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

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

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. 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