Total synthesis of sulfolipid-1

Article abstract of DOI:10.1039/c3cc48087a

Sulfolipid-1, a tetra-acylated sulfotrehalose from Mycobacterium tuberculosis, was isolated over 40 years ago. Being a main component of the mycomembrane of M. tuberculosis, its biosynthesis and function have been studied in depth, but the chemical synthesis of sulfolipid-1 has not been reported. The synthesis presented here is based on iterative catalytic asymmetric conjugate additions of methylmagnesium bromide for the preparation of the phthioceranic and hydroxyphthioceranic acid side chains, a double regioselective reductive ring-opening and a fivefold deprotection in the final step. The Royal Society of Chemistry 2014.

Full text of DOI:10.1039/c3cc48087a

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Total synthesis of sulfolipid-1†
Danny Geerdink‡§ and Adriaan J. Minnaard*§
Cite this: Chem. Commun., 2014,
50, 2286
Received 21st October 2013,
Accepted 4th January 2014
DOI: 10.1039/c3cc48087a
www.rsc.org/chemcomm
Sulfolipid-1, a tetra-acylated sulfotrehalose from Mycobacterium tuber-
culosis, was isolated over 40 years ago. Being a main component of the
mycomembrane of M. tuberculosis, its biosynthesis and function have
been studied in depth, but the chemical synthesis of sulfolipid-1 has not
been reported. The synthesis presented here is based on iterative
catalytic asymmetric conjugate additions of methylmagnesium bromide
for the preparation of the phthioceranic and hydroxyphthioceranic acid
side chains, a double regioselective reductive ring-opening and a five-
fold deprotection in the final step.
Mycobacterium tuberculosis (M. tuberculosis), isolated by Koch
130 years ago, is at present still the cause of the most widespread
bacterial infectious disease.1 Much of the molecular research on
M. tuberculosis, considerably stimulated by the sequencing of the
bacterial genome, has focused on the composition and biosynthesis
of its unusual waxy cell envelope.2 The outer membrane of the cell
envelope, the mycomembrane, is composed of long-tailed (glyco)
lipids and, as a consequence, this hydrophobic layer functions as a
very efficient barrier for antibiotics.3
Fig. 1 SL-1 and Ac2SGL.
spectrometry and 2D-NMR spectroscopy, that the composition,
A number of these mycomembrane glycolipids are known to abundance, and position of the fatty acids on the trehalose core
modulate the immune response of the host, and several specific of 1 varies per strain.6 The role of 1 in the pathogenicity of
glycolipids act as ligands for the CD1 immune system.4 One of the M. tuberculosis has not been demonstrated and in addition could
most prominent and most complex glycolipids present is sulfolipid-1 be species specific, a phenomenon not uncommon in mycobacterial
(1), a 2,3,6,60-tetraacyl-a,a-trehalose 20-sulfate, decorated with phthio- infections.7 Studies with knockout mutants, unable to produce the
ceranic, hydroxyphthioceranic, and palmitic acid residues (Fig. 1). negatively charged 1, indicate that 1 has a role in host–pathogen
Remarkably, 1 is only present in pathogenic mycobacteria but its interactions by mediating between a cationic human antimicrobial
function remains largely unknown. Sulfolipid-1 was isolated, and its peptide and the bacterium.8
structure elucidated using scrupulous degradation studies, by Goren
In a series of studies, the group of Bertozzi has unraveled the
more than 40 years ago.5 Recently, Gilleron et al. showed, using mass biosynthesis of 1, that starts with the sulfation of trehalose.9
In addition, its membrane transport is a topic of interest.9
A comprehensive view on the biosynthesis and role of trehalose-
Stratingh Institute for Chemistry, University of Groningen, Nijenborgh 7, NL-9747
containing lipids in mycobacteria is expected to originate from a
AG Groningen, The Netherlands. E-mail: a.j.minnaard@rug.nl
† Electronic supplementary information (ESI) available: Experimental proce- bioorthogonal chemistry approach.10 As far as synthetic precursors
dures, spectral data, and a HRMS-spectrum of synthetic 1. See DOI: 10.1039/
and model compounds have been involved in these studies, the
c3cc48087a
multimethyl-branched phthioceranic acid residues were replaced by
‡ Current address: Department of Organic Chemistry, University of Vienna,
mono- or disubstituted analogues for synthetic reasons.11
¨
Wahringer Straße 38, Vienna, Austria.
Also the hydroxy group in hydroxyphthioceranic acid was
§ Author contributions: The manuscript was written through contributions of both
authors. Both authors have given approval to the final version of the manuscript. omitted; in addition its stereochemistry was unknown.
2286 | Chem. Commun., 2014, 50, 2286--2288
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Scheme 2 Double EDC-promoted esterification.
Fig. 2 Phthioceranic and hydroxyphthioceranic acid.
esterification of 10 with (S)-methylbutyric acid under Yamaguchi
conditions, however, gave mainly a disappointing mixture of 6- and
60-monoacylated products. The desired diacylated 11 could, on the
other hand, be obtained in very good yield using an EDC-promoted
esterification (Scheme 2). Straightforward removal of the bis(diiso-
propylsilyl) ether with buffered TBAF, followed by regioselective
introduction of a 2,2,2-trichloroethyl-protected sulfate at the
2-OH,22 and removal of all protecting groups under hydro-
genolysis conditions, led to the corresponding analogue of 1
(see ESI,† compounds 15–17).
Recently, we reported the first total synthesis of Ac2SulfoGlycoLipid
(Ac2SGL, 2), a biosynthetic precursor of 1.12,13 Ac2SGL is a potent
antigen in M. tuberculosis-infected humans, specifically recognized by
antigen presenting cells expressing CD1b. This points to the potential
use of (analogues of) Ac2SGL in a TB vaccine.14 In these studies the
stereochemistry of the hydroxy group in hydroxyphthioceranic acid was
also established. Although still challenging, with the experience gained
in the synthesis of Ac2SGL, a total synthesis of 1 has come within reach.
This would potentially assist in the research on M. tuberculosis and in
addition serve as an illustration of the power of asymmetric catalysis in
the synthesis of complex deoxypropionates.15
In order to synthesize 1, 6 was efficiently acylated with 3 at the
3-position, by Yamaguchi esterification, to afford 7 (Scheme 3). Apply-
ing the optimized conditions for regioselective reductive ring-opening
with TMSOTf and BH3ꢀTHF, 8 was obtained in 59% yield, similar to the
model substrate. EDC-promoted double esterification of 8 using the
previously prepared benzyl ether-protected 5 initially proved rather
sluggish. Addition of an excess of EDC (8 eq.) and DMAP (8 eq.) was
required to obtain complete conversion of the starting material. This
diminished reactivity of long-chain fatty acids has been reported
before.23 Despite the formation of 10% of the tri-acyl product, tetra-
acyl 9 was obtained in 71% yield. Deprotection of the bis(diiso-
propylsilyl) ether under buffered conditions proved to be facile
affording the 20,30-diol 12 in 84% yield. Subsequent introduction of
the 2,2,2-trichloroethyl protected sulfate at the 20-OH using 13 turned
out to be not completely regioselective.22 In the synthesis of Ac2SGL,
only introduction of the TCE-sulfate at the 20-OH and minor amounts
of difunctionalization was observed. In the current case, also trace
amounts of, assumed, 30-OH sulfated products were obtained. This can
be subscribed to the diminished rigidity of the substrate as a con-
sequence of the previously effected benzylidene ring-opening. Never-
theless, pure 14 was isolated in 58% yield. To circumvent the
purification of highly polar and charged intermediates, we envisioned,
as our final step, the complete removal of all protecting groups using
hydrogenolysis. Previously, the fivefold deprotection of our model
system had shown to be straightforward. With a combination of
ammonium formate, Pd(OH)2/C and hydrogen at atmospheric pres-
sure, the benzyl groups on both the 4- and 40-OH, the benzyl group in
the side chain and the TCE group were removed.12 In 14, however, the
benzyl groups on the 4- and 40-OH were not removed under these
conditions. While 15 bar of hydrogen pressure did not lead to full
deprotection, prolonged hydrogenolysis at 30 bar hydrogen finally led
to 1 in a low, but delivering 15% yield. The low yield is a result of the
multiple attempts to remove all protecting groups causing desulfation
of the variety of intermediates and the product. Indeed, also a small
amount of the desulfated product was isolated.16 An explanation given
earlier,23b to account for the reluctant hydrogenolysis, is most likely the
steric hindrance caused by the long-tailed lipids that prevents the
Key to the successful synthesis of 1 is the regioselective esterification
and sulfation of trehalose, with minimal loss of the precious phthio-
ceranic acids 3 and 4 (Fig. 2). Commencing with intermediate 6,16
prepared from trehalose in three steps, esterification with 3 (Scheme 1),
followed by regioselective reductive ring-opening of the benzylidene
acetals, should produce 8 with the 6- and 60-OH liberated. Subsequent
esterification of both 6-positions with 5 should provide 9, which upon
desilylation, regioselective sulfation and overall deprotection would
afford 1.
In the synthesis of Ac2SGL, the preparation of 3 and 5 had already
been combined, thereby minimizing the total number of reactions
and purification steps.12 To study the reductive ring-opening of
the benzylidene acetals in 7, a less precious substrate carrying
(S)-methylbutyric acid was prepared.17 Whereas a combination of
borane and CoCl2, contrary to literature reports, gave no conver-
sion,18 its combination with Cu(OTf)219 and TMSOTf20 afforded 10,
albeit in low yield. The main side product in these reactions was
the fully deprotected 4,6-40,60-tetraol.21 Increasing the amount of
BH3ꢀTHF to 12 eq. in combination with TMSOTf gave the product
in a satisfactory 59% yield. Also the regioselectivity affording the free
6- and 60-OH was excellent using this approach. A successive double
Scheme 1 Regioselective esterification towards SL-1.
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Scheme 3 End game in the synthesis of sulfolipid-1.
7 A. Apt and I. Kramnik, Tuberculosis, 2009, 89, 195–198.
approach of the heterogeneous Pd-catalyst. Although natural 1 is iso-
lated as a mixture of homologues, the 1H-NMR spectrum of synthetic 1
is similar to the one reported for natural 1 (Fig. S1, ESI†). Sulfolipid-1
turned out unstable in CDCl3 solution (most probably desulfation takes
place),5a accounting for the minor differences. High resolution mass
analysis (ESI) confirmed the formation of the desired product.
In summary, forty years after its first isolation as a main compo-
nent of the mycomembrane of Mycobacterium tuberculosis, sulfolipid-1
has been synthesized and its structure confirmed. The availability of
synthetic 1 and its precursors will accommodate research on its
biosynthesis and function. Key steps in the synthesis are the prepara-
tion of hydroxyphthioceranic acid, the regioselective reductive ring-
opening of the benzylidene acetals and a final fivefold deprotection.
We thank Dr M. Gilleron for kindly providing a 1H-NMR
spectrum of natural, purified SL-1. Financial support from the
Netherlands Organization for Scientific Research (NWO-CW) is
gratefully acknowledged.
8 S. A. Gilmore, M. W. Schelle, C. M. Holsclaw, C. D. Leigh, M. Jain,
J. S. Cox, J. A. Leary and C. R. Bertozzi, ACS Chem. Biol., 2012, 7, 863–870.
9 J. C. Seeliger, C. M. Holsclaw, M. W. Schelle, Z. Botyanszki,
S. A. Gilmore, S. E. Tully, M. Niederweis, B. F. Cravatt, J. A. Leary
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11 (a) C. D. Leigh and C. R. Bertozzi, J. Org. Chem., 2008, 73, 1008–1017;
´
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2288 | Chem. Commun., 2014, 50, 2286--2288
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