An original strategy for Gln containing peptide synthesis using SPPS and Glu(OH)-1-OAll

Article abstract of DOI:10.1007/s10989-010-9213-0

Using multiple peptide synthesis in parallel, a series of 24 compounds analogues of tripeptide sequence Z-Leu-Phe-Gln-H, modified by imidazole moiety were synthesized. An effective and simple scheme for including imidazole heterocycle to C- and/or N-terminus of Gln residue was created by means of allyl group as α-COOH protecting group for Fmoc-Glu. The approach using Fmoc-Glu-1-OAll as a first amino acid linked to the resin could be useful for the synthesis of a large number of amino acids and/or heterocyclic moieties including compounds. Based on the preliminary biological trials we could conclude that the presence of imidazole heterocycle affect positively the antiviral activity against Coxsackieviruses B1 and Poliovirus type 1.

Full text of DOI:10.1007/s10989-010-9213-0

Int J Pept Res Ther (2010) 16:233–238
DOI 10.1007/s10989-010-9213-0
ORIGINAL PAPER
An Original Strategy for Gln Containing Peptide Synthesis Using
SPPS and Glu(OH)-1-OAll
•
Ivaylo N. Minchev Dantcho L. Danalev
•
•
•
Lyubomir T. Vezenkov Lyubomira Nikolaeva-Glomb
Angel S. Galabov
Accepted: 4 May 2010 / Published online: 25 May 2010
Ó Springer Science+Business Media, LLC 2010
Abstract Using multiple peptide synthesis in parallel, a
series of 24 compounds analogues of tripeptide sequence
Z-Leu-Phe-Gln-H, modified by imidazole moiety were
synthesized. An effective and simple scheme for including
imidazole heterocycle to C- and/or N-terminus of Gln
residue was created by means of allyl group as a-COOH
protecting group for Fmoc-Glu. The approach using Fmoc-
Glu-1-OAll as a first amino acid linked to the resin could
be useful for the synthesis of a large number of amino acids
and/or heterocyclic moieties including compounds. Based
on the preliminary biological trials we could conclude that
the presence of imidazole heterocycle affect positively the
antiviral activity against Coxsackieviruses B1 and Polio-
virus type 1.
represents a major challenge and is often unpredictable,
especially when obtaining peptides with high molecular
weight and difficult sequences. The creation of new syn-
thetic schemes for synthesis of peptides is provoked both
by the discovery of new methods for condensation and new
protective groups (alone or in combination with already
known ones) as well as the possibilities to apply various
strategies of realizing a peptide sequence using known
condensation methods.
A lot of experiments connected with compounds pos-
sessing inhibition activity against the cysteine-containing
human rhinovirus 3C protease (3CP) revealed that some
simple changes in the 3CP substrate H-His-Leu-Phe-
Gln-Gly-Pro-OH could transform it into low molecular
strong inhibitor Z-Leu-Phe-Gln-Michael acceptor (Fig. 1)
(Dragovich et al. 1998a, b).
Keywords Gln containing peptides Á
Allyl protecting group Á Peptidomimetics Á 3CP inhibitors
A series of similar inhibitors have been synthesized and
described in the literature in order to reveal the role of
different parts of the molecule on the antiviral activity
(Dragovich et al. 1998a, b; Dragovich 2001; Dragovich
and Prins 2003; Wang 1999).
Introduction
Despite the large progress in peptide chemistry due to solid
phase peptide synthesis (SPPS) discovered by Merrifield
(1963), the synthesis of each new peptide sequence
It was proven that Leu residue at P3 position is not
necessary for the substrate-enzyme interaction (Dragovich
et al. 1999). Therefore, a wide variety of substitutes are
tolerated at this position. Further studies showed that
aldehyde group as a Michael acceptor is not acceptable
because of their toxicity, lack of selectivity and stability
(Dragovich et al. 2000).
Electronic supplementary material The online version of this
article (doi:10.1007/s10989-010-9213-0) contains supplementary
material, which is available to authorized users.
We were challenged to synthesize a series of mimetics
of hexapeptide H-His-Leu-Phe-Gln-Gly-Pro-OH contain-
ing Gln, with potential antiviral activity, by means of
specifically protected Glu(OH)-1-OAll residue and rink
amide resin using SPPS. Herein, we report an original
strategy for synthesis of these compounds as well as some
preliminary antiviral tests.
I. N. Minchev Á D. L. Danalev (&) Á L. T. Vezenkov
Department of Organic Chemistry, University of Chemical
Technology and Metallurgy, 1756 Sofia, Bulgaria
e-mail: dancho_danalev@yahoo.com
L. Nikolaeva-Glomb Á A. S. Galabov
Bulgarian Academy of Science, Stephen Angeloff Institute of
Microbiology, Sofia, Bulgaria
123

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Int J Pept Res Ther (2010) 16:233–238
Fig. 1 Transformation model
of 3CP substrate into its
inhibitor
NH2
O
NH2
O
O
O
O
O
H
N
O
Η
H
N
Ν
H3N
N
H
N
H
N
N
H
N
H
Ο
O
Ο
O
HO
Materials and Methods
DCM and diisopropylcarbodiimide (DIC) (fivefold excess
compared to the resin) were mixed and the resulting solution
was stirred for 20 min at 0°C. Further the solvent was
evaporated under vacuum. The obtained oil was dissolved in
DMF and added to the resin followed by 4-N,N-dimethyl-
aminopyridine (DMAP) (0.1 equiv. compared to the resin).
2.5 h later the solvent was removed under vacuum and the
resin was washed with 39 DMF and 39 DCM, subsequently.
A standard Kaiser test was made. The negative Kaiser test
means that the reaction of condensation is successfully done.
The purity of the newly synthesized compounds were
monitored by the HPLC Perkin-Elmer Series 410 LC
BioPump, Diode Array detector LC 235, Spherisorb 5 ODS
250 mm 9 4.6 mm column, isocratic elution with 50%
MeCN/50% 0.02 M KH2PO4:K2HPO4, pH = 7, flow rate
1 ml/min, k = 220 nm.
The mass specters were recorded on the Micromas
Platform LC, cone voltage 70, heat 70°C and positive or
negative charge.
All syntheses were performed by multiple-peptide
syntheses in parallel using SPPS, Fmoc-strategy, Rink
Amide Resin (HCPam 02-1, 1% DVSB, 100–200 mesh).
2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexa-
fluorophosphate/1-Hydroxybenzotriazole/N,N-Diisopropyl-
ethylamine (HBTU/1-HOBt/DIPEA) were used as coupling
reagents. Three fold excess of the Fmoc-amino acid com-
pared to the resin was used.
B: General Procedure for Coupling of Amino Acids Using
HBTU (TCTU)/DIPEA Method
The Fmoc-amino acid (4 equiv. relative to resin loading),
HBTU (TCTU) (4 equiv. relative to resin loading) and
diisopropylethylamine (DIPEA) (4 equiv. relative to resin
loading) were dissolved in DMF. The reaction mixture was
stirred for 10 min and added to the peptide-resin solution in
DMF previously deprotected as regards the Fmoc N-ter-
minal function. The reaction mixture was stirred for 2 h.
The Kaiser test was made at the end of the reaction time.
The negative Kaiser test means that the reaction of con-
densation is successfully done.
A Pd(PPh3)4-Tetracis (3 equiv.)/CHCl3-AcOH-N-meth-
ylmorpholine (37:2:1) (15 ml per gram resin) under argon
stream was used for the 1-allyl group deblocking.
The imidazole condensation was performed by 3 equiv.
of imidazole to 1 equiv. resin and O-(6-Chloro-1-hydro-
cibenzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluo-
roborate (TCTU)/1-HOBt/DIEA (1:1.1:1.1 fold excess
compared to the resin).
C: Preparing of Peptide-Resin for Cleavage and Removing
the Peptide from the Resin
The coupling of 4-imidazole acetic acid was made by
means of TCTU/1-HOBt/DIPEA.
The obtained peptide-resin was washed by 39 DMF; 39
DCM; 39 i-propanol and 39 diethyl ether to shrink the
resin. The peptide-resin was then dried under high vacuum
for 4 h over NaOH. The dry peptide-resin was placed in a
flask and TFA/TIS/H2O (95:2.5:2.5) were added for 5 h. At
the end of the reaction time the obtained peptide was
collected by filtration and the removed resin was washed
with 39 TFA and 39 DCM, subsequently. The obtained
filtrates were collected and evaporated to dryness.
The final peptides were obtained by treatment with a
mixture of 2.5 ml of 95% TFA (2.4 ml), 2.5% H2O (0.06 ml)
and 2.5% Triisopropylsilane (TIS) (0.06 ml) for 2.5 h.
Chemical Part
A: Procedure for Attachment of Fmoc-Glu(OH)-OAll
to Rink Amide Resin
The Rink amide resin was pre-swollen with dichloromethane
(DCM) for 1 h and then washed thoroughly with N,N-
dimethylformamide (DMF). Separately 4 equiv. excess
compared to the resin of Fmoc-Glu(OH)-OAll dissolved in
D: Condensation of imAc and Imidazole
ImAc or imidazole (3 equiv. to the peptide-resin) dissolved
in DMF and TCTU/1-HOBt/DIPEA (1:1.1:1.1 equiv.
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Int J Pept Res Ther (2010) 16:233–238
235
excess compared to the resin) were added. The reaction
mixture was stirred for 5 h.
mm) of the zone of plaque inhibition and the zone of
cytotoxicity.
E: Allyl Group Deblocking
Results and Discussion
Pd(PPh3)4 (threefold excess to the peptide-resin) dissolved
in a mixture of CHCl3/AcOH/N-methylmorpholine (37:2:1)
(15 ml/g resin) under argon atmosphere was added to the
fully dried resin. The reaction mixture was stirred for 4.5 h
under argon and at the end of the reaction time the resin was
washed with 0.5% DIPEA in DMF and 0.5% sodium dieth-
yldithiocarbamate in DMF for catalyst removal.
In order to study the possibility to use our established
strategy for synthesis of Gln containing peptides and pep-
tides containing pseudopeptide bonds a series of 24 new
compounds with a general structure H(or imAc)-Aaa-Phe-
Gln-OH(or Im) (Aaa = Arg, Ile, Leu, His, Pro and Trp)
were synthesized.
The syntheses of the target peptides were performed by
means of solid phase based Multiple-peptide synthesis in
parallel.
Biological Trials
In all syntheses allyl ester as a protection for a-COOH
function of Glu was used. This type of esters was extremely
advanced in the last 2–3 years due to their specific prop-
erties. First of all, they are quite stable in acidic and
alkaline hydrolysis unlike the other ester groups such as
OMe, OEt, OBzl and OBut, which allows its preservation
in all necessary conditions for another protection groups
deblocking. Further, this function can be successfully
reduced thanks to its similarity with the normal functions
of ester to aldehyde group under the action of diisobutyl-
aluminium hydride or to free COOH function using
Pd(PPh)3/PhSiH3. So, this approach allows us to build the
peptide chain using the specific features of OAll protection
group either to its C- or N-terminus.
The CV-B1 and LSc-2ab viruses used for our biological
trials were kindly placed at our disposal by The Stephen
Angeloff Institute of Microbiology at the Bulgarian
Academy of Sciences. All procedures for virus and FL cells
cultivation were performed according to the literature
(Rada and Zavada 1962; Galabov and Galabov 1980;
Galabov and Iossifova 1996; Galabov and Nikolaeva 1998;
Ivanova and Milkova 1997).
The agar-diffusion plaque-inhibition test with cylinders
was used for the initial screening of antiviral activity and
was performed as previously described (Rada and Zavada
1962; Galabov and Galabov 1980; Galabov and Iossifova
1996; Galabov and Nikolaeva 1998; Ivanova and Milkova
1997). The presented results were obtained in two inde-
pendent experiments with three replications in each
experiment. The test compounds (0.1 ml of 20 mM in
ethanol) were added dropwise in the 6 mm glass cylinders
fixed in the agar overlay. The antiviral effect was registered
on the basis of the difference between the size (diameter in
Using Fmoc-Glu(OH)-1-OAll as a first amino acid
linked to the resin allowed us to synthesize the general
fragment Fmoc-Aaa-Phe-Glu(CONH-resin)-1-OAll that
was required for the final products obtaining (Scheme 1).
The main advantage of our strategy is that we use Glu as
a first amino acid in place of the Gln residue in the target
Fmoc
Scheme 1 Synthesis of Fmoc-
Aaa1–6-Phe-Glu(CONH-resin)-
1-OAll
NH-rink amide resin
1.20%piperidin/DMF
2.Fmoc-Glu(OH)-1-OAll
HBTU/ DIEA
NH-resin
CO
1.20%piperidine/ DMF
2.Fmoc- Phe-OH
HBTU/DIEA
Fmoc-Glu-1-OAll
NH-resin
CO
Fmoc-Phe-Glu-1-OAll
1.20%piperidine/ DMF
2.Fmoc-His(Trt)-OH
HBTU/DIEA
2.Fmoc-Ile-OH
HBTU/DIEA
2.Fmoc-Arg(Pbf)-OH
HBTU/DIEA
2.Fmoc-Trp(Boc)-OH
HBTU/DIEA
2.Fmoc-Pro-OH
HBTU/DIEA
2.Fmoc-Leu-OH
HBTU/DIEA
NH-resin
CO
NH-resin
CO
NH-resin
CO
Fmoc-Pro-Phe-Glu-1-OAll
Fmoc-Arg(Pbf)-Phe-Glu-1-OAll
Fmoc-His(Trt)-Phe-Glu-1-OAll
NH-resin
NH-resin
CO
CO
NH-resin
CO
Fmoc-Ile-Phe-Glu-1-OAll
Fmoc-Trp(Boc)-Phe-Glu-1-OAll
Fmoc-Leu-Phe-Glu-1-OAll
123

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Int J Pept Res Ther (2010) 16:233–238
Scheme 2 Synthesis of
compounds with general
structure: H-Aaa1–6-Phe-Gln-
im, imAc-Aaa1–6-Phe-Gln-OH
and imAc-Aaa1–6-Phe-Gln-im
NH-resin
Fmoc-Aaa1-6-Phe-Glu-1-OAll
20% piperidine/DMF
NH-resin
NH-resin
Pd(PPh3)4
H-Aaa1-6-Phe-Glu-1-OH
Cleavage
H-Aaa1-6-Phe-Glu-1-OAll
imAc
NH-resin
imAc-Aaa1-6-Phe-Glu-1-OAll
NH-resin
H-Aaa1-6-Phe-Gln-OH
NH-resin
Pd(PPh3)4
Imidazole
imAc-Aaa1-6-Phe-Glu-1-OH
Cleavage
imAc-Aaa1-6-Phe-Glu-1-im
Cleavage
imAc-Aaa1-6-Phe-Gln-OH
imAc-Aaa1-6-Phe-Gln-1-im
NH-resin
NH-resin
Imidazole
Pd(PPh3)4
Fmoc-Aaa1-6-Phe-Glu-1-im
Fmoc-Aaa1-6-Phe-Glu-1-OH
20% piperidine/DMF
NH-resin
Cleavage
H-Aaa1-6-Phe-Gln-im
H-Aaa1-6-Phe-Glu-1-im
Fmoc-Aaa1-6-OH: Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-His (Trt)-OH,
Fmoc-Trp(Boc)-OH;imA c: 4-Imidazole acetic acid hydrochloride; im: Imidazole;
Table 1 Analytical data for
newly synthesized compounds
1–24
No.
Peptide structure
Exact Mass
calculated
Mass found
RP-HPLC/Rt
(min)
1
H-Arg-Phe-Gln-OH
H-Arg-Phe-Gln-im
449.2387
499.2656
557.2710
607.2979
406.2216
456.2485
514.2540
564.2809
430.1965
480.2234
538.2288
588.2557
406.2216
514.2540
456.2485
564.2809
390.1903
440.2172
498.2227
548.2496
479.2169
529.2438
587.2492
637.2761
449.98
499.0
1.89
1.82
1.72
1.74
1.91
1.76
1.69
1.72
1.30
1.09
2.38
2.60
1.25
1.17
1.69
1.67
1.32
3.13
1.19
1.22
1.15
1.65
1.96
2.04
2
3
imAc-Arg-Phe-Gln-OH
imAc-Arg-Phe-Gln-im
H-Ile-Phe-Gln-OH
558.7
4
608.05
407.5
5
6
H-Ile-Phe-Gln-im
457.8
7
imAc-Ile-Phe-Gln-OH
imAc-Ile-Phe-Gln-im
H-His-Phe-Gln-OH
H-His-Phe-Gln-im
514.6
8
565.5
9
430.4
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
480.4
imAc-His-Phe-Gln-OH
imAc-His-Phe-Gln-im
H-Leu-Phe-Gln-OH
imAc-Leu-Phe-Gln-OH
H-Leu-Phe-Gln-im
539.5
588.6
407.97
514.62
456.5
imAc-Leu-Phe-Gln-im
H-Pro-Phe-Gln-OH
H-Pro-Phe-Gln-im
565.48
390.71
440.1
imAc-Pro-Phe-Gln-OH
imAc-Pro-Phe-Gln-im
H-Trp-Phe-Gln-OH
H-Trp-Phe-Gln-im
499.97
549.62
480.77
530.76
589.41
639.02
imAc-Trp-Phe-Gln-OH
imAc-Trp-Phe-Gln-im
All MS spectra are included as a
supporting information
peptides needed to link the peptide moiety to the
rink amide resin. What is more important is that it was
linked by its d-COOH function. The unblocking of Glu
using a standard ‘‘cocktail’’ of agents in the final stage of
synthesis led to obtaining the required Gln residue
(Scheme 2).
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Int J Pept Res Ther (2010) 16:233–238
237
Table 2 Antiviral activity screened by the agar-diffusion plaque-inhibition test for compounds 1–18
No.
6
Peptide structure
H-Ile-Phe-Gln-im
Virus
I Petri
II Petri
Average
D (mm)
Effect
LSc-2ab
CV-B1
LSc-2ab
CV-B1
LSc-2ab
CV-B1
LSc-2ab
CV-B1
LSc-2ab
CV-B1
LSc-2ab
CV-B1
LSc-2ab
CV-B1
14 [11]
0 [10]
14 [7]
0 [6.5]
17 [7]
0 [7.5]
38 [12]
36 [11]
–
17 [6.5]
0 [8]
15.5 [8.75]
0 [9]
6.75 mm
–
-
-
11
imAc-His-Phe-Gln-OH
imAc-Leu-Phe-Gln-im
16 [7]
0 [6.5]
17.5 [7.5]
0 [7]
15 [7]
0 [6.5]
17.25 [7.25]
0 [7.25]
34 [11]
34.5 [10.5]
–
8 mm
–
16
10 mm
–
-
Ref: OG
30 [10]
33 [10]
–
23 mm
24 mm
–
??
??
-
Ref: Disoxaril
Ref: Guanidine.HCl
Ref: HBB
25 [11]
–
25 [11]
–
25 [11]
–
14 mm
–
?
-
17 [6]
25 [6]
–
17 [6]
25 [6]
–
17 [6]
25 [6]
–
11 mm
19
?
?
–
-
(-): Dd B 5 mm; ( ): Dd C 6–10 mm; (?): Dd C 11–20 mm; (??): Dd C 21–40 mm
Further the compounds synthesized according to
Scheme 1 were used for obtaining new products including
imidazole heterocycle. The latter was chosen for the fol-
lowing reasons:
16 are active against CV-B1 and LSc-2ab tested by plaque-
inhibition test.
Conclusions
–
its incorporation in the desired molecule will allow us
to show the possibility to synthesize the peptide
moieties including pseudopeptide bonds by means of
our strategy,
Using multiple peptide synthesis in parallel, a new, original
and successful strategy for synthesis of Gln containing
peptides using Glu as a starting amino acid was demon-
strated and a series of 24 compounds analogues of tripep-
tide sequence Z-Leu-Phe-Glu-CHO, modified by imidazol
moiety were synthesized.
–
it has various biological features including antiviral
properties (Katritzky et al. 1984; Grimmett 1997;
Brown 1988; Pozharskii et al. 1997; Gilchrist 1985),
which could increase the antiviral properties of our
mimetics.
An effective and simple scheme for the inclusion of
imidazole heterocycle to C- and/or N-terminus of Gln
residue was created by using 1-allyl protecting group as a
protecting group for Fmoc-Glu. The approach by means of
Fmoc-Glu-1-OAll as a first amino acid linked to the resin
could be useful for the synthesis of a large number of
compounds consisting of different amids of dicarboxylic
amino acids and heterocyclic moieties. Based on the
obtained antiviral results we could conclude that some of
the newly synthesized compounds showed relatively good
antiviral activity against two tested viruses and that the
presence of imidazole heterocycle affects the antiviral
activity positively according to the tested type of viruses.
4-Imidazole acetic acid hydrochloride (imAc) was used
for the incorporation of imidazole moiety into N-terminus
of the basic structures. The imidazole (im) was used for the
C-terminus modifying (Scheme 2).
The purity of all the newly synthesized compounds was
monitored by RP-HPLC and their structure was confirmed
by MS spectroscopy (Table 1).
A preliminary antiviral test against Coxsackieviruses B1
(CV-B1) and Poliovirus type 1 (LSc-2ab) using agar-diffu-
sion plaque-inhibition test by means of Oxoglaucine,
Disoxaril, Guanidine.HCl and hydroxybenzylbenzimidazole
(HBB) as referent compounds (Table 2) were done. For all
compounds cytotoxicity was determined in FL cells by using
0.5 l g concentration.
All the rest of the synthesized compounds as well as
H-Leu-Phe-Gln-OH and Imidazole have no effect on these
two types of viruses according to the agar-diffusion plaque-
inhibition test. It was determined that compounds 6, 11 and
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