Simple synthesis of nodulation-factor analogues exhibiting high affinity towards a specific binding protein

Article abstract of DOI:10.1002/anie.200460275

A "Nod" in the right direction: Aromatic analogues of the lipooligosaccharidic nodulation (Nod) factors involved in the symbiosis of Sinorhizobium meliloti and legumes are easily assembled from very simple precursors by combining biotechnological and chem

Full text of DOI:10.1002/anie.200460275

Communications
strain and define the high degree of specificity in the legume–
rhizobia interaction. One of the most studied models is the
symbiosis of Sinorhizobium meliloti and alfalfa (Medicago
sativa) in which the major bacterial signal molecule 1
(Scheme 1) contains four d-glucosamine residues with a 6-
Scheme 1. Structures of the major nodulation factors produced by
Sinorhizobium meliloti.
O-acetyl group and an N-(2E,9Z)-hexadecadienoyl side chain
at the nonreducing residue and an essential 6-O-sulfate group
at the reducing unit which is the main determinant of the host
specificity in M. sativa.[2]
Glycolipid Mimics
Simple Synthesis of Nodulation-Factor Analogues
Although highly active, signal molecule 2 is approximately
ten times less active than the acetylated derivative 1 in
morphogenic activity. However, since it is more stable, 2 is
more suitable for biological studies. Structure–function rela-
tionship studies have shown that the correct nature of the
lipid chain, in totally synthetic molecules,[3] is essential for
triggering an optimal morphogenic activity in M. sativa
roots.[4] Moreover, the length and the structure of the lipid
chain play a role in the high-affinity binding of LCOs to a Nod
factor binding site (NFBS2) characterized in cell suspension
cultures of legumes.[5–7]
As a step forward in the search for modified molecules for
the study of the Nod factor perception mechanisms by the
plant, we now report a simple synthesis of analogues by using
metabolically engineered bacterial cells, a method that avoids
the complexity of total synthetic approaches from N-acetyl-d-
glucosamine monomers.[3,8] We further demonstrate that the
incorporation of a substituted benzamide (see B in Scheme 2)
provides new analogues able to interact with a putative LCO
receptor at nanomolar concentrations.
Exhibiting High Affinity towards a Specific
Binding Protein**
Nathalie Grenouillat, Boris Vauzeilles,
Jean-Jacques Bono, Eric Samain, and
Jean-Marie Beau*
Rhizobial glycolipids (lipochitooligosaccharides (LCOs), also
called nodulation (Nod) factors) are important signaling
molecules involved in the establishment of symbiosis with
legumes.[1] This association results in the formation of
rhizobia-infected nodules on plant roots in which atmospheric
nitrogen is reduced to ammonia for the benefit of the plant.
These nodulation factors represent a large family of lipo-
oligosaccharides consisting of a chitin fragment (three–five N-
acetyl-d-glucosamine units) with an N-acyl chain attached to
the nonreducing unit and a variety of additional substi-
tuents.[1a] The variations are characteristic of each bacterial
[*] N. Grenouillat, Dr. B. Vauzeilles, Prof. J.-M. Beau
UniversitØ Paris-Sud
Laboratoire de Synth›se de BiomolØcules
associØ au CNRS, Institut de Chimie MolØculaire et des MatØriaux
91405 Orsay Cedex (France)
Fax: (+33)1-6985-3715
E-mail: jmbeau@icmo.u-psud.fr
Scheme 2. An alkoxybenzamide (X=O) or benzylamine (X=H2)
structure B can mimic the natural a,b-unsaturated amide A.
Dr. J.-J. Bono
Pôle de Biotechnologie VØgØtale
UMR 5546 CNRS–UniversitØ Paul Sabatier
24, Chemin de Borde Rouge, BP 17 Auzeville
31326 Castanet-Tolosan (France)
The aromatic motif was selected because a) it leads to
benzamides that are potentially more stable chemically and
metabolically than the natural conjugated amides, b) it makes
it possible to introduce a photoactivatable group at a strategic
position, and c) it is readily prepared from commercial
precursors. Although replacement of unsaturated bonds in
lipids by aromatic derivatives is frequently used to produce
molecular probes and agonists or antagonists of bioactive
molecules, this substitution combining an aromatic motif and
Dr. E. Samain
Centre de Recherches sur les MacromolØcules VØgØtales
UPR CNRS 5301, BP 53
38041 Grenoble Cedex 9 (France)
[**] We are grateful to Rhone-Poulenc SA (contract no. 041313.00),
Aventis CropScience SA, and Bayer CropScience SA for the financial
support of this study.
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DOI: 10.1002/anie.200460275
Angew. Chem. Int. Ed. 2004, 43, 4644 –4646

Angewandte
Chemie
a heteroatom to mimic a carbon–carbon double bond and a
methylene group in the acyclic system has, to our knowledge,
never been described.[9]
We started our synthesis by preparing the unsaturated
fatty acid analogues 6, 9, and 10 as outlined in Scheme 3.
Scheme 3. Synthesis of the side-chain precursors. a) BuLi, HMPA (1 equiv),
THF, ꢀ788C then reflux, 4 h, 80%; b) Lindlar catalyst, quinoline, cyclohex-
ane, H2, 258C, 7 h, 94%; c) NaI (3 equiv), acetone, reflux, 24 h, 89%; d) 4
(1.1 equiv), K2CO3 (1.1 equiv), DMF, 908C, 4 h, 76%; e) NaOH (2 equiv),
MeOH, reflux, overnight, 96%; f) oxalyl chloride, cat. DMF, CH2Cl2, 258C,
2 h, 99%; g) LiAlH4 (2 equiv), Et2O, RT, 1.5 h, 99%; h) PCC (2 equiv),
CH2Cl2, reflux, 1 h, 99%; i) as (d), 7 h, 79%; j) as (e), 98%; k) as (f), 99%;
l) as (d), 8 h, 66%; m) as (e), 24 h, 99%; n) as (f), 99%. DMF= N,N-di-
methylformamide, HMPA=hexamethylphosphoramide, PCC=pyridinium
chlorochromate.
Scheme 4. Synthesis of nodulation factor analogues (for conditions,
see text.)
The synthetic analogues 12–15 were tested, and
compared with the NodSm-IV(S) molecule (2), for
their ability to interact with the binding protein NFBS2
associated with a membrane preparation of M. varia.
The results (Table 1) show that, in comparison with the
Coupling of lithiated 1-octyne with commercially available 1-
bromo-3-chloropropane (3), followed by a Lindlar reduction
and a Cl/I exchange gave the expected unsaturated iodide 4.
Alkylation of methyl 3-hydroxybenzoate (5) with iodide 4
provided ester 6, which could be transformed into acid
chloride 7 or aldehyde 8. The p- and o-isomeric acid chlorides
9 and 10 were also prepared by following the same synthetic
sequence from the corresponding p- and o-substituted
phenols.
Table 1: Affinity of analogues 12–15 for binding site NFBS2, as measured
by competitive inhibition of [35S]-NodSm-IV(S) binding, and the
chromatographic behavior of the analogues.
Compound
Ki [nm][a]
tR [min][b]
2
20 (ꢁ5%)
35 (ꢁ18%)
28 (ꢁ7%)
77 (ꢁ19%)
22 (ꢁ13%)
17.2
16.5
17.9
13.7
20.5
12
13
14
15
The carbohydrate segment, the sulfated tetramer 11
(Scheme 4), was easily prepared by cultivating, at high cell
density, recombinant Escherichia coli strains coexpressing the
nodBC genes (encoding the chitooligosaccharide synthase
and the chitooligosaccharide N-deacetylase) and the nodH
gene (encoding the chitooligosaccharide sulfotransferase)
from Sinorhizobium meliloti.[10] Glycerol was provided as
the carbon source.
The oligosaccharide recovered from the cell extracts by
using the published procedure[10b] contained about 25% of the
pentamer and was further purified by column chromatogra-
phy on silica gel. N-Acylation of tetrasaccharide 11 with acid
chlorides 7, 9, and 10 was performed in DMF containing water
and provided the corresponding benzamides 12–14 in 33, 40,
and 48% yields, respectively (60% conversion). Benzyl
amine 15 and its N-acetyl derivative 16 were also prepared
by reductive alkylation of amine 11 with aldehyde 8.
[a] Ki =inhibition constant. This is a measure of the affinity for NFBS2.
[b] tR =Retention times on a C18 HPLC column.
reference compound 2, the affinity for NFBS2 of analogues
12, 13 and 15 was high and in the same 20–30 nm range as the
natural signal molecule.
The slightly lower affinity exhibited for the ortho-sub-
stituted benzamide 14 (Ki = 77 nm) probably reflects a very
different local geometry close to the nonreducing-end sugar, a
geometry that positions the side chain in a different space
location. Moreover, compound 14 appeared less hydrophobic
than the other analogues, as assumed from its shorter
retention time when analyzed by reversed-phase chromatog-
raphy on a C18 column (Table 1). This observation is in
agreement with previous studies on the selectivity of NFBS2,
which showed that the overall hydrophobicity of the LCOs,
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Communications
Culimore, J.-J. Bono, Proc. Natl. Acad. Sci. USA 1999, 96, 4704;
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[7] By using a genetic approach, two genes involved in the
perception of Nod factors and encoding transmembrane-recep-
tor-like serine/threonine kinases with extracellular domains
containing modules potentially involved in the oligosaccharide
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Franken, P. Smit, J. Willemse, T. Bisseling, R. Geurts, Science
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Felle, Y. Umehara, M. Gronlund, S. Sato, Y. Nakamura, S.
Tabata, N. Sandal, J. Stougaard, Nature 2003, 425, 585; E. B.
Madsen, L. H. Madsen, S. Radutoiu, M. Olbryt, M. Rakwalska,
K. Szczyglowski, S. Sato, S. Kaneko, S. Tabata, N. Sandal, J.
Stougaard, Nature 2003, 425, 637.
[8] Synthesis of Nod factors from monomers: K. C. Nicolaou, N. J.
Bockovich, D. R. Carcanague, C. W. Hummel, L. F. Even, J. Am.
Chem. Soc. 1992, 114, 8701; S. Ikeshita, A. Sakamoto, Y.
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mainly due to the structure of the fatty acid chain, was
important for high-affinity binding.[6] The morphogenic
activity of these benzamide analogues is currently being
evaluated for potential agricultural applications, and bio-
logical details will be reported in the near future.[11]
Experimental Section
Preparation of 12: Sodium hydrogencarbonate (2 equiv) and a
solution of 7 in THF (0.82m, 1 equiv) were added to a solution of
tetrasaccharide 11 (17 mmol) in DMF/water (2.5:1, 350 mL). The
reaction mixture was warmed to 608C, then more acid chloride
solution (5 equiv) and sodium hydrogencarbonate (6 equiv) were
added in six portions over a period of 18 h. Flash chromatography
with ethyl acetate/methanol/water (7:2:1) as the eluent and deposi-
tion of the reaction residue at the top of the column in dichloro-
methane/methanol (5:1) gave 12 (6.5 mg, 33%). The purity of the
product was checked by elution at 1 mLminꢀ1 on a C18 HPLC column
with a linear gradient of 28!66% acetonitrile in 10 mm K2SO4
(pH 4.6) and detection at 210 nm; 1H NMR (CD3OD, 250 MHz):
d = 7.48–7.41 (m, 2H; ArH-2, ArH-6), 7.36 (dd, 1H, J5,6 = 7.8, J5,4
=
[9] Some examples of aromatic derivatives designed to resemble
unsaturated lipids: D. W. Snyder, P. R. Bernstein, Prostaglandins
1988, 35, 903; R. C. Larock, N. H. Lee, J. Org. Chem. 1991, 56,
6253; H. Hashizume, H. Ito, K. Yamada, H. Nagashima, M.
Kanao, H. Tomoda, T. Sunazuka, H. Kumagai, S. Omura, Chem.
Pharm. Bull. 1994, 42, 512; J. P. F. C. Rossi, J. M. Delfino, A. J.
Caride, H. N. Fernꢀndez, Biochemistry 1995, 34, 3802.
[10] a) E. Samain, S. Drouillard, A. Heyraud, H. Driguez, R. A.
Geremia, Carbohydr. Res. 1997, 302, 35; b) E. Samain, V.
Chazalet, R. A. Geremia, J. Biotechnol. 1999, 72, 33.
8.1 Hz; ArH-5), 7.07 (ddd, 1H, J4,2 ꢂ J4,6 = 1.4 Hz; ArH-4), 5.48–5.33
(m, 2H; CH CH), 5.03 (d, 0.8H, J1a,2 = 3.2 Hz; H1aI), 4.68/4.59/4.50
=
(3d, 3H, J1,2 = 8.7 Hz; H1II,III,IV), 4.56 (d, 0.2H, J1b,2 = 7.7 Hz; H1bI),
ꢀ
4.25–3.30 (m, 26H; CH2 OAr, other carbohydrate H atoms), 2.25 (td,
ꢀ
=
ꢀ
ꢀ
2H, J = 6.7, 6.2 Hz; CH2 CH CH CH2), 2.08–1.94 (m, 2H; CH2
=
ꢀ
CH CH CH2), 2.03/1.99/1.96 (3 s, 9H; 3 ” CH3CO), 1.83 (tt, 2H, J =
6.7 Hz; ArOCH2CH2CH2), 1.35–1.20 (m, 8H; 4 ” CH2), 0.88 ppm (t,
3H, J = 7.0 Hz; CH3); MS (ES): m/z: 1139.4 [MꢀNa]ꢀ.
Binding experiments were performed as previously described.[6]
Briefly, microsomal fraction protein (60 mg) was incubated in the
presence of 0.8 nm [35S]-NodSm-IV(S) in a binding buffer (total
volume 200 mL) with increasing amounts of the different analogues in
concentrations ranging between 0.25 nm and 1 mm. The nonspecific
binding component was determined in the presence of 2 mm NodSm-
IV(S). Incubations were performed for 1 h at 08C in 96-well
microtiter plates (Nunc), and the steps that followed were as
previously described. Binding data were analyzed by the RADLIG
software, Version 4 (Biosoft, Cambridge, UK).
[11] F. Maillet, J. DØnariØ, personal communication.
Received: April 8, 2004
Keywords: aromatic substitution · glycolipids · lipid analogues ·
.
nitrogen fixation · oligosaccharides
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