Solid phase synthesis of α-amino squaric acid-containing peptides

Article abstract of DOI:10.1039/c4ra10442k

A new method has been developed for the synthesis of 3-(1-aminoalkyl)-4-hydroxycyclobut-3-ene-1,2-dione [α-amino squaric acid (α-Asq)]-containing peptides using solid phase peptide synthesis according to an Fmoc protecting group strategy. FmocHN-Gly-[α-Asq]-Oi-Pr was successfully used as a coupling unit in this method, which allowed for the construction of α-Asq-containing hexapeptide libraries including Sq-Gly and Sq-Phe units using the Wang-resin. Peptides containing the α-Asq moiety exhibited inhibitory activity towards a digestive enzyme.

Full text of DOI:10.1039/c4ra10442k

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COMMUNICATION
Solid phase synthesis of a-amino squaric acid-
containing peptides†
Cite this: RSC Adv., 2014, 4, 50639
a
a
a
b
Kentaro Maeda, Yu-ichi Kiniwa, Yasufumi Ohfune, Shinichi Ishiguro,
b
c
c
a
Koichi Suzuki, Kazuya Murata, Hideaki Matsuda and Tetsuro Shinada*
Received 15th September 2014
Accepted 1st October 2014
DOI: 10.1039/c4ra10442k
www.rsc.org/advances
3
A new method has been developed for the synthesis of 3-(1-amino- and materials chemistry. It was envisaged that the incorpora-
alkyl)-4-hydroxycyclobut-3-ene-1,2-dione [a-amino squaric acid tion of a-Asq into peptides would allow for the preparation of
(a-Asq)]-containing peptides using solid phase peptide synthesis novel a-Asq-containing peptides, because the inherent reactivity
according to an Fmoc protecting group strategy. FmocHN-Gly- of the Sq group would allow this moiety to react with various
3,4
[
a-Asq]-Oi-Pr was successfully used as a coupling unit in this method, amines to form amide like N-Sq bonds. In our previous study,
which allowed for the construction of a-Asq-containing hexapeptide we reported for the rst time the synthesis of the Sq-Gly-con-
4b
libraries including Sq-Gly and Sq-Phe units using the Wang-resin. taining Leu-enkephalin analogue 3 (Fig. 1), which exhibited
Peptides containing the a-Asq moiety exhibited inhibitory activity promising binding activities towards opioid receptors. This
towards a digestive enzyme.
compound was also found to be stable under acidic conditions
i.e., 30% HBr/AcOH), as well as being stable to the conditions
required for its purication by HPLC (i.e., H O/MeCN/TFA), and
(
2
The solid phase synthesis of peptides using the Fmoc protecting
group (i.e., the Fmoc strategy) represents a robust technology
for the rapid construction of various peptides, which is also
its subsequent analysis by NMR spectroscopy in DMSO-d
6
and
4b
D
2
O. These observations led us to explore the possibility of
developing a method for the rapid construction of a-Asq-con-
taining peptides using the Fmoc strategy. Herein, we report the
1
amenable to the automated synthesis of peptide libraries. The
application of this technique to the synthesis of peptides con-
taining amino acid isosteres has recently attracted considerable
levels of attention because the resulting peptide analogues
exhibit improved levels of potency and selectivity towards bio-
logical targets, as well as enhanced metabolic and chemical
stabilities compared with the corresponding unmodied
peptides. Several amino acid isosteres have been developed for

rst solid phase synthesis of a-Asq-containing peptides and
their application to the construction of peptide analogue
libraries containing the a-Asq unit.
FmocHN-[a-Asq]-Oi-Pr 4 was selected as a coupling unit
for the Fmoc strategy used in this study (Scheme 1). The
synthesis of FmocHN-[Sq-Gly]-Oi-Pr 9 was commenced with
2
use in the solid phase synthesis of peptide analogues.
We recently reported 3-(1-aminoalkyl)-4-hydroxycyclobut-3-
ene-1,2-dione [a-amino squaric acid (a-Asq)] (1) as a novel
amino acid analogue, where the carboxylic acid moiety of the
a-amino acid had been replaced with a 4-hydroxy-2,3-dioxocy-
3
clobut-1-enyl (Sq) group (Fig. 1). The Sq group and squaric acid
2
have been studied extensively as carboxylic acid surrogates in
medicinal chemistry, bioorganic chemistry, chemical biology,
a
Graduate School of Science, Osaka City University, 3-3-138, Sugimoto, Sumiyoshi,
Osaka 558-8585, Japan. E-mail: shinada@sci.osaka-cu.ac.jp; Tel: +81-6-6605-2570
b
Office of Research Exchange, Iwate University, 3-18-8 Ueda, Morioka, Iwate 020-8550,
Japan
c
Faculty of Pharmacy, Kinki University, 3-4-1 Kowakae, Higashiosaka, Osaka 577-
8
†
1
502, Japan
Electronic supplementary information (ESI) available. See DOI:
0.1039/c4ra10442k
Fig. 1 Structures of a-amino acid, a-Asq (1), squaric acid (2), and
a-Asq-containing Leu-enkephalin (3).
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2
Scheme 2 Reaction conditions: (a) H /Pd–C (20 wt%), MeOH, rt, 3 h;
(
(
H
b) FmocHN-Gly-OH (1 eq.), EDCI (1 eq.), THF, rt, 3 h; (c) 12 N HCl
ꢀ
1 eq.), CH
N–Phe-OH (3 eq.), THF, rt, 3 h; (f) 20% Et
O, THF, rt, 0.5 h.
Cl
2 2
, rt, 3 h; (d) TFA (30 eq.), CH
2
Cl
2
, 0 C to rt, 12 h; (e)
2
NH/THF, rt, 0.5 h then
2
Ac
2
also investigated the use of a variety of different reagents and
Scheme 1 Reaction conditions: (a) H
2
/Pd–C (20 wt%), MeOH, rt, 3 h; conditions [e.g., Et NH, DBU, 2,2,6,6-tetramethyl piperidine,
2
(
(
2 2
b) FmocOSu (1 eq.), MeCN, rt, 4 h; (c) 12 N HCl (1 eq.), CH Cl , rt, 3 h;
d) TFA (30 eq.), CH Cl , 0 C to rt, 12 h.
2 2
lower temperature, different concentrations, in situ trapping
with Boc O and Ac O], but none of these reactions resulted in
the formation of the desired amine 12. 9-Methylideneuorene
11) was detected as major by-product in the complex mixture
ꢀ
2
2
(
4b
N-Cbz-hydroxycyclobutenone 5. Removal of the Cbz group resulting from this reaction, which indicated that the Fmoc
from 5 followed by protection of the resulting free amino group group was being liberated during the course of the reaction to
with the Fmoc group gave 7 in 81% yield, which was converted give amine 12, which must have underwent rapid
to N-Fmoc a-Sq glycine 8 in 84% yield using the conventional decomposition.
3,4
condition in the presence of a small amount of 12 N HCl.
It was envisaged that the protection of the free amine group
Subsequent removal of the t-butyl group in compound 8 as an amide such as dipeptide unit 13 would overcome the
provided the corresponding carboxylate, which underwent a decomposition issues observed above and allow for the use for
4
spontaneous Sq group-promoted decarboxylation reaction in the solid phase synthesis (Scheme 2). FmocHN-Gly-[Sq-Gly]-Oi-
situ to give FmocHN-[Sq-Gly]-Oi-Pr 9 in 76% yield.
Pr 16 was synthesized from compound 5 according to the route
4a
The Fmoc strategy used in the current study consists of a shown below. Compound 6 was coupled with FmocHN-Gly-
series of sequential transformations, including (i) linkage of the OH using EDCI to give 14 in 98% yield. Compound 14 was then
Fmoc-protected amino acids (FmocHN-AA-OH) to the acid- converted to 16 in a similar manner to that used for the
labile linker on solid phase; (ii) removal of the Fmoc protecting synthesis of 9 via a Sq group-promoted decarboxylation reac-
group from the AA; (iii) condensation of FmocHN-AA-OH; (iv) tion. FmocHN-Gly-[Sq-Gly]-Oi-Pr 16 was then condensed with
repetition of steps (ii) and (iii) to elongate the peptide chain;
and (v) treatment of the resulting peptide with acid to cleave the yield. Compound 17 was treated with Et
peptide from the resin. We initially investigated the compati- removal of the Fmoc protecting group, and the resulting amine
bility of FmocHN-[Sq-Gly]-Oi-Pr 9 under the proposed reaction was acetylated with Ac O to give the corresponding amide 18 in
conditions prior to the solid phase synthesis using a model 72% yield, without decomposition of the free amine (Scheme 2).
system involving the dipeptide analogue FmocHN-[Sq-Gly]-Phe- With a suitably stable linkage unit in hand, we proceeded to
OBn 10. Treatment of 9 with H N–Phe-OBn gave 10 in 71% yield evaluate the use of solid-supported 16 in a series of sequential
H
2
N–Phe-OBn to give the model tripeptide analogue 17 in 64%
2
NH to allow for the
2
2
(Scheme 1). Our initial attempt to remove the Fmoc group in 10 transformations, including (i) removal of the Fmoc group of the
using 20% piperidine in THF resulted in a complex mixture. We FmocHN-Phe-functionalised Merrield resin; (ii) treatment of
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the resulting amine with 2 equivalents of 16; and (iii) cleavage of
the resulting peptide from the resin by treatment with TFA
(Scheme 3).
The yield of FmocHN-Gly-[Sq-Gly]-Phe-OH 19 following the
cleavage of the product from the resin was used to evaluate the
linkage efficiency of the peptide to the resin. Unfortunately,
however, this protocol only resulted in the formation of a trace
amount of 19. Increasing the amount of 16, as well as extending
the reaction period or switching to trityl resin and Clear resin®
resulted in similarly low yields of the product. Following an
extensive period of investigation, we found that the Wang res-
in® gave the best results of all of the resins tested, with 19
being isolated in 43% yield following a single loading of 16.
The coupling efficiency could be further improved by twice
repeating the loading of 16. Under the optimized conditions, 19
was isolated in 80% yield following its cleavage from the resin.
The a-substituted a-Asq, Sq-Phe, could also be incorporated
Scheme 4 Synthesis of Sq-Phe-containing peptide. Reaction condi-
tions: (a) 20% piperidine/DMF, rt, 15 min; (b) 20 (4 eq.), DIEA (2 eq.),
2
AcOEt, rt, 60 h; (c) 20% Et NH/DMF, rt, 15 min; (d) FmocHN-Gly-OH
into the peptide using the Fmoc strategy. To shorten the (4 eq.), HOBt (4 eq.), HBTU (3.6 eq.), DIEA (8 eq.), DMF, rt, 1 h; (e) 20%
number of synthetic steps required of this protocol, we inves- Et
tigated the use of the dipeptide unit 20 bearing a –COOt-Bu
group at the a-position of Sq-Phe as the coupling unit. It was
2
NH/DMF, rt, 15 min; (f) FmocHN-Tyr(t-Bu)-OH (4 eq.), HOBt (4 eq.),
2
HBTU (3.6 eq.), DIEA (8 eq.), DMF, rt, 1 h; (g) 20% Et NH/DMF, rt, 15 min;
5
(h)TFA, rt, 3 h.
envisaged that the –COOt-Bu group could be readily removed
under the conditions used for the cleavage of the peptide from
the resin using TFA, which would also allow for the removal of
the t-Bu group and decarboxylation.
represents either Phe, Tyr, or His. The composition of each
peptide library (including 27 hexapeptide analogues) was
conrmed by MALDI-TOF MS analysis (Scheme SI-1†). The Sq-
Phe-containing peptide libraries 25, 26, and 27 were success-
fully synthesized in a similar manner (Scheme SI-2†).
Compound 20 was prepared from 15 in 90% yield following a
4c
benzylation reaction in the presence of Et N. The suitability of
3
20 for use in the solid phase synthesis protocol was evaluated by
the synthesis of Leu-enkephalin analogue 21. Treatment of the
Sq-Phe-containing dipeptide unit 20 (4 equiv.) with the Wang
resin in the presence of DIEA (2 equiv.) allowed for the dipeptide
to be efficiently immobilized on the resin. In this case, excess
amount of 20 could be recovered cleanly from the ltrate aer
coupling reaction. The remaining two amino acid residues Gly
and Tyr were successfully condensed with the resin-supported
peptide using HOBt (4 equiv.) and HBTU (3.6 equiv.). Treatment
of the peptide analogue-linked resin with TFA provided the Sp-
Phe-containing enkephalin analogue 21 in an overall yield of
30% (Scheme 4).
The synthetic strategy described above was extended to the
construction of an a-Asq-containing peptide library using the
dipeptide units 16 and 20 (Scheme 5). Starting from FmocHN-
Phe-Wang resin, a mixture of FmocHN-Phe-OH, FmocHN-Tyr(t-
Bu)-OH, FmocHN-His(Trt)-OH and dipeptide unit 16 were
linked on the solid phase. The three peptide libraries 22, 23,
and 24, which differed only in the positioning of the Gly-[Sq-Gly]
unit, were generated in a parallel manner. AA in the each library
Scheme
5 Syntheis of a-Asq-containing eptide libraries 22–27.
Scheme 3 Solid phase support of dipeptide unit 16.
Reagents and coupling sequences are depicted in ESI.†
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The degradation of biologically active peptides by digestive be inactive at this concentration. None of the libraries exhibited
enzymes represents a signicant limitation to the development inhibitory activity towards acetylcholinesterase at a dose of
À1
of peptide drugs. The incorporation of an amino acid isostere 100 mg mL
into biologically active peptides has emerged as an effective
.
In summary, we have developed a method for the solid phase
strategy for increasing the metabolic stability of peptides, and it synthesis of a-Asq-containing peptides and peptide libraries.
was envisaged that the use of the a-Asq moiety in this regard We examined the inhibitory effect of a-Asq towards protease
would enhance the stability of the resulting peptides towards using carboxypeptidase Y for the rst time. The a-Asq moiety
digestive enzymes. To evaluate the feasibility of this strategy, we was found to be resistant to carboxypeptidase Y. This results
developed a model study using peptide 28 and carboxypeptidase would aid to the design and synthesis of stable a-Asq-incorpo-
Y (Fig. 2). The a-Asq-containing peptide 28 was prepared using rating peptides. The biological screening of the libraries
solid phase synthesis (Scheme SI-3†) and subsequently sub- generated in this study revealed that 23 exhibited moderate
jected to an enzymatic hydrolysis reaction using carboxypepti- inhibitory activity towards dRLh-8 cells at 100 mM. Further
dase Y. The time course of the enzymatic hydrolysis reaction library screening studies, and studies directed towards the
6
was monitored by MALDI-TOF MS (Fig. 2). Aer 2 hours, all of application of novel peptide analogues in chemical biology, are
the starting peptide was converted to peptide 29 [m/z 880], currently underway in our laboratory.
where the three amino acid residues –Ser–Tyr–Phe-OH had been
cleaved from the C-terminal of 28 (Fig. 2(c)). These results
indicated that the a-Asq moiety was stable to carboxypeptidase
Acknowledgements
Y. The a-Asq-containing peptide libraries was employed to
This work was nancially supported by grants from the Ministry
random screening using two assay systems: (i) rat hepatoma
of Education, Culture, Sports, Science and Technology (MEXT)
cells (dRLh-8) proliferation effects and (ii) inihibitory effects
(
no. 23102009), and the Japan Society for the Promotion of
toward acetylcholinesterase (see SI†). Among them, peptide
library 23 exhibited moderate inhibitory activity towards dRLh8
cancer cells at 100 mM whereas the other libraries appeared to
Science (KAKENHI, Grant no. 23228001 & 25282233).
Notes and references
1
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