of 1-hydroxybenzotriazole (HOBt), as well as the synthesis of the correspond-
ing IAA-amino acid conjugates with free carboxylic group, together with full character-
ization data. The use of WSCIÁHCl offers the advantage of easy removal of the urea
byproduct in the water layer, while DCC coupling procedure requires an additional
purification step of the product.
Results and discussion
WSCIÁHCl has been successfully utilized for the coupling of protected amino acids in
[
19]
[20]
the synthesis of peptides,
the coupling of protected amino acids with amines
or
[
21]
protected dipeptides with acids.
In this work, coupling reactions of IAA with the
methyl esters of glycine (Gly), L-alanine (Ala), L-valine (Val), L-tryptophan (Trp),
L-phenylalanine (Phe), L-tyrosine (Tyr), L-methionine (Met), L-serine (Ser), L-aspartic
acid (Asp) and L-glutamic acid (Glu) were performed, as illustrated in Table 1.
Protection of the amino group of indole ring before coupling proved unnecessary.
The desired products were obtained in excellent yields and varied from 85% to 95%
(Table 1). Amide conjugates of IAA with the methyl esters of Gly, Val, Tyr, Ser, Phe,
Table 1. Coupling reaction of IAA and amino acid methyl esters.
HN
O
HN
O
OH
NH2
R
NH
·
HCl
O
O
R
WSCI·HCl, HOBt, Et N,CH Cl , r.t., o/n
3
2
2
O
O
1
2
Entry
Reactant
Product
IAA-Gly-OMe 2a
Yield (%)
1
2
3
4
5
6
7
8
9
H-Gly-OMeÁHCl 1a
89
83
92
90
95
95
87
85
90
93
H-L-Ala-OMeÁHCl 1b
H-L-Val-OMeÁHCl 1c
H-L-Trp-OMeÁHCl 1d
H-L-Phe-OMeÁHCl 1e
H-L-Tyr-OMeÁHCl 1f
H-L-Met-OMeÁHCl 1g
H-L-Ser-OMeÁHCl 1h
H-L-Asp(Me)-OMeÁHCl 1i
H-L-Glu(Me)-OMeÁHCl 1j
IAA-L-Ala-OMe 2b
IAA-L-Val-OMe 2c
IAA-L-Trp-OMe 2d
IAA-L-Phe-OMe 2e
IAA-L-Tyr-OMe 2f
IAA-L-Met-OMe 2g
IAA-L-Ser-OMe 2h
IAA-L-Asp(Me)-OMe 2i
IAA-L-Glu(Me)-OMe 2j
1
0

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Table 2. Cleavage of methyl ester group.
HN
O
HN
O
1
. NaOH 2N
NH
NH
MeOH, r.t., 90 min
O
HO
R
+
2
. H
R
O
O
2
3
Entry
Reactant
Product
Yield (%)
1
2
3
4
5
6
7
8
9
IAA-Gly-OMe 2a
IAA-L-Ala-OMe 2b
IAA-L-Val-OMe 2c
IAA-L-Trp-OMe 2d
IAA-L-Phe-OMe 2e
IAA-L-Tyr-OMe 2f
IAA-L-Met-OMe 2g
IAA-L-Ser-OMe 2h
IAA-L-Asp(Me)-OMe 2i
IAA-L-Glu(Me)-OMe 2j
IAA-Gly-OH 3a
99
97
98
95
95
92
98
96
93
96
IAA-L-Ala-OH 3b
IAA-L-Val-OH 3c
IAA-L-Trp-OH 3d
IAA-L-Phe-OH 3e
IAA-L-Tyr-OH 3f
IAA-L-Met-OH 3g
IAA-L-Ser-OH 3h
IAA-L-Asp(OH)-OH 3i
IAA-L-Glu(OH)-OH 3j
1
0
Met, Asp and Glu are characterized here for the first time. For the removal of the
methyl ester group, three different solvents were tested. IAA-amino acid methyl ester
conjugate was dissolved in dioxane, tetrahydrofuran (THF) or methanol (MeOH).
Sodium hydroxide was added and the reactions progression was monitored by TLC.
Dioxane and THF proved ineffective, since the saponification was not completed, while
cleavage of methyl ester group with sodium hydroxide 2 M in MeOH afforded the cor-
responded IAA-amino acid conjugates with free carboxylic group in excellent yields,
from 92% to 99% in 90 min (Table 2). Removal of the ester group in the presence of
NaOH is a conventional method for de-protection used in peptide chemistry and is
[
22]
known to be free of racemization.
1
Indole amide derivatives can be easily identified from their H NMR spectra in
DMDO-d , by the characteristic signals of acidic NH proton of indole at about
6
10.86 ppm, the five aromatic protons of indole moiety between 7.60 and 6.80 ppm and
the signal of the NHCO amide bond proton between 8.30 and 8.20 ppm. The method
described herein provides the IAA-amino acid conjugates with free carboxylic group in
[
18]
higher yields than the previously described benzotriazolyl esters method.
Full charac-
1
terization data for all synthesized compounds include optical rotation, IR, H NMR and
C NMR spectra and HRMS in negative ion mode and are presented in details in the
1
3
Supplementary material.
Conclusion
In conclusion, a simple and efficient coupling method involving WSCIÁHCl and
HOBt has been successfully applied in the synthesis of IAA-amino acid conjugates
either in the free carboxylic form and methyl ester form in excellent yields. This
method may also be applied for the synthesis of auxin conjugates with a variety of
amino acids.

4
P.-K. REVELOU AND V. CONSTANTINOU-KOKOTOU
Experimental
General procedure for the synthesis of IAA-amino acid methyl ester conjugates
ꢀ
A stirred solution of IAA (1.0 mmol) in dry CH Cl (10.0 ml) was cooled at 0 C Et N
2
2
3
(
(
0.3 ml, 2.2 mmol), 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride
WSCIÁHCl) (0.21 g, 1.1 mmol) and 1-hydroxybenzotriazole (HOBt) (0.14 g, 1.0 mmol)
were subsequently added. The amino acid methyl ester (1.0 mmol) was added and the
reaction mixture was stirred for 1 h at 0 C and then overnight at room temperature.
ꢀ
The reaction progress was monitored by TLC. After the completion of the reaction, the
solvent was evaporated under reduced pressure and ethyl acetate (EtOAc) (20 ml) was
added. The EtOAc layer was washed consecutively with brine, 5% citric acid, brine, 5%
NaHCO , brine and dried over Na SO . The organic solvent was evaporated under
3
2
4
reduced pressure and the product was purified by column chromatography using a mix-
ꢀ
ture of EtOAc–petroleum ether (bp 40–60 C) (7:3) as eluent.
General procedure for the cleavage of methyl ester group
To a stirring solution of IAA-amino acid methyl ester conjugate (0.35 mmol) in MeOH
(2.0 ml) 2 N NaOH (0.38 ml, 0.77 mmol) was added, at room temperature. The reaction
progress was monitored by TLC. The mixture was stirred for 90 min at room tempera-
ture. After the completion of the reaction, the mixture was extracted with Et O, acidi-
2
fied with 2 N HCl and extracted with EtOAc. The organic layer was washed with brine
and dried over Na SO .The organic solvent was evaporated under reduced pressure to
2
4
obtain pure product.
Disclosure statement
The authors declare that they have no conflict of interest. This research did not receive any spe-
cific grant from funding agencies in the public, commercial, or not-for-profit sectors.
References
[
1] Teale, W. D.; Paponov, I. A.; Palme, K. Auxin in Action: Signalling, Transport and the
Control of Plant Growth and Development. Nat. Rev. Mol. Cell Biol. 2006, 7, 847–859.
doi: 10.1038/nrm2020
[
2] Ludwig-M u€ ller, J. Auxin Conjugates: Their Role for Plant Development and in the
Evolution of Land Plants. J. Exp. Bot. 2011, 62, 1757–1773. doi: 10.1093/jxb/erq412
3] Ljung, K.; Hull, A. K.; Kowalczyk, M.; Marchant, A.; Celenza, J.; Cohen, J. D.; Sandberg,
G. Biosynthesis, Conjugation, Catabolism and Homeostasis of Indole-3-Acetic Acid in
Arabidopsis thaliana. Plant Mol. Biol. 2002, 49, 249–272. doi: 10.1023/A:1015298812300
4] P ꢀe n ꢀc ꢁı k, A.; Rol ꢀc ꢁı k, J.; Nov ꢁa k, O.; Magnus, V.; Bart ꢁa k, P.; Bucht ꢁı k, R.; Salopek-Sondi, B.;
Strnada, M. Isolation of Novel Indole-3-Acetic Acid Conjugates by Immunoaffinity
Extraction. Talanta 2009, 80, 651–655. doi: 10.1016/j.talanta.2009.07.043
[
[
[
5] Sohlberg, J. J.; Myrenås, M.; Kuusk, S.; Lagercrantz, U.; Kowalczyk, M.; Sandberg, G.;
Sundberg, E. STY1 Regulates Auxin Homeostasis and Affects Apical-Basal Patterning of
the Arabidopsis gynoecium. Plant J. 2006, 47, 112–123. doi: 10.1111/j.1365-313X.2006.
02775.x

V
R
SYNTHETIC COMMUNICATIONS
5
[
6] Yu, P.; Hegeman, A. D.; Cohen, J. D. A Facile Means for the Identification of Indolic
Compounds from Plant Tissues. Plant J. 2014, 79, 1065–1075. doi: 10.1111/tpj.12607
7] Chisnell, J. R.; Bandurski, R. S. Translocation of Radiolabeled Indole-3-Acetic Acid and
Indole-3-Acetyl-Myo-Inositol from Kernel to Shoot of Zea mays L. Plant Physiol. 1988,
[
86, 79–84. doi: 10.1104/pp.86.1.79
€
[
8] Ostin, A.; Kowalyczk, M.; Bhalerao, R. P.; Sandberg, G. Metabolism of Indole-3-Acetic
Acid in Arabidopsis. Plant Physiol. 1998, 118, 285–296. doi: 10.1104/pp.118.1.285
9] Cohen, J. D.; Gray, W. M. Auxin Metabolism and Signaling. In Annual Plant Reviews:
Plant Hormone Signaling, 1st ed.; Hedden, P., Thomas, S. G., Eds.; Blackwell Publishing
Ltd: Oxford, 2006; Vol. 24, pp 37–66.
[
[
10] Cohen, J. D.; Bandurski, R. S. The Bound Auxins: Protection of Indole-3-Acetic Acid
from Peroxidase-Catalyzed Oxidation. Planta 1978, 139, 203–208. doi: 10.1007/
BF00388631
[
11] Mollan, R. C.; Donnelly, D. M. X.; Harmey, M. A. Synthesis of Indole-3-Acetylaspartic
Acid. Phytochemistry 1972, 11, 1485–1488. doi: 10.1016/S0031-9422(00)90107-9
12] Toma ꢀs i ꢁc , A.; Berto ꢀs a, B.; Tomi ꢁc , S.; So ꢀs ki ꢁc , M.; Magnus, V. Binding Behavior of Amino
ꢀ
[
Acid Conjugates of Indole-3-Acetic Acid to Immobilized Human Serum Albumin.
J. Chromatogr. A 2007, 1154, 240–249. doi: 10.1016/j.chroma.2007.03.095
[
[
[
13] Weller, L. E.; Sell, H. M. Synthesis of 3-Indoleacetamides. J. Org. Chem. 1958, 23,
1776–1777. doi: 10.1021/jo01105a602
14] Good, N. E. The Synthesis of Indole-3-acetyl-D,L-Aspartic Acid and Related Compounds.
Can. J. Chem. 1956, 34, 1356–1358. doi: 10.1139/v56-172
15] Napoli, L. D.; Evidente, A.; Piccialli, G.; Santacroce, C.; Iacobellis, N. S.; Sisto, A.
Synthesis of N-e-(Indole-3-Acetyl)-l-Lysine. Phytochemistry 1993, 33, 13–16. doi: 10.1016/
0031-9422(93)85388-8
[
16] LeClere, S.; Tellez, R.; Rampey, R. A.; Matsuda, S. R. T.; Bartel, B. Characterization of a
Family of IAA-Amino Acid Conjugate Hydrolases from Arabidopsis. J. Biol. Chem. 2002,
2
77, 20446–20452. doi: 10.1074/jbc.M111955200
[
17] Ilic, N.; Magnus, V.; Ostin, A.; Sandberg, G. Stable-Isotope Labeled Metabolites of the
Phytohormone, Indole-3-Acetic Acid. J. Label. Compd. Radiopharm. 1997, 39, 433–440.
doi: 10.1002/(SICI)1099-1344(199705)39:5 < 433::AID-JLCR987 > 3.0.CO;2-H
[
18] Katritzky, A. R.; Khelashvili, L.; Munawar, M. A. Syntheses of IAA- and IPA-Amino Acid
Conjugates. J. Org. Chem. 2008, 73, 9171–9173. doi: 10.1021/jo8017796
[
19] Smyrniotou, A.; Kokotou, M. G.; Mouchlis, V. D.; Barbayianni, E.; Kokotos, G.; Dennis,
E. A.; Constantinou-Kokotou, V. 2-Oxoamides Based on Dipeptides as Selective Calcium-
Independent Phospholipase A2 Inhibitors. Bioorg. Med. Chem. 2017, 25, 926–940. doi:
10.1016/j.bmc.2016.12.007
[
[
[
20] Fotaras, S.; Kokotos, C. G.; Tsandi, E.; Kokotos, G. Prolinamides Bearing Thiourea
Groups as Catalysts for Asymmetric Aldol Reactions. Eur. J. Org. Chem. 2011, 2011,
1310–1317. doi: 10.1002/ejoc.201001417
21] Vasilakaki, S.; Pastukhov, O.; Mavromoustakos, T.; Huwiler, A.; Kokotos, G. Small
Peptides Able to Suppress Prostaglandin E2 Generation in Renal Mesangial Cells.
Molecules 2018, 23, 158. doi: 10.3390/molecules23010158
22] Psarra, A.; Kokotos, C. G.; Moutevelis-Minakakis, P. tert-Butyl Esters of Tripeptides Based
on Pro-Phe as Organocatalysts for the Asymmetric Aldol Reaction in Aqueous or Organic
Medium. Tetrahedron 2014, 70, 608–615. doi: 10.1016/j.tet.2013.12.007