How recent knowledge on furano-specific enzymes has renewed interest for the synthesis of glycofuranosyl-containing conjugates - Carbohydrate Chemistry: Chemical and Biological Approaches - page 407

membrane. Subsequently, a L -rhamnosyl unit (a- L -Rhap) is grafted to O-3
of the a- D -GlcNAcp so that the resulting lipidic disaccharide is indeed
necessary for the continuation of the polymerization with a- D -Galf
entities. Therefore, a first enzyme, GlfT1, ensures the introduction of a
first Galf on O-4 of a- L -Rhap, as well as the furanosylation of position 5 of
the previous furanoside. The action of the bifunctional GlfT1 is followed
by that of a dual-acting transferase/polymerase, the GlfT2. The resulting
approximately 30-residue long galactan 76,77 is a chain composed of Galf
residues alternatively linked to the 5 or 6 position of the preceding a- D -
Galf entity, thus affording a specific and structurally defined polymer.
Once again, the survival of M. tuberculosis is highly sensitive to the
presence of this galactan.
3.2 Chemical and biological tools to study transferases and
polymerase
In order to study the potential of transferases in mycobacteriae, a first
tool was proposed by Brennan and Besra, and their collaborators. 78 It
relies firstly on an overexpression of GlfT genes in non-pathogenic
M. smegmatis or Escherichia coli. Secondly, incubation of the resulting
membrane extract, that contains all necessary biological machinery to
synthesized furanosyl-containing compounds, with UDP-[U- 14 C]a- D -Galp
and synthetic acceptors and/or inhibitors allowed measure of transferase
activity by thin layer chromatography/autoradiography of the extracted
carbohydrates. This technique indeed accounts for the full biosynthesis
of the cell wall galactan of mycobacteria by the membrane extract that
contains the whole of the necessary biological machinery, i.e. UGM and
of course both furanosyl transferases GlfT1 and GlfT2. 74 It was further
applied to study the impact of disaccharides, 79 hexofuranose-like
iminosugars 80,81 and UDP-derivatives, 82 on the first steps of the cell wall
biosynthesis of M. smegmatis (Fig. 4). For the disaccharides, MIC
(minimum inhibitory concentration) values were moderate and best
results were observed for 23-25 (12.8 o MIC o 128 mg/mL). With imino-
sugars, 40% inhibition was obtained for compound 27 at 8 mM. Using
the isomers 29 and 30, the authors have demonstrated that the L -Galf
analogue 30 surprisingly presented significant better inhibition activity
than 29 at 8 mM. Inhibition of galactan growth from the membrane ex-
tracts was however very ecient starting from UDP-furanosides bearing
chemical modulations on the side arm. Indeed, it was observed severe
impact on the related pathways at concentrations of 9 mM(5mg/mL) and
we could also differentiate effect on both transferases GlfT1 and GlfT2
according to the nature of the chemical variations. Some nucleotides-
derivatives were indeed substrates of transferases so that the Galf entity,
or its analogue, was incorporated into the growing galactan. But because
of the absence of a required hydroxyl function in 5- or 6-position,
subsequent furanosylation was not possible. Consequently, the
formation of the full-length galactan was stopped by short ''dead-end''
intermediates. 82 Some results are summarized in Fig. 5. It was also
shown that pure nucleotide-furanose inhibitors are ecient on whole
M. tuberculosis cells. 83