On-Bead Peptoid Dimerization Induced by Incorporation of Glycosylated Bridging Units in Submonomer Solid-Phase Approach to Glycopeptoids

Article abstract of DOI:10.1021/acs.orglett.9b01242

A study on submonomer solid-phase synthesis of S-glycopeptoids has been carried out by screening different parameters. Dimeric species, featuring glycosylated bridging amino monomers, were found under suitable conditions. These dimers arise from an on-res

Full text of DOI:10.1021/acs.orglett.9b01242

Letter
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
pubs.acs.org/OrgLett
On-Bead Peptoid Dimerization Induced by Incorporation of
Glycosylated Bridging Units in Submonomer Solid-Phase Approach
to Glycopeptoids
Daniela Comegna,* Annarita Del Gatto,†,‡ Michele Saviano,§,‡ and Laura Zaccaro*,†,‡
,†
†
Institute of Biostructures and Bioimaging-CNR, Via Mezzocannone 16, 80134 Naples, Italy
‡
Interdepartmental Center of Bioactive Peptide, University of Naples Federico II, Via Mezzocannone 16, 80134 Naples, Italy
§
Institute of Crystallography-CNR, Via Amendola 122/O, 70126 Bari, Italy
*
S Supporting Information
ABSTRACT: A study on submonomer solid-phase synthesis of
S-glycopeptoids has been carried out by screening different
parameters. Dimeric species, featuring glycosylated bridging
amino monomers, were found under suitable conditions. These
dimers arise from an on-resin cross-linking reaction occurring
with the incorporation of a glycoamino submonomer into the
growing chain and subsequent nucleophilic attack of the
resulting secondary amine to a still unreacted bromoacetylated
unit. The arising byproduct can be regarded as a novel dimeric
peptoid type.
arbohydrate−amino acid bonding entails significant
prepared are useful tools in the comprehension of multivalent
carbohydrate−protein interactions.
C
modifications to biological functions of proteins and
natural peptides. Indeed, glycosides are known to provide both
structural and functional features contributing to protein
folding and stability and play pivotal roles in a wide set of
However, only a few examples of glycopeptoid syntheses are
8
strictly based on the solid-phase submonomer approach
9
developed by Zuckermann et al., which still remains the most
1
recognition events.
straightforward and versatile strategy for oligopeptoid prep-
aration by the use of a wide range of primary amines and
monobromoacetic acid (mba) in a two-step iterative procedure
(Figure 1).
Therefore, the introduction of a carbohydrate moiety into a
protein or a peptide-like structure is currently a very intriguing
topic in organic chemistry. So far, a large effort has been
addressed to the elaboration of streamlined synthetic methods
Toward this goal, we have previously employed readily
synthesized 2-aminoethyl peracetyl thioglycosides for the
attachment of S-linked sugars onto various N-phenylmethyl
peptoid backbones under submonomer solid-phase condi-
2
for accessing glycosylated proteins, peptides, and peptidomi-
3
metics such as peptoids.
Peptoids are based on an oligoglycine scaffold deriving from
the shift of the side chain from the α-carbon to the nitrogen
atom throughout the peptide backbone. This simple
modification leads to important changes to peptide features,
mostly in magnification of serum stability and flexibility so that
the bioactive action of the molecule can be improved and,
more, giving the chance to an easier preparation of a great
variety of peptoids adorned with diverse side chains. These
advantages make it very attractive molecules suited to different
8b
tions. This approach is the first reported synthetic access to
S-glycopeptoids; it is founded on the fast preparation of
suitable glycosylated building blocks accomplished by a two-
step conversion of peracetylated carbohydrates into glycosyl
1
0
11
thiolates via glycosyl iodides. Nucleophilic glycosyl
thiolates are then entrapped in situ with 2-bromoethylamine
to provide the thioglycosyl ethylamines suited to submonomer
synthesis and featuring an N-homocysteine linkage.
4
The choice to develop a synthetic method for the access to
S-glycopeptoids was spurred by the fact that sulfur is isosteric
with respect to oxygen and forms an enzymatically more stable
biological applications.
To date, different classes of peptoids have been already
5
6
reported, such as linear and cyclic α-peptoids, β-peptoids,
12
7
glycosidic linkage, as witnessed by the numerous reported
and α-alkyl β-peptoids. Furthermore, different synthetic
strategies to O-, N-, C-, and S-linked glycopeptoids have
been developed both in solution and on solid phase, by
monomer and submonomer strategies, as well as by
13
methods for the assembly of S-glycopeptides.
Received: April 9, 2019
3b
postchemical glycoconjugation. Glycopeptidomimetics thus
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.9b01242
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
Table 1. Stack Chromatograms of Crudes from Nme-Ngalte
Dipeptoid Submonomer Synthesis at Different Loadings of
2
-Chlorotrityl Resin (TRT) (a−d) and of Other Resin
Types (e, f)
a
entry
resin
loading (mmol/g)
product ratio (%) (I/II)
a
b
c
d
e
f
TRT
TRT
TRT
TRT
TGR
TGT
1.6
1.0
0.6
0.1
0.25
0.16
25/75
40/60
60/40
84/16
88/12
97/3
a
Product ratio in a percentage calculated by integration of the each
HPLC at 210 nm and normalized by the titration curve.
Figure 1. Submonomer solid-phase approach.
Herein, we present a new synthetic opportunity offered by
the reported method of glycopeptoid assembly that was
unveiled upon varying different reaction parameters such as the
peptoid sequence, the nature of the resin, and its loading.
Typically, peptoids were synthesized on medium-loaded
resins (0.4−0.9 mmol/g) such as polystyrene-bound Rink
amide or PEG-based Tentagel resins. In order to obtain free C-
terminal glycopeptoids, we used 2-chlorotrityl resin, partic-
ularly useful also to suppress diketopiperazine formation. This
polystyrene-based resin is characterized by high-loading
functionalization, up to 1.6 mmol/g.
In attempting the preparation of an alternated N-(2-
methoxyethyl)glycine (Nme) and N-(galactosylthio)ethyl
glycine (Ngalte) peptoid sequence on highly loaded 2-
chlorotrityl resin (1.6 mmol/g), we had to stop the synthesis
at the dipeptoid stage. In fact, surprisingly, only traces of the
expected dipeptoid 1 (I) (Figure 2), with 581 m/z value was
found (peak 1 (I), Table 1, entry a), and instead, a major
product with a higher mass of 754 m/z was observed (peak 2
Table 2. Stack Chromatograms of Crudes from the Npm-
Ngalte Dipeptoid Submonomer Synthesis at Different
Loadings of 2-Chlorotrityl Resin (TRT)
a
entry
resin
loading (mmol/g)
product ratio (%) (I/II)
a
b
c
TRT
TRT
TRT
TRT
1.6
1.0
0.6
0.1
73/27
83/17
85/15
96/4
(II), Table 1, entry a).
d
a
Product ratio in a percentage calculated by integration of the each
HPLC at 210 nm and normalized by the titration curve.
Figure 2. Expected products of the submonomer synthesis (1 and 3)
and their correlated dimeric byproducts (2 and 4).
Intrigued by this observation, we repeated the same
procedure for the preparation of a dipeptoid composed of N-
phenylmethyl glycine (Npm) and Ngalte, since it was already
successfully accomplished in previous work. As a matter of
fact, the expected dipeptoid 3 (I), with 613 m/z value, was
formed in good yield, although a byproduct with a higher mass
Isolated byproducts 2 and 4 were characterized by NMR
and mass analysis. Integration of proton signals in the H
NMR spectra indicated that the species with a higher mass are
bearing a single sugar unit, and the corresponding mass values
were perfectly coherent with the dimeric structures reported in
Figure 2.
1
8b
(
m/z = 818) was again found (peak 4 (II), Table 2, entry a).
B
DOI: 10.1021/acs.orglett.9b01242
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
With this evidence in hand, we put forward a possible
mechanism accounting for the dimer formation: during the
preparation of the dipeptoid oligomer, a nucleophilic
substitution by the newly formed glycosyl secondary amine
can occur for the still unreacted bromoacylated precursor with
formation of a dimeric species featuring the glycosidic moiety
in the middle of the molecule (Scheme 1).
better than that obtained on TRT resin at a lower loading
(Table 1, entries e and f).
To confirm this result, we performed the synthesis of a
longer S-glycopeptoid containing N-methoxyethyl glycines on
a TGT resin. The introduction of three sugar moieties was
successfully accomplished, the triglycosylated hexapeptoid
being obtained in very good yield (Scheme 2, 5).
Scheme 1. Reaction Mechanism of the Dimer Formation in
the Submonomer Approach
Scheme 2. Submonomer Synthesis of Peptoid 5 on Novasyn
TGT Resin
With the aim of deeply investigating the cross-linking
reaction, we performed the synthesis of a commonly prepared
homopentapeptoid (Nme) on highly loaded 2-chlorotrityl
5
resin (1.6 mmol/g). As expected, the synthesis was smoothly
accomplished and afforded the pentapeptoid in a very high
yield (Figure 3, 7). However, the LC−MS spectra of the crude
This intermolecular side reaction leads to chain termination
and can heavily affect the efficiency of the normal
oligomerization. Thus, in order to prevent dimerization, we
first tested different resin loadings. Checking the outcome of
the solid-phase procedure at dipeptoid stage, we found out
that, in the case of Npm-Ngalte peptoid synthesis, the
monomeric species (I) was formed in a larger amount than
the dimeric species (II) even with a very high loading rate (1.6
mmol/g); expectedly, by using lower loading rates the dimer
was observed in trace amounts (Table 2, entries a−d).
Considering that the dimer absorbs twice respect with the
monomer, as measured by a titration curve (see Supporting
Information), we determined the overall yield normalizing the
integration peak ratios. In this way we found that the yield of
the monomeric species could range from good (73%) to
excellent (96%), in accordance with the results already
8b
reported for the synthesis of this sequence.
Conversely, the formation of the dimer was markedly
pronounced in the presence of the methoxyethyl side chain,
much less hindered than the benzyl group. As a matter of fact,
the yield of Nme-Ngalte peptoid synthesis was drastically
reduced by the use of high to medium loaded 2-chorotrytil
resins (Table 1, entries a−c).
Figure 3. Distribution of products in the submonomer synthesis of
the peptoid 7 (entry a) and the glycopeptoid 11 (entry b) using 2-
chlorotrytil resin (loading 1.6 mmol/g). Product ratio in a percentage
calculated by integration of the each HPLC at 210 nm and normalized
by the titration curve.
To afford a very good yield for the Nme-Ngalte peptoid
sequence we had to lower the loading of 2-chlorotrityl resin
down to 0.1 mmol/g, eventually observing the full inversion in
the abundance of the two peaks (Table 1, entry d). Thus, the
higher the loading, the lower the efficiency of the
oligomerization, and this trend confirmed the hypothesis that
interchain reactions occurred on the same resin bead.
These results prompted us to compare the outcome of
analogous syntheses also on alternative resins. Commonly used
product disclosed the formation of dimers at each nucleophilic
substitution step (Figure 3, entry a, compounds 6, 8,
9 and 10), even if in a very tiny amount. Instead, on
attempting the synthesis of hexapeptoid 11 (Figure 3) from pen-
pentapeptoid 7 by introducing the thiogalactosylethyl unit by
the same submonomer pathway and reaction conditions, the
prevalent formation of the dimer 12 (Figure 3) was found.
1
4
SPPS supports, such as TGT alcohol and TGR PEG-
polystyrene based resins, were therefore tested. Fortunately,
with pegylated resins dimerization was mostly suppressed and
the yield of the monomeric species was excellent and even
C
DOI: 10.1021/acs.orglett.9b01242
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Organic Letters
Letter
a
Scheme 3. Differently Glycosylated Dipeptoids and Their Correlated Dimers Obtained on 2-Chlorotrityl Resin
a
b
All reactions were carried out by using 2-chlorotrityl resin loaded 1.0 mmol/g. Product ratio in a percentage calculated by integration of each
HPLC at 210 nm and normalized by the titration curve.
These results highlighted the leading role of the thioglycosyl
amine that works, regardless of the oligomer length, to favor
significantly the dimer formation. In contrast, when common
unglycosylated primary amines were used, the dimer formation
was a totally negligible byreaction, as witnessed by the fact that
no cross-reaction was mentioned so far, in spite of the
countless examples of submonomer solid-phase synthesis
reported.
A systematic study was thus carried out to decipher the
peculiar behavior of the thiogalactosyl ethylamine on the solid
phase.
First, we prepared different S-glycosyl amines to be
employed in the submonomer solid-phase route to N-
methoxyethyl peptoids on a medium-functionalized 2-chloro-
trityl resin. Collected data revealed a similar behavior for
glucosyl, fucosyl, and lactosyl analogues, showing that the
cross-reaction is not dependent on the sugar configuration
resin the dimer formation is highly promoted contrary to what
happens on pegylated solid supports.
In perspective, the on-resin interchain reaction can be
exploited as a route for the access to peptoid dimers
comprising a sugar moiety. High-yielding dimerization can be
contemplated for less sterically hindered peptoid sequences
prepared on high-loaded polystyrene resins. This method is
clearly more straightforward with respect to classical
dimerization methods on the solid phase which typically entail
the construction of the product step by step. Thus, with the
use of the described strategy the dimer preparation takes half
the time and requires a lower reagent amount. In addition, it
returns dicarboxylated compounds ready for further function-
alizations.
In conclusion, we have detected and characterized the
byproduct featuring a bridge-sugar moiety formed during the
submonomer solid-phase route to S-glycopeptoids and
resulting from a chain termination reaction. Different reaction
conditions to modulate or totally avoid the formation of the
dimeric product have been investigated. It was found that
dimerization takes place only when the glycosyl amine is added
and occurs at every position of the growing chain during the
submonomer synthesis. The bridge-sugar dimer is obtained in
good yield on highly loaded polystyrene-supported resin, and it
is completely suppressed on medium-loaded PEG-based solid
supports.
(
Scheme 3, 13−18) or on the length of the linker bearing the
sugar (Scheme 3, 19 and 20). Suspecting a role of the sulfur
atom in a possible anchimeric assistance of the cross-reaction,
we also tested the O-analogue, employing 2-aminoethyl
peracetyl-O-galactose in the dipeptoid route. In this case, we
found a slight reduction of the cross-reaction obtaining a
similar amount of the monomer and the dimer (Scheme 3, 21
and 22). Even if a small effect of the sulfur cannot be ruled out,
it is evident that other factors are responsible for the cross-
reaction. Interestingly, upon use of a free thiogalactosyl
ethylamine the dimer formation was even higher than with
its peracetylated analogue (Scheme 3, 23 and 24).
ASSOCIATED CONTENT
Supporting Information
■
*
S
On these bases, the reactivity of the thioglycosyl ethylamines
on the solid phase seems to be mainly correlated to the nature
of the solid support and the polarity of the sugar
functionalities. Actually, it is known that many carbohydrate-
binding proteins can interact with carbohydrate ligands with
aromatic amino acid residues in their binding sites via CH/π
interactions arising from a stacking geometry. Likewise, the
hydrophobic face of the sugars could tender to adhere to the
flat faces of polystyrene support by interacting via CH/π
interaction and promoting the interchain nucleophilic
substitution. Thus, with polystyrene-supported 2-chlorotrityl
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.9b01242.
Full experimental details, characterization of all com-
1
13
pounds, H and C NMR spectra, and LC−MS profiles
of all compounds (PDF)
15
AUTHOR INFORMATION
Corresponding Authors
■
*E-mail: daniela.comegna@unina.it.
D
DOI: 10.1021/acs.orglett.9b01242
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
E-mail: lzaccaro@unina.it.
Letter
*
Menchise, V.; Saviano, S.; De Luca, S. Chem. - Eur. J. 2018, 24, 6231−
6
(
238.
ORCID
14) Palomo, J. M. RSC Adv. 2014, 4, 32658−32672.
Daniela Comegna: 0000-0001-7367-1233
Michele Saviano: 0000-0001-5086-2459
Laura Zaccaro: 0000-0001-6843-0152
(15) Spiwok, V. Molecules 2017, 22, 1038.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful to Mr. Leopoldo Zona (Institute of
Biostructures and Bioimaging-CNR, Naples) for NMR
technical assistance and Mr. Luca De Luca (Institute of
Biostructures and Bioimaging-CNR, Naples) for computer
assistance. This study has been supported by grants from
MIUR, Programma Operativo Nazionale Ricerca e Compet-
itivita
Nazionale Ricerca e Competitivita
1078.
̀
2007−2013 PON 01/02388, and Programma Operativo
̀
2007−2013 PON 01/
0
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