Skip to main content
NCBI home page
ACS Medicinal Chemistry Letters logoLink to ACS Medicinal Chemistry Letters
. 2017 Dec 27;9(1):6–10. doi: 10.1021/acsmedchemlett.7b00234

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

Takuya Kobayakawa †, Yudai Matsuzaki †, Kentaro Hozumi ‡, Wataru Nomura †, Motoyoshi Nomizu ‡, Hirokazu Tamamura †,*
PMCID: PMC5767888  PMID: 29348803

Abstract

graphic file with name ml-2017-00234j_0007.jpg

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.

Keywords: Chloroalkene dipeptide isostere, peptidomimetic, solid-phase peptide synthesis, cyclic RGD peptide

During the last quarter-century, various biologically active peptides have been discovered and characterized. These bioactive peptides influence and control physiological functions through interaction with their various receptors, and the number of natural and modified peptides that are used as therapeutics 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 limited by several factors, including low metabolic stability toward proteolysis and undesired activity resulting from interactions of peptides with various receptors.1,2

Alkene dipeptide isosteres (ADIs), which are designed based on the partial double-bond character of the native peptide bond in its ground state conformation, have been expected to be structure units as they have ideal 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.3−11 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. In addition, many ADIs cannot be supplied efficiently due to problems associated with their synthesis.

Our research group has focused on the chloroolefin structures in chloroalkene dipeptide isosteres (CADIs), which can be used to replace an amide bond in peptides as shown in Figure 1. Replacement of a peptide bond by the chloroolefin moiety can also be considered as mimicking steric restriction resulting from the pseudo-1,3-allylic strain by a chlorine atom, which is larger than a carbonyl oxygen.11,12

Figure 1.

Figure 1

Open in a new tab

Native peptide bonds and chloroalkene dipeptide isosteres.

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 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 the lack of efficient methods or limitation of substrates for synthesis of CADIs.

Our group has developed synthetic methods for various type CADIs (Bus-Xaa-ψ[(Z)-CCl=CH]-Yaa-OEt) utilizing organocopper reagents and switching the olefin geometry of the allylic gem-dichlorides that are used as chloroalkene precursors.16−19 In addition, a Boc- or Fmoc-protected dipeptide (Boc- or Fmoc-Xaa-ψ[(Z)-CCl=CH]-Yaa-OH) can be easily prepared for peptide synthesis from a common intermediate Bus-protected dipeptide (Bus-Xaa-ψ[(Z)-CCl=CH]-Yaa-OH) in a few steps and with high total yield. In this report, we describe the introduction of a CADI into a cyclic pentapeptide, cyclo[-Arg-Gly-Asp-d-Phe-Val-] 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 evaluation of a CADI-containing cyclic RGD peptide 2 utilizing Fmoc-based solid-phase peptide synthesis (SPPS),22 and the peptidomimtic was biologically evaluated (Figure 2).

Figure 2.

Figure 2

Open in a new tab

Newly designed RGD peptidomimetic.

Initially, Fmoc-d-Phe-ψ[(Z)-CCl=CH]-Val-OH 3 was produced by published synthetic methods.16−19 As shown in Scheme 1, the γ,γ-dichloro-α,β-unsaturated ester 6, which has been reported as a precursor in CADI synthesis,13 was prepared.

Scheme 1. Synthesis of Fmoc-d-Phe-ψ[(Z)-CCl=CH]-Val-OH.

Scheme 1

Open in a new tab

Diastereoselective allylic alkylation utilizing organocopper reagents, prepared from 30 mol % CuCl and 2-propylzinc bromide, 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 protection, led to the ester 8. The ester group of 8 was reduced to the corresponding aldehyde with DIBAL at −78 °C, and this was followed by Pinnick oxidation to provide the desired Fmoc-protected carboxylic acid 3 in 81% yield from the Bus-protected ester 7 without decrease in diastereoselectivity or appreciable olefin isomerization. These 10 steps proceeded smoothly to provide the desired compound from starting materials 4 and 5,23 and in this way, the Fmoc-protected carboxylic acid 3 became available on a gram-scale synthesis in 38% overall yield.13

Finally, the synthesis of the CADI-containing RGD peptide was performed by established protocols (Scheme 2).24 A protected peptide resin 10 was constructed by Fmoc-based SPPS on a glycinyl 2-chlorotrityl (Clt) resin 9, which can provide side chain-protected peptides by subsequent mild acidic treatment.24 Exposing the resin 10 to AcOH/TFE/CH2Cl2 (1:1:3) provided the protected peptide 11 without removal of the protecting groups in the aspartic acid and arginine residues, which was then cyclized using HATU and HOAt25 to give the protected CADI-containing 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 d-Phe-ψ[(Z)-CCl=CH]-Val-type isostere, in 72% yield from the resin 9. The metabolic stability of the obtained peptidomimetic 2 was shown by no detectable decomposition in human serum.13

Scheme 2. Synthesis of the CADI-Containing Cyclic RGD Peptide Utilizing Fmoc-Based Solid-Phase Peptide Synthesis.

Scheme 2

Open in a new tab

The CADI-containing cyclic RGD peptide 2 was evaluated for its inhibitory effect against integrin-mediated cell attachment, and the results are shown in Table 1. The CADI-containing peptide 2 showed approximately 20-fold 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 CADI-containing cyclic RGD peptides have 7.74–11.5-fold higher inhibitory activity than other pseudopeptides, the ADI-containing peptide 13, and a (E)-tetrasubstituted alkene dipeptide isostere (TADI)-containing peptide 14 (entries 3, 4).26 According to this result, the stabilized structure of the CADI-containing 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 chloroalkene (Figure 3a) or the 1,3-allylic strain exerted by the chlorine atom, which is higher than that associated with the parent amide bond (Figure 3b).13 The structural analysis of the CADI-containing 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 4a, the superimposed peptide 1 and CADI-containing 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 4b,c). 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 region involving the RGD sequence because the d-Phe-Val sequence is located outside of the interactions between the RGD sequence and αVβ3.13,29 In addition, some groups have reported that hydrogen bond-like properties can be observed in O–H···Cl or N–H···Cl interactions.30,31 An intermolecular hydrogen bond or H2O-mediated hydration with a chlorine atom might occur. Therefore, it is considered that the CADI-containing RGD peptide was more potent than the TADI-containing peptide.

Table 1. Inhibitory Effect of Cyclic RGD Peptides against Human Dermal Fibroblast (HDF) Attachment to Vitronectin.

entry cyclo[-Arg-Gly-Asp-d-Phe-Ψ-Val-] IC50 (nM)
1 Ψ = −C(O)NH– (1) 10.8 ± 6.76a
(6.80 ± 2.70)b
2 Ψ = −ψ[(Z)-CCl=CH]– (2) 0.497 ± 0.149a
3 Ψ = −ψ[(E)-CH=CH]– (13) 3.60 ± 1.30b
4 Ψ = −ψ[(E)-CMe=CH]– (14) 2.4 ± 0.33b
Open in a new tab
a

IC50 values are the concentrations for 50% inhibition of the against integrin-mediated cell attachment.

b

Fujii and co-workers’ reported values.26

Figure 3.

Figure 3

Open in a new tab

Stabilized whole structure by the chloroalkene structure.

Figure 4.

Figure 4

Open in a new tab

(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.

In this Letter, we describe utilization of an Fmoc-based SPPS to complete a Gram-scale synthesis of Fmoc-d-Phe-ψ[(Z)-CCl=CH]-Val-OH 3, the first chemical synthesis of a CADI-containing cyclic RGD peptide. In addition, the synthetic CADI-containing cyclic RGD peptide as a peptidomimetic was shown to be an effective inhibitor of integrin-mediated cell attachment, superior to the parent peptide.

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. Kentaro Takayama (Tokyo University of Pharmacy and Life Sciences), 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, Grant-in-Aid for Scientific Research (B) (15H04652 to H.T.); Research Program on HIV/AIDS, Japan Agency for Medical Research and Development (AMED); JSPS Core-to-Core Program, A. Advanced Research Networks; and the Platform for Drug Discovery, Informatics, and Structural Life Science of MEXT, Japan. T.K. was supported by JSPS Research Fellowships for Young Scientists (15J04754).

Glossary

ABBREVIATIONS

ADI

alkene dipeptide isostere

Bus

N-tert-butylsulfonyl

CADI

chloroalkene dipeptide isostere

Clt

2-chlorotrityl

DIBAL

diisobutylaluminum hydride

DIPEA

N,N-diisopropylethylamine

Fmoc

9-fluorenylmethyloxycarbonyl

HATU

1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate

HDF

human dermal fibroblast

HOAt

1-hydroxy-7-azabenzotriazole

MOE

Molecular Operating Environment

MS

mass spectrometry

SPPS

solid-phase peptide synthesis

Pbf

2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl

TADI

(E)-tetrasubstituted alkene dipeptide isostere

TFA

trifluoroacetic acid

TFE

2,2,2-trifluoroethanol

THF

tetrahydrofuran

Supporting Information Available

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmedchemlett.7b00234.

  • Experimental procedures and characterization data including NMR charts (PDF)

The authors declare no competing financial interest.

Supplementary Material

ml7b00234_si_001.pdf (2.6MB, pdf)

References

  1. Veber D. F.; Freidinger R. M. The design of metabolically-stable peptide analogs. Trends Neurosci. 1985, 8, 392–396. 10.1016/0166-2236(85)90140-7. [DOI] [Google Scholar]
  2. Grauer A.; König B. Peptidomimetics: A Versatile Route to Biologically Active Compounds. Eur. J. Org. Chem. 2009, 2009, 5099–5011. 10.1002/ejoc.200900599. [DOI] [Google Scholar]
  3. Tamamura H.; Hiramatsu K.; Ueda S.; Wang Z. X.; Kusano S.; Terakubo S.; Trent J. O.; Peiper S. C.; Yamamoto N.; Nakashima H.; Otaka A.; Fujii N. Stereoselective synthesis of [L-Arg-L/D-3-(2-naphthyl) alanine]-type (E)-alkene dipeptide isosteres and its application to the synthesis and biological evaluation of pseudopeptide analogues of the CXCR4 antagonist FC131. J. Med. Chem. 2005, 48, 380–391. 10.1021/jm049429h. [DOI] [PubMed] [Google Scholar]
  4. Fu Y. W.; Bieschke J.; Kelly J. W. E-olefin dipeptide isostere incorporation into a polypeptide backbone enables hydrogen bond perturbation: Probing the requirements for Alzheimer’s amyloidogenesis. J. Am. Chem. Soc. 2005, 127, 15366–15367. 10.1021/ja0551382. [DOI] [PubMed] [Google Scholar]
  5. Misu R.; Oishi S.; Yamada A.; Yamamura T.; Matsuda F.; Yamamoto K.; Noguchi T.; Ohno H.; Okamura H.; Ohkura S.; Fujii N. Development of Novel Neurokinin 3 Receptor (NK3R) Selective Agonists with Resistance to Proteolytic Degradation. J. Med. Chem. 2014, 57, 8646–8651. 10.1021/jm500771w. [DOI] [PubMed] [Google Scholar]
  6. McKinney B. E.; Urban J. J. Fluoroolefins as Peptide Mimetics. 2. A Computational Study of the Conformational Ramifications of Peptide Bond Replacement. J. Phys. Chem. A 2010, 114, 1123–1133. 10.1021/jp9094535. [DOI] [PubMed] [Google Scholar]
  7. Narumi T.; Hayashi R.; Tomita K.; Kobayashi K.; Tanahara N.; Ohno H.; Naito T.; Kodama E.; Matsuoka M.; Oishi S.; Fujii N. Synthesis and biological evaluation of selective CXCR4 antagonists containing alkene dipeptide isosteres. Org. Biomol. Chem. 2010, 8, 616–621. 10.1039/B917236J. [DOI] [PubMed] [Google Scholar]
  8. Villiers E.; Couve-Bonnaire S.; Cahard D.; Pannecoucke X. The fluoroalkene motif as a surrogate of the amide bond: syntheses of AA-ψ[(Z) and (E)-CF=CH]-Pro pseudodipeptides and an Enalapril analogue. Tetrahedron 2015, 71, 7054–7062. 10.1016/j.tet.2015.06.093. [DOI] [Google Scholar]
  9. Reddy G. S. K. K.; Ali A.; Nalam M. N. L.; Anjum S. G.; Cao H.; Nathans R. S.; Schiffer C. A.; Rana T. M. Design and Synthesis of HIV-1 Protease Inhibitors Incorporating Oxazolidinones as P2/P2′ Ligands in Pseudosymmetric Dipeptide Isosteres. J. Med. Chem. 2007, 50, 4316–4328. 10.1021/jm070284z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kobayashi K.; Oishi S.; Hayashi R.; Tomita K.; Kubo T.; Tanahara N.; Ohno H.; Yoshikawa Y.; Furuya T.; Hoshino M.; Fujii N. Structure-Activity Relationship Study of a CXC Chemokine Receptor Type 4 Antagonist, FC131, Using a Series of Alkene Dipeptide Isosteres. J. Med. Chem. 2012, 55, 2746–2757. 10.1021/jm2016914. [DOI] [PubMed] [Google Scholar]
  11. Bondi A. van der Waals Volumes and Radii. J. Phys. Chem. 1964, 68, 441–451. 10.1021/j100785a001. [DOI] [Google Scholar]
  12. Batsanov S. S. Van der Waals Radii of Elements. Inorg. Mater. 2001, 37, 871–885. 10.1023/A:1011625728803. [DOI] [Google Scholar]
  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 domain resulting in highly potent analogues of hPTH(1–36) fragment. Bioorg. Med. Chem. Lett. 1996, 6, 1151–1156. 10.1016/0960-894X(96)00188-6. [DOI] [Google Scholar]
  15. Cottens S.; Fliri H.; Lier E.; Weidmann B.. WO1993005011A1, 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 Organocuprate-Mediated Reduction/Alkylation. Org. Lett. 2012, 14, 4490–4493. 10.1021/ol301988d. [DOI] [PubMed] [Google Scholar]
  17. Kobayakawa T.; Narumi T.; Tamamura H. Remote Stereoinduction in the Organocuprate-Mediated Allylic Alkylation of Allylic gem-Dichlorides: Highly Diastereoselective Synthesis of (Z)-Chloroalkene Dipeptide Isosteres. Org. Lett. 2015, 17, 2302–2305. 10.1021/acs.orglett.5b00611. [DOI] [PubMed] [Google Scholar]
  18. Kobayakawa T.; Tamamura H. Efficient synthesis of Xaa-Gly type (Z)-chloroalkene dipeptide isosteres via organocuprate mediated reduction. Tetrahedron 2016, 72, 4968–4971. 10.1016/j.tet.2016.06.080. [DOI] [Google Scholar]
  19. Kobayakawa T.; Tamamura H. Stereoselective Synthesis of Xaa-Yaa Type (Z)-Chloroalkene Dipeptide Isosteres via Efficient Utilization of Organocopper Reagents Mediated Allylic Alkylation. Tetrahedron 2017, 73, 4464–4471. 10.1016/j.tet.2017.06.003. [DOI] [Google Scholar]
  20. Aumailley M.; Gurrath M.; Muller G.; Calvete J.; Timpl R.; Kessler H. Arg-Gly-Asp constrained within cyclic pentapeptides. Strong and selective inhibitors of cell adhesion to vitronectin and laminin fragment P1. FEBS Lett. 1991, 291, 50–54. 10.1016/0014-5793(91)81101-D. [DOI] [PubMed] [Google Scholar]
  21. Mas-Moruno C.; Fraioli R.; Rechenmacher F.; Neubauer S.; Kapp T. G.; Kessler H. αvβ3- or α5β1-Integrin-Selective Peptidomimetics for Surface Coating. Angew. Chem., Int. Ed. 2016, 55, 7048–7067. 10.1002/anie.201509782. [DOI] [PubMed] [Google Scholar]
  22. Coin I.; Beyermann M.; Bienert M. Solid-phase peptide synthesis: from standard procedures to the synthesis of difficult sequences. Nat. Protoc. 2007, 2, 3247–3256. 10.1038/nprot.2007.454. [DOI] [PubMed] [Google Scholar]
  23. Robak M. T.; Herbage M. A.; Ellman J. A. Synthesis and Applications of tert-Butanesulfinamide. Chem. Rev. 2010, 110, 3600–3740. 10.1021/cr900382t. [DOI] [PubMed] [Google Scholar]
  24. Barlos K.; Gatos D. 9-Fluorenylmethyloxycarbonyl/tbutyl-based convergent protein synthesis. Biopolymers 1999, 51, 266–278. . [DOI] [PubMed] [Google Scholar]
  25. Manzoni L.; Belvisi L.; Arosio D.; Civera M.; PilkingtonMiksa M.; Potenza D.; Caprini A.; Araldi E. M. V.; Monferini E.; Mancino M.; Podesta F.; Scolastico C. Cyclic RGD-Containing Functionalized Azabicycloalkane Peptides as Potent Integrin Antagonists for Tumor Targeting. ChemMedChem 2009, 4, 615–632. 10.1002/cmdc.200800422. [DOI] [PubMed] [Google Scholar]
  26. Oishi S.; Miyamoto K.; Niida A.; Yamamoto M.; Ajito K.; Tamamura H.; Otaka A.; Kuroda Y.; Asai A.; Fujii N. Application of tri- and tetrasubstituted alkene dipeptide mimetics to conformational studies of cyclic RGD peptides. Tetrahedron 2006, 62, 1416–1424. 10.1016/j.tet.2005.11.033. [DOI] [Google Scholar]
  27. Molecular Operating Environment (MOE), 2016.08; Chemical Computing Group Inc., Montreal, QC, Canada, 2016. [Google Scholar]
  28. Labute P. LowModeMD — Implicit Low-Mode Velocity Filtering Applied to Conformational Search of Macrocycles and Protein Loops. J. Chem. Inf. Model. 2010, 50, 792–800. 10.1021/ci900508k. [DOI] [PubMed] [Google Scholar]
  29. Xiong J. P.; Stehle T.; Zhang R.; Joachimiak A.; Frech M.; Goodman S. L.; Arnaout M. A. Crystal Structure of the Extracellular Segment of Integrin αVβ3 in Complex with an Arg-Gly-Asp Ligand. Science 2002, 296, 151–155. 10.1126/science.1069040. [DOI] [PubMed] [Google Scholar]
  30. Zhu Y.-Y.; Yi H.-P.; Li C.; Jiang X.-K.; Li Z.-T. The N—H···X (X = Cl, Br, and I) Hydrogen-Bonding Pattern in Aromatic Amides: A Crystallographic and 1H NMR Study. Cryst. Growth Des. 2008, 8, 1294–1300. 10.1021/cg7010293. [DOI] [Google Scholar]
  31. Metz A. E.; Podlesny E. E.; Carroll P. J.; Klinghoffer A. N.; Kozlowski M. C. Axial Chiral Bisbenzophenazines: Solid-State Self-Assembly via Halide Hydrogen Bonds Triggered by Linear Alkanes. J. Am. Chem. Soc. 2014, 136, 10601–10604. 10.1021/ja506137j. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

ml7b00234_si_001.pdf (2.6MB, pdf)

Articles from ACS Medicinal Chemistry Letters are provided here courtesy of American Chemical Society

RESOURCES