Traceless Photolabile Linker Expedites the Chemical Synthesis of Complex Oligosaccharides by Automated Glycan Assembly

Article abstract of DOI:10.1021/jacs.9b03769

Automated glycan assembly (AGA) aims at accelerating access to synthetic oligosaccharides to meet the demand for defined glycans as tools for molecular glycobiology. The linkers used to connect the growing glycan chain to the solid support play a pivotal

Full text of DOI:10.1021/jacs.9b03769

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Article
Traceless Photolabile Linker Expedites Chemical Synthesis of
Complex Oligosaccharides by Automated Glycan Assembly
Kim Le Mai Hoang, Alonso Pardo-Vargas, Yuntao Zhu, Yang
Yu, Mirco Loria, Martina Delbianco, and Peter H Seeberger
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b03769 • Publication Date (Web): 15 May 2019
Downloaded from http://pubs.acs.org on May 15, 2019
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Traceless Photolabile Linker Expedites Chemical Synthesis of
Complex Oligosaccharides by Automated Glycan Assembly
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Kim Le Mai Hoang,† Alonso Pardo-Vargas,†,‡ Yuntao Zhu,† Yang Yu,†,‡ Mirco Loria,‡ Martina
Delbianco,† and Peter H. Seeberger*,†,‡.
†Department of Biomolecular Systems, Max-Planck-Institute of Colloids and Interfaces, Am Mühlenberg 1, 14476
Potsdam, Germany
‡Institute of Chemistry and Biochemistry, Freie Universität Berlin, Arnimallee 22, 14195 Berlin, Germany
ABSTRACT: Automated glycan assembly (AGA) aims at accelerating access to synthetic oligosaccharides to meet the de-
mand for defined glycans as tools for molecular glycobiology. The linkers used to connect the growing glycan chain to the
solid support play a pivotal role for the synthetic strategy as they determine all chemical conditions used during the syn-
thesis and the form of the glycan obtained at the end of it. Here, we describe a traceless photolabile linker to prepare car-
bohydrates with a free reducing end. Modification of the o-nitrobenzyl scaffold of the linker is key to high yields and
compatibility with the AGA workflow. The assembly of an asymmetrical bi-antennary N-glycan from oligosaccharide
fragments prepared by AGA and linear as well as branched β-oligoglucans are described to illustrate the power of the
method. These substrates will serve as standards and biomarkers to examine the unique specificity of glycosyl hydrolases.
compatible with a wide range of protecting groups such
as carbonates (9-flourenylmethoxycarbonyl, Fmoc), esters
INTRODUCTION
The procurement of complex oligosaccharides in satisfac-
tory quantity and quality as tools for glycobiology remains
challenging. Harvesting from natural sources is untena-
ble, because the post-translational glycosylation pathway
is heavily influenced by minute changes in the environ-
ment of the living cell, leading to multiple glycoforms.1 At
present, chemical and enzymatic approaches, or combina-
tions thereof, are often the only reliable methods to ac-
cess pure glycans.2 Efforts to reduce the time and re-
sources spent on traditional chemical syntheses have fo-
cused mainly on automated glycan assembly (AGA)3 and
computer-assisted one-pot synthesis.4
(levulinoyl, Lev and benzoate, Bz) as well as ethers (ben-
zyl, Bn). These linkers are cleaved to afford glycans carry-
ing an aminoalkyl spacer at the reducing end. The result-
ing oligosaccharides, after global deprotection, are ready
for conjugation to carrier proteins to give glycoconjugate
vaccine candidates,9 or to be immobilized on microarrays
for high-throughput analysis of protein-carbohydrate in-
teractions.10
Here, we report the design and use of the spacer-free
photolabile linker 5 that retains the excellent chemical
orthogonality and enables access to protected glycans
with a free reducing end. These in turn can be trans-
formed into the corresponding glycosyl fluorides, phos-
phates, and trichloroacetimidates. Oligosaccharide frag-
ments produced in this way can be united in block cou-
plings to prepare more complex polysaccharides. Fur-
thermore, free reducing glycans are valuable analytical
standards to inspect fragmentation patterns in tandem
mass spectrometry,11 or for glycosynthase assays that de-
mand linker-free glycan epitopes as precursors. Photola-
bile linker 5 offers flexibility compared to previous meth-
ods,12 which prepared glycans with a benzyl group at the
reducing terminus. The new linker 5 is illustrated for the
AGA of a penta- and a trisaccharide that are combined in
a [5+3] glycosylation strategy to obtain an asymmetric bi-
antennary N-glycan fragment. Additionally, several struc-
turally defined linear and branched laminarin β-(1,3)-
oligoglucans are prepared, which will be instrumental to
elucidate the hydrolytic mechanism of various hydrolases
AGA relies on the delivery of building blocks to a sus-
pension of solid support that is equipped with a linker,
followed by addition of a suitable activating solution us-
ing either home-built instruments or the commercial Gly-
coneer 2.1 glycan synthesizer.5 Precise control of im-
portant reaction parameters such as temperature and
concentration under an inert atmosphere allows for near
quantitative coupling of building blocks to the resin. Re-
petitive cycles consisting of glycosylation, capping and
selective deprotection steps extend the glycan chain to
the desired structure. Different linkers for AGA were ex-
plored (Figure 1), including the metathesis-labile linker 1,6
the base-labile linker 2,7 and the photocleavable linkers 3
and 4.8 Photocleavable linkers such as 3 and 4 are often
referred to as “traceless” since the on-resin removal of
photosensitive groups only require exposure to light to
release the product into the solution. They have proven
stable under both acidic and basic conditions and are
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Figure 1. Linkers for AGA.
from marine bacteria. Investigations into these highly
specific processes are hampered by the lack of high purity
-glucans from seawater that contain heterogenous gly-
cans released by different microbial species.
trophotometry15 and revealed loading values ranging from
0.32 to 0.36 mmol/g.
Linker 8 was prepared by reduction of 5-hydroxy-2-
nitrobenzaldehyde 9 and immobilization on Merrifield
resin by heating with Cs2CO3 in the presence of catalytic
amounts of TBAI (Scheme 1A). Linker 3 was prepared
starting from the reductive condensation of 9 with 5-
amino-pentan-1-ol to yield 11 in 85% yield.8 Addition of
Cbz to the secondary amine was followed by immobiliza-
tion onto Merrifield resin to obtain the linker-equipped
resin 3 (Scheme 1B).
RESULTS AND DISCUSSION
Design and Synthesis of Photolabile Linkers. One of
the most frequently used photolabile groups13 to date is
the methyl-6-nitroveratryl (MeNV) that introduces two
modifications to the o-nitrobenzyl (oNB) scaffold: the α-
methyl group at the benzylic carbon and two methoxy
groups on the aromatic ring. The methyl group was
thought to improve the chemical yield by the release of a
ketone byproduct instead of an aldehyde, which is less
prone to side-reactions.13 Meanwhile, the introduction of
electron donating substituents on the aromatic ring in-
creases the absorption coefficient at longer wavelengths,
to match the peak emission at 366 nm from a mercury UV
lamp.14 A design based on the MeNV motif should im-
prove the photocleavage process. To provide an anchor
point to the solid support, one of the methoxy groups of
the MeNV was substituted. Three different MeNV-type
linkers 5, 6 and 7 (Figure 2) were prepared starting from
apocynin, an abundant plant extract. After photocleavage,
the resin equipped with linker 5 will produce spacer-free
reducible glycans while linker 6 returns glycans with the
aminoalkyl spacer and linker 7 produces benzyl-protected
glycans at the reducing end. For a direct comparison, the
oNB-type linkers 8 and 3 were prepared to provide the
same products as linkers 5 and 6. Resin loading was de-
termined for each batch of resin by glycosylation of 50 mg
resin with 6-O-Fmoc mannose building block A on the
synthesizer (Scheme 2). Cleavage of the Fmoc group pre-
sent on the solid support was quantified by UV-vis spec-
Figure 2. Merrifield resins functionalized with photolabile
linkers for AGA.
Syntheses of linkers containing the MeNV scaffold
started from allyl-protected apocynin 12 that was selec-
tively nitrated with KNO3 in TFA. This reaction is very
sensitive to the initial temperature16 as di-nitrated prod-
ucts were formed when a slight excess of KNO3 was intro-
duced at 25 C. Reactions initiated at 0 C produced only
trace amounts of 13. The reaction proceeded best when
KNO3 was added portion-wise at exactly 10C, followed by
heating at 60 C for four hours to give mono-nitro 13 se-
lectively (59% conversion starting from 12). Next, reduc-
tion of the ketone and cleavage of the allyl group employ-
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ing Pd(PPh3)4 proceeded smoothly to provide precursor Evaluation of Photolabile Linkers for AGA. With the
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15, ready for conjugation with the resin to give linker 5
(Scheme 1C). Utilizing intermediates 13 and 14, the N-
linked and O-benzylether-linked variations of linker 5
were prepared. The reductive amination of ketone 13 was
known for its poor yield.17 Reasonable conversion was
achieved by using Ti(OiPr)4 in an one-pot transformation
of 13 to the Cbz-protected compound 16 (39% over three
steps).18 Removal of the allyl group and on-resin function-
alization afforded linker 6 (Scheme 1D). Double deallyla-
tion of 17 to linker 7 proceeded best when 1,3-
dimethylbarbituric acid replaced K2CO3 as scavenger, as
the latter failed to completely remove the protecting
groups (Scheme 1E).
photocleavable linkers in hand, evaluation of the support-
bound photolytic process was based on the quantity of
fully protected oligosaccharides released after cleavage
(Scheme 2). Recovery of cleaved product is an important
parameter that emphasizes the practical utility of each
functionalized resin. The automated synthesis of tetra-
mannose 22 proceeded as follows: functionalized resin 3
(40 mg = 0.013 mmol) was placed in the reaction vessel
before the acidic wash module I was executed (TMSOTf
in CH2Cl2 at -20 C for 3 min) to remove any residual base
and water from the resin. Next, module IIa delivered 6.5
equivalents of mannose A and a solution of activator
(NIS/TfOH in CH2Cl2/dioxane, -20 C for 5 min, then 0 C
for 20 min) to the reaction vessel. Capping module III
(MsOH in Ac2O/CH2Cl2, 20 min) masked any unreacted
hydroxyl groups, followed by cleavage of the Fmoc car-
bonate on the C-6 hydroxyl group with module IVa (20%
piperidine in DMF, 5 min). The module sequence I-IIa-
III-IVa was repeated four times to obtain the resin-bound
tetra-mannose 22. Cleavage of 22 from the solid support
was achieved using a continuous flow photoreactor.19
Mono-mannose, tetra-α-(1,6)-mannose and hexa--(1,4)-
glucose were assembled on resins equipped with different
linkers. All linkers proved to be compatible with all
standardized AGA protocols. No trace of deletion se-
quences was observed in any of the HPLC chromatograms
of the crude products after photocleavage. Thus, differ-
ences in the isolated yield should correlate exclusively
with the photolytic sensitivity of each linker. Linkers 5
and 8, for example, both delivered the free reducing end
sugars, but differed significantly in their photolytic effi-
ciency. For oNB-type linker 8, the highest conversion ob-
served was monomer 18 at only 34%, with even lower
yields for tetra-mannose (21, 22%) and hexa-glucose (24,
17%). In contrast, MeNV-type linker 5 consistently deliv-
ered the same products in 65-70% yield. O-linked resin 5
performed best in this study, surpassing both the N-
linked resin 6 and O-Bn-linked resin 7.
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Scheme 1. Synthesis of photolabile linkers.
The resin equipped with N-linked oNB 3 outperformed
the N-linked MeNV-type resin 6 in all experiments to
procure 5-aminopentyl mannoside 19, tetra-mannoside 22
and hexa-glucoside 25. Still, the resin with linker 6 re-
leased more product than with linker 8. The photolytic
sensitivity of O-Bn-linked resin 7 was found to be be-
tween that of the N-linked resins 3 and 6. The following
order of photocleavage performance was observed: 5 > 3 >
7 > 6 > 8 (direct O-linked MeNV-type > N-linked oNB-
type > O-Bn-linked MeNV-type > N-linked MeNV-type >
direct O-linked oNB-type). This seemingly counterintui-
tive series highlights one of the caveats when picking a
suitable photocleavable scaffold for a specific leaving
group. The photolytic process depends both on nature of
the leaving groups and the photo-labile core.20 Hence, to
prepare reducible glycans by AGA, the use of MeNV-type
linkers 5 or 7 is most efficient. For conjugation-ready oli-
gosaccharides carrying a terminal amine, the best option
is oNB-type linker 3.
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Scheme 2. Comparison of Photolabile Linkers.
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Assembly of an Asymmetric N-glycan Fragment.
AGA of asymmetrically branched octasaccharide 27, pre-
sent in complex-type N-glycans served to illustrate the
utility of the new linker (Figure 3). The complexity of N-
glycans and their biological importance has rendered
them attractive targets to test chemical25-28 and chemoen-
zymatic synthesis methods.29-32 Following the convergent
strategy employed by most solution-phase syntheses of
bi-antennary N-glycans, we embarked on a [5+3] glycosyl-
ation strategy. Target oligosaccharide 27 was constructed
by union of trisaccharide donor 28 and pentasaccharide
acceptor 29, that were both prepared by AGA (Figure 3).
Resins with photocleavable linkers 5 and 3 and six com-
mercially available building blocks (C-H) were employed
in the automated syntheses.
The automated synthesis of donor 28 commenced with
glucosamine C (Figure 4) and the orthogonal removal of
levulinoyl ester at C-3 position was achieved by executing
module IVb (N2H4.HOAc in pyridine). Stereoselective α-
fucosylation was secured by running the glycosylation
module IIa twice for fucose D at lower temperature (-40
C for 5 min, then -20 C for 20 min). The Fmoc group at
the C-4 position of C was removed by module IVa (20%
piperidine in DMF). Next, the modules I-IIa-IVa-III were
executed to introduce galactose F as the third sugar. By
capping after the deprotection module, the Fmoc group at
C-6 of galactose E was converted to the more resilient
acetate group. At any point during the automated pro-
cess, a small amount of resin could be extracted, irradiat-
ed using a portable UV lamp (6 W, 366 nm) for ten
minutes to release the glycans from the solid support.
This convenient real time monitoring of the reaction pro-
gress via HPLC and MALDI analysis helped to optimize
the reaction conditions and identify potential problems.
Photocleavage from the solid support afforded trisaccha-
ride 30 as a mixture of α/-anomers in 52% yield. Fluori-
nation of 30 by Deoxo-Fluor yielded glycosyl fluoride 28
Figure 3. Retrosynthetic analysis of octasaccharide 27.
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each compound revealed that the main glycan was accep-
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tor 29 and the minor its diastereoisomer having an aber-
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rant Man--(1,3)-Man glycosidic bond with a JC,H value of
160 Hz at 95.8 ppm (Figure 5A, circled in red). To support
this assignment, AGA of glycan sequence F2GH2E pro-
ceeded smoothly to afford the congener tetrasaccharide
as the sole product (see ESI, compound S6). The poor
stereo-selectivity (α/ = 3:2) of the last glycosylation cycle
leading to 29 was unexpected since the preliminary
screening36 of all building blocks showed excellent selec-
tivity. The “double stereodifferentiation” effect, also re-
ferred to as the “matched and mismatched” principle,
may account for the stereochemical scrambling in the
synthesis of -(1,3)-glucans.37 A similar mechanism could
be responsible for the substantial ratio of -isomer of 29.
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With both glycosyl donor 28 and glycosyl acceptor 29 in
hand, the glycosylation reaction promoted by AgOTf/
Cp2HfCl2 furnished octasaccharide 27 in 21% isolated yield
(Scheme 3). An aliquot taken from the reaction vial after
four hours was analyzed by analytical HPLC and showed
near complete disappearance of donor 20. Product 27 was
Figure 4. Automated synthesis of trisaccharide donor 28. A
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typical glycosylation cycle included the module sequence I-
IIa-III-IVa, represented by the letter of the building block
being used, e.g. C. Changes in the sequence or parameters
were indicated with break line and box.
in 89% yield after HPLC purification. Glycosyl fluorides
are glycosyl donors for both chemical33 and enzymatic34
trans-glycosylation reactions.
With glycosyl donor 28 in hand, the automated synthe-
sis of glycosyl acceptor 29 was initiated (Figure 5).
Branching at the C-6 position of mannose F was achieved
via step-wise introduction of mannose G, glucosamine H,
and galactose E with an on-resin capping of C-6 as acetyl
ester (module IVa, then III). The Lev ester on mannose F
was cleaved to reveal the C-3 hydroxyl group, ready for
branching with mannose building block G. Fmoc removal
on G concluded the automated sequence before photolyt-
ic release furnished acceptor 29 (Figure 5A). However, the
building block sequence FGHEG was not crowned by
success. Analysis of the crude mixture by HPLC and
MALDI revealed the presence of several deletion se-
quences and regioisomeric products (Figure 5B). Deletion
sequences resulting from incomplete couplings were
avoided by performing the glycosylation modules twice
on unreactive building blocks such as F and H. Fmoc
cleavage using piperidine was previously found to be ac-
companied by partial migration of Lev to the liberated
hydroxyl group.35 This unwanted byproduct was not
formed when Et3N was used as base. Thus, we introduced
the deprotection module IVc (20% Et3N in DMF) to re-
place module IVa. Glycosylation module IIa was per-
formed twice for building blocks F and H. The coupling
time to introduce building block H and E was increased
from 20 to 40 min. Gratifyingly, the refined building block
sequence F2GH2EG significantly improved the synthesis,
as evident from the HPLC traces of the crude product
showing only two peaks (Figure 5C). Structural analysis of
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Figure 5. (A) Automated synthesis of pentasaccharide accep-
tor 29 using building blocks E-H. (B) Standard AGA condi-
tion led to complicated HPLC traces of crude products. (C)
Optimized condition revealed glycan 29 as a mixture of dia-
stereoisomers.
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found to elude in close proximity to glycosyl acceptor 29. Automated Synthesis of Oligo-β-Glucans. Marine al-
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The fraction containing only glycan 27 was collected and
1H-, 13C-, and coupled HSQC-NMR experiments confirmed
the homogeneity of the sample, indicating a successful -
glucosamidation. The synthesis of the asymmetrical N-
glycan octasaccharide 27 from glycan fragments prepared
by AGA highlights the utility of the traceless photolabile
linker 5 for the convergent synthesis of complex oligosac-
charides.
gae are major carbon sink that convert carbon dioxide
into carbohydrate materials such as laminarin, an oligo-
saccharide comprising of -(1,3)-linked glucose with vari-
able degrees of -(1,4)- and -(1,6)-glucose residues in the
backbone and the branches. Studies concerning the lami-
narin hydrolase mechanism require homogenous -(1,3)-
glucans. Traceless resin 5 expedites the automated syn-
thesis of oligosaccharides that serve as analytical stand-
ards and substrates to investigate the ecological roles of
laminarin.
9
Scheme 3. Assembly of octasaccharide 27.
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Convergent solution-phase syntheses of -(1,3)-glucans
were hampered by low yields and aberrant α-linkage for-
mation due to “double stereo-differentiation” effects, as
well as limitation in chain length, position and degree of
branching. AGA of linear and branched -(1,3)-glucans
carrying an aminoalkyl spacer at the reducing end was
achieved earlier using glycosyl phosphate building blocks
and photolabile linker 3.38. The unprotected and spacer-
free laminarin derivatives 31-34 were chosen as model
substrates to compare the linker performance to the earli-
er study (Scheme 4). In addition to linear -(1,3)-
heptaglucose 31, different branched -glucans such as
penta-glucose 32, as well as octa-glucoses 33 and 34 were
prepared (Scheme 4, circled in red). The more reactive
phosphate building blocks K and L required 4.0 equiva-
lents for the AGA cycle. Extension of the linear β-(1,3)-
glucose backbone employed building block K bearing
Fmoc group at the C-3 position. For structures with a β-
(1,6)-branch, the linear backbone was first built up using
the pre-defined sequence of glycosyl phosphates K and L.
Building block L contains a C-3 Fmoc protected hydroxyl
group and a Lev ester on the C-6 hydroxyl. Selective re-
moval of the levulinoyl ester allowed for the extension of
the β-(1,6)-branch. Module IVa (20% piperidine in DMF)
was used to remove Fmoc, except when Lev was present,
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Time (mins)
Scheme 4. Automated Synthesis of Oligo-β-Glucans.
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in which case module IVc (20% Et3N in DMF) was em-
REFERENCES
ployed. Further optimization36 resulted in a 20% reduc-
tion of building block consumption. The methanolysis of
base-labile protecting groups directly on the solid support
was more effective than in solution. The partially protect-
ed oligosaccharides were photocleaved from the resin and
immediately debenzylated via hydrogenation using Pd/C
catalyst (60 psi in under one hour, module VII). With this
protocol, only a single purification with reverse-phase
HPLC was needed to yield oligosaccharides 31 and 32 in
30% and 22% yield respectively. Branched octasaccharides
33 and 34 were isolated in lower quantity as these glycans
unexpectedly fragmented during hydrogenation to create
mixtures of truncated glycans.
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(1) Varki, A.; Cummings, R. D.; Esko, J. D.; Freeze, H. H.; Stan-
ley, P.; Bertozzi, C. R.; Hart, G. W.; Etzler, M. E. Essentials of
Glycobiology; Cold Spring harbor Laboratory Press: 2009.
(2) Hsu, C.-H.; Hung, S.-C.; Wu, C.-Y.; Wong, C.-H. Toward
Automated Oligosaccharide Synthesis. Angew. Chem., Int. Ed.
2011, 50, 11872-11923.
(3) (a) Seeberger, P. H. The Logic of Automated Glycan As-
sembly. Acc. Chem. Res. 2015, 48, 1450-1463. (b) Pardo-Vargas,
A.; Delbianco, M.; Seeberger, P. H. Automated Glycan Assembly
as an Enabling Technology. Curr. Opin. Chem. Biol. 2018, 46, 48-
55.
(4) Wang, Y.; Ye, X.-S.; Zhang, L.-H. Oligosaccharide Assem-
bly by One-Pot Multi-Step Strategy. Org. Biomol. Chem. 2007, 5,
2189-2200.
(5) Hahm, H. S.; Schlegel, M. K.; Hurevich, M.; Eller, S.;
Schuhmacher, F.; Hofmann, J.; Pagel, K.; Seeberger, P. H. Auto-
mated Glycan Assembly Using the Glyconeer 2.1 Synthesizer.
Proc. Natl. Acad. Sci. USA. 2017, 114, E3385-E3389.
(6) (a) Love, K. R.; Seeberger, P. H. Automated Solid-Phase
Synthesis of Protected Tumor-Associated Antigen and Blood
Group Determinant Oligosaccharides. Angew. Chem., Int. Ed.
2004, 43, 602-605. (b) de Jong, A. R.; Volbeda, A. G.; Hagen, B.;
van den Elst, H.; Overkleeft, H. S.; van der Marel, G. A.; Codée, J.
D. C. A Second-Generation Tandem Ring-Closing Metathesis
Cleavable Linker for Solid-Phase Oligosaccharide Synthesis. Eur.
J. Org. Chem. 2013, 29, 6644-6655.
(7) (a) Krock, L.; Esposito, D.; Castagner, B.; Wang, C.-C.;
Bindschadler, P.; Seeberger, P. H. Streamlined Access to Conju-
gation-Ready Glycans by Automated Synthesis. Chem. Sci. 2012,
3, 1617-1622. (b) Wu, X.; Grathwohl, M.; Schmidt, R. R. A New
Phenoxyacetate-Based Linker System for the Solid-Phase Syn-
thesis of Oligosaccharides. Org. Lett. 2001, 3, 747-750. (c)
Czechura, P.; Guedes, N.; Kopitzki, S.; Vazquez, N.; Martin-
Lomas M.; Reichardt, N.-C. A New Linker for Solid-Phase Syn-
thesis of Heparan Sulfate Precursors by Sequential Assembly of
Monosaccharide Building Blocks. Chem. Commun. 2011, 47,
2390-2392.
(8) (a) Eller, S.; Collot, M.; Yin, J.; Hahm, H. S.; Seeberger, P.
H. Automated Solid-Phase Synthesis of Chondroitin Sulfate Gly-
cosaminoglycans. Angew. Chem., Int. Ed. 2013, 52, 5858-5861. (b)
Wilsdorf, M.; Schmidt, D.; Bartetzko, M. P.; Dallabernardina,
P.;Schuhmacher, F.; Seeberger, P. H.; Pfrengle, F. A Traceless
Photocleavable Linker for the Automated Glycan Assembly of
Carbohydrates with Free Reducing Ends. Chem. Commun. 2016,
52, 10187-10189.
(9) (a) Schumann, B.; Hahm, H. S.; Parameswarappa, S. G.;
Reppe, K.; Wahlbrink, A.; Govindan, S.; Kaplonek, P.; Pirofski, L.
A.; Witzenrath, M.; Anish, C.; Pereira, C. L.; Seeberger, P. H. A
Semisynthetic Streptococcus Pneumoniae Serotype 8 Glycocon-
jugate Vaccine. Sci. Transl. Med. 2017, 9, eaaf5347. (b) Weis-
haupt, M. W.; Matthies, S.; Hurevich, M.; Pereira, C. L.; Hahm,
H. S.; Seeberger, P. H. Automated Glycan Assembly of a S.
Pneumoniae Serotype 3 CPS Antigen. Beilstein J. Org. Chem.
2016, 12, 1440-1446.
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CONCLUSION
Traceless photolabile linkers for automated glycan as-
sembly were developed to prepare complex oligosaccha-
rides with a free reducing end. For oligosaccharides with
an aminoalkyl spacer at the reducing end, o-NB type link-
er 3 offers the best photocleavage efficiency, whereas for
spacer-free hemiacetal glycans, MeNV-type linker 5 is the
best choice. The new linker enabled the convergent syn-
thesis of an asymmetrically branched N-glycan octasac-
charide and of pure laminarins. Levulinoyl ester migra-
tion during Fmoc cleavage is suppressed by using Et3N as
base. AGA of four -(1,3)-glucans was achieved using 20%
less building block than previously. A new protocol for
the global deprotection of oligosaccharides with free re-
ducing end was developed. AGA using the new linkers
enables access to glycans that can be converted into gly-
cosylating agents to be used on block couplings. The gly-
cans with a free reducing end are valuable standards for
mass spectrometry and biological assays.
ASSOCIATED CONTENT
Supporting Information.
The supporting information is available free of charge via the
Internet at http://pubs.acs.org.
Synthetic procedures, AGA modules, and characterization
data of glycans, including HPLC chromatograms and NMR
spectra
AUTHOR INFORMATION
Corresponding Author
*Peter.Seeberger@mpikg.mpg.de
ORCID
Peter H. Seeberger: 0000-0003-3394-8466
(10) (a) Geissner, A.; Reinhardt, A.; Rademacher, C.; Johanns-
sen, T.; Monteiro, J.; Lepenies, B.; Thépaut, M.; Fieschi, F.; Mráz-
ková, J.; Wimmerova, M.; Schuhmacher, F.; Götze, S.; Grünstein,
D.; Guo, X.; Hahm, H. S.; Kandasamy, J.; Leonori, D.; Martin, C.
E.; Parameswarappa, S. G.; Pasari, S.; Schlegel, M. K.; Tanaka, H.;
Xiao, G.; Yang, Y.; Pereira, C. L.; Anish, C.; Seeberger, P. H.
Microbe-Focused Glycan Array Screening Platform. Proc. Natl.
Acad. Sci. USA. 2019, 116, 1958-1967. (b) Seeberger, P. H. Glycan
Arrays and Other Tools Produced by Automated Glycan Assem-
bly. Perspect. Sci. 2017, 11, 11-17. (c) Broecker, F.; Seeberger, P. H.
Synthetic Glycan Microarrays. Methods Mol. Biol. 2017, 1518, 227-
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
We thank the Max-Planck Society and EU ITN Marie-Curie
program (IMMUNOSHAPE Grant No. 642870) for generous
financial support. We thank Eva Settels and Olaf Niemeyer
for technical support.
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240. (d) Dallabernardina, P.; Ruprecht, C.; Smith, P. J.; Hahn, M.
Number and Degree of Branching Cooperate to Regulate Cell
Proliferation and Differentiation. Cell, 2007, 129, 123-134.
G.; Urbanowicz, B. R.; Pfrengle, F. Automated Glycan Assembly
of Galactosylated Xyloglucan Oligosaccharides and Their Recog-
nition by Plant Cell Wall Glycan-Directed Antibodies. Org. Bio-
mol. Chem. 2017, 15, 9996-10000. (e) Park, S.; Gildersleeve, J. C.;
Blixt, O.; Shin I. Carbohydrate Microarrays. Chem. Soc. Rev. 2013,
42, 4310-4326. (f) Rillahan C.D.; Paulson, J. C. Glycan Microarrays
for Decoding the Glycome. Annu. Rev. Biochem. 2011, 80, 797-
823.
(11) (a) Gagarinov, I. A.; Li, T.-H.; Toraño, J. S.; Caval, T.; Sri-
vastava, A. D.; Kruijtzer, J. A. W.; Heck, A. J. R.; Boons, G.-J.
Chemoenzymatic Approach for the Preparation of Asymmetric
Bi-, Tri-, and Tetra-Antennary N-Glycans from a Common Pre-
cursor. J. Am. Chem. Soc. 2017, 139, 1011-1018. (b) North, S. J.;
Hitchen, P. G.; Haslam, S. M.; Dell, A. Mass Spectrometry in the
Analysis of N-linked and O-linked Glycans. Curr. Opin. Struct.
Biol. 2009, 19, 498-506.
(12) (a) Boltjie, T. J.; Kim, J.-H.; Park, J.; Boons, G.-J. Chiral-
Auxiliary-Mediated 1,2-Cis-Glycosylations for the Solid-
Supported Synthesis of a Biologically Important Branched α-
Glucan. Nat. Chem. 2010, 2, 552-557. (b) Wu, X.; Grathwohl, M.;
Schmidt, R. R. Efficient Solid-Phase Synthesis of a Complex,
Branched N-Glycan Hexasaccharide: Use of a Novel Linker and
Temporary-Protecting-Group Pattern. Angew. Chem., Int. Ed.
2002, 41, 4489-4493.
1
2
3
4
5
6
7
8
(25) (a) Chiesa, M. V.; Schmidt, R. R. Synthesis of an Aspara-
gine-Linked Heptasaccharide - Basic Structure of N-Glycans.
Eur. J. Org. Chem. 2000, 21, 3541-3554. (b) Jonke, S.; Liu, K. G.;
Schmidt, R. R. Solid-Phase Oligosaccharide Synthesis of a Small
Library of N-Glycans. Chem. Eur. J. 2006, 12, 1274-1290.
(26) (a) Walczak, M. A.; Hayashida, J.; Danishefsky, S. J. Build-
ing Biologics by Chemical Synthesis: Practical Preparation of Di-
and Triantennary N-Linked Glycoconjugates. J. Am. Chem. Soc.
2013, 135, 4700-4703. (b) Wang, P.; Zhu, J.; Yuan, Y.; Danishefsky,
S. J. Total Synthesis of the 2,6-Sialylated Immunoglobulin G
Glycopeptide Fragment in Homogeneous Form. J. Am. Chem.
Soc. 2009, 131, 16669-16671. (c) Wu, B.; Hua, Z.; Warren, J. D.;
Raganathan, K.; Wan, Q.; Chen, G.; Tan, Z.; Chen, J.; Endo, A.;
Danishefsky, S. J. Synthesis of the Fucosylated Biantennary N-
Glycan of Erythropoietin. Tetrahedron Lett. 2006, 47, 5577-5579.
(27) (a) Manabe, Y.; Shomura, H.; Minamoto, N.; Nagasaki,
M.; Takakura, Y.; Tanaka, K.; Silipo, A.; Molinaro, A.; Fukase, K.
Convergent Synthesis of a Bisecting N-Acetylglucosamine (Glc-
NAc)-Containing N-Glycan. Chem. Asian J. 2018, 13, 1544-1551. (b)
Nagasaki, M.; Manabe, Y.; Minamoto, N.; Tanaka, K.; Silipo, A.;
Molinaro, A.; Fukase, K. Chemical Synthesis of a Complex-Type
N-Glycan Containing a Core Fucose. J. Org. Chem. 2016, 81,
10600-10616.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(13) Klán, P.; Šolomek, T.; Bochet, C. G.; Blanc, A.; Givens, R.;
Rubina, M.; Popik, V.; Kostikov, A.; Wirz, J. Photoremovable
Protecting Groups in Chemistry and Biology: Reaction Mecha-
nisms and Efficacy. Chem. Rev. 2013, 113, 119-191.
(14) Holmes, C. P. Model Studies for New o-Nitrobenzyl Pho-
tolabile Linkers:ꢀ Substituent Effects on the Rates of Photochem-
ical Cleavage. J. Org. Chem. 1997, 62, 2370-2380.
(15) Gude, M.; Ryf, J.; White, P. D. An Accurate Method for the
Quantitation of Fmoc-Derivatized Solid Phase Supports. Lett.
Pept. Sci. 2002, 9, 203-206.
(16) Hoffmann, J.; Kazmaier, U. Development of a New
NPPOC-Derived Photolabile Protecting Group Suitable for Cy-
clizations via Ring Closing Metathesis. Curr. Org. Synth. 2015, 12,
475-483.
(17) Wilcox, M.; Viola, R. W.; Johnson, K. W.; Billington, A. P.;
Carpenter, B. K.; McCray, J. A.; Guzikowski, A. P.; Hess, G. P.
Synthesis of Photolabile Precursors of Amino Acid Neurotrans-
mitters. J. Org. Chem. 1990, 55, 1585-1589.
(18) Salmi, C.; Letourneux, Y.; Brunel, J. M. Efficient Synthesis
of Various Secondary Amines Through a Titanium(IV) iso-
propoxide-Mediated Reductive Amination of Ketones. Lett. Org.
Chem. 2006, 3, 396-401.
(19) Bartetzko, M. P.; Schuhmacher, F.; Hahm, H. S.;
Seeberger, P. H.; Pfrengle, F. Automated Glycan Assembly of
Oligosaccharides Related to Arabinogalactan Proteins. Org. Lett.
2015, 17, 4344-4347.
(20) Šolomek, T.; Mercier, S.; Bally, T.; Bochet, C. G. Photoly-
sis of Ortho-Nitrobenzylic Derivatives: The Importance of the
Leaving Group. Photochem. Photobiol. Sci. 2012, 11, 548-555.
(21) (a) Ohtsubo, K.; Marth, J. D. Glycosylation in Cellular
Mechanisms of Health and Disease. Cell, 2006, 126, 855-867. (b)
Lauc, G.; Pezer, M.; Rudan, I.; Campbell, H. Mechanisms of Dis-
ease: The Human N-Glycome. Biochim. Biophys. Acta, 2016,
1860, 1574-1582.
(28) (a) Mꢀnnich, M.; Eller, S.; Karagiannis, T.; Perkams, L.;
Luber, T.; Ott, D.; Niemietz, M.; Hoffman, J.; Walcher, J.; Berger,
L.; Pischl, M.; Weishaupt, M.; Wirkner, C.; Lichtenstein, R. G.;
Unverzagt, C. Highly Efficient Synthesis of Multiantennary Bi-
sected N-Glycans Based on Imidates. Angew. Chem., Int. Ed.
2016, 55, 10487-10492. (b) Ott, D.; Seifert, J.; Prahl, I.; Niemietz,
M.; Hoffman, J.; Guder, J.; Mꢀnnich, M.; Unverzagt, C. Modular
Synthesis of Core Fucosylated N-Glycans. Eur. J. Org. Chem.
2012, 26, 5054-5068. (c) Eller, S.; Schuberth, R.; Gundel, G.; Sei-
fert, J.; Unverzagt, C. Synthesis of Pentaantennary N-Glycans
with Bisecting GlcNAc and Core Fucose. Angew. Chem., Int. Ed.
2007, 46, 4173-4175. (d) Schuberth, R.; Unverzagt, C. Synthesis of
a N-glycan nonasaccharide of the bisecting type with additional
core-fucose. Tetrahedron Lett. 2005, 46, 4201-4204.
(29) (a) Li, T.; Liu, L.; Wei, N.; Yang, J.-Y.; Chapla, D. G.;
Moremen, K. W.; Boons, G.-J. An Automated Platform for the
Enzyme-Mediated Assembly of Complex Oligosaccharides. Nat.
Chem. 2019, 11, 229-236. (b) Liu, L.; Prudden, A. R.; Capicciotti, C.
J.; Bosman, G. P.; Yang, J.-Y.; Chapla, D. G.; Moremen, K. W.;
Boons, G.-J. Streamlining the Chemoenzymatic Synthesis of
Complex N-Glycans by a Stop and Go Strategy. Nat. Chem. 2019,
11, 161-169. (c) Wang, Z.; Chinoy, Z. S.; Ambre, S. G.; Peng, W.;
McBride, R.; de Vries, R. P.; Glushka, J.; Paulson, J. C.; Boons, G.-
J. A General Strategy for the Chemoenzymatic Synthesis of
Asymmetrically Branched N-Glycans. Science, 2013, 341, 379-383.
(30) Koizumi, A.; Matsuo, I.; Takatani, M.; Seko, A.; Hachisu,
M.; Takeda, Y.; Ito, Y. Top-Down Chemoenzymatic Approach to
a High-Mannose-Type Glycan Library: Synthesis of a Common
Precursor and Its Enzymatic Trimming. Angew. Chem., Int. Ed.
2013, 52, 7426-7431.
(31) Li, L.; Liu, Y.; Ma, C.; Qu, J.; Calderon, A. D.; Wu, B.; Wei,
N.; Wang, X.; Guo, Y.; Xiao, Z.; Song, J.; Sugiarto, G.; Li, Y.; Yu,
H.; Chen, X.; Wang, P. G. Efficient Chemoenzymatic Synthesis of
an N-Glycan Isomer Library. Chem. Sci. 2015, 6, 5652-5661.
(32) (a) Shivatare, S. S.; Chang, S.-H.; Tsai, T.-I.; Ren, C.-T.;
Chuang, H.-Y.; Hsu, L.; Lin, C.-W.; Li, S.-T.; Wu, C.-Y.; Wong, C.-
H. Efficient Convergent Synthesis of Bi-, Tri-, and Tetra-
antennary Complex Type N-Glycans and Their HIV-1 Antigenici-
ty. J. Am. Chem. Soc. 2013, 135, 15382-15391. (b) Shivatare, S. S.;
Chang, S.-H.; Tsai, T.-I.; Tseng, S. Y.; Shivatare, V. S.; Lin, Y.-S.;
Cheng, Y.-Y.; Ren, C.-T.; Lee, C.-C. D.; Pawar, S.; Tsai, C.-S.; Shih,
H.-W.; Zeng, Y.-F.; Liang, C.-H.; Kwong, P. D.; Burton, D. R.;
(22) Freeze, H. H. Genetic Defects in the Human Glycome.
Nat. Rev. Genet. 2006, 7, 537-551.
(23) Zhao, Y.-Y.; Takahashi, M.; Gu, J.-G.; Miyoshi, E.; Matsu-
moto, A.; Kitazume, S.; Taniguchi, N. Functional Roles of N-
Glycans in Cell Signaling and Cell Adhesion in Cancer. Cancer
Sci. 2008, 99, 1304-1310.
(24) Lau, K. S.; Partridge, E. A.; Grigorian, A.; Silvescu, C. I.;
Reinhold, V. N.; Demetriou, M.; Dennis, J. W. Complex N-Glycan
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Wu, C.-Y.; Wong, C.-H. Modular Synthesis of N-glycans and
Arrays for the Hetero-Ligand Binding Analysis of HIV Antibod-
ies. Nat. Chem. 2016, 8, 338-346.
1
2
(33) Mukaiyama, T.; Jona, H. Glycosyl fluoride – A Superb Gly-
cosyl Donor in Glycosylation. Proc. Japan Acad. 2002, 78, 73-83.
(34) (a) Lougheed, B.; Ly, H. D.; Wakarchuk, W. W.; Withers,
S. G. Glycosyl Fluorides Can Function as Substrates for Nucleo-
tide Phosphosugar-dependent Glycosyltransferases. J. Biol.
Chem. 1999, 274, 37717-37722. (b) Williams, S. J.; Withers, S. G.
Glycosyl Fluorides in Enzymatic Reactions. Carbohydr. Res.
2000, 327, 27-46.
(35) Markad, S. D.; Schmidt, R. R. Temporary Carbohydrate
Diol Protection with Ester Groups – Orthogonality under Solid-
Phase Oligosaccharide Synthesis Conditions. Eur. J. Org. Chem.
2009, 29, 5002-5011.
(36) (a) Delbianco, M.; Kononov, A.; Poveda, A.; Yu, Y.;
Diercks, T.; Jiménez-Barbero, J.; Seeberger, P. H. Well-Defined
Oligo- and Polysaccharides as Ideal Probes for Structural Stud-
ies. J. Am. Chem. Soc. 2018, 140, 5421-5426. (b) Yu, Y.; Kononov,
A.; Delbianco, M.; Seeberger, P. H. A Capping Step During Au-
tomated Glycan Assembly Enables Access to Complex Glycans in
High Yield. Chem. Eur. J. 2018, 24, 6075-6078.
(37) (a) Bohé, L.; Crich, D. Double Diastereoselection, Regi-
oselectivity, and the Importance of Donor-Acceptor Comple-
mentarity in the Stereoselectivity of Glycosylation Reactions.
Trends Glycosci. Glyc. 2010, 22, 1-15. (b) Vetvicka V.; Novak, M.
Beta-Glucan, Structure, Chemistry and Specific Application; Ben-
tham Science Publishers: 2013.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(38) (a) Weishaupt, M. W.; Hahm, H. S.; Geissner, A.;
Seeberger, P. H. Automated Glycan Assembly of Branched β-
(1,3)-glucans to Identify Antibody Epitopes. Chem. Commun.
2017, 53, 3591-3594. (b) Weishaupt, M. W.; Matthies, S.;
Seeberger, P. H. Automated Solid-Phase Synthesis of a β-(1,3)-
Glucan Dodecasaccharide. Chem. Eur. J. 2013, 19, 12497-12503.
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Table of Contents artwork
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