Synthesis of orthogonally protected and functionalized bacillosamines

Article abstract of DOI:10.1039/d0ob00256a

2,4-Diamino-2,4,6-trideoxyglucose (bacillosamine) is a monosaccharide found in many pathogenic bacteria, variation in the functionalities appended to the amino groups occurs depending on the species the sugar is derived from. We here report the first synt

Full text of DOI:10.1039/d0ob00256a

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DOI: 10.1039/D0OB00256A.
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DOI: 10.1039/D0OB00256A
ARTICLE
Synthesis of orthogonally protected and functionalized
bacillosamines
Jeanine van Mechelen, Jim Voorneveld, Hermen S. Overkleeft, Dmitri V. Filippov, Gijsbert A. van der
Marel,* Jeroen D. C. Codée*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
2,4-Diamino-2,4,6-trideoxyglucose (bacillosamine) is a monosaccharide found in many pathogenic bacteria variation in the
functionalities appended to the amino groups occurs depending on the species the sugar is derived from. We here report
the first synthesis of bacillosamine synthons that allow for the incorporation of two different functionalities at the C-2-N-
acetyl and C-4-amines. We have developed chemistry to assemble a set of conjugation ready Neisseria meningitidis C-2-N-
acetyl bacillosamine saccharides, carrying either an acetyl or (R)- or (S)-glyceroyl at the C-4 amine. The glyceroyl
bacillosamines have been further extended at the C-3-OH with an α-D-galactopyranose to provide structures that occur as
post-translational modifications of N. meningitidis PilE proteins, which make up the bacterial pili.
O
O
Introduction
*
X =
OH
O
O
RHN
HO
Bacillosamine (1, Bac, Figure 1) is a characteristic bacterial
monosaccharide that is present in various bacterial capsular
polysaccharides and glycopeptides.1 It is a 2,4-diamino-2,4,6-
trideoxyhexose (DATDH), that is most commonly functionalized
with N-acetyl groups at C-2 and C-4 (2,4-di-NAcBac, 2). This
residue is found at the reducing end of oligosaccharide chains
of both O- and N-linked glycoproteins of Campylobacter jejuni2
and Neisseria gonorrhoeae.3 Recently, a differently modified
bacillosamine has been discovered in Neisseria meningitidis,4
where it is decorated, besides a single acetamide, with an N-
glyceroyl group, thought to reside at the C4-amine (3, Figure 1).5
This Bac-residue represents one of various post-translational
modifications (PTMs) that can occur on N. meningitides pilE
proteins, the constituent protein monomers of the type IV pili.
It can be further decorated with different mono- or
disaccharides (see Figure 1). The role of these PTMs is currently
OH
OH
RHN
1, R = H, Bacillosamine (Bac)
2, R = Ac, diNAcBac
XHN
RO
O
O
NH
AcHN
3
R = H or
α
-
D
-Gal or
α
-
D
-Glc or
α
-
D
-Gal-(1-4)-α-D-Gal
OH
OH
O
O
RHN
HO
HO
HO
O
NH2
OH
AcHN
6
O
HO
O
O
AcHN
HN
4a R = Ac
NH2
O
4b R = (R)-glyceroyl
4c R = (S)-glyceroyl
5
6
Figure 1 Bacillosamine
found in N. meningitidis pili (
and ).
(
1
), di-N-acetylbacillosamine
(2), bacillosamine PTMs
3
) and the synthetic targets of the current study (
4
5
not known but it is clear that the type IV pili play an important bacillosamine synthon, of which all functional groups can be site
role in the pathogenicity of the bacterium. Also, the exact selectively addressed. We here report the first synthesis of such
structure of the Bac-residue present on the N. meningitides pili an orthogonally protected bacillosamine building block and we
remains elusive as the stereochemistry of the glyceroyl group show the synthesis of spacer-equipped bacillosamines bearing
has not been established.5
different acyl functionalities (4b-4c, Figure 1) and the extension
of these towards a relevant conjugation-ready disaccharide (5,
To aid in the structure elucidation of the functionalized Bac Figure 1).
residues in the N. meningitidis pili, its detection and the role of
these differentially decorated bacillosamines in virulence, we
reasoned that the availability of synthetic Bac-residues would
provide powerful tools. We therefore set out to develop an
effective route of synthesis for an orthogonally protected
a. Bioorganic synthesis group, Leiden University, Einsteinweg 55, 2333 CC Leiden,
Leiden, The Netherlands.
† Footnotes relating to the title and/or authors should appear here.
Electronic Supplementary Information (ESI) available: [synthetic procedures and
experimental data]. See DOI: 10.1039/x0xx00000x
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Results and discussion
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DOI: 10.1039/D0OB00256A
Different syntheses of bacillosamine6 and bacillosamine
containing oligosaccharides7 and glycopeptides8 have been
reported, but none of these allow for the discrimination of the
C-2 and C-4 amino groups and therefore the development of a
new synthetic route was required. We thus set out to target key
synthon 7, bearing two orthogonally protected amino groups at
C-2 and C-4, as well as a 2-napthylmethyl ether that masks the
C-3-OH and an anomeric selenophenyl group for glycosylation
purposes (See Scheme 1). Retrosynthetically, this building block
can be derived from a suitably protected 6-deoxy galactosamine
derivative 8, by a substitution reaction of the C-4-O-mesylate.
Selenophenyl galactosamine 8 can be readily accessed from D-
fucal through an azidoselenation event9 and subsequent
transformation of the azide into the protected C-2-aminosugar.
Accordingly, 3,4-di-O-acetyl-D
-fucal 910 was subjected to
Scheme 1 Retrosynthetic analysis for the assembly of target compounds 4 and 5.
azidoselenation conditions to provide azidofucose 1011 (See
Scheme 2). Saponification of the two acetates in 10 was then
followed by regioselective installation of the C-3-
naphthylmethyl ether and subsequent mesylation of the C-4-
OH. Because we aimed to use azide as nucleophile for the
introduction of the C-4-amine functionality, we had to
transform the C-2-azide in 11 to achieve orthogonality.
Although amines are commonly avoided during manipulations
on carbohydrate building blocks, we reasoned that inorganic
azide anion should be sufficiently nucleophilic to outcompete
the C-2 amine in mesylate displacement. We therefore decided
to reduce the azido group in 11 to the corresponding amine and
invert the C-4-O-mesylate in the resulting fucosamine, without
masking the C-2-amine. Thus, fucosazide 11 was exposed to 1,3-
propanedithiol to deliver fucosamine 12, which was treated
with NaN3 and TBABr in DMF at elevated temperature to
provide the desired bacillosamine 13, bearing a fully orthogonal
functional group pattern, in 81%. Next, the C-2-amine was
masked with a trichloroacetyl (TCA) group to provide fully
protected bacillosamine 14. Reduction of the azide with 1,3-
propanedithiol then set the stage for the differentiation of the
C-4-amino group into either an acetamide (towards 4a) or the
two glyceroyl substituted bacillosamines 4b and 4c. During the
reduction of the azide in 14, the TCA moiety was partially
reduced as well to the corresponding C-2-dichloroacetamide
(DCA), yielding compound 15. As the DCA group can be removed
under similar conditions as the TCA, the synthetic strategy did
not need any adjustment and we continued with 4-N-acylation.
Trichloroacetylation provided bacillosamine synthon 16a while
Scheme 2 Synthesis of differentially functionalized bacillosamines. Reagents and
conditons: a) (PhSe)2, PhI(OAc)2, TMS-N3, DCM, -20 °C to rt., 93%. b) i) NaOMe,
MeOH. ii) Bu2SnO, toluene, 110 °C. iii) TBABr, NapBr, 82%. c) MsCl, pyr. 0 °C to rt.,
93%. d) 1,3-propanedithiol, TEA, DMF, 92% for12, 82% for 15
DMF, 80 °C, 81%. f) TCA-Cl, DCM:pyr., 0 °C, 89% for 14, 88% for 16a
azidohexanol, NIS, TMSOTf, DCM, 0 °C, 70% for 17a, 74% for 17b, 74% for 17c
2,2-dimethyl-1,3-dioxolane-4-carboxylic acid (R for 16b, S for 16c), HCTU, DIPEA,
DMF, °C to rt., 83% for 16b 80% for 16c i) Pd(OH)2/C 20wt%, H2.
H2O/THF/tBuOH, 63% j) i) H2O/THF/AcOH, 80 °C. ii) Pd(OH)2/C 20wt%, H2,
H2O/THF/tBuOH, 79% for 4b, 81% for 4c
.
e) NaN3, TBABr,
g) 6-
h)
.
.
0
,
.
.
.
reaction of the C-4-amine with either R- or S-isopropylidene-
glyceric acid under the agency of HCTU, provided bacillosamines
16b and 16c. Next, the three building blocks were equipped
with an azidohexanol spacer, in glycosylation reactions
promoted by N-iodosuccinimide (NIS) and trimethylsilyl
trifluoromethanesulfonate (TMSOTf) to provide 17a, 17b and
17c. The products were obtained as single diastereoisomers and
no oxazoline side products were detected, indicating that the
dichloroacetyl groups provided effective anchimeric
assistance.12 With the fully protected spacer- equipped
bacillosamines in hand, deprotection of the intermediates was
undertaken. We first tried to unmask the TCA and DCA groups
in 17a using Cs2CO3 in DMF13, but this did not lead to the desired
C-2,4-diamine. Instead bicyclic urea 18 was formed.14 We
therefore switched to a hydrogenation reaction, aiming to
unmask the spacer azide, C-2-N-DCA, C-3-O-Nap and C-4-N-TCA
2 | J. Name., 2012, 00, 1-3
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groups in one step. After the first hydrogenation event using
Pearlman’s catalyst, LCMS analysis of the crude reaction
mixture revealed the presence of three chlorides and therefore
the mixture was resubjected to the hydrogenation conditions
until all chlorides had been removed. This delivered spacer-
equipped di-N-acetyl bacillosamine 4a in 63% yield. After the
acidic hydrolysis of the glyceroyl isopropylidene ketal in 17b and
17c, deprotection under the same conditions delivered the C-2-
N-acetyl-C-4-N-glyceroyl bacillosamines 4b and 4c in 79% and
81% yield, respectively.
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DOI: 10.1039/D0OB00256A
With a successful route of synthesis towards differentially
functionalized bacillosamines established, we set out to probe
the generation of a N. meningitidis disaccharides, featuring an
additional a-D-galactopyranose attached to the C-3-OH of the
bacillosamine. To install the required 1,2-cis-galactosyl linkage
we used a 4,6-di-tert-butyl silylidene protected galactose donor
19 as 4,6-silylidene functionalized galactopyranosides are
amongst the most reliable donors for the installation of cis-
galactosyl linkages.15 Because the presence of amides in
glycosyl acceptors can lead to problematic glycosylation
reactions16 we decided to attach the
a-galactoside prior to the
introduction of the N-glyceroyl. Thus, we prepared C-4-azide
acceptor 20 by DDQ-mediated unmasking of the C-3-hydroxyl
function in 14. In a chemoselective glycosylation reaction,
imidate donor 1917 and bacillosamine selenoglycoside acceptor Scheme 3 Synthesis of galactosylated C4-N-glyceroyl bacillosamines. Reagents
and conditions: a) DDQ, DCM/H2O, 80%. b) compound 20, TMSOTf, DCM, 0 °C,
20 were then reacted to provide, in
a completely
91%. c) 1,3-propanedithiol, TEA, DMF, 48%. d) (S)-2,2-dimethyl-1,3-dioxolane-4-
carboxylic acid for, HCTU, DIPEA, DMF, 0 °C to rt., 30%. e) 6-azidohexanol, NIS,
TMSOTf, DCM, 0 °C, 57%, f) i) HF.pyr, THF, 64% ii) H2O/THF/AcOH, 80 °C. iii)
Pd(OH)2/C 20wt%, H2, H2O/THF/tBuOH, 41%.
stereoselective manner, the desired disaccharide 21 in 91%
yield. Before installation of the azidohexanol spacer, the C-4-N-
glyceroyl groups had to be incorporated and we therefore
reduced the C-4-azide in 21, which again led to the conversion
of the C-2-N-TCA amide into the corresponding DCA-group.
Amine 22 was obtained in 48% yield and condensed with (S)-
isopropylidene-glyceric acid using HCTU as the condensation
agent to furnish the glyceroyl bacillosamine disaccharide 23 in
30%. It was observed that both the reduction and the amide
bond forming reactions proceeded significantly slower in the
disaccharide than the corresponding reactions on the
monosaccharides described above. This may be attributed to
increased steric hindrance of the appended galactopyranose
with the neighboring C4-nitrogen. Next the azidohexanol spacer
was introduced using NIS/TMSOTf to activate the seleno
disaccharide to afford the fully protected disaccharide 24 in 57%
yield. At this stage,
all protecting groups in disaccharide 24 were removed by the
following procedure. First, the silylidene group was removed by
treatment of HF.Pyr in THF yielding the diol in 64% yield. Next,
the isopropylidene group in the glyceroyl moiety was
hydrolysed with AcOH/H2O/THF. Finally, the hydrogenation
procedure, as described above, was undertaken to remove the
benzyl ethers and transform the dichloroacetamide into the
corresponding acetamide and the spacer azide into a primary
amine. The hydrogenation reaction was monitored by LC-MS
analysis, and repeated when required. In this manner
disaccharide 5 was obtained in 41% yield.
Conclusions
In conclusion, we have here disclosed the first synthesis
towards an orthogonally protected bacillosamine synthon, that
allows for the differentiation of the two amino functionalities,
and its use in the generation of a N. meningitidis PilE
disaccharide bearing a C-3-a-D-galactose appendage and a C-4-
N-glyceroyl. The chemistry developed here can be used to
generate differentially functionalised bacillosamines and pave
the way to the assembly of larger bacillosamine containing
oligosaccharides as well as glycopeptides. The set of
conjugation ready bacillosamines can be used to generate
antibodies directed at these N. meningitidis PilE post-
translational modifications for both therapeutic and diagnostic
purposes.
Conflicts of interest
There are no conflicts to declare
Acknowledgements
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
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Journal Name
This work was financially supported by The Netherlands
Organization for Scientific Research (NWO).
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DOI: 10.1039/D0OB00256A
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