Chemical synthesis of asparagine-linked archaeal N-glycan from Methanothermus fervidus

Article abstract of DOI:10.1002/chem.201304950

Several N-linked glycoproteins have been identified in archaea and there is growing evidence that the N-glycan is involved in survival and functioning of archaea in extreme conditions. Chemical synthesis of the archaeal N-glycans represents a crucial step

Full text of DOI:10.1002/chem.201304950

DOI: 10.1002/chem.201304950
Communication
&
Glycoproteins
Chemical Synthesis of Asparagine-Linked Archaeal N-Glycan from
Methanothermus fervidus
Someswara Rao Sanapala and Suvarn S. Kulkarni*[a]
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Abstract: Several N-linked glycoproteins have been identi-
fied in archaea and there is growing evidence that the N-
glycan is involved in survival and functioning of archaea
in extreme conditions. Chemical synthesis of the archaeal
N-glycans represents a crucial step towards understanding
the putative function of protein glycosylation in archaea.
Herein the first total synthesis of the archaeal l-asparagine
linked hexasaccharide from Methanothermus fervidus is re-
ported using a highly convergent [3+3] glycosylation ap-
proach in high overall yields. The synthesis relies on effi-
cient preparation of regioselectively protected thioglyco-
side building blocks for orthogonal glycosylations and late
stage N-aspartylation.
Figure 1. Structure of N-glycan 1 from Methanothermus fervidus.
pure and structurally well characterized N-glycan 1 is essential
for biological studies aimed at probing the exact role of the N-
glycoprotein in M. fervidus. Remarkable advances in the synthe-
sis of eukaryotic and bacterial N-glycoproteins have been
made over the past few years.[10] However, to the best of our
knowledge, no studies towards the synthesis of archaeal N-gly-
cans are reported so far. Herein we report the first total synthe-
sis of l-asparagine linked hexasaccharide 1 using a highly con-
vergent [3+3] glycosylation approach.
Protein glycosylation was once believed to be restricted to eu-
karyotes. However, it is now well established that this impor-
tant post-translational modification is also common in prokar-
yotes, including bacteria and archaea.[1] Intriguingly, all the
archaeal glycoproteins isolated so far are found to be N-glyco-
sylated whereas O-glycosylation is more prevalent in bacterial
glycoproteins.[2] Archaea are the most abundant group
amongst the three domains of life and are closely associated
with humans and other living organisms. Methanogenic arch-
aea are able to colonize and survive in humans.[3] Moreover,
there is growing evidence that they are engaged in syntrophic
relationship with other disease-causing microbes in polymicro-
bial diseases, such as chronic periodontitis,[4] colon cancer and
diverticulosis, and may have a role as human pathogens.[5] One
of the distinct features of archaea is that they thrive in harsh
environmental conditions of extreme temperature, salinity, pH
and pressure. Recent studies indicate that the glycan structures
help the archaea in survival under extreme environments.[6]
Furthermore, the archaeal glycoproteins show a great diversity
in glycan structures, which reflects species-specific means of
coping with the diverse surroundings.[7] The N-glycans are also
essential for cell motility.[8] To date, there are only thirteen N-
glycan structures isolated and characterized from archaea[2b]
and none of them has been synthesized yet. Chemical synthe-
sis of the archaeal N-glycans is regarded as a crucial step to-
wards delineating the role of protein glycosylation in archaea.
In 1993, Kꢁrcher et al.[9] isolated the N-glycan 1 from the pu-
rified S-layer glycoprotein of hyperthermophile Methanother-
mus fervidus and proposed its structure as [a-d-3-O-Me-Manp-
(l!6)-a-d-3-O-Me-Manp-((l!2)-(a-d-Manp)3-(l!4)-a-d-GalNAc]
using methylation analysis, plasma desorption mass spectrom-
etry, and high-field NMR spectroscopy (Figure 1). They also
speculated that the N-linked hexasaccharide could be involved
in the stabilization of this surface protein at high temperatures
and may play a role in cell aggregation. Access to chemically
The N-glycan 1 comprises a unique 3-O-methyl mannopyra-
nose containing a-(1!6) linked end disaccharide motif con-
nected in a-(1!3) manner to a a-(1!2) linked trimannoside,
which is in turn connected a-(1!4) to GalNAc at the reducing
end further attached through the nitrogen of l-asparagine
(Figure 1). Retrosynthetically, a convergent approach involving
a [3+3] glycosylation employing selectively protected thiogly-
cosides and subsequent N-aspartylation seemed appropriate
for the assembly of hexasaccharide 1.
Our synthesis began with preparation of 3-O-methyl manno-
pyranosyl building blocks for constructing the nonreducing
end disaccharide. For this purpose, a 4,6-O-benzylidenation of
thiomannoside followed by tin-mediated regioselective O3
methylation seemed straightforward. However, selective 4,6-O-
benzylidene protection of d-mannopyranoside is usually ac-
companied by the formation of unwanted 2,3-O-benzylidene
side product. As the 2,3-diol in mannopyranosides is oriented
in cis fashion, competing acetalation takes place at C-2/C-3.
Moreover, often the 2,3-acetal is formed as a mixture of exo/
endo diastereomers. The concomitant formation of the 2,3:4,6-
di-O-benzylidene product makes this reaction complex and te-
dious column purification results in low yields of the desired
product. Nevertheless, a few methods have been reported for
the selective preparation of monobenzylidene acetal of man-
nopyranosides under a variety of conditions with varying effi-
ciencies and yields.[11] In the course of our studies we observed
that employment of acetonitrile as solvent in place of DMF
dramatically increases the rate of the reaction and cleanly gen-
erates the 4,6-monobenzylidene derivative obviating the for-
mation of the 2,3-O-acetal. Thus, easily accessible a-thioman-
noside 2[12] upon treatment with benzaldehyde dimethylacetal
and (ꢀ)-10-camphorsulfonic acid (CSA) in acetonitrile afforded
monobenzylidene protected compound 3 (94%) exclusively
(Scheme 1). The reaction was complete in 5 min and the prod-
uct was precipitated out from acetonitrile solution. Compound
[a] S. R. Sanapala, Prof. Dr. S. S. Kulkarni
Department of Chemistry, Indian Institute of Technology Bombay
Powai, Mumbai, 400076 (India)
E-mail: suvarn@chem.iitb.ac.in
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201304950.
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Scheme 2. Synthesis of d-mannosyl 3-OH acceptor 9.
Scheme 1. Synthesis of 3-O-methyl thiomannoside building blocks 5 and 6.
3 could be easily purified by
a simple aqueous work-up and
precipitation in hexane. The
product so obtained was homo-
geneous on TLC and was free
from any detectable impurities
as judged by 1H NMR spectros-
copy. The efficient reaction could
be carried out on 20 g scale.
Tin-mediated
regioselective
methylation at O3 position of d-
mannopyranosides has been
studied well.[13] We could achieve
better yields of 3-O-methyl deriv-
ative 4 by modification of the
earlier reported procedure.[14]
Scheme 3. Synthesis of left-hand side trisaccharide donor 12.
Thus, diol 3 was refluxed in tolu-
ene with dibutyltin oxide to form the 2,3-stannylene acetal,
which was subsequently treated with methyl iodide in DMF at
508C to obtain 4 in 86% yield. Benzoylation of the remaining
2-OH group provided fully protected building block 5. A highly
regioselective reductive ring opening of 4,6-O-benzylidene
acetal in compound 5 with borane-tetrahydofuran complex
and catalytic amount of TMSOTf[15] afforded compound 6
(92%), which was used as an acceptor for ensuing glycosyla-
tion with donor 5 to construct the end disaccharide. Prepara-
tion of the 3-OH mannose building block 9 is shown in
Scheme 2. For this purpose, diol 3 was similarly transformed
into a stannylene acetal and subsequently treated with naph-
thylmethyl bromide and stoichiometric amount of cesium fluo-
ride to afford 3-O-naphthylmethyl protected derivative 7 (93%
over 2 steps) in a highly regioselective manner.[16] Hydrolysis of
the benzylidene group in 7 followed by benzylation provided
the fully protected building block 8 (77% over 2 steps), which
upon concomitant removal of naphthylmethyl group using
DDQ furnished 3-OH acceptor 9 (79%).
phenyl glycoside by NBS to obtain the corresponding hemiace-
tal and its treatment with trichloroacetonitrile and DBU in 89%
yield over two steps. Orthogonal coupling of donor 11 with 3-
OH mannopyranosyl acceptor 9 in the presence of TMSOTf fur-
nished the a-linked trisaccharide 12 in 98% yield.
The construction of the right-hand trisaccharide entailed
preparation of O2 differentiated mannose building blocks. For
this purpose, we resorted to tri-O-benzyl orthobenzoate 13, as
a suitable starting material, which could be easily prepared on
a multigram scale from d-mannose by using the procedure re-
ported by Seeberger and co-workers.[17]
Compound 13 underwent a smooth nucleophilic attack of
thiophenol[18] to afford 2-O-benzoyl thioglycoside 14 (70%;
Scheme 4). Deprotection of the benzoyl group was effected
using NaOMe in MeOH to obtain 2-OH building block 15
With the key building blocks 5, 6 and 9 in hand, the nonre-
ducing end trisaccharide 12 was assembled in a highly efficient
manner as shown in Scheme 3. First thioglycoside 5 was trans-
formed into the corresponding glycosyl bromide, which was
subsequently glycosylated with acceptor 6 in the presence of
silver triflate to obtain 3-O-methyl mannose containing a-
linked disaccharide 10 (60% over 2 steps). The thioglycoside
10 was then efficiently transformed into a-trichloroacetimidate
11 through a two step sequence involving hydrolysis of thio-
Scheme 4. Synthesis of building blocks 15 and 17 from orthobenzoate 13.
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(92%), which could act as an acceptor in the ensuing glycosy-
lation. On the other hand, nucleophilic ring opening of 13
with water in the presence of p-toluenesulfonic acid as acid
catalyst[19] generated hemiacetal 16 (80%), which was convert-
ed to imidate 17 (91%) by treatment with trichloroacetonitrile
and DBU.
For the preparation of the d-galactosamine building block,
we opted for C4 epimerization of the cheaply available d-glu-
cosamine derivative by Lattrell–Dax reaction.[20] Strategically,
the use of N-trichloroethoxycarbonyl (N-Troc) group was
deemed superior as it has been shown to enhance the reactivi-
ty of 4-OH in glucosamine series as compared to other N-pro-
tecting groups.[21] Also, in order to allow installation of the as-
paragine unit at the anomeric position, b-linked azido handle
was appropriate. The synthesis of suitably protected 4-OH d-
galactosamine derivative is shown in Scheme 5. The known d-
Scheme 6. Synthesis of right-hand side trisaccharide acceptor 29.
charide 28 in 79% yield. Removal of the chloroacetate group
by treatment with thiourea in pyridine afforded trisaccharide
acceptor 29 (81%).
The two trisaccharide fragments were joined together using
a [3+3] glycosylation to access the desired hexasaccharide
(Scheme 7). Glycosylation of donor 12 and acceptor 29 in the
Scheme 5. Synthesis of 4-OH GalNHTroc building block 24.
glucosamine derivative 18[22] was first treated with titanium(IV)
chloride[23] to afford a-glycosyl chloride 19,[24] which was
smoothly converted into b-glycosyl azide 20[25] (J1,2 =9.2 Hz) by
nucleophilic displacement under phase transfer conditions[26]
in 97% yield. Deprotection of the acetates in 20 using 20%
Et3N in MeOH[27] followed by subsequent 4,6-O-benzylidene
acetal formation afforded compound 21, which was benzoylat-
ed to obtain the fully protected d-glucosamine derivative 22.
A highly regioselective reductive ring opening of benzylidene
acetal using a combination of triethylsilane and trifluoroacetic
acid[28] afforded 4-OH glucosamine 23 (85%) and set the stage
for C4 epimerization. Inversion of the 4-OH in 23 by triflation
followed by nucleophilic displacement of the formed triflate
with nitrite ion smoothly delivered the requisite d-galactos-
amine derivative 24 in 79% yield over two steps.
With all the desired building blocks in hand, the reducing
end trisaccharide 29 was efficiently assembled as outlined in
Scheme 6. Glycosylation of imidate 17 and acceptor 15 using
TMSOTf as an activator afforded the required a-linked disac-
charide 25 in 86% yield. At this stage, the O2’ benzoyl protect-
ing group in 25 was replaced by a chloroacetyl group to form
27. Coupling of thioglycoside 27 and GalNHTroc acceptor 24
under NIS/TMSOTf promotion cleanly furnished a-linked trisac-
Scheme 7. Synthesis of N-glycan 1.
presence of NIS and TMSOTf at ꢁ108C afforded hexasaccharide
30 in 90% yield. The stage was now set for N-linked aspartyla-
tion, which is a challenging task and an important transforma-
tion in the synthesis of glycoproteins. The common problems
encountered in the coupling are: 1) competing formation of
aspartimide[29] under the conditions, and 2) the glycosyl amine
product formed by reduction of azide is relatively unstable and
is prone to anomerize[30] to give a mixture of anomers. An effi-
cient synthesis of N-linked glycosyl amino acid was achieved
by first reducing the azide to amine by treatment of com-
pound 30 with 1,3-propanedithiol in the presence of Hꢂnig’s
base[31] and then carrying out amide bond formation with
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known l-aspartic acid derivative[32] Boc-Asp-OtBu in the pres-
ence of 1m solutions of EDCI and HOBt in NMP.[33] The proce-
dure involved preactivation of aspartic acid for 1 h followed by
the addition of the amine to obtain the coupling product 31.
It should be noted that our earlier attempts to obtain the re-
quired product using other coupling reagents namely HBTU/
DIPEA[34] and TBTU/DIPEA in DMF as a solvent failed. A one-pot
Troc deprotection of 31, followed by acetylation employing
Zn/Ac2O in acetic acid[27] afforded compound 32 (81%). Global
deprotection of N-glycan 32 was accomplished in three steps.
Debenzoylation with 2N NaOMe in methanol followed by the
removal of tert-butyl ester and tert-butyl carbamate with 80%
TFA and finally debenzylation and benzylidene deprotection
under hydrogenation conditions using Pd(OH)2 in 50% acetic
acid furnished the target N-glycan 1 in 77% overall yield. All
the compounds were thoroughly characterized using spectral
means and all the newly generated mannosidic linkages after
each glycosylation were determined as a on the basis of the
1JCH values which were in the range of 168.8 to 175.0 Hz (see
the Supporting Information).
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In conclusion, we have successfully achieved the first total
synthesis of archaeal l-asparagine linked hexasaccharide from
Methanothermus fervidus by a highly convergent [3+3] glycosy-
lation route. The synthesis involves efficient preparation of re-
gioselectively protected thioglycoside building blocks for or-
thogonal glycosylations in high selectivity and yields and a late
stage N-aspartylation.
Acknowledgements
This work was supported by the Department of Science and
Technology (Grant No. SR/S1/OC-40/2009) and Council of Sci-
entific and Industrial Research (Grant No. 01(2376)/10/EMR-II).
S.R.S. thanks UGC-New Delhi for a fellowship.
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Keywords: archaea
· glycosylation · glycoproteins · N-
aspartylation · N-glycans
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Received: December 18, 2013
Revised: February 2, 2014
Published online on March 11, 2014
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