Solid-phase insertion of N-mercaptoalkylglycine residues into peptides

Article abstract of DOI:10.3390/molecules24234261

N-mercaptoalkylglycine residues were inserted into peptides by reacting N-free amino groups of peptides, which were initially synthesized on 2-chlorotrityl resin (Cltr) using the Fmoc/tBu method, with bromoacetic acid and subsequent nucleophilic replacement of the bromide by reacting with S-4-methoxytrityl- (Mmt)/S-trityl- (Trt) protected aminothiols. The synthesized thiols containing peptide-peptoid hybrids were cleaved from the resin, either protected by treatment with dichloromethane (DCM)/trifluoroethanol (TFE)/acetic acid (AcOH) (7:2:1), or deprotected (fully or partially) by treatment with trifluoroacetic acid (TFA) solution using triethylsilane (TES) as a scavenger.

Full text of DOI:10.3390/molecules24234261

molecules
Article
Solid-Phase Insertion of N-mercaptoalkylglycine
Residues into Peptides
Spyridon Mourtas 1,2, Dimitrios Gatos 1 and Kleomenis Barlos 1,3,
*
1
Department of Chemistry, University of Patras, 26510 Rio Patras, Greece; s.mourtas@upatras.gr (S.M.);
d.gatos@upatras.gr (D.G.)
Institute of Chemical Engineering Sciences of the Foundation for Research and Technology
Hellas (FORTH/IEC-HT), 26504 Rio Patras, Greece
CBL-Patras, Patras Industrial Area, Block 1, 25018 Patras, Greece
Correspondence: barlos@cblpatras.gr; Tel.: +30-2610-647600; Fax: +30-2610-647316
2
3
*
Academic Editor: Theodore Tselios
Received: 1 November 2019; Accepted: 20 November 2019; Published: 22 November 2019
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: N-mercaptoalkylglycine residues were inserted into peptides by reacting N-free amino
groups of peptides, which were initially synthesized on 2-chlorotrityl resin (Cltr) using the Fmoc/tBu
method, with bromoacetic acid and subsequent nucleophilic replacement of the bromide by
reacting with S-4-methoxytrityl- (Mmt)/S-trityl- (Trt) protected aminothiols. The synthesized thiols
containing peptide–peptoid hybrids were cleaved from the resin, either protected by treatment with
dichloromethane (DCM)/trifluoroethanol (TFE)/acetic acid (AcOH) (7:2:1), or deprotected (fully or
partially) by treatment with trifluoroacetic acid (TFA) solution using triethylsilane (TES) as a scavenger.
Keywords: aminothiols; oxidation; peptoids; solid-phase synthesis; 2-chlorotrityl resin
1. Introduction
Peptides as pharmaceutical compounds suffer from unfavorable pharmacokinetics such as slow
uptake into cells and rapid proteolytic cleavage, among other factors. N-alkylation of peptides
can overcome these limitations, thus, structures such as N-alkylated/methylated peptides [
1,2] and
N-substituted glycine residues (peptoids) [ ] are of great importance. Oligomers of N-substituted
3,4
glycine residues (peptoids) differ from peptides in that the side chains are connected to amide nitrogen
atoms rather than carbon atoms (Figure 1). They are classified as unnatural molecules that confer a
high degree of resistance to proteolytic degradation, while the absence of any backbone hydrogen
bonding means that peptoids exhibit a high degree of conformational flexibility [
present a broad variety of biological activities [ 16]. They also present bio-nanotechnological and
bio-medicinal interest as novel therapeutics [17 20], while peptide–peptoid hybrids (peptomers) are
also interesting structures which mimic biologically active peptides [21 23]. All of the aforementioned
5,6], while they also
7–
–
–
points have contributed to peptoids increasingly being investigated in the field of medicinal chemistry
as an option to replace peptides in the development of new therapeutics [24].
SH
SH
O
O
R
N
O
O
H
N
N
N
H-peptide2
peptide1-OH
R
C: thiol-containig peptide-peptoid hybrids
A: peptide
B: peptoid
thiol-peptoid
Figure 1. Comparison of a peptide (
A
), a peptoid (B), and the newly synthesized thiol-containing
peptide–peptoid structure (C).
Molecules 2019, 24, 4261; doi:10.3390/molecules24234261
www.mdpi.com/journal/molecules

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The solid-phase synthesis (SPS) of peptoids was a major breakthrough because it greatly increased
the synthetic efficiency, synthesis yields, and available side chain diversity, thereby dramatically
reducing time and costs [3]. Since then, several methods have been used for the solid-phase synthesis
(SPS) of peptoids [25–28] including the use of 2-chlorotrityl resin (Cltr) [29,30].
The thiol group is a structural element which is contained in important biologically active
compounds, like peptides, proteins, coenzymes (acetyl coenzyme A), and vitamins (biotin).
Examples of FDA-approved drugs include the enzyme inhibitors captopril (which is approved
as an Angiotensin-Converting Enzyme (ACE) inhibitor for the treatment of hypertension) [31,32] and
thiorphan (the active metabolite of the antidiarrheal racecadotril (acetorphan), which prevents the
degradation of endogenous enkephalins by acting as an enkephalinase inhibitor) [33], tiopronin (another
approved drug against heavy metal poisoning, liver diseases, or cystenuria), N-acetylcysteine (used
to treat paracetamol (acetaminophen) overdose and to loosen thick mucus in individuals with cystic
fibrosis or chronic obstructive pulmonary disease), and amifostine (a cytoprotective adjuvant used in
cancer chemotherapy and radiotherapy) [34], among others.
It is therefore of great interest to develop methods for the solid-phase synthesis (SPS) of
thiol-containing peptide–peptoid hybrids in order to mimic cysteine-like structures. For this reason,
we prepared S-protected aminothiols, which were linear or derived from naturally occurring aminoacids,
and applied using solid-phase peptide synthesis (SPPS), which is the most common method of peptide
production [35] by applying common methods of peptoid synthesis.
In general, peptoid submonomers are synthesized using a two-step procedure. The first step is an
acylation step with bromoacetic acid, using N,N0-diisopropylcarbodiimide (DIC) as the condensing
agent, while the second step is nucleophilic displacement using a monosubstituted amine, resulting in
the peptoid submonomer B (Figure 1).
By using S-4-methoxytrityl- (Mmt)/S-trityl- (Trt) protected aminothiols, we envisioned the
incorporation of the peptoid submonomer into peptide–peptoid hybrids like molecule
C (Figure 1).
Solid-phase peptide synthesis (SPPS) using Cltr and fluorenylmethoxycarbonyl (Fmoc)/tert-butyl (tBu)
was the selected method, which is now the most commonly used methodology for the preparation of
peptides [35]. The use of the S-Mmt and S-Trt protecting groups is of high importance, since S-Mmt
derivatives can be removed selectively in the presence of tBu-type protecting groups, thereby enabling
selective derivatization via alkylation, pegylation, labeling of the liberated thiol, or synthesis of
cystine analogues of peptide–peptoid hybrids after oxidation of the liberated thiols, as examples.
Thus, besides the general method for the preparation of the hybrid
C, we also provide selected
examples for the applicability of our method in the synthesis of the protected or deprotected (fully or
partially) thiol-containing peptide–peptoid hybrids, and the preparation of inter-disulfide bridged
peptide–peptoid structures based on the oxidation of different thiol groups of peptoid submonomers
that are present on the peptide chain.
2. Results and Discussion
Peptoids are usually synthesized using the submonomer synthetic approach consisting of
two steps. The first is an acylation step of the available amine group by bromoacetic acid and
diisopropylcarbodiimide (DIC) (which acts as an activator of the bromoacetic acid carboxylic group),
whilst the second involves nucleophilic displacement using a monosubstituted amine. This procedure
results in peptoid structure
B (Figure 1). It is of high importance that no racemization was observed by
the application of the submonomer method for peptoid synthesis [36–38].
By using S-4-methoxytrityl- (Mmt)/S-trityl- (Trt) protected aminothiols, we envisioned the
solid-phase synthesis of peptoid–peptide hybrids like molecule
C (Figure 1). For this, we first
synthesized S-Mmt- and S-Trt-protected derivatives of cysteamine or aminothiols derived from
optically active aminoacids [39] by their reaction with trityl- or 4-methoxytrityl chloride in the presence
of N,N-diisopropylethylamine (DIPEA) as a base (Scheme 1). The derived S-Mmt/Trt-protected
aminothiols were applied in the synthesis of thiol-containing peptide–peptoid hybrids (Figure 1)

Molecules 2019, 24, 4261
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using solid phase peptide synthesis (SPS) and fluorenylmethoxycarbonyl (Fmoc)/tert-butyl (tBu)
methodology and 2-chlorotrityl resin (Cltr), which is now the most commonly used methodology for
the production of peptides [38].
H2N
SH
R1
H2N
S
X
Cl
X
DIPEA
R1
=Pr
X=H (Trt)
X = OMe (Mmt)
Scheme 1. Synthesis of S-Mmt/S-Trt aminothiols. R1 = H or the side chain of naturally
occurring aminoacids.
For this, resin-bound peptide
N-Fmoc-amino acids and 1-hydroxy benzotriazole (HOBt)/DIC for their activation. Then, bromoacetic
acid was coupled on the N-terminal function of using DIC in N-methyl-2-pyrrolidone (NMP).
The obtained bromoacetyl resin-bound peptide was further reacted with the aminothiol , which was
protected at its thiol function with the acid-sensitive Trt and Mmt groups. In all cases, the nucleophilic
replacement of the bromine atom in with the aminothiol was completed within 12 h at room
1 was assembled on 2-chlorotrityl resin [40] (Scheme 2) using
1
2
3
2
3
temperature, resulting in the thiol-containing resin-bound peptide 4.
S-Pr
S-Pr
O
R1
R1
O
O
DCM/TFE/AcOH
R2
N
R2
N
peptide1-OH
peptide1-O
acylation
O
5
6
S-Pr
O
H2N
S Pr
R1
O
3
Br-CH2-CO2H
R1
Br
HN
P
H-peptide
Cl
O
peptide1-O
peptide1-O
DIC
DIPEA
1
2
4
= H-peptide-O
SPPS /
Fmoc/tBu
method
R1=H
S-Pr
S-Pr
R1
R1
O
O
N
DCM/TFE/AcOH
N
Fmoc-peptide2
peptide1-OH
Fmoc-peptide2
peptide1-O
8
7
Scheme 2. Method for the synthesis of peptide–peptoid hybrids on 2-chlorotrityl resin. Pr = Mmt, Trt;
R1 = H or amino acid side chain; R2 = Ac or Ar.
At this step of the proposed synthetic procedure, two options existed. In the first option, acylation
of
4
with acetic anhydride (Ac2O), acyl (Ac) chlorides, and aroyl (Ar) chlorides in NMP/DIPEA gave
5
within 2 h at room temperature. Treatment of 5 with dichloromethane (DCM)/trifluoroethanol
(TFE)/acetic acid (AcOH) (7:2:1) for 15 min at room temperature gave the acylated mercaptoalkyl
peptoid . By applying this method, the peptoids 12 (Figure 2) were prepared and analyzed by
LC-MS (Table 1). The second option included the effective acylation by the coupling reaction of
with acids such as bromoacetic acid in NMP using DIC as the condensing agent, thereby enabling
6
9–
4

Molecules 2019, 24, 4261
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the insertion of another peptoid submonomer in the peptide chain or the continuation of peptide
synthesis. In the latter case, resin-bound aminothiol containing peptide was chain-elongated
by using DIC/HOBt or 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate
(HBTU)/DIPEA/HOBt [41 42], but only in the case of using S-protected cysteamine as the mercaptoalkyl
moiety (R1=H). In contrast, the coupling of Fmoc-amino acids with the more steric-hindered aminothiol
containing peptide could not be completed under these conditions, regardless of the use of excess
amino acid or excess activation reagent. At the end of the synthesis, treatment of with DCM/TFE/AcOH
(7:2:1) gave the peptide–peptoid hybrid . By applying this method, the peptoids 13 16 (Figure 2) were
4
,
4
7
8
–
prepared and analyzed by LC-MS (Table 1). High purities (>88%) were seen using LC-MS analysis for
all synthesized derivatives (9–16), as can be seen by the summarized results in Table 1.
S-Trt
O
S-Mmt
O
S-Trt
O
S-Trt
O
S-Trt
O
N
N
N
N
N
Ac
Phe
Tyr(tBu)-Leu-OH
Ac
Asp(tBu)-Leu-OH
Ac
N
Asp(tBu)-Leu-
OH
O
9
10
11
S-Mmt
S-Trt
O
S-Trt
S-Mmt
O
O
Fmoc-Phe
N
N
Ala-OH
N
N
Bz
12
Asp(tBu)-OH
Fmoc-Asp(tBu)-Ala
Pro-OH
O
13
14
S-Mmt
S-Mmt
O
N
Fmoc-Tyr(tBu)-Thr(tBu)-Asp(tBu)
Thr(tBu)-Glu(tBu)-Ser(tBu)-Gly-OH
15
S-Mmt
O
S-Mmt
O
N
N
Tyr(tBu)-Thr(tBu)-Asp(tBu)
Thr(tBu)-Gln(Trt)-Ser(tBu)-Gly-OH
Fmoc-Ala
16
Figure 2. The synthesized S-Mmt/S-Trt/tBu-protected peptide–peptoid hybrids.
Table 1. Retention time (tR), yield of the crude product (%), HPLC purity of the crude products (%),
m/z (calculated and found) of the peptide–peptoid hybrids 9–17.
tR
Crude Yield
(%)
HPLC Purity c
(%)
m/z
a/a
(min)
Calculated
Found
26.7 b
23 a
760.4 d
1330.63 d
1536.71 d
709.33 d
969.41 d
1249.52 d
1719.89 d
2365.13 d
1004.45 d
760.6 d
665.9 e
768.9 a
709.3 d
969.4 d
625.4 e
855.0 e
-
9
92
94
92
89
95
93
93
90
96
91
93
89
88
93
90
89
92
92
10
11
12
13
14
15
16
17
34 b
27.2 b
32 b
18.8 a
19.3 a
26.9 a
28.3 b
1004.4 d
a Column: Zorbax SB-C18, 3.5
acetonitrile within 15 min; flow rate: 0.4 mL/min. Column: Lichrospher RP-8, 5
µm; 2.1
×
30 nm; gradient: from 50% to 100% acetonitrile in water within 15 min, 100%
b
µm; 4
×
150 mm; gradient: from
c
d
20% to 100% acetonitrile in water within 30 min; flow rate: 1 mL/min. HPLC purity determined at 265 nm. [M +
e
H]+. [M + 2H]+2
.
Besides bromoacetic acid, coupling with the resin-bound peptide
1 and subsequent replacement
of the halogen was also performed using the m- and p-chloromethylbenzoic acid. In one example of

Molecules 2019, 24, 4261
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this approach, H-Glu(tBu)-2-Cltr resin reacted with p-chloromethylbenzoic acid in the presence of
DIC. The derived product was then coupled with Fmoc–Leucine in the presence of HOBt/DIC, and the
derived hybrid was cleaved from the resin in its protected form using DCM/TFE/AcOH (7:2:1) to the
hybrid 17 (Scheme 3), which was also analyzed by LC-MS (Table 1).
Cl
CO2H
O
Cl
O
S-Mmt
H2N
Glu(tBu)-O
H-Glu(tBu)-O
Glu(tBu)-O
HN
DIPEA
DIC
S-Mmt
Fmoc-Leu-OH
HOBt/DIC
O
O
DCM/TFE/AcOH
Fmoc-Leu
Fmoc-Leu
Glu(tBu)-OH
N
Glu(tBu)-O
N
17
S-Mmt
S-Mmt
Scheme 3. Synthesis of peptide–peptoid hybrid 17 using m-chlorobenzoic acid.
As an example of the HPLC analysis chromatograms of the different products 9–17, we present
the synthesis of the S-Mmt-protected peptide–peptoid hybrids 10 and 17. These were obtained after
treatment of the resin-bound peptide–peptoid hybrids with DCM/TFE/AcOH (7:2:1), which resulted in
the cleavage of the S-Mmt-protected peptide–peptoid hybrids from the resins (Figure 3A,B). In addition,
in order to investigate the effect of different acidic conditions, we treated the resin-bound hybrid 18
with 65% trifluoroacetic acid (TFA)/triethylsilane (TES) (95:5) (Scheme 4). Under these conditions,
the hybrid was cleaved from the resin, while the S-Mmt and tBu groups were simultaneously removed
to form hybrid 19. This was prepared in high purity (Figure 3C).
Figure 3. HPLC analysis of peptide–peptoid hybrids: 10
gradient: from 50% to 100% acetonitrile in water within 15 min, 100% acetonitrile within 15 min;
flow rate: 0.4 mL/min); 17
) (column: Lichrospher RP-8 (4 × 150 mm, 5 µm); gradient: from 20%
to 100% acetonitrile in water within 30 min; flow rate: 1 mL/min); 19 ) (column: Lichrospher
(A) (column: Zorbax SB-C18, 3.5 µm; 2.1 × 30 nm;
(B
(
C
RP-8 (4 × 150 mm, 5 µm); gradient: from 20% to 100% acetonitrile in water within 30 min; flow rate:
1 mL/min). All chromatograms were detected at 265 nm.
SH
S-Mmt
O
O
65% TFA/TES
N
N
Fmoc-Asp-Ala
Pro-OH
Fmoc-Asp(tBu)-Ala
Pro-O
19
18
Scheme 4. Synthesis of S-free/completely unprotected peptide–peptoid 19.

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Disulfides in proteins play an important role in the maintenance of biological activity and
conformational stability, thus, their formation is often a crucial final stage in peptide synthesis [43].
For this reason, we were also interested in evaluating the applicability of our method in the
synthesis of thiol-bridged peptoid–peptide hybrids. As an example, we synthesized the resin-bound
peptide-peptoid hybrid 20 (Scheme 5), which contained two S-Mmt-protected peptoid submonomers
and tBu-protected amino acids. This was treated with 1.1% TFA in DCM/TES (95:5) for 5 min at room
temperature, thereby enabling the cleavage of the hybrid from the resin and selectively removing the
S-Mmt groups, while the tBu-groups remained unaffected.
S-Mmt
O
S-Mmt
O
N
N
Fmoc-Gly
Tyr(tBu)-Thr(tBu)-Asp(tBu)
Thr(tBu)-Glu(tBu)-Ser(tBu)-Gly-O
20
1.1% TFA/TES
SH
SH
O
O
N
N
Fmoc-Gly
Tyr(tBu)-Thr(tBu)-Asp(tBu)
21
Thr(tBu)-Glu(tBu)-Ser(tBu)-Gly-OH
DMSO/H2O (7:3)
S
S
O
O
N
N
Fmoc-Gly
Tyr(tBu)-Thr(tBu)-Asp(tBu)
Thr(tBu)-Glu(tBu)-Ser(tBu)-Gly-OH
22
Scheme 5. Synthesis of thiol-bridged peptoid–peptide hybrid 22.
t
Via this procedure, the partially Bu-protected hybrid 21 was obtained and further oxidized
using dimethyl sulfoxide (DMSO), which is known as a mild oxidant. The progress of the oxidation
was followed by LC-MS (Figure 4). As can be seen, the treatment of the resin 20 with 1.1% TFA
in DCM/TES (95:5) gave the S-free (tBu-protected) hybrid 21. HPLC analysis of the crude hybrid
showed the existence of Mmt-H (identified by ESI-MS), which was the product of the reaction of
TES with Mmt cations formed during cleavage/deprotection. Hybrid 21 was further oxidized to the
tBu-protected peptide–peptoid hybrid 22. Under these mild conditions, oxidation was completed in
48 h without the formation of any by-products, which was shown by LC-MS analysis (Figure 4B).
Moreover, by following the HPLC analysis, we were able to observe the conversion of 21 to 22. Thus,
the oxidation of 21 was completed at about 50% in 12 h (Figure 4A), while after 48 h, 21 was completely
oxidized to 22 under the applied conditions (Figure 4B). Both products were identified by MS analysis
(see Figure 4C for 22).
Figure 4. LC-MS analysis of the oxidation of crude hybrid 21 in DMSO, after 12 h (
A) and 48 h (B).
Column: Nucleosil C8, 7 m, 125 4 mm; flow rate: 1 mL/min; gradient: from 50 to 100% acetonitrile
µ
×
in water within 30 min; detection at 265 nm. (C
) ES-MS analysis of the oxidized dimer 22. [M + Na]+
calc: 1655.80; [M + Na]+ found: 1655.6.

Molecules 2019, 24, 4261
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In order to investigate the selective deprotection of the S-Mmt over S-Trt protecting groups,
we studied the gradual removal of the protecting groups of the S-Mmt/S-Trt type. For this reason we
prepared the resin-bound hybrid 23 and initially treated it with 1.1% TFA in DCM/TES (95:5) for 5 min
at room temperature (Scheme 6).
S-Trt
O
Fmoc-Phe
N
N
Ala-O
O
23
S-Mmt
DCM/TFE/AcOH
1.1% TFA/TES
65% TFA/TES
SH
S-Trt
O
S-Trt
O
O
Fmoc-Phe
N
Fmoc-Phe
N
Fmoc-Phe
N
N
Ala-OH
N
Ala-OH
N
Ala-OH
O
O
O
14
24
25
S-Mmt
SH
SH
Scheme 6. Synthesis of S-Mmt/S-Trt-protected peptide–peptoid hybrid 14, partially S-free
peptide–peptoid hybrid 24, and S-free peptide–peptoid hybrid 25.
Under these conditions, the fully S-Mmt/S-Trt-protected hybrid 14 was synthesized in high
selectivity, as shown by HPLC analysis (Figure 5A). In addition, the resin-bound hybrid 23 was also
treated with 1.1% TFA in DCM/TES (95:5). Under these conditions, the direct cleavage of the hybrid
from the resin and removal of the S-Mmt groups enabled the synthesis of the S-free/S-Trt-protected
hybrid 24 (Figure 5B). Finally, the resin-bound hybrid 23 was treated with 65% TFA in DCM/TES (95:5),
which resulted in the cleavage of the hybrid from the resin and the simultaneous removal of both
S-Mmt/S-Trt groups to form the fully unprotected hybrid 25 (Figure 5C).
Figure 5. HPLC analysis of hybrids 14
2.1 × 30 nm; gradient: ( ) from 50% to 100% acetonitrile in water within 15 min, 100% acetonitrile
within 15 min; ( ) from 20% to 100% acetonitrile in water within 30 min; ( ) from 20% to 100%
(A), 24 (B), and 25 (C). Column: Zorbax SB-C18, 3.5 µm;
A
B
C
acetonitrile in water within 30 min; flow rate: 0.4 mL/min; detection at 265 nm.
3. Materials and Methods
3.1. Materials
All chemicals were purchased from Sigma-Aldrich OM, Athens, Greece, except 2-chlorotrityl
polystyrene resin (Cltr) and Fmoc-protected amino acids, which were gifted from CBL Patras S.A.
(Industrial area of Patras, Building block 1, GR-25018, Patras, Greece). All chemicals were used without
further purification.

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3.2. Analytical Methods
Thin layer chromatography (TLC) was performed on precoated silica gel 60 F254 plates (Merck,
Darmstadt, Germany) and spot detection was carried out by UV light or by charring with a ninhydrin
solution. HPLC analysis was performed on a Waters 600E multisolvent delivery system (Milford, MA,
USA), combined with Waters 991 photodiode array detector, using the following methods: (A) column:
Nucleosil C8, 7
water within 30 min; (B): column: Zorbax SB-C18, 3.5
acetonitrile in water within 15 min, 100% acetonitrile within 15 min or from 20% to 100% acetonitrile in
µ
m, 125
×
4 mm; flow rate: 1 mL/min; gradient: from 50 to 100% acetonitrile in
m; 2.1 30 nm; gradient: from 50% to 100%
µ
×
water within 30 min or from 20% to 100% acetonitrile in water within 30 min; flow rate: 0.4 mL/min;
(C): Lichrospher RP-8 (4
×
150 mm, 5 µm); gradient: from 20% to 100% acetonitrile in water within
30 min; flow rate: 1 mL/min. All chromatograms were detected at 265 nm. ES-MS spectra were
recorded on a Micromass Platform L.C. (Manchester, UK) at 30 V.
3.3. Synthetic Procedures
3.3.1. General Protocol for the Synthesis of S-trityl (Trt) and S-4-methoxytrityl (Mmt) Aminothiols
Cysteamine or aminothiols derived from optically active amino acids (1 mol) [34] were dissolved
in the highest possible concentration in dichloromethane (DCM), and diisopropyletheylamine (DIPEA)
(2.5 mol) was added. A 1 M solution (with respect to the aminothiol) of trityl chloride (1.2 mol) or
4-methoxytrityl chloride (1.2 mol) in DCM was prepared. This was added dropwise to the stirred
aminothiol solution. The completion of the reaction was checked by TLC. The DCM was removed by
rotary evaporation and diethylether (DEE) was added to the residual oil. The precipitated solid was
filtered, washed with DEE, and dried in vacuo.
3.3.2. Solid-Phase Peptide Assembly, General Protocol
Solid-phase peptide synthesis was carried out manually in plastic syringes equipped with porous
polypropylene frits at room temperature using the fluorenylmethoxycarbonyl (Fmoc)/tert-butyl (tBu)
strategy [44].
Pre-Activation of Fmoc-Amino Acids
Fmoc-amino acid (4.5 mmol) and 1-hydroxybenzotriazole hydrate (HOBt) (5.8 mmol) were
dissolved in N-methyl-2-pyrrolidone (NMP) (4.5 mL) and cooled to 5 ◦C. Then, diisopropylcarbodiimide
(DIC) (5.0 mmol) was added and the mixture was stirred for 15 min at 5 ◦C.
Coupling
The solution of the pre-activated amino acid was added to the resin-bound amino-acid or peptide
or peptide–peptoid hybrid (5.0 g, loading 0.3 mmol/g) and shaken for 3 h at room temperature.
A sample was taken to check the reaction completion by Kaiser test. In case of incomplete coupling
(positive Kaiser test), recoupling was performed with a fresh solution of activated amino acid (6 mmol).
Fmoc-Group Removal and Fmoc-Removal Test
The resin-bound Fmoc-protected amino acids, peptides, or hybrids were treated twice with 25%
piperidine in NMP (25 mL each) for 30 min at room temperature. To check the completion of the
Fmoc-removal, 25% piperidine in NMP (20
µ
L) was added to a resin probe (approximately 2 mg) and
the mixture was heated for 5 min at 100 ◦C. From the resulting solution, 10
µL was spotted on a TLC
plate and checked under a UV lamp for UV-absorbing material. Alternatively, the solution was injected
on HPLC and the Fmoc-material produced and released into solution was quantified at 265 nm. If the
Fmoc-removal test remained positive (violet spot under UV) the piperidine treatment was prolonged

Molecules 2019, 24, 4261
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or repeated until a negative test was achieved. The resin was filtered and washed with NMP ( 5),
×
DCM (×3), and DEE (×2), and dried in vacuo; otherwise, it was used directly in the next step.
3.3.3. General Procedure for the Solid-Phase Synthesis of Bromoacylated Peptides
Haloacid (3-fold molar excess over the resin bound peptide) was dissolved in NMP. The solution
was cooled on ice and then DIC (equivalent to 3 moles) was added. The reaction mixture was added to
the resin-bound peptide and allowed to react for 1–3 h at 25 ◦C. The resin was filtered and washed
with NMP (
next step.
×
5), DCM (
×
3), and DEE ( 2), and dried in vacuo; otherwise, it was used directly in the
×
3.3.4. General Procedure for Peptoid Formation
A solution of the S-Mmt and S-Trt aminothiols (2-fold molar excess over the resin bound peptide)
in NMP and DIPEA (equivalent to 2.2 moles) was added and the mixture was left to react for 2–12
h at 25 ◦C. The reaction could be easily followed by TLC and HPLC analysis. Thus 2–3 mg of resin
samples were taken and the protected peptides were cleaved from the resin using a 15 min treatment
with DCM/trifluoroethanol (TFE)/acetic acid (AcOH) (7:2:1). The resins were then applied to TLC and
HPLC analysis. After completion of the synthesis, the resins were extensively washed with NMP (
×
5),
DCM ( 3), and DEE ( 2), and then dried in vacuo; otherwise, they were used directly in the next step.
×
×
The obtained resins were either reacted with acetic anhydride (3 mol), or acyl chlorides (3 mol),
or aroyl chlorides (3 mol) in NMP and DIPEA (6 mol) for 1–2 h at 25 ◦C, or subjected to SPS/SPPS,
as previously described. The peptoid formation was easily followed by TLC and HPLC analysis.
Thus, 2–3 mg of resin samples were taken and the protected peptides were cleaved from the resin
using a 15 min treatment with DCM/TFE/AcOH (7:2:1) and were applied to TLC and HPLC analysis.
After completion of the synthesis, the resin was extensively washed with NMP (
and DEE (×2), and then dried in vacuo.
×
5), DCM ( 3),
×
3.3.5. General Procedure for the Cleavage of the Peptide–Peptoid Hybrids from the Resin
Resin (0.5 g) was treated with 5 mL of a mixture of DCM/TFE/AcOH (7:2:1) for 15 min at 25 ◦C,
and washed with DCM (3 5 mL). The combined filtrates were concentrated by rotary evaporation.
×
The fully protected crude peptides (S-Mmt/S-Trt/tBu-protected) were precipitated by the addition of
DEE, filtered, washed with DEE, and dried in vacuo. Treatment with 1.1% trifluoroacetic acid (TFA) in
DCM/triethylsilane (TES) (95:5) gave the S-free (S-Trt/tBu-protected) hybrids (by removal of the S-Mmt
protecting group), while treatment with 65% TFA in DCM/TES (95:5) gave the completely unprotected
peptides (by removal of the S-Mmt/S-Trt/tBu protecting groups).
3.3.6. General Procedure for the Oxidation of Mercaptoalkyl Peptoid Side Chains
◦
100 mg of resin was treated with 1 mL of 1.1% TFA in DCM/TES (95:5) for 10 min at 25 C.
This treatment resulted in the cleavage of the protected peptide–peptoid hybrid from the resin and the
simultaneous removal of the S-Mmt groups. In the case of the S-Trt protecting groups, a solution of
65% TFA in DCM/TES (95:5) for 1 h at 25 ◦C was necessary for the complete removal of the S-Trt and
tBu-type protecting groups. The resin was filtered off and 0.2 mL methanol (MeOH) was added to the
filtrates. The mixture was left for 5 min at room temperature and then evaporated. Dimethylsulfoxide
(DMSO)/water (H2O) (7:3) was added to the oily residue and the mixture was left at room temperature
for 48 h. The progress of the oxidation was followed by HPLC analysis.
4. Conclusions
Peptoids are very important peptide-mimicking compounds, since they can resist proteolytic
degradation, thereby retaining a high degree of conformational flexibility. Thiols are very important
chemical groups found in drugs and peptides with very important biological activities. Thus,

Molecules 2019, 24, 4261
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we synthesized thiol-containing peptide–peptoid hybrids using standard methods of solid-phase
synthesis (SPS). For this, suitably protected aminothiols (S-4-methoxytrityl (Mmt)/S-trityl (Trt)),
linear and derived from amino acids, were used, and peptide synthesis was performed on 2-chlorotrityl
resin (Cltr) using the fluorenylmethoxycarbonyl (Fmoc)/tert-butyl (tBu) strategy. This approach allowed
for the selective deprotection of the side groups, giving many possibilities regarding peptide synthesis,
including (a) the preparation of peptide–peptoid hybrids with selected thiol-free peptoid moieties
(which have the ability to act as new nucleophiles) and (b) the synthesis of peptide–peptoid hybrids
with inter- and intra-thiol-containing bridges.
Author Contributions: Conceptualization, K.B.; K.B., D.G. and S.M. designed the synthetic methodologies;
S.M. performed the experiments; K.B. and S.M. wrote the manuscript. All authors approved the final version of
the manuscript.
Funding: This research received no external funding.
Acknowledgments: The authors acknowledge C.B.L. Patras S.A., Patras, Greece, for financial support.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of all compounds are available.
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