Chemical synthesis of scyllo-inosamine and catabolism studies in Sinorhizobium meliloti

Article abstract of DOI:10.1016/j.bmc.2008.06.040

Rhizopines such as scyllo-inosamine (SIA) and l-3-O-methyl-scyllo-inosamine (3-O-MSI) play an intricate role as nutritional mediators during the establishment of the symbiotic relationship between legumes and rhizobia. The mechanism of action is not well

Full text of DOI:10.1016/j.bmc.2008.06.040

Bioorganic & Medicinal Chemistry 16 (2008) 7838–7842
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Chemical synthesis of scyllo-inosamine and catabolism studies
in Sinorhizobium meliloti
a
b
b
Elke Schoffers a, , Sing R. Gurung , Petra R. A. Kohler , Silvia Rossbach
*
a Department of Chemistry, Western Michigan University, 3425 Wood Hall, Kalamazoo, MI 49008-5413, USA
b Department of Biological Sciences, Western Michigan University, Kalamazoo, MI 49008-5410, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Rhizopines such as scyllo-inosamine (SIA) and
L
-3-O-methyl-scyllo-inosamine (3-O-MSI) play an intricate
Received 22 April 2008
Revised 14 June 2008
Accepted 20 June 2008
Available online 25 June 2008
role as nutritional mediators during the establishment of the symbiotic relationship between legumes
and rhizobia. The mechanism of action is not well understood. One challenge is the availability of rhizo-
pines, which occur in only minute amounts in plant nodules. We herewith report an efficient synthesis of
scyllo-inosamine and its biochemical activity in specific bacteria. SIA was prepared in 7 steps and 32%
overall yield from readily available myo-inositol. The chemically synthesized SIA was tested to determine
whether it can serve as sole carbon and nitrogen source for Sinorhizobium meliloti wild-type strain L5-30
and for strains carrying mutations in the rhizopine degradation (moc) genes. The analysis of the pheno-
type of the mutant strains revealed that the moc genes previously shown to be essential for the break-
down of the rhizopines isolated from root nodules are also essential for the utilization of the
chemically synthesized SIA.
Keywords:
scyllo-Inosamine
Rhizopine
Sinorhizobium meliloti
Published by Elsevier Ltd.
1. Introduction
symbiotic association between bacteria and legume plants such
as alfalfa, pea, and bean. Bacteria of the genus Rhizobium and Sino-
The cyclitol myo-inositol (MI, 1, Fig. 1) and its derivatives play
important roles in the cellular metabolism of animals, plants, and
microorganisms.1 For example, in biological membranes, phospho-
inositides serve either as precursors for the second messenger ino-
sitol 1,4,5-triphosphate or act directly as ligands for protein
signaling.2 In plant cells, myo-inositol serves as precursor for cell
wall carbohydrates and has been found to be conjugated with
the phytohormone auxine.1 Various methyl ethers of inositols are
commonly found in tissues of different plant species and have been
shown to specifically accumulate under different ecological condi-
tions, such as water stress. Inositol also serves as phosphate stor-
age compound when esterified with orthophosphate yielding
phytic acid.1
In microbiology, myo-inositol is of interest because it can serve
as sole carbon source for many bacteria, including Aerobacter aer-
ogenes (now Klebsiella pneumoniae),3 Salmonella,4 Bacillus subtilis,5
and Rhizobium species.6–8 Moreover, diaminocyclitols are compo-
nents of aminoglycoside antibiotics, such as streptomycin, hygro-
mycin B, and spectinomycin.9
rhizobium colonize the roots of legume plants and induce the for-
mation of root nodules. Once inside, bacterial nitrogenase fixes
atmospheric nitrogen which benefits the plant as a nutrient. SIA
and 3-O-MSI were first isolated from the nodules of alfalfa plants
(Medicago sativa) that were induced by one particular Sinorhizo-
bium meliloti strain, L5-30.10 It is believed that SIA and 3-O-MSI
can serve as proprietary growth substrate for the rhizobial strain
inducing the nodules. In analogy to agrobacterial opines, these
chemicals have been named ‘opine-like compounds’ or ‘rhizo-
pines’,10–12 but spectral analysis for the proposed structures of
the compounds extracted from nodules was not given in the origi-
nal literature. Rhizopines have been implicated to give a competi-
tive advantage in nodulation for strains that are able to synthesize
and catabolize rhizopines.13–16
Bacterial genes required for the synthesis (mos) and catabolism
(moc) of rhizopines have been cloned and sequenced.11,17–19 Some
of the rhizopine degradation genes show homology to myo-inositol
degradation genes, but the exact function of gene products en-
coded by mos and moc genes remains unknown.7,19 This is in part
due to the limited availability of rhizopines.
We have a long standing interest in two specific inosamine
derivatives, scyllo-inosamine (SIA, 4a) and
inosamine (3-O-MSI, 5). Both compounds are relevant in the
L
-3-O-methyl-scyllo-
Since 1948, only three publications address the chemical syn-
thesis of scyllo-inosamine (SIA),20–22 while Yokoyama et al.24 used
an aminotransferase to afford unknown amounts of SIA. Several
investigators prepared N-acyl and hexaacetyl derivatives.20–23,25–29
The first synthesis of the 3-methoxyderivative (3-O-MSI) was
* Corresponding author. Tel.: +1 269 387 2265; fax: +1 269 387 2909.
E-mail address: elke.schoffers@wmich.edu (E. Schoffers).
0968-0896/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmc.2008.06.040

E. Schoffers et al. / Bioorg. Med. Chem. 16 (2008) 7838–7842
7839
R
1
R=OH myo-inositol (MI)
HO
HO
OH
OH
2a R=NH2 myo-inosamine, inosamine-SA
2b R=NHAc N-acetyl-myo-inosamine
OH
R1
3
R1=OH, R2=H, scyllo-inositol
4a R1=NH2, R2=H, scyllo-inosamine, SIA, inosamine-SB
4b R1=NHAc, N-acetyl-scyllo-inosamine
HO
HO
OH
4c R1=NH3Cl, R2=H, scyllo-inosamine hydrochloride
OR2
OH
5
R1=NH2, R2=CH3, 3-O-methyl-scyllo-inosamine, 3-O-MSI
Figure 1. scyllo-Inosamine and related derivatives.
reported in 2004.30 Unfortunately, spectroscopic evidence for these
inosamines has been sparse. Carter, who coined the term ‘inosa-
mine’ to describe ‘analogues of inositol’, utilized oxime and phen-
ylhydrazine intermediates (7, Scheme 1).21 They were derived
from scyllo-inosose (6) and reduced with Raney nickel to produce
an epimeric mixture of myo(2a)- and scyllo(4a)-inosamine in 70%
yield. Extraction and recrystallization methods allowed for the iso-
lation of pure SIA, but in only 21% yield. Carter et al. did not iden-
tify the configuration of the new C–N bond and arbitrarily named
their products ‘inosamine-SA’ and ‘inosamine-SB’. The stereochem-
istry of the latter as SIA was not assigned until Anderson and Lar-
dy20 carried out a catalytic, stereoselective reduction of the oxime
with sodium amalgam affording a mixture of both epimers as N-
acetyl derivatives (2b/4b, 27: 73) in 57% yield.31 After deprotection
and purification pure SIA was isolated as a hemihydrate, decreas-
ing the overall yield to 28% from the oxime. The stereochemistry
of ‘inosamine-SB’ was inferred through comparison with preferred
‘cis alcohols’ obtained from the reduction of scyllo-inosose with
hydrogen over platinum.32 The stereochemistry of the product
from this procedure was never established with modern analytical
methods, yet some biochemical studies using SIA relied on this
reduction.33,34
intermediates and the final product. Our approach entails the over-
all stereoselective conversion of the myo to the scyllo configuration.
The quantities obtained were sufficient to carry out in vitro cata-
bolic studies using different S. meliloti strains, including those with
transposon insertions in the rhizopine catabolic genes. We show
that the same moc genes that are essential for the breakdown of
the rhizopine isolated from root nodules are also essential for the
utilization of the chemically synthesized SIA.
2. Results and discussion
myo-Inositol (1) served as inexpensive starting material
(Scheme 3). Protection of the cis diol,35,36 subsequent benzyla-
tion,36 and acetonide removal36 furnished diol 9. Selective benzyla-
tion using dibutyltin oxide37,38 yielded alcohol 10, which was
converted to azide 11 via its triflate.39 Initial efforts entailed the
conversion of the azide to its amine 12 followed by treatment with
BBr3 and K2CO3. However, after testing a variety of catalytic hydro-
genation procedures, we optimized the reaction conditions. The
use of medium pressure hydrogen in combination with Pd or
Pd(OH)2 on carbon under acidic conditions provided a practical
one-step method for the reduction of the azide and the removal
of five benzyl groups to afford pure scyllo-inosamine as HCl salt
(4c) in 87% yield. (These hydrogenation conditions are also applica-
ble to amine 12.) A small amount of product (4c) was converted
into the hexaacetate using standard conditions. SIA and its acetate
were fully characterized via mp, 1H NMR, 13C NMR, IR, and EA.
In order to test the chemically synthesized scyllo-inosamine for
its biological efficacy, we used various S. meliloti Moc+ and Mocꢀ
wild-type and transposon-induced mutant strains for a catabolism
assay. The growth of the different strains in liquid, chemically de-
fined medium with 0.2% scyllo-inosamine as sole carbon (C) and
nitrogen (N) source, was determined by measuring the absorption
of all cultures at 600 nm. The Moc+ wild-type strain L5-30 grew to
an optical density of 1.4 with scyllo-inosamine as sole C- and N-
source, whereas, as expected, the Mocꢀ wild-type strain 1021 did
not grow at all (Fig. 2A). The same mutant strains that were re-
ported earlier as being unable to degrade the rhizopine purified
from nodules,7,19 namely the mocA (M20), mocB (M21), mocC
(M11), and mocR (M22, M23) mutants, were also not able to grow
in minimal medium with the chemically synthesized scyllo-inosa-
mine as sole C- and N-source (Fig. 2A). Mutant strains with inser-
tions in mocD (M24, M25), mocE (M30), and mocF (M32) grew to
almost the same final optical density as the wild type, whereas
A more recent report by Delgado and co-workers22 utilized
epoxide 8 as key intermediate to afford SIA in four steps via ring
opening with sodium azide (Scheme 2). The epoxide itself was ob-
tained in 9 steps from methyl-a-D-glucopyranoside; a Ferrier rear-
rangement converted the carbohydrate to a carbocycle. SIA was
prepared in 24% over 13 steps. The last step involved the deprotec-
tion of five benzyl groups with BCl3. Such reaction conditions are
problematic without subsequent purification because polar side
products are difficult to separate from the final inosamine.
We herewith describe a practical method that affords pure SIA
in 7 steps and 34% overall yield from myo-inositol. Moreover, we
provide relevant spectroscopic characterization data for the key
X
HO
HO
OH
OH
a or b
2a and 4a
or
2b and 4b
OH
6 X=O; 7a X=NOH; 7b X=NNHPh
Scheme 1. Reduction of oxime and phenylhydrazine intermediates. Reagents and
condition: (a) Na/Hg; (b) H2, Raney Ni, 100–150 atm.
NH2
OH
HO
HO
HO
OH
4 steps
76%
9 steps
O
O
BnO
HO
HO
BnO
H
OH
32%
HO
OBn
OH
OCH3
8
4a
Scheme 2. Synthesis of scyllo-inosamine from methyl-a-D-glucopyranoside.

7840
E. Schoffers et al. / Bioorg. Med. Chem. 16 (2008) 7838–7842
R
NH3Cl
OH
OH
OH
BnO
BnO
OBn
OBn
HO
HO
BnO
BnO
OH
BnO
BnO
OBn
OBn
a-c
d
e, f
h
1
OH
OBn
OBn
OH
OBn
OBn
9
10
11 R=N3
12 R=NH2
4c
g
Scheme 3. Synthesis of scyllo-inosamine from myo-inositol. Reagents and conditions: (a) 2,2-dimethoxypropane, p-TsOH, DMSO, NEt3, 74%; (b) BnBr, NaOH, 95%; (c) HCl,
CH3OH, 89%; (d) Bu2SnO, Bu4NI, BnBr, CH3CN, 83%; (e) Tf2O, pyr, CH2Cl2, 93%; (f) NaN3, DMF, 81%; (g) Ph3P, THF, H2O, 96%; (h) H2/Pd, MeOH/CH2Cl2/H2O, HCl, 56 psi, 82%.
the mutant L56 grew with myo-inositol as sole C-source (Fig. 2B).
As an additional control, we found that all strains grew with 0.2%
proline as sole C- and N- source (data not shown). Thus, our results
confirm that the mocABCR genes are essential for the degradation
of scyllo-inosamine.
The location of the Tn5 transposons in the mutant strains used
for the catabolism assay had been mapped previously by restric-
tion enzyme analysis.7,19 Under best circumstances, mapping can
determine the location of transposon insertions with accuracy of
50 base pairs. For this study, it was desirable to confirm the exact
locations of the Tn5 transposons in the different moc genes (Table 1).
We constructed oligonucleotides (Table 2) based on the published
DNA sequence of the rhizopine catabolism genes (Genbank Acces-
sion Nos. X78503 and AF076471) and used them as primers in PCR.
The purified PCR products were subjected to DNA sequencing, and
for each mutant strain the location inside the targeted open read-
ing frame was confirmed (Fig. 2C).
Table 1
Sinorhizobium meliloti wild-type and mutant strains, shown with the exact locations
of the transposon insertions in the inositol dehydrogenase (idhA) and the rhizopine
catabolism (moc) genes
Strain Phenotype
Tn5 insertion n bp after
start/total gene length in bp
1021
Sinorhizobium meliloti wild-type,
No transposon insertion
streptomycin resistant derivative of 2011,
Mosꢀ, Mocꢀ,40
L5-30 S. meliloti wild-type, SmR, Mos+, Moc+,41
L56
No transposon insertion
387/993
S. meliloti L5-30 idhA::Tn5 Mutant, SmR,
KmR,7
M11
M13
M16
M20
M21
M22
M23
M24
M25
M30
M32
S. meliloti L5-30 mocC::Tn5, SmR, KmR
S. meliloti L5-30 ORF334::Tn5, SmR, KmR
S. meliloti L5-30 ORF293::Tn5, SmR, KmR
S. meliloti L5-30 mocA::Tn5, SmR KmR
S. meliloti L5-30 mocB::Tn5, SmR, KmR
S. meliloti L5-30 mocR::Tn5, SmR, KmR
S. meliloti L5-30 mocR::Tn5, SmR, KmR
S. meliloti L5-30 mocD::Tn5, SmR, KmR
S. meliloti L5-30 mocD::Tn5, SmR, KmR
S. meliloti L5-30 mocE::Tn5, SmR, KmR
S. meliloti L5-30 mocF::Tn5, SmR, KmR
841/978
633/1005
541/882
418/954
539/930
452/1482
910/1482
263/1086
667/1086
133/318
972/1227
Figure 2. Growth of Sinorhizobium meliloti wild-type and mutant strains in minimal
medium with (A) 0.2% scyllo-inosamine as sole carbon and nitrogen source and (B)
0.2% myo-inositol as carbon and 0.1% KNO3 as nitrogen source. The growth was
determined spectrophotometrically at 600 nm (OD600). Strain designations are as
follows: L5-30, Moc+ wild-type; 1021, Mocꢀ wild-type; L56, idhAꢀ; M11, mocCꢀ;
M13, Tn5 insertion in ORF334; M16, Tn5 insertion in ORF 293; M20, mocAꢀ; M21,
mocBꢀ; M22, mocRꢀ; M23, mocRꢀ; M24, mocDꢀ; M25, mocDꢀ; M30, mocEꢀ; M32,
mocFꢀ. Bars represent the average values from duplicate cultures of one typical
experiment. Error bars denote standard deviations. (C) Structure of the moc operon
in Sinorhizobium meliloti L5-30. The horizontal arrows indicate the location of the
open reading frames in the moc region. The locations of the Tn5 insertions are
marked with vertical arrows, and labeled with the strain designations. The scyllo-
inosamine catabolic phenotype is indicated by + (catabolism) and ꢀ (no catabo-
lism).
Table 2
Oligonucleotides used as primers in this study
Primer
Sequence
idhA Forward
mocA Forward
mocB Forward
mocC Forward
mocD Forward
mocF Forward
mocR Forward
ORF293 Forward
ORF334 Forward
Tn5Ext
50 GCC GAC GGA TAC TCA TGC CGA TCT C 30
50 CCT TAA TTT GGC GGG CAA TGG GAT 30
50 CAA GTT CGG AAC CCT GCT AC 30
50 GCG GGA TGT GGA TGC AGA ATT TGA 30
50 CGA GAT TAC AGT CTG CTT GGT CG 30
50 GAG CCG TAT TGT GAT CGT TGG CGC G 30
50 CAT CTC AAG GCT GAC ACA CG 30
mutants with insertions in ORF334 (M13) and ORF293 (M16) grew
to around half of the optical density as the wild-type strain. The
L56 mutant strain, which carries a transposon insertion in the idhA
gene of S. meliloti L5-30,7 did not grow with the scyllo-inosamine as
sole C- and N-source. As control, the strains were grown on 0.2%
myo-inositol as sole carbon source. As expected, all strains but
50 GTT GAG AAA AGC GAC AGC G 30
50 CGC AAT CGC CGT TGA GAA GGT TAT 30
50 GAA AGT TAC CAT GTT AGG AGG TC 30

E. Schoffers et al. / Bioorg. Med. Chem. 16 (2008) 7838–7842
7841
In conclusion, we present an efficient chemical method for the
preparation of scyllo-inosamine in 7 steps and 32% overall yield.
Moreover, we provide detailed spectroscopic data for the charac-
terization of SIA and combine these chemical data with biological
results to unequivocally prove that the S. meliloti mocABCR genes
are necessary for the utilization of SIA as sole carbon and nitrogen
source.
chromatography (hexane/ethyl acetate, 2:1) to afford 1.02 g
(96%) of amine 12. Mp 120–121 °C; 1H NMR (CDCl3, 400 MHz) d
7.44–7.34 (m, 25H), 5.21–4.80 (m, 10 H), 3.74–3.73 (m, 3H), 3.46
(m, 2H), 3.05 (t, J = 9.88 Hz, 1H), 1.86 (br s, NH2); 13C NMR (CDCl3,
100 MHz) d 138.6, 138.5, 128.8, 128.7, 128.6, 128.1, 128.03, 128.0,
127.9, 127.8, 84.5, 83.7, 83.3, 76.2, 76.0, 75.9, 55.6. Anal. calcd for
C41H43NO5: C, 78.19; H, 6.88; N, 2.22. Found: C, 77.83; H, 6.77;
N, 2.41.
3. Experimental
3.3. scyllo-Inosamine hydrochloride (4c)
Commercial chemicals and reagents in 98+ purity were used
without further purification. Solvents were purchased as reagent
grade. Dichloromethane, DMF, and methanol were purified and
dried using standard procedures. Melting points were measured
in open capillaries using a Thomas–Hoover Unimelt instrument.
NMR spectra were recorded using a 400 MHz Jeol Eclipse nuclear
magnetic resonance instrument. IR spectra were obtained from
Bruker Equinox 55 and Perkin Elmer 1710 Fourier Transform Infra-
red Spectrometers. Elemental analyses were carried out by Nume-
ga Resonance Labs, Inc. in San Diego, CA.
To the suspension of 10% Pd/C (200 mg, 0.188 mmol) in MeOH/
H2O (2:1, 9 mL) was added a solution of azide 11 (794 mg,
1.212 mmol) in CH2Cl2 (9 mL). Concentrated HCl (1 mL) was added
to the mixture which was hydrogenated at 56 psi for 48 h. The reac-
tion mixture was filtered through Celite and rinsed with MeOH/H2O
(4:1). The filtrate was concentrated and the residue was partitioned
between H2O and EtOAc layers. The aqueous layer was concentrated
to give 215 mg (82%) of a white solid. A white precipitate was col-
lected. Using the same procedure hydrogenation of 49 mg of amine
12 gave 13 mg (76%) of scyllo-inosamine hydrochloride 4c. Mp
290 °C (dec.); 1H NMR (CDCl3, 400 MHz) d 3.51 (app t, J = 9.72 Hz,
2H), d 3.39 (app t, J = 9.16 Hz, 2H), d 3.32 (t, J = 9.16 Hz, 1H), d 3.07
(t, J = 10.44 Hz, 1H); 13C NMR (CDCl3, 100 MHz) d 74.4, 74.2, 70.02,
55.90; IR (KBr) 3400, 3045, 1929, 1626, 1576, 1505, 1347, 1036;
Anal. calcd for C6H14ClNO5: C, 33.42, H, 6.54, N, 6.50. Found: C,
33.45, H, 6.23; N, 6.76. A small sample was reacted with acetic anhy-
dride in dry pyridine at room temperature for 48 h to afford the cor-
responding hexaacetate. Mp 299 °C; 1H NMR (CDCl3, 400 MHz) d
5.71 (d, J = 9.88 Hz, 1H), d 5.28 (app t, J = 9.90 Hz, 2H), d 5.17 (t,
J = 9.88 Hz, 1H), d 5.05 (app t, J = 10.26 Hz, 2H), d 4.42 (app dd, J1
= 10.48 Hz, J2 = 21.06 Hz, 1H), d 2.02 (s, 6H), d 2.00 (s, 6H), d 1.99 (s,
3H), d 1.89 (s, 3H); 13C NMR (CDCl3, 100 MHz) d 170.86, 170.16,
169.76, 169.36, 70.54, 70.48, 70.42, 51.35, 23.13, 20.58, 20.53,
20.50; IR (KBr) 3426, 2939, 1733, 1687, 1521, 1381, 1234, 1037;
Anal. calcd for C18H25NO11: C, 50.12; H, 5.84; N, 3.25. Found: C,
50.51; H, 5.80; N, 3.19.
3.1. 1,3,4,5,6-Penta-O-benzyl-2-O-deoxy-2-azido-scyllo-inositol
(11)
Azide 11 was prepared from alcohol 10 using modified litera-
ture procedures.35–38 To a solution of 1,3,4,5,6-penta-O-benzyl-
myo-inositol 10 (3.45 g, 5.47 mmol) in anhydrous dichloromethane
(200 mL) was added anhydrous pyridine (1.33 mL, 16.41 mmol) at
ꢀ60 °C under argon. Triflic anhydride (2.27 mL, 13.67 mmol) was
added dropwise to the solution, and the reaction mixture was al-
lowed to warm to room temperature. TLC (hexane/ethyl acetate,
2:1) revealed the reaction completed after 26 h. After quenching
with water, the reaction mixture was extracted with CH2Cl2, and
subsequently washed with water, saturated aqueous NaHCO3,
and brine. The organic layer was dried (MgSO4) and concentrated
to afford a thick oil (3.89 g, 93%) which was used without purifica-
tion for the next step. The crude triflate (3.75 g, 4.92 mmol) and so-
dium azide (5.11 g, 78.65 mmol) were dissolved in dry DMF
(300 mL). The reaction mixture was stirred vigorously at 80 °C
for 5.5 h while monitoring by TLC (heptane/ethyl acetate, 4:1).
The undissolved sodium azide was filtered off and the residue con-
centrated. An excess of water was added and the mixture was ex-
tracted with CH2Cl2. The organic layer was washed with a 5%
sodium chloride solution, dried with MgSO4, and concentrated to
yield 95% of crude yellow solid. Recrystallization from hot metha-
nol yielded pure azide 11 (2.60 g, 81%) which was previously de-
scribed as oil.22 mp 95–96 °C; 1H NMR (CDCl3, 400 MHz) d 7.42–
7.2 (m, 25H), 4.93–4.88 (m, 10H), 3.63–3.49 (m, 4H), 3.38 (app t,
J = 9.16 Hz, 2H); 13C, NMR (CDCl3, 100 MHz) d 138.37, 138.34,
137.9, 128.62, 128.58, 128.4, 128.1, 127.98, 127.91, 127.88, 83.4,
82.7, 81.2, 76.2, 76.11, 76.08, 67.1; IR (KBr) 3062, 2904, 2200,
2102; Anal. calcd for C41H41N3O5: C, 75.09; H, 6.30; N, 6.41. Found:
C, 75.10; H, 6.11; N, 6.34.
3.4. Bacterial strains, media, and growth conditions
Bacterial strains used are shown in Table 1. The complete med-
ium used was TY42 and the defined medium was Minimal M med-
ium.19 Antibiotics were used in the following concentrations:
streptomycin (Sm), 250 lg/ml, kanamycin (Km), 200 lg/ml.
3.5. Catabolism assays
Sinorhizobium meliloti precultures were grown in TY for 48 h
and then inoculated 1:100 into glass tubes containing 2 ml mini-
mal medium with either 0.2% myo-inositol as sole carbon (C)
source and 0.1% KNO3 as nitrogen (N) source or 0.2% proline or
0.2% scyllo-inosamine as sole C- and N- source. The cultures were
shaken (200 rpm) at 28 °C. After 72 h, the bacterial growth was
determined by measuring the absorbance at 600 nm (OD600) with
a Beckman DU 640 spectrophotometer.
3.2. 1,3,4,5,6-Penta-O-benzyl-2-O-deoxy-2-amino-scyllo-
inositol (12)
3.6. Determination of transposon insertions sites by PCR and
DNA sequencing
Azide 11 (1.10 g, 1.677 mmol) and triphenylphosphine (0.484 g,
1.845 mmol) were dissolved in THF (100 mL). The solution was
stirred at room temperature for 2 h and water (5 mL) was added.
The reaction mixture was refluxed at 80 °C for 24 h until comple-
tion as monitored by TLC (hexane/ethyl acetate, 4:1). The reaction
mixture was concentrated and the residue was extracted with
ethyl acetate. The organic layer was dried (MgSO4) and concen-
trated to obtain the crude product which was purified by column
The exact insertion site of the Tn5 insertions in the mutant
strains was determined via PCR. As template, 1 microliter of a li-
quid bacterial culture was used in 50-ll reaction mixtures contain-
ing (as final concentrations): 1ꢁ Red-Taq PCR Buffer (Sigma–
Aldrich Corp., St. Louis, MO), a 0.2 mM concentration of each
deoxyribonucleotide, 50 pmol of each forward and the Tn5Ext pri-
mer (see Table 2), and 1.5 U of Red-Taq DNA polymerase (Sigma–

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E. Schoffers et al. / Bioorg. Med. Chem. 16 (2008) 7838–7842
13. Gordon, D. M.; Ryder, M. H.; Heinrich, K.; Murphy, P. J. Appl. Environ. Microbiol.
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Since the submission of our manuscript another publication re-
ported the synthesis of SI in 23% yield using 5 steps from the
monoorthoformate of myo-inositol.
43
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Acknowledgments
We acknowledge the support of the Herman Frasch Foundation
(543-HF02), and Western Michigan University through its Office of
the Vice President for Research (FRACASF) and the Graduate
College.
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