Synthesis of Oligo-(alkyne-triplet)peptide Constructs

Article abstract of DOI:10.1021/acs.orglett.7b03231

Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click synthesis of an Fmoc-(trispropargyl)amino acid building block for solid phase peptide synthesis (SPPS) of oligo-(trialkyne)peptide constructs is reported. These can carry potentially indefinite

Full text of DOI:10.1021/acs.orglett.7b03231

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Synthesis of Oligo-(alkyne-triplet)peptide Constructs
Nicolai Stuhr-Hansen, Christian Risinger, Ebbe Engholm, and Ola Blixt*
Department of Chemistry, Chemical Biology, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark
S
* Supporting Information
ABSTRACT: Copper(I)-catalyzed azide−alkyne cycloaddition
(CuAAC) click synthesis of an Fmoc-(trispropargyl)amino acid
building block for solid phase peptide synthesis (SPPS) of oligo-
(trialkyne)peptide constructs is reported. These can carry
potentially indefinite numbers of inherent alkyne-triplets, which
are click derivatized with GlcNAc-azide into the corresponding
glycopeptides.
ultivalent interactions are of high interest and govern
coupling of a trisalkyne carboxylic acid succeeded by triple click
Mmany interactions between proteins and its ligands on forming a tetrapeptide construct, and more recently13
cell membranes.1 A multivalent ligand is composed of a scaffold
analogously for synthesis of tris-glyco peptides by performing
three final click attachments of the corresponding azido-
glycans. The reported technologies for tripod-glycosylation of
peptides are limited to introduction of only one single tris-glyco
moiety at the N-terminal or by reversible attachment at a
cysteine moiety by thiol/maleimide conjugation.
The introduction of oligo(trisalkyne) moieties in peptides
used for click generation of heavily glycosylated constructs is an
extension of our recently described procedures for glycocalyx-
ification of giant unilamellar vesicles.14,15 Thus, to gain access to
trisalkyne molecular systems incorporated into a peptide
sequence, preferably by SPPS, a strategy for developing the
synthesis of an amino acid alkyne-derivative suitable for SPPS
was required.
The present paper describes a facile procedure for the
indirect oligo triplet-glycosylation of peptides by introduction
of trispropargyl amino acid units in peptides. The key in this
study was the development of an easily synthesizable Fmoc-
chemistry compatible trisalkyne-containing Fmoc-amino acid,
which could be used as a standard amino acid in SPPS
succeeded by attachment of multiple azidoglycans by CuAAC16
click chemistry17 generating the title oligo-(triplet-glyco)
peptides (Scheme 1). The versatility of the methodology was
demonstrated by synthesis of tris- and hexakis(GlcNAc)peptide
constructs.
Introduction of oligo-trisalkyne moieties into peptides
required monofunctionalization of a tetrakisalkyne with
azidopeptide derivatives. These must be protected and
adaptable to peptide synthesis, for which artificial azido-lysine
derivatives were considered appropriate, due to good
compatibility with SPPS. The initial aim was therefore to
design azido-amino acid constructs generating the most optimal
or backbone to which a particular number (valency) of identical
or nonidentical epitopes are attached. Dimers have been shown
to display up to more than thousand times higher inhibitory
activities toward their respective receptors than their
monovalent analogues.2 Trivalency is involved in substrate
binding of sialic acid to the influenza and adenovirus receptors3
and β-galactose derivatives to the asialoprotein receptor.4,5
Strategies to obtain multivalent trimeric ligands can be
performed via three consecutive copper(I) catalyzed 1,3-dipolar
cycloaddition reactions between azides and alkynes and
represent versatile building blocks as exemplified by recent
methods for the synthesis of α-triazido keto compounds.6 In a
reverse click approach toward triple point assembly, trispro-
pargyl amine has been used as the tripod branching system,
thereby safely connecting three azido sugar entities.3 However,
utilization of this technique leaves no possibilities for further
functionalization. 2-Amino-2-(hydroxymethyl)-1,3-propanediol
has been used for a triple connection of three glyco-units,7
affording constructs with a free amino group for further
derivatization. The importance of the ability to generate
multialkyne analogues with functional handles was highlighted
by Papp et al.,8 generating thiol functionalized quadruple sialic
acid constructs for adhesions to gold nanoparticles. However,
besides producing nonsymmetrical constructs, this method
requires presynthesis of oligo-propargyl precursors with limited
applicability for general constructions of multialkyne deriva-
tives. Trisalkyne derivatives have huge potential in material
science due to their cross-linking capabilities.9
The concept of mono-click functionalization of a tetraki-
salkyne was first applied in peptide science10 via the
introduction of trisalkyne systems at a cysteine moiety situated
at single chain variable fragments (scFv)11 by thiol/maleimide
conjugation. Introduction of trisalkyne units in peptides as
direct extension of SPPS was performed12 by the terminal
Received: October 17, 2017
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.7b03231
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
3 is the first example of a side-chain functionalized tris-alkyne
amino acid derivative. Only by chromatography on new
stationary dry silica columns eluting with dry methanol
mixtures 3 was isolated in as high as a 62% yield, because
application of old silica/wet methanol induced severe tailing
due to the PEG-carboxylic acid characteristics of 3 manifested
by dramatic lowering of the yield caused by a substantial
amount of residual product stuck on the column. Glaser
product formation was not observed presumably since only
slight exposure to a dilute acidic aqueous environment was
needed during quenching. Logically, the yield of 3 was lowered
due to formation from basically statistical reactions with the
highest probability for reaction between 1 and 2, but with
parallel truncating reactions for instance between 2 and 3. The
yield of 3 was improved by using an excess of 1; however, above
3 equiv no further attenuation of the yield was observed.
Utilization of the reagent couple CuSO4/ascorbate16 instead of
CuI18 did not seem to have a beneficial effect, since TLC
revealed substantial quantities that were unreacted after 24 h. In
terms of commercialization, besides the yield of 3 possibly
being optimized by fine-tuning of chromatographic procedures,
it is highly advantageous that the applied excess of 1 can be
quantitatively separated in a pure fraction and recycled.
Scheme 1. General Strategy for the Generation of Oligo-
(tris-alkyne)peptides by Incorporating an Fmoc-
(trisalkyne)amino Acid as a Standard Amino Acid in SPPS
Succeeded by Post-SPPS Click Introduction of e.g. Glycans
Introduction of regiopure trispropargyl moieties into
peptides was achieved by utilizing 3 as a standard amino acid
into an SPPS-sequence as an N-terminal extension of the
peptide transportan 10 (TP10), which due to its reported
GUV-membrane inserting properties18 may be applicable as
artificial GUV-glycocalyx components upon glycosylation.
Trispropargyl containing 4a could be obtained in satisfactory
yield after cleavage from the resin with TFA−TES−H2O
(18:1:1). Due to absence of copper salts, Glaser alkyne-
dimerization seemed completely absent, despite exposure to
strong acidic conditions. In order to extend the scope for
alkynylation of one peptide construct, introduction of multiple
trisalkynes was attempted on TP10 by allowing consecutive
HBTU couplings with 3. No complications were observed
during synthesis of a peptide with two, three, or four
neighboring trispropargyl units 4b−4d, respectively. CuAAC
of 4a/4b with azido-ethyl-GlcNAc afforded the (GlcNAc)-
peptides 5a/5b after HPLC-purification (Scheme 3).
Synthesis of the oligo-tris-alkyne peptides 4 was considered
as valuable proof that the potential applications of 3 completely
analogous to normal Fmoc-amino acids in SPPS had been fully
explored also in terms of random insertion into peptides, since
for instance the construct 4d contains an alkyne triplet on the
fourth amino acid position from the N-terminal. When aiming
for densely glycosylated peptide constructs, easy installation of
several neighboring alkyne-triplet units might be highly
advantageous, and furthermore, because copper traces had
been removed during presynthesis steps multialkyne peptides
can be cleaved from resin under strong acidic conditions
without formation of Glaser alkyne dimers.
trisalkyne systems, with an emphasis on developing a trisalkyne
Fmoc-amino acid derivative.
In a first direct solid phase approach for introduction of a
tris-alkyne unit in amino acid derivatives, the resin bound
peptide Fmoc-Lys(N3)-Gly-2ClTrt-resin was reacted with 1, in
the presence of copper(I) iodide at room temperature, and the
major products were intramolecularly dimerized Glaser diynes
after standard acidic resin cleavage conditions (see Supporting
Information). We then envisioned that copper(I)-catalyzed
solution phase click reactions of azido-amino acid derivatives
with an excess of 1 would generate isolable trisalkyne constructs
free of Glaser byproducts by avoiding handling with a
concentrated strong acid. During recent work on side-chain
cholesterylation of amino acid derivatives, we developed a facile
procedure18 for side-chain click functionalization of Fmoc-
amino acids mediated by CuI/DIPEA. By adapting these
conditions, Fmoc-Lys(N3)-OH 2 was smoothly reacted with 3
equiv of 1 forming a trispropargyl lysine moiety by copper(I)
iodide catalysis in DIPEA/DCM, facilitating isolation of the
symmetrical trisalkyne substituted Fmoc-amino acid 3 (Scheme
2).
Scheme 2. Synthesis of a Trispropargyl Lysine Containing
Fmoc-Amino Acid
The synthesized GlcNAc peptide constructs were then used
in a lectin microarray, thereby proving the potential application
as non-natural glycoconjugates. The three TP10 peptides
bearing a GlcNAc-triplet 5a, a mono-GlcNAc unit, and a non-
glyco hexaalkyne system 4b, respectively, were immobilized on
NHS-activated glass slides.19 After incubation with Cy3 labeled
wheat germ agglutinin (WGA), the binding of the lectin specific
binding to GlcNAc was evaluated by measuring the resulting
fluorescence. The compound having no GlcNAc at all only
showed a weak background signal, whereas the other two
B
DOI: 10.1021/acs.orglett.7b03231
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
were smoothly generated upon copper(I) catalyzed click
attachment of a GlcNAc-azide derivative. Upon immobilizing
a tris-GlcNAc-peptide on a glass slide array studies gave positive
indications of lectin binding to GlcNAc-triplets.
Scheme 3. Synthesis of Oligo-trispropargylpeptides
Functionalized into Corresponding Oligo-GlcNAc-peptides
by CuAAC Click Chemistry
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.7b03231.
Experimental procedures for all compounds including
NMR spectra (PDF)
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: olablixt@chem.ku.dk.
ORCID
Ola Blixt: 0000-0003-4143-6276
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors acknowledge support by the Danish Research
Council (Innovationsfonden) within the framework of the EU
ERASynBio project SynGlycTis, and the support from the
Stiftelsen for Strategisk Forskning (SSF). 2-Azidoethyl N-
acetylglucosamine was received from Consortium for Func-
tional Glycomics (CFG).
conjugates showed a clear fluorescence signal that increased
with the numbers of GlcNAc. This clearly demonstrated the
applicability utilizing the oligo-trispropargylpeptides derived
glycopeptide constructs as functional glycan mimetics (Scheme
4).
REFERENCES
■
Scheme 4. Microarray WGA-lectin Binding Studies: (a, c,
and e) GlcNAc-spacer-amine (See Supporting Information;
Positive Controls); (b) tris-GlcNAc-TP10 5a; (d) mono-
GlcNAc-TP10 (See Supporting Information); (f) Hexakis-
alkyne-TP10 4b (non-Glyco Negative Control)
(1) Shuker, S. B.; Hajduk, P. J.; Meadows, R. P.; Fesik, S. W. Science
1996, 274, 1531−1534.
(2) Tamiz, A. P.; Bandyopadhyay, B. C.; Zhang, J.; Flippen-
Anderson, J. L.; Zhang, M.; Wang, C. Z.; Johnson, K. M.; Tella, S.;
Kozikowski, A. P. J. Med. Chem. 2001, 44, 1615−1622.
(3) Caraballo, R.; Saleeb, M.; Bauer, J.; Liaci, A. M.; Chandra, N.;
Storm, R. J.; Frangsmyr, L.; Qian, W.; Stehle, T.; Arnberg, N. Org.
̈
Biomol. Chem. 2015, 13, 9194−9205.
́
(4) Mamidyala, S. K.; Dutta, S.; Chrunyk, B. A.; Preville, C.; Wang,
H.; Withka, J. M.; McColl, A.; Subashi, T. A.; Hawrylik, S. J.; Griffor,
M. C. J. Am. Chem. Soc. 2012, 134, 1978−1981.
(5) Thapa, B.; Kumar, P.; Zeng, H.; Narain, R. Biomacromolecules
2015, 16, 3008−3020.
(6) Klahn, P.; Erhardt, H.; Kotthaus, A.; Kirsch, S. F. Angew. Chem.,
Int. Ed. 2014, 53, 7913−7917.
(7) Nair, J. K.; Willoughby, J. L.; Chan, A.; Charisse, K.; Alam, M. R.;
Wang, Q.; Hoekstra, M.; Kandasamy, P.; Kel’in, A. V.; Milstein, S. J.
Am. Chem. Soc. 2014, 136, 16958−16961.
(8) Papp, I.; Sieben, C.; Ludwig, K.; Roskamp, M.; Bottcher, C.;
̈
Schlecht, S.; Herrmann, A.; Haag, R. Small 2010, 6, 2900−2906.
(9) Ishiwata, T.; Furukawa, Y.; Sugikawa, K.; Kokado, K.; Sada, K. J.
Am. Chem. Soc. 2013, 135, 5427−5432.
(10) Natarajan, A.; Du, W.; Xiong, C.-Y.; DeNardo, G. L.; DeNardo,
S. J.; Gervay-Hague, J. Chem. Commun. 2007, 695−697.
(11) Schellinger, J. G.; Kudupudi, A.; Natarajan, A.; Du, W.;
DeNardo, S. J.; Gervay-Hague, J. Org. Biomol. Chem. 2012, 10, 1521−
1526.
(12) Schellinger, J. G.; Danan-Leon, L. M.; Hoch, J. A.; Kassa, A.;
Srivastava, I.; Davis, D.; Gervay-Hague, J. J. Am. Chem. Soc. 2011, 133,
3230−3233.
(13) Hsieh, H. W.; Davis, R. A.; Hoch, J. A.; Gervay-Hague, J. Chem. -
Eur. J. 2014, 20, 6444−6454.
In conclusion, a trispropargyl substituted Fmoc-amino acid
was synthesized and utilized as a standard amino acid in solid
phase peptide synthesis (SPPS) generating oligo-(trialkyne)-
peptides containing tris-propargyl moieties, ranging from tris-,
hexakis-, nonakis-, and dodecakisalkynepeptide constructs. The
corresponding tris- and hexakis-GlcNAc-peptide constructs
C
DOI: 10.1021/acs.orglett.7b03231
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
(14) Stuhr-Hansen, N.; Madl, J.; Villringer, S.; Aili, U.; Romer, W.;
̈
Blixt, O. ChemBioChem 2016, 17, 1403−1406.
(15) Stuhr-Hansen, N.; Vagianou, C.-D.; Blixt, O. Chem. - Eur. J.
2017, 23, 9472−9476.
(16) Tornøe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002,
67, 3057−3064.
(17) Kolb, H. C.; Finn, M.; Sharpless, K. B. Angew. Chem., Int. Ed.
2001, 40, 2004−2021.
(18) Islam, M. Z.; Ariyama, H.; Alam, J. M.; Yamazaki, M.
Biochemistry 2014, 53, 386−396.
́
(19) Blixt, O.; Clo, E.; Nudelman, A. S.; Sørensen, K. K.; Clausen, T.;
Wandall, H. H.; Livingston, P. O.; Clausen, H.; Jensen, K. J. J. Proteome
Res. 2010, 9, 5250−5261.
D
DOI: 10.1021/acs.orglett.7b03231
Org. Lett. XXXX, XXX, XXX−XXX