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Amino Propynyl Benzoic Acid Building Block in Rigid Spacers of ...

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Bioorganic & Medic<strong>in</strong>al Chemistry Letters 13 (2003) 1241–1244<strong>Am<strong>in</strong>o</strong> <strong>Propynyl</strong> <strong>Benzoic</strong> <strong>Acid</strong> <strong>Build<strong>in</strong>g</strong> <strong>Block</strong> <strong>in</strong> <strong>Rigid</strong> <strong>Spacers</strong> <strong>of</strong>Divalent Ligands B<strong>in</strong>d<strong>in</strong>g to the Syk SH2 Doma<strong>in</strong>s with EquallyHigh Aff<strong>in</strong>ity as the Natural LigandFrank J. Dekker, Nico J. de Mol, Marcel J. E. Fischer and Rob M. J. Liskamp*Department <strong>of</strong> Medic<strong>in</strong>al Chemistry, Utrecht Institute <strong>of</strong> Pharmaceutical Sciences, Utrecht University,PO Box 80.082, 3508 TB Utrecht, The NetherlandsReceived 5 November 2002; revised 14 January 2003; accepted 31 January 2003Abstract—The construction <strong>of</strong> rigid spacers composed <strong>of</strong> am<strong>in</strong>o propynyl benzoic acid build<strong>in</strong>g blocks is described. These spacerswere used to l<strong>in</strong>k two phosphopeptide ligand sites towards obta<strong>in</strong><strong>in</strong>g divalent ligands with a high aff<strong>in</strong>ity for Syk tandem SH2doma<strong>in</strong>s, which are important <strong>in</strong> signal transduction. The spacer conta<strong>in</strong><strong>in</strong>g two <strong>of</strong> those rigid build<strong>in</strong>g blocks led to a ligand whichwas as active as the natural ligand, <strong>in</strong>dicat<strong>in</strong>g that this build<strong>in</strong>g block can be used <strong>in</strong> the design and synthesis <strong>of</strong> high aff<strong>in</strong>itydivalent constructs that can successfully <strong>in</strong>terfere with crucial prote<strong>in</strong>–prote<strong>in</strong> <strong>in</strong>teractions.# 2003 Elsevier Science Ltd. All rights reserved.In eukaryotic cells, <strong>in</strong>tracellular signal<strong>in</strong>g prote<strong>in</strong>s playa major role as biomolecular switches between receptoractivation and ultimately changes <strong>in</strong> cell growth, metabolismor differentiation. 1,2 An <strong>in</strong>trigu<strong>in</strong>g example is theSyk prote<strong>in</strong> tyros<strong>in</strong>e k<strong>in</strong>ase (p72 syk ), which comprisestwo tandemly arranged Src homology 2 (SH2) doma<strong>in</strong>sand a tyros<strong>in</strong>e k<strong>in</strong>ase doma<strong>in</strong>. This k<strong>in</strong>ase plays animportant role <strong>in</strong> the mast cell, 3,4 where it acts as abiomolecular switch between antigenic activation <strong>of</strong> thehigh aff<strong>in</strong>ity receptor for immunoglobul<strong>in</strong> E (FceRIreceptor) and mast cell degranulation, result<strong>in</strong>g <strong>in</strong>release <strong>of</strong> <strong>in</strong>flammatory mediators. 5,6 Mast cell degranulationmay lead to excessive and undesired immuneresponses like asthma and hay fever.A crucial step <strong>in</strong> mast cell activation is diphosphorylation<strong>of</strong> the immunoreceptor tyros<strong>in</strong>e-based activationmotif (ITAM) <strong>in</strong> the g-cha<strong>in</strong> <strong>of</strong> the FceRIreceptor. This ITAM features the consensus sequencepTyr - Xxx - Xxx - Leu - (Xxx) 6-7 - pTyr - Xxx - Xxx - Leu(pTyr=phosphotyros<strong>in</strong>e, Xxx=undef<strong>in</strong>ed am<strong>in</strong>o acid).The ITAM b<strong>in</strong>ds upon phosphorylation to the tandemSH2 doma<strong>in</strong>s <strong>of</strong> Syk tyros<strong>in</strong>e k<strong>in</strong>ase <strong>in</strong> a divalent mode<strong>in</strong> which the underl<strong>in</strong>ed tetrapeptides are ma<strong>in</strong>lyresponsible for the <strong>in</strong>teraction. This divalency results <strong>in</strong>*Correspond<strong>in</strong>g author. Tel.: +31-30-253-7396; Fax: +31-30-253-6655; e-mail: r.m.j.liskamp@pharm.uu.nla 5000-fold enhanced aff<strong>in</strong>ity compared to the monovalent<strong>in</strong>teraction. 7 9 The divalent <strong>in</strong>teraction is thesimplest form <strong>of</strong> multivalency, which is one <strong>of</strong> nature’sfasc<strong>in</strong>at<strong>in</strong>g mechanisms to significantly <strong>in</strong>crease aff<strong>in</strong>ity<strong>of</strong> <strong>in</strong>dividual weakly <strong>in</strong>teract<strong>in</strong>g modules. 10 Development<strong>of</strong> peptidomimetic molecular constructs thatselectively <strong>in</strong>terfere with this divalent <strong>in</strong>teraction mayprovide valuable tools for biomolecular research andcan ultimately lead to a new class <strong>of</strong> therapeutic compounds(Fig. 1).Recently, 11 we described peptoid–peptide hybrids <strong>of</strong> themonophosphorylated Syk b<strong>in</strong>d<strong>in</strong>g tetrapeptide AcpTyr-Glu-Thr-Leu-NH2 4. However, the aff<strong>in</strong>ity <strong>of</strong>Figure 1. (A) Interaction <strong>of</strong> the Syk tandem SH2 doma<strong>in</strong>s with thedouble phosphorylated ITAM from the g-cha<strong>in</strong> <strong>of</strong> the FceRI receptor.(B) Interference with this <strong>in</strong>teraction by a divalent construct with aflexible spacer. 12 (C) Interference with this <strong>in</strong>teraction by a molecularconstruct with a rigid spacer.0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.doi:10.1016/S0960-894X(03)00117-3


1242 F.J.Dekker et al./ Bioorg.Med.Chem.Lett.13 (2003) 1241–1244these monovalent peptidomimetic constructs rema<strong>in</strong>edmodest. Thus, we <strong>in</strong>troduced divalency by l<strong>in</strong>k<strong>in</strong>g twomonophosphorylated peptides with an oligoethyleneglycol spacer result<strong>in</strong>g <strong>in</strong> a peptidomimetic construct 3with a 500-fold <strong>in</strong>creased aff<strong>in</strong>ity. 12 In order to improvethe aff<strong>in</strong>ity <strong>of</strong> the Syk b<strong>in</strong>d<strong>in</strong>g peptidomimetic constructsfurther, the rigidity <strong>of</strong> the non-peptide spacer hasto be <strong>in</strong>creased to avoid unfavorable entropy loss uponb<strong>in</strong>d<strong>in</strong>g. For this purpose, a special rigid am<strong>in</strong>o acidbuild<strong>in</strong>g block was synthesized (Scheme 1), which caneasily be <strong>in</strong>corporated <strong>in</strong>to peptides by solid phase peptidesynthesis (vide <strong>in</strong>fra) lead<strong>in</strong>g to a peptidomimeticconstruct with enhanced aff<strong>in</strong>ity for the Syk tandemSH2 doma<strong>in</strong>s.DesignDifferent spacers were designed <strong>in</strong> model<strong>in</strong>g studies 12based on the crystal structure <strong>of</strong> the Syk tandem SH2doma<strong>in</strong>s complexed with the ITAM peptide. 13 Model<strong>in</strong>gshowed that the <strong>in</strong>terven<strong>in</strong>g am<strong>in</strong>o acids betweenthe <strong>in</strong>teract<strong>in</strong>g tetrapeptides can be replaced by tworigid am<strong>in</strong>o acid build<strong>in</strong>g blocks 1 lead<strong>in</strong>g to a rigidspacer as <strong>in</strong> molecular construct 5 (Scheme 2). However,for b<strong>in</strong>d<strong>in</strong>g <strong>of</strong> rigid molecules small spatial mismatchesbetween ligand and receptor are very likely to occurthus result<strong>in</strong>g <strong>in</strong> low aff<strong>in</strong>ity. This problem might becircumvented by a comb<strong>in</strong>ation <strong>of</strong> flexible and rigidbuild<strong>in</strong>g blocks as <strong>in</strong> molecular construct 6 conta<strong>in</strong><strong>in</strong>gtwo glyc<strong>in</strong>e residues flank<strong>in</strong>g the rigid am<strong>in</strong>o acid derivative.ChemistryFor the synthesis <strong>of</strong> rigid build<strong>in</strong>g block 1 4-iodobenzoicacid was converted to the methyl ester andpropargylam<strong>in</strong>e was tert-butyloxycarbonyl (Boc) protectedus<strong>in</strong>g Boc 2 O and sodium hydroxide (Scheme 1).The result<strong>in</strong>g 4-iodobenzoic acid methyl ester and Bocprotectedpropargylam<strong>in</strong>e were coupled by Sonogashiracoupl<strong>in</strong>g 14 (Scheme 1) us<strong>in</strong>g tetrakis(triphenylphosph<strong>in</strong>e)palladium(0)(Pd(PPh 3 ) 4 ), cupper(I)iodide andtriethylam<strong>in</strong>e followed by saponification <strong>of</strong> the methylester. F<strong>in</strong>ally the Boc-group was removed and the allyloxycarbonyl-group(Alloc) was <strong>in</strong>troduced to give 4-(3-allyloxycarbonylam<strong>in</strong>o-prop-1-ynyl)-benzoic acid 1. 15Alloc protection was used <strong>in</strong>stead <strong>of</strong> Fmoc protection,because the Fmoc protected build<strong>in</strong>g block was poorlysoluble <strong>in</strong> organic solvents. The Alloc-group is nicelycompatible with Fmoc-based peptide chemistry.This rigid build<strong>in</strong>g block was now used for the assembly<strong>of</strong> the designed peptide molecular constructs us<strong>in</strong>gFmoc based peptide chemistry on R<strong>in</strong>k (amide) res<strong>in</strong>,followed by purification as described previously(Scheme 2). 11,12 The pure compounds were characterizedby high resolution mass spectroscopy and 1 H500 MHz NMR. 16 Removal <strong>of</strong> the Alloc-group waseffected with 20 mol% Pd(PPh 3 ) 4 and 8 equiv sodiumparatoluene sulf<strong>in</strong>ate <strong>in</strong> methanol/tetrahydr<strong>of</strong>uran. 17Aff<strong>in</strong>ity MeasurementThe rigid and semi-rigid peptide constructs 5 and 6 wereevaluated for their b<strong>in</strong>d<strong>in</strong>g to the Syk tandem SH2doma<strong>in</strong>s us<strong>in</strong>g a surface plasmon resonance assay (SPR)as was described previously. 11,12 In this competitionassay a Syk b<strong>in</strong>d<strong>in</strong>g ITAM peptide was immobilized ona sensor-chip surface on which competition experimentswere performed <strong>in</strong> triplicate (Fig. 2). From theseexperiments the IC 50 values were obta<strong>in</strong>ed and shown <strong>in</strong>Table 1 ([Syk tandem SH2] was 100 nM). The dissociationconstant for the <strong>in</strong>teraction <strong>of</strong> the Syk tandem SH2doma<strong>in</strong>s with the ITAM on the sensor-chip surface was40 nM (n=2, measured at equilibrium), which was usedto calculate dissociation b<strong>in</strong>d<strong>in</strong>g constants (K d )<strong>of</strong>thedifferent peptides and peptide-hybrids <strong>in</strong> solution. 18 Forcomparison the monovalent and divalent Syk b<strong>in</strong>d<strong>in</strong>gligands 4 and 2 and peptide-oligoethylene glycol hybrid3 were <strong>in</strong>cluded <strong>in</strong> this study. 12In Table 1 the 5000-fold difference between the monophosphorylatedpeptide 4 and the diphosphorylatedpeptide 2 is shown, 7 9 which clearly demonstrates themagnitude <strong>of</strong> the divalency effect <strong>in</strong> this <strong>in</strong>teraction. Theconstruct with the ethylene glycol spacer 3 showed thata complete different spacer can replace the seven <strong>in</strong>terven<strong>in</strong>gam<strong>in</strong>o acids between the <strong>in</strong>teract<strong>in</strong>g phosphotetrapeptides<strong>of</strong> the ITAM peptide. 12 S<strong>in</strong>ce this is amolecular construct conta<strong>in</strong><strong>in</strong>g a flexible spacer, it provideda start<strong>in</strong>g po<strong>in</strong>t to evaluate the effect <strong>of</strong> rigidspacers. If a more rigid construct has the proper geometryfor <strong>in</strong>teraction with the Syk SH2 doma<strong>in</strong>s, theaff<strong>in</strong>ity is expected to be higher due to a more favorableTable 1. Aff<strong>in</strong>ities <strong>of</strong> the peptide and peptide-hybrids for the Syktandem SH2 doma<strong>in</strong>sCompd IC 50 , mM K d , mMScheme 1. Synthesis <strong>of</strong> the rigid am<strong>in</strong>o acid build<strong>in</strong>g block; (a) Boc 2 O,NaOH, dioxane/H 2 O; (b) H 2 SO 4 , MeOH; (c) Pd(PPh 3 ) 4 , CuI, Et 3 N,CH 3 CN; (d) NaOH, dioxane/H 2 O; (e) HCl <strong>in</strong> Et 2 O, ethylacetate; (f)allylchlor<strong>of</strong>ormate, Et 3 N, dioxane/H 2 O.2 0.13 (0.01) 0.0373 1.2 (0.2) 0.344 570 (50) 1665 0.10 (0.007) 0.0296 62 (12) 4.0


F.J.Dekker et al./ Bioorg.Med.Chem.Lett.13 (2003) 1241–1244 1243Scheme 2. Synthesis <strong>of</strong> the peptide molecular constructs <strong>in</strong> three ma<strong>in</strong> steps: i=Coupl<strong>in</strong>g cycle : Fmoc-Xxx-OH, BOP, DiPEA, NMP; 20% piperid<strong>in</strong>e/NMP(Fmoc deprotection) or Pd(PPh 3 ) 4 with sodium paratoluene sulf<strong>in</strong>ate <strong>in</strong> MeOH/THF (Alloc deprotection); 17 ii=Acetylation : Ac 2 O,DiPEA, HOBt, NMP; iii=F<strong>in</strong>al cleavage and deprotection: TFA, EDT, Tis, H 2 O (90/2.5/2.5/5); (a) reference 12 (b) (i) 1, 1, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tyr(PO(OBzl)OH)-OH; (ii) and (iii) (c) (i) Fmoc-Gly-OH, 1, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH,Fmoc-Thr(tBu)-OH, Fmoc-Tyr(PO(OBzl)OH)-OH; (ii) and (iii)


1244 F.J.Dekker et al./ Bioorg.Med.Chem.Lett.13 (2003) 1241–1244used for b<strong>in</strong>d<strong>in</strong>g to- widely- separated multivalent<strong>in</strong>teraction sites prevalent <strong>in</strong> many prote<strong>in</strong>–prote<strong>in</strong><strong>in</strong>teractions.AcknowledgementsWe thank Isabel Catal<strong>in</strong>a (Dept. <strong>of</strong> Biomolecular MassSpectrometry) for mass spectrometry analysis and Dr.Johan Kemm<strong>in</strong>k for 500 MHz NMR spectroscopyanalysis.References and NotesFigure 2. Aff<strong>in</strong>ities <strong>of</strong> the peptide and peptide-hybrids for the Syktandem SH2 doma<strong>in</strong> as measured by SPR <strong>in</strong> competition experiments.entropic contribution. Molecular construct 6 conta<strong>in</strong><strong>in</strong>gthe semi-rigid spacer shows a 10-fold reduced aff<strong>in</strong>itycompared to the oligoethylene glycol spaced construct3. The comb<strong>in</strong>ation <strong>of</strong> flexible and rigid build<strong>in</strong>g blocks<strong>in</strong> compound 6 was not effective. Apparently, the length<strong>of</strong> the spacer was too small and the proper geometry foroptimal position<strong>in</strong>g <strong>of</strong> the <strong>in</strong>teract<strong>in</strong>g tetrapeptidescould not be obta<strong>in</strong>ed. Molecular construct 5 with thetwo rigid spac<strong>in</strong>g build<strong>in</strong>g blocks showed a 10-fold<strong>in</strong>creased aff<strong>in</strong>ity compared to the molecular constructconta<strong>in</strong><strong>in</strong>g the flexible oligoethylene glycol spacer,thereby reach<strong>in</strong>g an aff<strong>in</strong>ity that is equal to the aff<strong>in</strong>ity<strong>of</strong> the native diphosphorylated ITAM peptide 2.Clearly, this construct has a more optimal geometry for<strong>in</strong>teraction with the Syk tandem SH2 doma<strong>in</strong>s. The<strong>in</strong>teraction is expected to be facilitated by the rigidity <strong>of</strong>the spacer. F<strong>in</strong>ally, the entropic contribution related t<strong>of</strong>lexibility is only one <strong>of</strong> the factors play<strong>in</strong>g a role <strong>in</strong>b<strong>in</strong>d<strong>in</strong>g thermodynamics. A more detailed thermodynamicstudy will be subject <strong>of</strong> further research, as wellas the ability <strong>of</strong> these constructs to <strong>in</strong>terfere with cellularprocesses.To our knowledge this is the first time that two <strong>in</strong>teract<strong>in</strong>gphosphopeptides have been l<strong>in</strong>ked by a rigidspacer to give rise to a divalent <strong>in</strong>teraction with an aff<strong>in</strong>itythat is both better than a molecular construct conta<strong>in</strong><strong>in</strong>ga flexible l<strong>in</strong>ker and equally high to that <strong>of</strong> thenatural ligand. We th<strong>in</strong>k that this and other rigid am<strong>in</strong>oacid derivatives can be more generally applied as versatilebuild<strong>in</strong>g blocks connect<strong>in</strong>g ligands, which can be1. Lim, W. A. Curr.Op<strong>in</strong>.Struct.Biol.2002, 12, 61.2. Pawson, T.; Gish, G. D.; Nash, P. Trends Cell Biol. 2001,11, 504.3. Sada, K.; Takano, T.; Yanagi, S.; Yamamura, H. J.Biochem.(Tokyo)2001, 130, 177.4. Turner, M.; Schweigh<strong>of</strong>fer, E.; Colucci, F.; Di Santo, J. P.;Tybulewicz, V. L. Immunol.Today 2000, 21, 148.5. Benhamou, M.; Ryba, N. J. P.; Kihara, H.; Nishikata, H.;Siraganian, R. P. J.Biol.Chem.1993, 268, 23318.6. Taylor, J. A.; Karas, J. L.; Ram, M. K.; Green, O. M.;Seidel-Dugan, C. Mol.Cel.Biol.1995, 15, 4149.7. Chen, T.; Repetto, B.; Chizzonite, R.; Pullar, C.; Burghardt,C.; Dharm, E.; Zhao, Z.; Carroll, R.; Nunes, P.; Basu,M.; Danho, W.; Visnick, M.; Kochan, J.; Waugh, D.; Gilfillan,A. M. J.Biol.Chem.1996, 271, 25308.8. Grucza, R. A.; Bradshaw, J. M.; Mitaxov, V.; Waksman,G. Biochemistry 2000, 39, 10072.9. Ott<strong>in</strong>ger, E. A.; Botfield, M. C.; Shoelson, S. E. J.Biol.Chem. 1998, 273, 729.10. Mammen, M.; Choi, S.; Whitesides, G. M. Angew.Chem.Int.Ed.1998, 37, 2754.11. Ruijtenbeek, R.; Kruijtzer, J. A. W.; van de Wiel, W.;Fischer, M. J. E.; Flu¨ck, M.; Redegeld, F. A. M.; Liskamp,R. M. J.; Nijkamp, F. P. ChemBioChem. 2001, 2, 171.12. Dekker, F. J.; de Mol, N. J.; van Ameijde, J.; Fischer,M. J. E.; Ruijtenbeek, R.; Redegeld, F. A. M.; Liskamp, R. M.J. ChemBioChem. 2002, 2-3, 238.13. Fu¨tterer, K.; Wong, J.; Grucza, R. A.; Chan, A. C.;Waksman, G. J.Mol.Biol.1998, 281, 523.14. Sonogashira, K.; Tohda, Y.; Hagihara, N. TetrahedronLett. 1975, 50, 4467.15. Compound 1: 1 H NMR (300 MHz, CD 3 OD), d=4.15(s, 2H),4.57(d, 6H), 4.97(br. 2H), 5.18–5.35(m, 2H) 5.90–6.00(m, 1H),7.49(d, 2H), 7.97(d, 2H). 13 CNMR(75MHz,CD 3 OD) d=31.8,66.7, 82.5, 89.8, 117.7, 128.9, 130.7, 131.5, 132.6, 134.3, 169.1.16. All protons could be assigned <strong>in</strong> TOCSY and NOESY 1 H500 MHz NMR experiments. High resolution mass spectroscopyanalysis for compound 5 [M+2H+] calcd 1475.53;found 1475.52 for compound 6 [M+2H+] calcd 1432.52;found 1432.52.17. Honda, M.; Morita, H.; Nagakura, I. J.Org.Chem.1997,62, 8932.18. de Mol, N. J.; Gillies, M. B.; Fischer, M. J. E. 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