Synthesis of BODIPY-Labeled Cholesterylated Glycopeptides by Tandem Click Chemistry for Glycocalyxification of Giant Unilamellar Vesicles (GUVs)

Article abstract of DOI:10.1002/chem.201702104

The glycocalyx cover membrane surfaces of all living cells. These complex architectures render their interaction mechanisms on the membrane surface difficult to study. Artificial cell-sized membranes with selected and defined glycosylation patterns may serve as a minimalistic approach to systematically study cell surface glycan interactions. The development of a facile general synthetic procedure for the synthesis of BODIPY-labeled cholesterylated glycopeptides, which can coat cell-size giant unilamellar vesicles (GUVs), is described. These peptide constructs were synthesized by: 1) solid-phase peptide synthesis (SPPS) using cholesterylated Fmoc-amino acids (Fmoc=9-fluorenylmethoxycarbonyl) followed by tandem click reactions, 2) attachment of a BODIPY-bicyclononyne (BCN) (prepared by Mitsunobu chemistry via novel aryl BCN-ethers) in the absence of a catalyst, and 3) glycosylation by means of copper(I)-catalyzed click reaction of an azidoglycan. Seven different GUV-glycoforms were prepared and four of these were evaluated with their corresponding four specific anti-glycan binding lectins.

Full text of DOI:10.1002/chem.201702104

A Journal of
Accepted Article
Title: Synthesis of BODIPY-Labelled Cholesterylated Glycopeptides
by Tandem Click Chemistry for Glycocalyxification of Giant
Unilamellar Vesicles (GUVs)
Authors: Nicolai Stuhr-Hansen, Charikleia-Despoina Vagianou, and
Ola Blixt
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.201702104
Link to VoR: http://dx.doi.org/10.1002/chem.201702104
Supported by

Chemistry - A European Journal
10.1002/chem.201702104
COMMUNICATION
Synthesis of BODIPY-Labelled Cholesterylated Glycopeptides by
Tandem Click Chemistry for Glycocalyxification of Giant
Unilamellar Vesicles (GUVs)
[
a]
[a]
[a]
Nicolai Stuhr-Hansen , Charikleia-Despoina Vagianou , and Ola Blixt*
Abstract: The glycocalyx cover membrane surfaces of all living cells. click approach using azidoglycans and an alkyne amino acids as
common precursors for incorporation into cholesterylated
These complex archtechtures render their interaction mechanisms
modules. However, this path required a different strategy for the
on the membrane surface difficult to study. Artificial cell-sized
introduction of the fluorophore since the rhodamine group used
membranes with selected and defined glycosylation patterns may
previously was found to be incompatible with Cu(I)/base click
serve as a minimalistic approach to systematically study cell surface
glycan interactions. Here we developed a facile general synthetic
procedure for the synthesis of BODIPY-labelled cholesterylated
glycopeptides, which can coat cell-size giant unilamellar vesicles
[7]
conditions. Instead, the BODIPY-system was utilized due to its
[
8]
high (photo)chemical stability. Sequential introduction of both
BODIPY and sugar moieties via click chemistry requires that first
one copper-free click reaction occurs, utilizing most practically
[
9]
bicyclononyne (BCN) substituted BODIPY-derivatives.
(
GUVs). These peptide constructs were synthesized by SPPS using
cholesterylated Fmoc-amino acids followed by tandem click
reactions; firstly attaching BODIPY-bicyclononyne (BCN)
prepared by Mitsunobu chemistry via novel aryl BCN-ethers) in
a
BODIPY
(
H
O
absence of a catalyst and secondly by copper(I) catalyzed click
reaction of an azidoglycan. Seven different GUV-glycoforms were
prepared and four of these were evaluated with their corresponding
four specific anti-glycan binding lectins.
N
N
H
Glycan
N3
N
N
N
N
1
. BCN-BODIPY
. Glycan-N3, Cu+
2
SPPS
PEPTIDE
PEPTIDE
Nature's escape from genetic imprisonment are post-
translational modifications (PTM), where glycosylation is
structurally considered the far most complex modification (>70%
of all proteins are glycosylated); glycosylation has critical
influences on cell-adhesion, pathogen recognition and cellular
N
N
N
N
N
N
Cholesteryl
Cholesteryl
[
1]
differentiation. Synthetic and analytic glycomic advances have
facilitated a small revolution in the past decade in terms of better
Figure 1. Double functionalization of cholesterylated peptide constructs by
site-specific tandem click chemistry: Copper-free click attachment of
[
2]
understanding of the biological roles of individual glycans.
a
bicyclononyne-BODIPY moiety succeeded by copper(I)-catalyzed click
reaction of an azidoglycan.
However, most glycan displaying techniques employ fairly static
and individual display of glycans on mediums such as gold-,
[
3]
plastic- and glass microarrays. Relatively few studies have
considered adhesion of lipidated glycans when integrated or
[
4]
Beginning with an azido-peptide containing a propargyl moiety,
we implemented a strategy to introduce a BODIPY-moietiy via
copper-free click chemistry. This was followed by a copper (I)-
catalyzed click reaction with an azidoglycan. This synthesis
strategy was[2a] explored due to the ready availability of
embedded cellular or artificial membranes. The Bertozzi group
[
5]
introduced cholesterylated GalNAc-polypeptides onto cells and
successfully mimicked the glycocalyx of living cells by using the
cholesteryl functionality as a lipid anchor for adhesion to the cell
membrane; this extensively exposed the glycan to the cell-
exterior. However, in terms of physical visualization, this
methodology does not easily allow for introduction of
fluorophores onto the polypeptide backbones. Recently, we
showed that rhodamine-labeled cholesterylated O-GalNAc-
peptide modules prepared by solid-phase peptide synthesis
azidoglycans.
In current reports regarding the introduction of
BCN-systems into peptides, the BCN-functionality was installed
[10]
as a separate post-SPPS step
The preparation of BCN-
peptides by SPPS implementing BCN-containing Fmoc amino
acids seemed inappropriate due to lability during terminal acidic
cleavages (see Supporting Information, Section S3). However,
(
SPPS) had the capacity to efficiently be integrated in giant
bacterial expressions of BCN-peptides via BCN-amino acids has
unilamellar vesicles (GUVs) with a dispersed distribution around
[11]
been a[1c2]complished
azides.
accompanied by click labelling with
[
6]
the lipid membrane. The O-GalNAc-residues were introduced
during SPPS utilizing a Fmoc protected GalNAc-threonine amino
acid, a procedure that limits the glycan diversity.
Little attention has been paid to synthesizing BCN-BODIPY
[
13]
derivatives , and no general procedures for preparation have
been reported despite their huge labeling potentials. In this study,
we have developed facile procedures for synthesis of BODIPY-
BCN substrates via introductions of yet unreported BCN-
Aiming for a more flexible strategy for introduction of the the
glyco-moieties into analogous peptide modules, we selected a
[
14]
arylethers. This allows aryl-BODIPY, cholesteryl, or any azide
functionality to be assembled into glyco-modules suitable for
GUV-membrane insertion. We demonstrated that the glycans
GlcNAc (GN) attached via a short as well as a long spacer unit,
[
a]
Dr. N. Stuhr-Hansen, Ms C.-D. Vagianou. Prof. Dr. O. Blixt*
*corresponding author). Department of Chemistry, Chemical
Biology, University of Copenhagen, Thorvaldsensvej 40, 1871
Frederiksberg C, Denmark.
(
Neu5Acα(2,3)Galβ(1,4)GlcNAc-
(3'SLN),
and
E-mail: olablixt@chem.ku.dk
Neu5Acα(2,6)Galβ(1,4)GlcNAcβ(1,3)Galβ(1,4)GlcNAc-
.
(
6'SLNLN), could be efficiently assembled, displayed and
recognized by glycan-recognizing lectins.
Supporting information for this article is given via a link at the end of
the document.
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201702104
COMMUNICATION
To synthesize of BODIPY-labelled cholesterylated glycopeptides, (DIAD) and triphenyl phosphine to generate 4-(((1R,8S,9S)-
two orthogonally reactive alkyne moieties were reacted under
copper-free click conditions, succeeded by a copper(I)-catalyzed
click reaction attaching an azide-substituted glycan (Figure 1). It
was attempted to utilize amino acids with BCN-functionalities in
SPPS, but in synthesized peptide constructs these moieties
decomposed during terminal acidic cleavage steps. However,
BCN-containing Fmoc-amino acids, such as Fmoc-Lys(BCN)-
OH , were prepared and could be incorporated in peptide
sequences. Furthermore, our Mitsunobu strategy for the
synthesis of BCN-ethers (ibid.) was extended to tyrosine
derivatives such as the protected BCN-amino acid analogue
Fmoc-Tyr(BCN)-OBu-t (see supporting information), During
TFA-based cleavages of the tert-butyl ester functionality for
releasing the free C-terminal the compound largely decomposed
representing degradation during acidic cleavage of a BCN-
peptide from resin. It therefore seemed more feasible to
introduce the BCN-BODIPY system into the peptide as a post-
SPPS step.
bicyclo[6.1.0]non-4-yn-9-yl)methoxy)benzaldehyde
2
representing the first BCN-arylethers. Treatment of 2 with
kryptopyrrole/TFA was succeeded by an in situ oxidation with p-
chloranil. This was followed by a complexation reaction with
3
BF /triethylamine producing BCN-OPh-BODIPY 3 (Scheme 1).
[
15]
Utilizations of our recently published peptide cholesterylation
[6]
technology allowed for facile synthesis of peptides bearing an
alkyne, an azide and a cholesteryl moiety by utilizing Fmoc-
3
Lys(N )-OH, Fmoc-Gly(Propargyl)-OH (Fmoc-Pra-OH) and
Fmoc-Ala(CholesterylTriazole)-OH, respectively, as standard
Fmoc-amino acids in SPPS. Accordingly, a cholesterylated
azido-propargylpeptide 4 was prepared by SPPS. A site-specific
copper-free click reaction with 3 produced the corresponding
BODIPY-labelled cholesterylated propargylpeptide module. This
was then reacted independently with seven different
azidoglycans under copper(I)-catalyzed click conditions in the
presence of copper(I) iodide, producing the BODIPY-labelled
cholesterylated glycopeptides 5a-5g after HPLC purification
(Scheme 2). The azidoglycans utilized for making the
For the synthesis of a BODIPY-BCN derivative the Mitsunobu-
chemistry was employed. An alkoxy-benzaldehyde precursor
was generated by utilizing alcohols as phenol-alkylating agents.
[
2a]
compounds 5a to 5e were reported in an earlier publication.
The two azidoglycans used as precursors for synthesis of 5f to
5
g were prepared from their corresponding glycoesters; these
[17]
F
F
were converted to amides upon reaction with ethylenediamine
creating free amine groups, which were then converted into
desired azidoglycans by HBTU-coupling of 6-azidohexanoic acid
,
B
N
N
O
(
see Supporting Information, Section S2). The synthesis of the
O
compounds 5a-5g exemplify the first site-specific copper-free
click reactions of BCN-moieties with azide groups in the
presence of a non-reacted propargyl functionalities, which are
i
ii, iii, iv
subsequently clicked with
However, consecutive click chemistry of internally similar, non-
strained alkynes has been reported.
of these reactions proved the orthogonal reactivity of the two
different BCN and propargyl alkyne moieties, demonstrating no
copper-free reactivity of the latter towards azides.
a new set of azidocompounds.
O
O
OH
[18]
Thorough investigations
1
H
H
H
H
2
BCN-OPh-BODIPY 3
An alternative procedure for copper-free introduction of a
BODIPY-flourophore into a peptide construct while leaving a
propargyl unit free for further click derivatization was
investigated. A BCN-propargyl-OPh-BODIPY was clicked onto
an azidopeptide prepared similarly to the synthesis of 3 via the
corresponding BCN-propargyl-benzaldehyde. This created an
inherently clickable alkyne peptide. This technique established
the means for conversion of an azide functionality into a
propargyl structural unit while during simultaneous introducing of
Scheme 1. Synthesis of a bicyclononyne-BODIPY derivative via
Mitsunobu chemistry. Reaction conditions. (i) BCN-OH, DIAD,
3
PPh , THF, rt, 4h, (55%); (ii) Kryptopyrrole, TFA (cat.), DCM, rt,
16h; (iii) p-Chloranil, DCM, rt, 2h; (iv) Boron trifluoride etherate,
triethylamine, DCM, rt, 2h, (26% over-all).
[
16]
Several oligo(propargyloxy)aryl systems have been employed
in routes to propargyl-BODIPY systems; however exchange with
BCN systems has not been described until this point, which
further motivated our research towards this important class of
compounds. 4-Hydroxy-benzaldehyde 1 was reacted with BCN-
OH in the presence of the reagents diisopropyl azadicarboxylate
a
third moiety such as a fluorophore (see Supporting
Information). However, despite the ease in tandem click
insertion, enhanced hydrophobicity of these specific peptide
constructs due to propargyl-fluprophore addition made them
difficult to purify, even after successive glycan click-attachment.
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201702104
COMMUNICATION
N3
N
Cholesteryl
N
N
O
O
SPPS
H
N
H
N
O
O
H
2
N
O
N
H
O
EPKPEKPEGPA
N
H
APP-OH
O
O
O
4
H
OPh-BODIPY
CLICK 1
N
N
H
CLICK 2
i. BCN-OPh-BODIPY 3
N
ii. Glycan-N
3
/Cu+
N
N
Cholesteryl
Glycan
N
N
N
N
O
O
H
N
H
N
O
O
H
2
N
O
N
H
O
EPKPEKPEGPA
N
H
APP-OH
O
O
O
5
Compd.
Glycan
Compd.
Glycan
OH
OH
OH
HO
COOH
O
5
a
HO
O
HO
O
5
e
AcHN
OH
NHAc
O
OH
O
OH OH
OH
HO
O
O
O
HO
OH OH
OH
OH
HO
O
HO
O
O
O
HO
COOH
OH
OH
O
O
NHAc
OH
NHAc
HO
O
5
b
O
O
O
O
HO
OH
OH
AcHN
HO
OH
O
HO
O
H
N
HO
HO
5
f
O
N
H
OH OH
NHAc
COOH
O
O
OH
O
O
5
c
HO
AcHN
O
HO
O
OH
OH
OH
NHAc
O
HO
HO
OH OH
OH
O
OH
5g
O
OH
O
OH
O
H
O
N
O
O
HO
HO
O
N
H
O
O
O
HO
O
NHAc
O
OH
OH
O
5
d
OH
OH
HO
HO
OH
O
HO
HO
Scheme 2. Synthesis of BODIPY-labelled cholesterylated glycopeptides by tandem specific copper-free click-reaction succeeded by copper(I)-catalyzed click-
reaction. Reaction conditions. (i) SPPS on 2-chlorotrityl resin using Fmoc-Gly(Propargyl)-OH, Fmoc-Ala(CholesterylTriazole)-OH and Fmoc-Lys(N )-OH; (ii)
BCN-OPh-BODIPY 3, DIPEA-MeOH (1:4), rt, 16h; (iii) Glycan-Spacer-N , CuI, DIPEA-MeOH (1:4), rt, 16h.
3
3
Globoside 5g) were also functional, but these results will be
published elsewhere in other contexts. We have previously
reported that lectin binding with non-glycosylated peptide chain
does not occur.
The seven different azido-glycans that were succesfully inserted
into the cholesterylated peptide constructs (Scheme 2) were
assembled into artificial membranes by using electroformation to
produce GUVs in the presence of the corresponding BODIPY-
[
6]
labelled cholesterylated glycopeptides. Mixtures of DOPC,
cholesterol, the membrane marker Rhodamine-B-DHPC and
four different cholesterylated glycopeptides were used for GUV
production. All four modules were incorporated into GUVs,
indicated by the BODIPY-FL marker showing an equal
distribution around the lipid membrane as seen is Figure 2. The
viability and accessibility of these four cholesterylated glyco-
moities on the GUV surface were examined by addition of lectins.
Fluorescently labeled Wheat Germ Agglutinin (WGA-AF405),
Maackia Amurencis lectin II (MALII-AF405) and Sambucus Nigra
lectin (SNA-AF405) showed binding to GN, 3SLN and 6LNLN
moieties respectively (Figure 2). To ensure that the lectins were
binding to their corresponding glyco-moiety and not to the lipid
membrane, GUVs formed from a mixture of DOPC, cholesterol,
Rhodamine-B-DHPE and cholesterylated GN-peptide were
In conclusion, a technology for the synthesis of fluorescently
labelled, cholesterylated glycopeptides by specific tandem click
reactions was employed. A sequential copper-free click reaction
between a BODIPY-BCN and a cholesterylated azido-peptide
was succeeded by a copper(I)-catalyzed click reaction of an
azido-glycan with an alkyne-moiety pre-installed in the peptide.
Preparations of BCN-substituted BODIPYs were based upon
Mitsunobu-chemistry via new BODIPY-arylether starting
materials that hold huge potentials in synthesizing new types of
BCN-derivatives directly from
BODIPY fluorophore was co-introduced in a copper-free click
reaction with an azido-peptide, leaving copper-clickable
a phenol. A BCN-propargyl-
a
monoalkyl-alkyne for reaction with another azido-compound.
The methodology is of broad scope, since it may be generally
applicable in click-handle ‘swapping’ when it is desirable to
convert an azido group into an alkyne functionality. A small
library of seven BODIPY-labelled cholesterylated glycopeptides
was prepared by click attachment of azido-glycans onto the
corresponding cholesterylated BODIPY-alkyne peptides. This
was then studied as an artificial glycocalyx by cholesteryl
incubated with SNA-AF405 and imaged in
a
confocal
microscope; no significant interactions were observable (see
Supporting Information, Section S10). Notably, the GN-moiety
with a long linker 5f seemed to be recognized a bit better than
the shorter 5a, suggesting that care should be taken to the
spacing and positioning of the glycans for lectin detection. The
additional glyco-GUV-modules (Blood group A 5d and
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201702104
COMMUNICATION
anchoring to a GUV-membrane accomplished by recognition by
appropriate lectins that binds exclusively to their corresponded
glycan structure, as no binding between lectin and non-
corresponded glycans was observed. The unnatural spacer
substituting the naturally occurring O-glycosyl linkage between
the glycan and peptide does not have a huge impact on lectin
binding allowing for simple preparation of target glycopetide
constructs without necessitating complicated synthesis of O-
glycan substituted analogues. Besides being versatile building
blocks tolerating insertions of numerous functional groups the
peptide unit primarily served as a structural assembling unit and
we showed that the simple cholesteryl-glyco constructs with
short spacers also displayed lectin binding when inserted into
GUV-membranes.
The authors acknowledge support by the Danish Research
Council (Innovationsfonden) and in the framework of the EU
ERASynBio project SynGlycTis, and the support from the
Stiftelsen for Strategisk Forskning (SSF). Prof Ole Hindsgaul is
acknowledged for generous contribution of synthetic
carbohydrate compounds, Consortium fo Funtional Glycomics
(
CFG) for azido glycans and Assoc. Prof. Nikos Hatzakis and
Mr. Matias Moses for access and guidance during confocal
microscope experiments.
Keywords: Giant Unilamellar Vesicles • Glycocalyx •
Glycosylation • Lectin • Solid-Phase Peptide Synthesis •
Copper-free click chemistry
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Lec5nꢀdetec5onꢀ
ꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀ(membraneꢀmarker)ꢀꢀꢀꢀꢀꢀꢀꢀ(Glycan)ꢀ
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’SLNLN(ꢁe)ꢀSNAꢀ
[8]
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6
[
[
9]
10]
[
11]
Figure 2. Glyco-decoration of GUVs by electroformation co-production of
GUVs and cholesterylated glycopeptides succeeded by recognition with
appropriate lectins. DOPC/RhoB-DHPE/Cholesterol mixes were used for
vesicle formation containing no (a), or 4.9 mol% GlcNAc-linked module
[12]
[13]
[14]
short
spacer
(b),
GlcNAc-linked
module
module
long
spacer
(d)
(c),
and
Neu5Acα(2,3)Galβ(1,4)GlcNAc-linked
Neu5Acα(2,6)Galβ(1,4)GlcNAcβ(1,3)Galβ(1,4)GlcNAc-linked Module (e).
The GlcNAc-moiety, the Neu5Aca(2,3)-moiety and the Neu5Aca(2,6)-
moiety on the glycodecorated GUVs were accessible for WGA-AF405,
MALII-AF405 and SNA-AF405 (c ≈ 44 nM) respectively, although with
different efficiency for different variants.
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Acknowledgements
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Chemistry - A European Journal
10.1002/chem.201702104
COMMUNICATION
Entry for the Table of Contents (Please choose one layout)
Layout 2:
COMMUNICATION
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[a]
F
F
Nicolai Stuhr-Hansen , Charikleia Vagianou , and Ola
B
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Blixt*
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Page No. – Page No.
Synthesis of BODIPY-Labelled Cholesterylated
Glycopeptides by Tandem Click Chemistry for
Glycocalyxification of Giant Unilamellar Vesicles (GUVs)
H
O
CLICK 1
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N
H
GLYCAN
N
N
N
CLICK 2
N
PEPTIDE
N
pre-SPPS
CLICK
N
N
CHOLESTERYL
Double functionalization of cholesterylated peptide constructs by site-specific
tandem click chemistry: Copper-free click attachment of bicyclononyne
fluorophore succeeded by copper(I)-catalyzed clicking on an azido-glycan.
a
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