First synthesis of 3-O-methyl-scyllo-inosamine, a natural product which favors the Rhizobium-Leguminosae symbiosis

Article abstract of DOI:10.1016/j.tetlet.2003.12.048

Rhizopine, extracted in small amounts from nodules induced by Sinorhizobium meliloti strain L5-30 infection of alfalfa, was proved to possess the 3-O-methyl-scyllo-inosamine gross structure.

Full text of DOI:10.1016/j.tetlet.2003.12.048

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 1461–1463
First synthesis of 3-O-methyl-scyllo-inosamine, a natural product
which favors the Rhizobium–Leguminosae symbiosis
Alain Krief,a,* Willy Dumont,a Denis Billen,a Jean-Jacques Letesson,b Pascal Lestrate,b
a
Peter J. Murphyc andDamien Lacroix
aLaboratoire de Chimie Organique de Synthꢀese, Facultꢁes Universitaires N.-D de la Paix, 61 rue de Bruxelles, Namur B-5000, Belgium
bLaboratoire d’Immunologie et de Microbiologie, Unitꢁe de Recherche en Biologie Molꢀeculaire Facultꢁes Universitaires, N.-D de la Paix,
61 rue de Bruxelles, Namur B-5000, Belgium
cDepartment of Applied and Molecular Ecology, Waite Campus, University of Adelaide, PB 1 Glen Osmond, South Australia 5064,
Australia
Received16 July 2003; revised24 November 2003; accepted9 December 2003
Abstract—Rhizopine, extracted in small amounts from nodules induced by Sinorhizobium meliloti strain L5-30 infection of alfalfa,
was provedto possess the 3- O-methyl-scyllo-inosamine gross structure.
Ó 2003 Publishedby Elsevier Ltd.
Members of the bacterial genera Rhizobium, Azorhizo-
bium, and Sinorhizobium form symbiotic associations
with the plant family Leguminosae allowing the con-
version of atmospheric nitrogen into an ammonium salt
that the plant can utilize (symbiotic nitrogen fixation).1
This process is important in agriculture since it favors
the growth of Leguminosae in soil with low bioactive
nitrogen content.
meliloti strain L5-30 (S. meliloti) infection of alfalfa
(3.3 ng/g fresh weight, 21 days after inoculation).2 It was
detected by high voltage electrophoresis on Whatman
4
3MM paper stainedwith silver nitrate. Furthermore it
has been suggested4 that it possesses the L-3-O-methyl-
scyllo-inosamine structure (3-O-MSI) 1 but this has not
yet been firmly established(Fig. 1).
The structure determination for rhizopine relies on the
GC–MS of a poorly purifiedextract, which was sub-
jectedto extensive acetylation (see below). It has a
Both partners benefit from the interaction, which
involves the invasion of the root hair by the Rhizobia,
which enters into the plant, differentiates into bacteroids
and leads to nitrogen-fixing nodules.2
2
molecular weight of 403, but the molecular ion was not
observed5 andhas been characterizedby the presence of
peaks at m=z 223 (loss of three acetic acidmolecules),
181 (most abundant base peak, loss of one ketene
group), 136 (loss of another ketene group). These ions
along with several others, including that at m=z 167,
Rhizopines are opine-like compounds produced only by
the bacteroid in the nodule. They are neither detected in
other parts of the plant nor in free-living bacteria.3 They
are known to favor the growth of free-living Rhizobia,
2
have been usedas a fingerprint to identify rhizopine.
which are not only able to catabolize them but also use
1;3;4
them as a source of carbon andnitrogen.
Genes
We disclose the first synthesis of 3-O-methyl-scyllo-
inosamine 1 andits fully acetylatedderivative whose
involvedin rhizopine catabolism ( moc genes) andsyn-
thesis (mos genes) have been recently identified and
characterized.1;4
One of the rhizopines (rhizopine) has been extractedin
small amounts from nodules induced by Sinorhizobium
Me
O
HO
H2N
OH
1
OH
HO
* Corresponding author. Tel.: +32-81-724539; fax: +32-81-724536;
e-mail: alain.krief@fundp.ac.be
Figure 1. Structure of rhizopine: 3-O-methyl-scyllo-inosamine.
0040-4039/$ - see front matter Ó 2003 Publishedby Elsevier Ltd.
doi:10.1016/j.tetlet.2003.12.048

1462
A. Krief et al. / Tetrahedron Letters 45 (2004) 1461–1463
physical properties favorably compare with those of
2
authentic samples of rhizopine isolatedfrom alfalfa.
group with the requiredaxial orientation. This was
effectively achievedin two successive steps involving
oxidation of the equatorial hydroxyl group to a ketone
followedby its reductive amination.
The synthetic strategy developed is reminiscent to that
6
of Kirby for a relatedcompound. It uses commercially
available myo-inositol 2 as starting material andtakes
advantage of the conformational rigidity of the corre-
sponding orthoformate 3 in which two hydroxyl groups
are axially orientedwhereas the remaining one is equa-
torial [2 mol equiv HC(OEt)3, Amberlyst 15, DMF,
100 °C, 3 h, 90% yield, Scheme 1].7
The first step, performedwith tetrapropylammonium
perruthenate (TPAP) in the presence of N-methyl mor-
pholine N-oxide (NMO) surprisingly gives, beside the
desired ketone 7, the epimeric alcohol 6 with the
hydroxyl group in an axial position (80/20 ratio).8
Reaction of the crude mixture with ammonium acetate
and sodium cyanoborohydride6;9 leads to the required
amine 8, which was separatedfrom the accompanying
alcohol 6 by taking advantage of the insolubility of its
ammonium salt in ether (Scheme 1, step viii). Pure
amine 8 was finally obtainedin 45% yieldafter a basic
workup.
Alkylations, sequentially carriedout with soiudm
hydride in DMF and methyl iodide then with benzyl
bromide led to the monomethyl–mono benzyl derivative
5 as well as some dibenzylated compound from which it
couldbe easily separated(Scheme 1). The selective
methylation of one of the axially orientedhyrdoxyl
group has been attributedto the easier formation of the
corresponding alkoxide due to intramolecular stabil-
ization from the remaining alcohol.6 The choice of the
sequence is crucial since reversing the order of alkylation
dramatically lowers the overall yield due to the com-
peting formation of the dibenzyl ether in addition to the
desired monobenzyl derivative.
The presence of the axial alcohol 6 as well as the
ketone 7 was unexpected. Its formation was also
observed when the oxidation was carried out under
Swern conditions.5 Furthermore we have been unable
to oxidize 6 under conditions, which allow the oxi-
dation of its epimer 5 andnot even when using PDC
or PCC instead.
It was decided to take advantage of the better accessi-
bility of reagents from the b-face to introduce the amino
The structure andstereochemistry of 8 has been ascer-
tainedon the basis of X-ray crystallography of its 3,5-
dinitrobenzoate 12.10
OH
O
O
3-O-Methyl-scyllo-inosamine hydrochloride 10 was
synthesizedfrom 8 by (i) debenzylation using sodium in
liquidammonia [Scheme 1, step x, 86% yieldof 9]1 and
(ii) acid hydrolysis of the tripode [Scheme 1, step xi, 90%
yieldof 10].
O
O
O
O
HO
HO
(i)
(iv, v)
48%
(ii, iii)
63%
HO
OH
OH
HO
90%
OH
O
Me
OH
OH
OH
2
3
4
O
O
O
O
O
6
O
6
The electrophoretic mobilities of natural rhizopine 1
extracted from nodules induced by S. meliloti strain L5-
30 andour sample of racemic-3- O-methyl-scyllo-inos-
amine are similar.
O
Me
O
O
O
Me
Ph
O
Ph
OH
O
O
(vii)
OH
(vi)
+
HO
+
6/7: 4/1
45%
O
O
O
O
O
Me
Ph
O
O
O
7
8
5
O
Me
O
Me
O
Ph
Ph
Both samples remain unaffectedwhen incubatedwith
S. meliloti Rm1021 (moc)) strain missing the gene
coding for rhizopine catabolism and are catabolized
when incubatedwith S. meliloti L5-30 (moc+) strains. It
is surprising that our racemic sample was fully catabo-
lizedby ( moc+) type Rhizobium, which wouldbe
expectedto react with only one of the two antipodes.
O
NH2
O
OAc OAc
OAc
OH
OH
OH
O
O
O
(xii)
45%
(x)
(xi)
90%
Cl
O
Me
86%
O
Me
O
Me
AcHN
OAc
NH3 OH
NH2 OH
O
O
O
(viii)
(ix)
9
10
11
O
Me
O
O
Ph
NH2
O
Furthermore, not only are the IR spectra of both the
synthetic product and the natural rhizopine superim-
posable (Fig. 2) but in addition the GC mass spectra of
the fully acetylated3- O-methyl-scyllo-inosamine 11
(Fig. 3) exhibits strong similarities with that of an
O
O2N
8
12
O
Me
Ph
NH
O
O
O2N
2
acetylatedauthentic sample recently published.
Scheme 1. Synthesis of racemic 3-O-methyl-scyllo-inosamine from
myo-inositol. (i) 2 mol equiv HC(OEt)3, Amberlyst 15, DMF, 100 °C,
3 h; (ii) NaH, DMF, 25 °C, 1 h; (iii) MeI, 25 °C, 15 h; (iv) NaH, DMF,
25 °C, 2 h; (v) BnBr, 25 °C, 15 h; (vi) 1.5 equiv NMO, 0.2 equiv TPAP,
CH2Cl2, molecular sieves; (vii) 10 equiv NH4OAc, 1.05 equiv
NaBH3CN, 20 °C, 72 h; (viii) HCl, ether; (ix) 1 M NaOH; (x) 4 equiv
Na, liq. NH3, t-BuOH, THF, )78 to 20 °C, 18 h; (xi) HCl (g), meth-
anol, 20 °C, 3 h; (xii) Ac2O, AcONa, 20 °C, 1.5 h.
The data described above strongly support the struc-
tural identification of 3-O-methyl-scyllo-inosamine 1 as
rhizopine. We are planning the synthesis of scalemic-3-
O-methyl-scyllo-inosamine to understand its biological
activity andto determine the absolute stereochemistry of
rhizopine.

A. Krief et al. / Tetrahedron Letters 45 (2004) 1461–1463
1463
Figure 2. IR spectra of rac-3-O-methyl-scyllo-inosamine andrhizopine.
ꢁ
ꢁ
moleculaire, Universite dÕOrsay, (France) for his valu-
able suggestions.
References and notes
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Rhizobium symbiosis. In Molecular Signals in Plant-
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€
7. (a) Baudin, G.; Glanzer, B. I.; Swaminathan, K. S.;
Figure 3. Mass spectra of natural rhizopine (A) andof our sample of
racemic 3-O-methyl-scyllo-inosamine (B).
Vasella, A. Helv. Chim. Acta 1988, 71, 1367–1378; (b) Lee,
H. W.; Kishi, Y. J. Org. Chem. 1985, 50, 4402–4404.
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10. The X-ray structure of 12 will be publishedelsewhere.
Acknowledgements
A.K. thanks Dr. Boris Vauzeilles, Laboratoire de
ꢀ ꢁ
Synthese des Biomolecules, UMR 8614-Glycochimie