Synthesis of new asparagine-based glycopeptides for future scanning tunneling microscopy investigations

Article abstract of DOI:10.3762/bjoc.16.80

For investigations on the biological functions of oligosaccharides and peptidomimetics, new asparagine-based mono- and disaccharides containing glycopeptides were prepared in solution. The applicability of two common peptide coupling reagents, using an or

Full text of DOI:10.3762/bjoc.16.80

Synthesis of new asparagine-based glycopeptides for future
scanning tunneling microscopy investigations
Laura Sršan and Thomas Ziegler*
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2020, 16, 888–894.
Institute of Organic Chemistry, University of Tübingen, Auf der
Morgenstelle 18, 72076 Tübingen, Germany
doi:10.3762/bjoc.16.80
Received: 09 March 2020
Accepted: 21 April 2020
Published: 30 April 2020
Email:
Thomas Ziegler* - thomas.ziegler@uni-tuebingen.de
* Corresponding author
Associate Editor: S. Flitsch
Keywords:
© 2020 Sršan and Ziegler; licensee Beilstein-Institut.
License and terms: see end of document.
amino acids; asparagine; carbohydrates; glycopeptides;
peptidomimetics
Abstract
For investigations on the biological functions of oligosaccharides and peptidomimetics, new asparagine-based mono- and disaccha-
rides containing glycopeptides were prepared in solution. The applicability of two common peptide coupling reagents, using an
orthogonal Fmoc/t-Bu strategy along with acetyl protecting groups for the carbohydrate moiety, was studied. Thus, the prepared
libraries of glycopeptides were designed as model systems of cell surfaces for future investigations by combined preparative mass
spectroscopy and scanning tunneling microscopy (STM) using soft-landing electrospray beam deposition (ES-IBD), on metal sur-
faces.
Introduction
Glycopeptides are generally found on virtually every eukary- hampered isolation of these structures in a pure form from bio-
otic and prokaryotic cell surface. So far, the study of glycopep- logical material, there is a great interest in the synthesis of
tide interactions with other proteins gave some insight into im- structurally defined peptidomimetic libraries for further biologi-
portant biological functions, for instance, intracellular commu- cal and medicinal investigation [4,8]. Recently, the characteri-
nication, cell–cell recognition, immune response, and pathogen- zation of heterogeneous mixtures of glycoconjugates was
esis [1-4]. Glycosylation is also considered to be one of the simplified by the development of novel MS-supported methods
most important post-translational modification (PTM) since [9-11]. Nevertheless, the complexity of the aforementioned
more than half of all human proteins are glycopeptides or glyco- macromolecules caused by PTM still constitutes a technical
proteins [5]. Therefore, understanding how glycopeptides challenge. They can only be applied to small amounts of pure
interact on an intra- and intermolecular level is highly impor- material, which is insufficient for extended biological investiga-
tant for resolving some of the most challenging topics in medic- tions [4,6]. Hitherto, a variety of glycopeptides are used for me-
inal chemistry [4,6,7]. Due to the microheterogeneity of natu- dicinal applications, for example, in anti-HIV therapy, MUC1-
rally occurring glycopeptides, which is the reason for the based antitumor vaccines, or as antibiotics [12-14]. Especially
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Beilstein J. Org. Chem. 2020, 16, 888–894.
glycans bearing noncanonical amino acids, which can only be would require an additional protection orthogonal to the Fmoc/
introduced into a peptide by organic synthesis, are suitable for t-Bu protocol and the acetyl groups of the sugar moieties. How-
cancer therapy since they show better resistance to enzymatic ever, numerous other natural or unnatural amino acids, functio-
degradation in comparison with naturally occurring amino acids nalized or not, could be used for this reaction protocol.
[14]. Thus, there is still a great effort in finding new potential
drugs derived from glycopeptides [4,15].
Results and Discussion
For the synthesis of the anticipated glycopeptides, we started
By using preparative mass spectrometry (pMS) combined with from the respective fully acetylated β-ᴅ-glycosyl azides 1a–f in
STM on submolecular-resolution peptides and carbohydrates the gluco, galacto, manno, cello, lacto, and malto series
can be investigated regarding their self-assembly on metal sur- (Scheme 1). These glycosyl azides were prepared from the cor-
faces [16,17]. For such novel MS applications, a new technolo- responding halogenoses by the treatment with 1.2 equivalents of
gy, i.e., ES-IBD, has been developed. ES-IBD furthermore sodium azide in aqueous acetone according to literature proce-
allows for the integrity of the material [18]. The application of dures [22-26]. In the case of 2,3,4,6-tetra-O-acetyl-α-ᴅ-
these new techniques to synthetic glycopeptides is expected to mannopyranosyl bromide, however, this procedure only
provide new insight into the mechanisms of the interaction of resulted in a complete hydrolysis of the halogenose. Under opti-
glycopeptides with the cell surface [16].
mized reaction conditions using Moyle’s procedure (2.1 equiva-
lents NaN3 in DMF) [23], the azide 1c could be obtained in
Our focus lay on the preparation of glycopeptide libraries con- 33% yield (compared to the reported 13%). Attempts to prepare
taining ʟ-asparagine since many saccharides on the cell surface the corresponding α-ᴅ-mannosyl azide under various previ-
are N-glycosidically linked to this amino acid [8]. The most ously described conditions [27,28] failed in our hands though.
abundant sugars found in these asparagine-linked structures are
N-acetylglycosylamines, glucose, galactose, mannose, Next, the glycosyl azides 1a–f were converted into the corre-
cellobiose, lactose, and maltose [19]. Therefore, we intended to sponding glycosylamines 2a–f by hydrogenation with Pd on
prepare glycopeptide structures containing these sugars in a charcoal in ethyl acetate. Since an anomeric mixture of
stepwise way, up to tripeptides. An orthogonal Fmoc/t-Bu glycosylamines was obtained in most cases, with a strong pre-
protecting group strategy was chosen along with the frequently dominance of the corresponding β-anomers, and TLC analysis
used onium salts HBTU and HATU as coupling reagents [20]. showed no formation of other unwanted side products during
The canonical amino acids ʟ-phenylalanine, ʟ-tryptophane, and hydrogenation, the latter amines were used for the next step
ʟ-alanine were chosen in order to allow for different side chain without further purification. In all cases, only the β-anomeric
motifs that, in turn, would increase the suitability for the glycosylpeptides were obtained from the anomeric mixtures of
following investigation via STM. Due to the aromatic back- amines (see below).
bone and known interaction of the compounds with Cu(100)
and Au(111), ʟ-Phe and ʟ-Trp should be distinguishable in STM The glycosylated aspartic acids 3 (except for 3d and 3f) were
from the remaining building blocks [16,18,21]. ʟ-Ala seemed to prepared previously with the help of different types of coupling
be an ideal supplement for the synthesis of these new glycocon- reagents, such as benzotriazol-1-yloxytris(dimethylamino)phos-
jugates since there was no additional functional group that phonium hexafluorophosphate (BOP) or diisopropyl carbo-
Scheme 1: Description of the starting materials 1a–f and 2a–f.
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Beilstein J. Org. Chem. 2020, 16, 888–894.
diimide (DIC) and additives, such as 1-hydroxybenzotriazole pling protocol with O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetra-
(HOBt) or triethylphosphine. [29-31] However, in our hands, methyluronium hexafluorophosphate (HATU) or with 2-(1H-
none of the described methods reached the high yields that we benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophos-
previously obtained with the onium salt-mediated peptide cou- phate (HBTU, Scheme 2 and Table 1) [6,32]. Here, we com-
Scheme 2: Peptide coupling reactions, including the previous Fmoc cleavage.
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Beilstein J. Org. Chem. 2020, 16, 888–894.
Table 1: Yields of the peptide coupling reactions and comparison of HBTU and HATU.
entry
compound
HBTU
HATU
entry
compound
HBTU
HATU
1
2
3
4
5
6
3a
3b
3c
3d
3e
3f
91%
92%
77%
61%
63%
56%
82%
84%
80%
79%
61%
86%
19
20
21
22
23
24
6a
6b
6c
6d
6e
6f
82%
76%
64%
75%
50%
50%
75%
72%
60%
59%
70%
78%
7
4a
4b
4c
4d
4e
4f
81%
67%
72%
68%
76%
78%
76%
73%
80%
80%
79%
67%
25
26
27
28
29
30
7a
7b
7c
7d
7e
7f
60%
76%
76%
60%
75%
58%
68%
79%
58%
68%
65%
50%
8
9
10
11
12
13
14
15
16
17
18
5a
5b
5c
5d
5e
5f
53%
52%
89%
38%
78%
59%
66%
75%
62%
59%
66%
73%
pared the condensation with HBTU and HATU for the prepara- The treatment of the glycosylated tripeptides 6a–f and 7a–f
tion of the glycosyl amino acids 3a–f since previous studies with a mixture of TFA, DCM, and H2O (10:10:1) [31] afforded
showed HATU to be more efficient than HBTU in similar cases the partially deprotected acids 8a–f and 9a–f in quantitative
[33-35]. Table 1 summarizes the obtained yields for conver- yields. The final deprotection of both the base-labile acetyl and
sions 2→3, 3→4, 3→5, 4→6, and 5→7 (Scheme 2). As can be Fmoc-protecting groups was achieved by the treatment with
seen in Table 1, the yields for all condensations with HBTU and 7 M NH3 in MeOH, which afforded the free glycopeptides
HATU are in a comparable range. Only for the cellobiosyl Asp 10a–f and 11a–f in good to excellent yields (Scheme 3 and Ta-
amino acid 3f (Table 1, entry 6), the lactosylated Asp–Phe–Ala ble 2). Since chromatographic purification of the unprotected
tripeptide 6e (Table 1, entry 23), and the galactosylated glycopeptides on silica gel would have been rather difficult due
Asp–Trp dipeptide 5b (Table 1, entry 14), HATU gave a signif-
icantly higher yield than HBTU.
Table 2: Final deprotection yields of 10a–f and 11a–f.
The Fmoc cleavage of the compounds 3–5 was performed with
entry
compound
yield
piperidine in DMF (20 vol %) [36]. The reaction products were
not isolated and purified but instead immediately used for the
succeeding peptide coupling step to give the dipeptides 4 and 5
from the glycosylated asparagines 3, and the tripeptides 6 and 7
from 4 and 5, respectively (Scheme 2). The yields of the glyco-
sylated di- and tripeptides were moderate to good (Table 1,
entries 7–30). For the coupling of Fmoc-Trp instead of Fmoc-
Phe, an improved yield could be observed when using HATU
instead of HBTU. Although it is well known that Fmoc-pro-
tected tryptophane can lead to unwanted side products when the
NH group in the indole ring remains unprotected, we decided to
refrain from additional protection of the indole moiety because
the removal of that protecting group would have reduced the
overall yield in the end.
1
10a
10b
10c
10d
10e
10f
98%
96%
66%
61%
80%
99%
92%
99%
77%
49%
99%
87%
2
3
4
5
6
7
11a
11b
11c
11d
11e
11f
8
9
10
11
12
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Beilstein J. Org. Chem. 2020, 16, 888–894.
Scheme 3: Cleavage of the fully protected peptides 6 and 7.
to their zwitterionic character, our simple deprotection strategy through simple filtration. After washing the residue with petro-
with NH3 in MeOH allowed the crude product to precipitate leum ether, the compounds 10 and 11 were obtained in pure
directly from the reaction mixture, followed by the isolation form.
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Beilstein J. Org. Chem. 2020, 16, 888–894.
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Conclusion
We described the efficient chemical synthesis of a series of new
glycopeptides containing asparagine, tryptophane, alanine, and
phenylalanine linked to various saccharides, such as glucose,
galactose, mannose, cellobiose, lactose, and maltose. We further
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Supporting Information File 1
General methods, experimental procedures, and product
characterization data of the compounds 1a–f to 11a–f.
[https://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-16-80-S1.pdf]
Supporting Information File 2
NMR spectra of the compounds 1a–f to 11a–f.
[https://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-16-80-S2.pdf]
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Acknowledgements
We would like to thank Dr. Dorothee Wistuba and her team for
the measurement of the mass spectra and Petra Schülzle for per-
forming the elemental analysis. We also want to thank Dr.
Sabine Abb and Prof. Dr. Klaus Kern from the Max Planck
Institute for Solid State Research for help in the field of
ES-IBD. We also thank http://www.pixabay.com for providing
parts of our graphical abstract.
doi:10.1146/annurev-anchem-071015-041633
18.Abb, S.; Harnau, L.; Gutzler, R.; Rauschenbach, S.; Kern, K.
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Funding
We would like to thank the Baden-Württemberg Stiftung for
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financial support.
21.Rauschenbach, S.; Rinke, G.; Gutzler, R.; Abb, S.; Albarghash, A.;
Le, D.; Rahman, T. S.; Dürr, M.; Harnau, L.; Kern, K. ACS Nano 2017,
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ORCID® iDs
Laura Sršan - https://orcid.org/0000-0001-6151-5858
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