Synthesis of a Chloroalkene Dipeptide Isostere-Containing Peptidomimetic and Its Biological Application

Article abstract of DOI:10.1021/acsmedchemlett.7b00234

The first rapid and efficient chemical synthesis of a cyclic Arg-Gly-Asp (RGD) peptide containing a chloroalkene dipeptide isostere (CADI) is reported. By a developed synthetic method, an N-tert-butylsulfonyl protected CADI was obtained utilizing diastereoselective allylic alkylation as a key reaction. This CADI was also transformed into an N-Fmoc protected CADI in a few steps. The CADI was used in Fmoc-based solid-phase peptide synthesis. The first synthesis of a CADI-containing cyclic RGD peptide was successful, and the synthesized CADI-containing peptidomimetic was found to be a more potent inhibitor against integrin-mediated cell attachment than the parent cyclic peptide.

Full text of DOI:10.1021/acsmedchemlett.7b00234

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Letter
Synthesis of a Chloroalkene Dipeptide Isostere-
Containing Peptidomimetic and Its Biological Application
Takuya Kobayakawa, Yudai Matsuzaki, Kentaro Hozumi,
Wataru Nomura, Motoyoshi Nomizu, and Hirokazu Tamamura
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.7b00234 • Publication Date (Web): 27 Dec 2017
Downloaded from http://pubs.acs.org on December 30, 2017
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Synthesis of a Chloroalkene Dipeptide IsostereꢀContaining Pepꢀ
tidomimetic and Its Biological Application
Takuya Kobayakawa,† Yudai Matsuzaki,† Kentaro Hozumi,‡ Wataru Nomura,† Motoyoshi Nomizu,‡
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and Hirokazu Tamamura*,†
†Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Chiyodaꢀku, Tokyo 101ꢀ0062, Japan
‡School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo 192ꢀ039, Japan
KEYWORDS: chloroalkene dipeptide isostere, peptidomimetic, solidꢀphase peptide synthesis, cyclic RGD peptide
ABSTRACT: The first rapid and efficient chemical synthesis of a cyclic ArgꢀGlyꢀAsp (RGD) peptide containing a chloroalkene
dipeptide isostere (CADI) is reported. By a developed synthetic method, an Nꢀtertꢀbutylsulfonyl protected CADI was obtained utiꢀ
lizing diastereoselecꢀ
NH
H
tive allylic alkylation
as a key reaction.
This CADI was also
transformed into an
Bn
H
N
O
iPr
3
Solid-phase
peptide synthesis
NH2
NH
Cl iPr
O
10 steps
N
H
Bn
+
O
S
H
CO2H
O
NꢀFmoc
protected
total yield 36%
(average: 90%) Fmoc
72% yield from
an H-Gly-Clt-resin
H
Cl
NH
H
O
CADI in a few steps.
The CADI was used
in Fmocꢀbased solidꢀ
phase peptide synꢀ
thesis. A first synꢀ
thesis of a CADIꢀ
N
tBu
NH2
O
N
H
Bn
CO2H
Fmoc-D-Phe- -Val-OH
cyclo[-Asp-D-Phe- -Val-Arg-Gly-]
[(Z)-CCl=CH]
IC50 = 0.497 0.149 nM
containing cyclic RGD peptide was successful, and the synthesized CADIꢀcontaining peptidomimetic was found to be a more poꢀ
tent inhibitor against integrinꢀmediated cell attachment than the parent cyclic peptide.
During the last quarterꢀcentury, various biologically active
peptides have been discovered and characterized. These bioacꢀ
tive peptides influence and control physiological functions
through interaction with their various receptors and the numꢀ
ber of natural and modified peptides that are used as therapeuꢀ
tics continues to increase. Many bioactive peptides have been
developed and have been involved in the discovery of novel
therapies. However, the use of peptides as therapeutics is limꢀ
ited by several factors, including low metabolic stability toꢀ
wards proteolysis and undesired activity resulting from interꢀ
actions of peptides with various receptors.1,2
In addition, many ADIs cannot be supplied efficiently due to
problems associated with their synthesis.
Alkene dipeptide isosteres (ADIs), which are designed
based on the partial doubleꢀbond character of the native pepꢀ
tide bond in its ground state conformation, have been expected
to be structure units as ideal the amide bond mimetics in the
original dipeptides. Practically, many groups have attempted
to replace the amide bonds in peptides with several types of
dipeptide isosteres.3ꢀ11 In addition, the metabolic stability of
ADIs was improved.5 However, bioactive peptides containing
ADIs do not always function effectively as peptidomimetics
because they may possess a smaller dipole moment as a result
of changes in the electronegativity. Furthermore, these ADIs
lack the steric restriction between the carbonyl oxygen and the
side chain of the amino acid due to their van der Waals radius
(VDR), which is smaller than that of the original amide bond.
Figure 1. Native peptide bonds and chloroalkene diꢀ
peptide isosteres
Our research group has focused on the chloroolefin strucꢀ
tures in chloroalkene dipeptide isosteres (CADIs) which can
be used to replace an amide bond in peptides as shown in Figꢀ
ure 1. Replacement of a peptide bond by the chloroolefin moiꢀ
ety can also be considered as mimicking steric restriction reꢀ
sulting from the pseudoꢀ1,3ꢀallylic strain by a chlorine atom
which is larger than a carbonyl oxygen.11,12
In addition, while the direction of the vector of the dipole
moment in the chloroolefin is similar to that of an amide, the
vector of the dipole moment in the fluoroolefin is significantly
different.13 Thus, it is expected that CADIs might compensate
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ACS Medicinal Chemistry Letters
for the drawbacks associated with ADIs. Few reports however,
have been available on application of chloroalkene structures
as peptidomimetics.14,15 This is possibly due to lack of effiꢀ
cient methods or limitation of substrates for synthesis of CAꢀ
DIs.
Page 2 of 5
protection led to the ester 8. The ester group of 8 was reduced
to the corresponding aldehyde with DIBAL at ꢀ78 ºC, and this
was followed by Pinnick oxidation to provide the desired
Fmocꢀprotected carboxylic acid 3 in 81% yield from the Busꢀ
protected ester 7 without decrease in diastereoselectivity or
appreciable olefin isomerization. These 10 steps proceeded
smoothly to provide the desired compound from starting mateꢀ
rials 4 and 5,23 and in this way the Fmocꢀprotected carboxylic
acid 3 became available on a gramꢀscale synthesis in 38%
overall yield.13
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8
Our group has developed synthetic methods for various type
CADIs (BusꢀXaaꢀψ[(Z)ꢀCCl=CH]ꢀYaaꢀOEt) utilizing organoꢀ
copper reagents and switching the olefin geometry of the alꢀ
lylic gemꢀdichlorides that are used as chloroalkene precurꢀ
sors.16ꢀ19 In addition, a Bocꢀ or Fmocꢀprotected dipeptide
(Bocꢀ or FmocꢀXaaꢀψ[(Z)ꢀCCl=CH]ꢀYaaꢀOH) can be easily
prepared for peptide synthesis from a common intermediate
Busꢀprotected dipeptide (BusꢀXaaꢀψ[(Z)ꢀCCl=CH]ꢀYaaꢀOH)
in a few steps and with high total yield. In this report, we deꢀ
scribe the introduction of a CADI into a cyclic pentapeptide,
cyclo[ꢀArgꢀGlyꢀAspꢀDꢀPheꢀValꢀ] 1, which was reported by
Kessler et al. as a highly bioactive αVβ3 integrin antagonist.20,21
We report the first chemical synthesis and biological evaluaꢀ
tion of a CADIꢀcontaining cyclic RGD peptide 2 utilizing
Fmocꢀbased solidꢀphase peptide synthesis (SPPS),22 and the
peptidomimtic was biologically evaluated (Figure 2).
9
Finally, the synthesis of the CADIꢀcontaining RGD peptide
was performed by established protocols (Scheme 2).24 A proꢀ
tected peptide resin 10 was constructed by Fmocꢀbased SPPS
on a glycinyl 2ꢀchlorotrityl (Clt) resin 9, which can provide
side chainꢀprotected peptides by subsequent mild acidic treatꢀ
ment.24 Exposing the resin 10 to AcOHꢀTFEꢀCH2Cl2 (1:1:3)
provided the protected peptide 11 without removal of the proꢀ
tecting groups in the aspartic acid and arginine residues, which
was then cyclized using HATU and HOAt25 to give the proꢀ
tected CADIꢀcontaining cyclic pentapeptide 12. In the final
step, deprotection of the cyclic peptide 12 with 87% TFA was
carried out, and the obtained crude peptide was then purified
by HPLC to provide the desired cyclic peptide 2 containing a
DꢀPheꢀψ[(Z)ꢀCCl=CH]ꢀValꢀtype isostere, in 72% yield from
the resin 9. The metabolic stability of the obtained pepꢀ
tidomimetic 2 was shown by no detectable decomposition in
human serum.13
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Initially, FmocꢀDꢀPheꢀψ[(Z)ꢀCCl=CH]ꢀValꢀOH 3 was proꢀ
duced by published synthetic methods.16ꢀ19 As shown in
Scheme 2. Synthesis of the CADIꢀcontaining cyclic
RGD peptide utilizing Fmocꢀbased solidꢀphase peptide
synthesis
Figure 2. A newly designed RGD peptidomimetic inꢀ
cluding
Scheme 1, the γ,γꢀdichloroꢀα,βꢀunsaturated ester 6, which has
been reported as a precursor in CADI synthesis13 was preꢀ
pared.
Diastereoselective allylic alkylation utilizing organocopper
reagents, prepared from 30 mol% CuCl and 2ꢀpropylzinc broꢀ
mide, afforded the desired chloroalkene product 7 in high
yield and with excellent diastereoselectivity. Deprotection of
the Bus group with AlCl3 and anisole, followed by the Fmoc
Scheme 1. Synthesis of FmocꢀDꢀPheꢀψ[(Z)ꢀCCl=CH]ꢀ
ValꢀOH
The CADIꢀcontaining cyclic RGD peptide 2 was evaluated
Table 1. The inhibitory effect of cyclic RGD peptides
against human dermal fibroblast (HDF) attachment to
vitronectin
compound
entry
1
IC50 (nM)
cyclo[ꢀArgꢀGlyꢀAspꢀDꢀPheꢀΨꢀValꢀ]
Ψ = ꢀC(O)NHꢀ (1)
10.8 ± 6.76a
(6.80 ± 2.70)b
2
3
4
Ψ = ꢀψ[(Z)ꢀCCl=CH]ꢀ (2)
Ψ = ꢀψ[(E)ꢀCH=CH]ꢀ (13)
Ψ = ꢀψ[(E)ꢀCMe=CH]ꢀ (14)
0.497± 0.149a
3.60 ± 1.30b
2.4± 0.33b
aIC50 values are the concentrations for 50% inhibition of the
against integrinꢀmediated cell attachment. bFujii and coꢀ
workers reported values.26
2
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ACS Medicinal Chemistry Letters
7.74ꢀ11.5 fold higher inhibitory activity than other pseudopepꢀ
1
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tides, the ADIꢀcontaining peptide 13 and a (E)ꢀtetrasubstituted
alkene dipeptide isostere (TADI)ꢀcontaining peptide 14 (enꢀ
tries 3, 4).26 According to this result, the stabilized structure of
the CADIꢀcontaining cyclic RGD peptide 2 interacts with αVβ3
integrin more strongly than the parent RGD peptide. This
might be due to the more highly rigid structure of the chloroꢀ
alkene (Figure 3ꢀa) or the 1,3ꢀallylic strain exerted by the
chlorine atom, which is higher than that associated with the
parent amide bond (Figure 3ꢀb).13 The structural analysis of
the CADIꢀcontaining peptide 2 was performed utilizing a
LowModeMD method in Molecular Operating Environment
(MOE) with reflection of the data from 2D NMR spectra
(Supporting Information) (Figure 4).27,28 As shown in Figure
4ꢀa, the superimposed peptide 1 and CADIꢀcontaining peptide
2 have similar conformations. Furthermore, comparison of the
superimposed 10 lowest energy structures of peptide 1 and
peptidomimetic 2 demonstrated a more rigid structure of 2
(Figure 4ꢀb and 4ꢀc). In fact, the root mean square deviation
(RMSD) values of peptide 1 and peptidomimetic 2 were 1.91
and 1.45, respectively. It is expected that the CADI moiety
might be mainly contributed to the restriction of the other reꢀ
gion involving the RGD sequence because the DꢀPheꢀVal seꢀ
quence is located outside of the interactions between the RGD
sequence and αVβ3.13,29 In addition, some groups have reported
that hydrogen bondꢀlike properties can be observed in Oꢀ
H···Cl or NꢀH···Cl interactions.30,31 An intermolecular hydroꢀ
gen bond or H2Oꢀmediated hydration with a chlorine atom
might be occurred. Therefore, it is considered that the CADIꢀ
containing RGD peptide was more potent than the TADIꢀ
containing peptide.
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Figure 3. The stabilized whole structure by the chloroꢀ
alkene structure
for its inhibitory effect against integrinꢀmediated cell attachꢀ
ment, and the results are shown in Table 1. The CADIꢀ
containing peptide 2 showed approximately 20ꢀfold higher
inhibitory activity (IC50 = 0.497 nM, entry 2) compared with
Kessler’s peptide 1 (IC50 = 10.9 nM, entry 1). This result
shows that the CADIꢀcontaining cyclic RGD peptides have
(a)
In this paper, we describe utilization of an Fmocꢀbased
SPPS to complete a gramꢀscale synthesis of FmocꢀDꢀPheꢀ
ψ[(Z)ꢀCCl=CH]ꢀValꢀOH 3, the first chemical synthesis of a
CADIꢀcontaining cyclic RGD peptide. In addition, the synꢀ
thetic CADIꢀcontaining cyclic RGD peptide as a pepꢀ
tidomimetic was shown to be an effective inhibitor of integrinꢀ
mediated cell attachment, superior to the parent peptide.
(b)
ASSOCIATED CONTENT
Supporting Information
Experimental procedures and characterization data including
NMR charts. The supporting information is available free of
charge on the ACS Publications website at DOI: .
AUTHOR INFORMATION
Corresponding Author
(c)
*Eꢀmail: tamamura.mr@tmd.ac.jp
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We are grateful to Dr. Takaaki Mizuguchi (Tokyo Medical and
Dental University) for his assistance in peptide synthesis. We
would like to extend our thanks to Prof. Yoshio Hayashi, Dr. Kenꢀ
taro Takayama (Tokyo University of Pharmacy and Life Sciencꢀ
es) and Ms. Kei Toyama (Tokyo Medical and Dental University)
for their assistance in behavior experiments in human serum. This
work was supported in part by KAKENHI, GrantꢀinꢀAid for Sciꢀ
entific Research (B) (15H04652 to H. T.); Research Program on
Figure 4. (a) Superimposition of the structures of peptide 1
and peptidomimetic 2, which are shown as orange and
green colors, respectively; (b) superimposition of the 10
lowest energy structures of peptide 1; (c) superimposition
of the 10 lowest energy structures of peptidomimetic 2.
3
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1964, 68, 441–451.
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(13) See supporting information.
(14) Waelchli, R.; Gamse, R.; Bauer, W.; Meigel, H.; Lier, E.; Feyen,
J. H. M. Dipeptide mimetics can substitute for the receptor activation
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HIV/AIDS, Japan Agency for Medical Research and Developꢀ
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search Networks; and the Platform for Drug Discovery, Informatꢀ
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ported by JSPS Research Fellowships for Young Scientists
(15J04754).
1
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ABBREVIATIONS
7
8
9
ADI, alkene dipeptide isostere; Bus, Nꢀtertꢀbutylsulfonyl; CADI,
chloroalkene dipeptide isostere; Clt, 2ꢀchlorotrityl; DIBAL, diisoꢀ
butylaluminum hydride; DIPEA, N,Nꢀdiisopropylethylamine;
(15) Cottens, S.; Fliri, H.; Lier, E.; Weidmann, B. WO1993005011
A1, 1993.
(16) Narumi, T.; Kobayakawa, T.; Aikawa, H.; Seike, S.; Tamamura,
H. Stereoselective Formation of Trisubstituted (Z)ꢀChloroalkenes
Adjacent to a Tertiary Carbon Stereogenic Center by Organocuprateꢀ
Mediated Reduction/Alkylation. Org. Lett. 2012, 14, 4490–4493.
(17) Kobayakawa, T.; Narumi, T.; Tamamura, H. Remote Stereoinꢀ
duction in the OrganocuprateꢀMediated Allylic Alkylation of Allylic
gemꢀDichlorides: Highly Diastereoselective Synthesis of (Z)ꢀ
Chloroalkene Dipeptide Isosteres. Org. Lett. 2015, 17, 2302–2305.
(18) Kobayakawa, T.; Tamamura, H. Efficient synthesis of XaaꢀGly
type (Z)ꢀchloroalkene dipeptide isosteres via organocuprate mediated
reduction. Tetrahedron 2016, 72, 4968–4971.
(19) Kobayakawa, T.; Tamamura, H. Stereoselective Synthesis of
XaaꢀYaa Type (Z)ꢀChloroalkene Dipeptide Isosteres via Efficient
Utilization of Organocopper Reagents Mediated Allylic Alkylation.
Tetrahedron 2017, 73, 4464–4471.
(20) Aumailley, M.; Gurrath, M.; Muller, G.; Calvete, J.; Timpl, R.;
Kessler, H. ArgꢀGlyꢀAsp constrained within cyclic pentapeptides.
Strong and selective inhibitors of cell adhesion to vitronectin and
laminin fragment P1. FEBS Lett. 1991, 291, 50−54.
(21) MasꢀMoruno, C.; Fraioli, R.; Rechenmacher, F.; Neubauer, S.;
Kapp, T. G.; Kessler, H. αvβ3ꢀ or α5β1ꢀIntegrinꢀSelective Pepꢀ
tidomimetics for Surface Coating. Angew. Chem. Int. Ed. 2016, 55,
1535−1539.
(22) Coin, I.; Beyermann, M.; Bienert, M. Solidꢀphase peptide synꢀ
thesis: from standard procedures to the synthesis of difficult sequencꢀ
es. Nat. Protoc. 2007, 2, 3247–3256.
(23) Robak, M. T.; Herbage, M. A.; Ellman, J. A. Synthesis and Apꢀ
plications of tertꢀButanesulfinamide. Chem. Rev. 2010, 110,
3600−3740.
(24) Barlos, K.; Gatos, D. 9ꢀFluorenylmethyloxycarbonyl/tbutylꢀ
based convergent protein synthesis. Biopolymers 1999, 51, 266−278.
(25) Manzoni, L.; Belvisi, L.; Arosio, D.; Civera, M.; PilkingtonMikꢀ
sa, M.; Potenza, D.; Caprini, A.; Araldi, E. M. V.; Monferrini, E.;
Mancino, M.; Podesta, F.; Scolastico, C. Cyclic RGDꢀContaining
Functionalized Azabicycloalkane Peptides as Potent Integrin Antagoꢀ
nists for Tumor Targeting. ChemMedChem 2009, 4, 615−632.
(26) Oishi, S.; Miyamoto, K.; Niida, A.; Yamamoto, M.; Ajito, K.;
Tamamura, H.; Otaka, A.; Kuroda, Y.; Asai, A.; Fujii, N. Application
of triꢀ and tetrasubstituted alkene dipeptide mimetics to conformaꢀ
tional studies of cyclic RGD peptides. Tetrahedron 2006, 62, 1416–
1424.
(27) Molecular Operating Environment (MOE), 2016.08; Chemical
Computing Group Inc., 1010 Sherbooke St. West, Suite #910, Monꢀ
treal, QC, Canada, H3A 2R7, 2016.
(28) Labute, P. LowModeMD — Implicit LowꢀMode Velocity Filterꢀ
ing Applied to Conformational Search of Macrocycles and Protein
Loops. J. Chem. Inf. Model. 2010, 50, 792−800.
Fmoc,
9ꢀfluorenylmethyloxycarbonyl;
HATU,
1ꢀ
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
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35
36
37
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40
41
42
43
44
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60
[bis(dimethylamino)methylene]ꢀ1Hꢀ1,2,3ꢀtriazolo[4,5ꢀ
b]pyridinium 3ꢀoxid hexafluorophosphate; HDF, human dermal
fibroblast; HOAt, 1ꢀhydroxyꢀ7ꢀazabenzotriazole; MOE, Molecuꢀ
lar Operating Environment; MS, mass spectrometry SPPS, solidꢀ
phase
peptide
synthesis,
Pbf,
2,2,4,6,7ꢀ
pentamethyldihydrobenzofuranꢀ5ꢀsulfonyl;
TADI,
(E)ꢀ
tetrasubstituted alkene dipeptide isostere; TFA, trifluoroacetic
acid; TFE, 2,2,2ꢀtrifluoroethanol; THF, tetrahydrofuran
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Assembly via Halide Hydrogen Bonds Triggered by Linear Alkanes
J. Am. Chem. Soc. 2014, 136, 10601−10604.
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