Global Transformation of OBOC Combinatorial Peptide Libraries into OBOC Polyamine and Small Molecule Libraries

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  • Global Transformation of OBOC Combinatorial Peptide Librariesinto OBOC Polyamine and Small Molecule Libraries

    Joseph C. Kappel, Yi C. Fan, and Kit S. Lam*

    DiVision of Hematology and Oncology, Department of Internal Medicine, UniVersity of California DaVisCancer Center, 4501 X Street, Sacramento, California 95817

    ReceiVed October 12, 2007

    In one-bead-one-compound (OBOC) combinatorial chemistry, a compound-bead library with hundredsof thousands to millions of diversities can be rapidly generated such that each bead displays only one chemicalentity. The highly efficient libraries-from-libraries approach involves the global transformation of a peptidelibrary into many small molecule solution-phase mixture libraries, but this approach has never beensuccessfully applied to OBOC libraries. Here we report a novel approach that allows us to combine thesetwo enabling technologies to efficiently generate OBOC encoded small molecule bead libraries. By usinga topologically segregated bilayer bead and a ladder-synthesis method, we can prepare peptide librarieswith the peptide on the bead surface and a series of peptide ladders in the bead interior. Various globaltransformation reactions can then be employed to transform the starting peptide library into a variety ofpeptidomimetic libraries. During the transformation reactions, the peptide ladders in the bead interior arealso transformed in a predictable manner. As a result, individual compound bead can be decoded by analyzingthe hydrogen fluoride-released encoding tags with matrix-assisted laser desorption ionization Fourier transformmass spectrometry. Using this novel approach, a random encoded dipeptide library was prepared andsubsequently transformed into polyamine and poly-N-acetylamine sublibraries. Random beads isolated fromthese sublibraries were reliably decoded.


    Combinatorial chemistry describes the rapid synthesis oflarge numbers of structurally similar molecules and, inconjunction with high-throughput screening strategies, offersa convenient way to identify ligands with high-bindingaffinities.17 In the one-bead-one-compound (OBOC)method,8 a split-mix synthetic approach810 is used toprepare thousands to millions of compounds on individualbeads, each of which contains a single chemical entity. On-bead screening assays against various biological targets ofinterest are then used to identify positive beads which canthen be removed for decoding. OBOC libraries have beenused to identify ligands for different biological targetsincluding protein kinase substrates and inhibitors,11,12 pro-tease substrates and inhibitors,1317 cell surface receptors,1823

    and artificial enzymes.24,25

    Structural determination of hit compounds from fullydeprotected, R-amino acid-containing peptide and peptoidOBOC libraries is straightforward by direct Edman microse-quencing which sequentially identifies the constituent aminoacid residues of the molecule. This technique restricts thecomposition of library compounds to R-amino acid buildingblocks, thus other decoding strategies are required for non-R-amino acid-containing peptides, branched peptides, pep-tidomimetics, and small molecules. For these nonsequence-able libraries, chemical encoding methods are often used in

    which a coding tag provides a record describing the synthetichistory of each bead. We have reported chemical encodingstrategies based on Edman microsequencing and massspectrometry (MS) for nonsequenceable libraries.2629

    In chemically encoded OBOC libraries, at least twocompounds are present on each bead, the coding tags andthe target (testing) compound. In biological screening assays,the interaction between receptors and resin-bound ligandsoccurs at the bead surface. To minimize interference fromthe coding tags, it is preferable to prepare beads with ahomogeneous outer layer containing only the testing com-pound and segregate the coding tags in the inner layers wherethe binding receptors do not penetrate. Thus, a key aspectof our encoding strategies is the use of topologicallysegregated bilayer beads. Two methods have been developedthat are routinely used in our laboratory for this purpose.The first of these methods is the partial amine-protection

    * To whom correspondence should be addressed. E-mail:

    Figure 1. PAP and PAD bilayer bead synthetic approaches.Reagents and conditions: (i) swell in water for 24 h; (ii) Fmoc-OSu (0.3 equiv), DIEA (0.6 equiv), DCMEt2O (55:45), 30 min;(iii) Alloc-OSu (3 equiv), DIPEA (6 equiv), 1 h; (iv) swell in waterfor 5 h; (v) Pd(PPh3)4 (0.24 equiv), PhSiH3 (20 equiv), DCM, 9min.

    J. Comb. Chem. 2008, 10, 333342 333

    10.1021/cc700165s CCC: $40.75 2008 American Chemical SocietyPublished on Web 01/09/2008

  • (PAP) bilayer approach.27 Using Tentagel resin, biphasicsolvent conditions are used to selectively protect the aminofunctionalities on the outer layer of the beads, while the innercore remains underivitized (Figure 1). Partial alloc-depro-tection (PAD) is another approach for making bilayerbeads.29 In this method, resin beads with alloc groupprotection are swollen in water and then treated withPd(Ph3)4/PhSiH3 for a certain amount of time, resulting indeprotection of the outer-layer amino groups (Figure 1).

    The ability to generate bilayer beads at any step in areaction sequence allows for the possibility of using aladder-synthesis encoding approach. Ladder synthesis usingthe PAD method has been used to encode OBOC libraries.29

    A cleavable linker (CL) is first inserted on the inner layerof the beads, and a multicomponent MS tag is assembledon the linker. Peptide synthesis with repetitive PAD cyclesis used to assemble the peptide and corresponding ladders.The ladders are chemically cleaved, and the mass differencebetween each of the ladders is used to decode the compoundstructure (Figure 2).

    A powerful technique for expanding the potential ofcombinatorial chemistry is the libraries-from-librariesmethod.30,31 This concept describes the transformation ofresin-bound amino acids, peptides, and peptidomimetics intolow molecular weight heterocyclic and acyclic compounds.Global transformation reactions (reduction, oxidation, alkyla-tion, and acylation) are performed on a parent library,modifying a particular functional group, to generate a

    sublibrary. This process can be repeated in turn so that aseries of diverse libraries can be rapidly synthesized fromthe parent library. Figure 3 illustrates these possibilitiesstarting from resin-bound tripeptide library. Exhaustivereduction of the peptide backbone yields a polyamine library,which treated with isocyanates or isothiocyanates generatesa polyurea (X ) O) or polythiourea (X ) S) library,respectively. Alternatively, peralkylation of tripeptide gener-ates a peptidomimetic library, which gives a polyaminesublibrary after reduction. In addition to these acyclicsublibraries, the libraries-from-libraries approach has beenused to prepare low molecular weight heterocycles.

    Methodology to access to peptidomimetic and smallmolecules is an important tool for drug discovery. Althoughpeptides can be routinely synthesized and have high bindingspecificity, they are limited as drug candidates because of alack of in vivo stability, susceptibility to proteolysis, andpoor bioavailability.3236 In contrast, compounds synthesizedwith the libraries-from-libraries approach have vastly dif-ferent physiochemical properties with respect to the originalpeptide library offering a means for discovering ligands morelikely suited for drug development. This paper describes ourefforts to combine the inherent power of the OBOC methodwith the libraries-from-libraries concept using chemicalencoding as a means for discovering high-affinity drug-likeligands.

    Results and Discussion

    MS-based chemical encoding strategies require an ad-ditional step, in which the coding molecules are removedfrom the solid support for analysis. A cleavable linkerfacilitates this process and is chosen or designed for aparticular synthesis according to the following criteria: itshould be (i) selectively cleavable, (ii) stable to the conditionsof the library synthesis, and (iii) capable of facilitating MSanalysis and peak identification. We based our cleavablelinker on the MBHA resin, and a literature search revealedN-tertbutoxy-carbonyl-aminomethyl(R-phenyl) phenoxy ace-tic acid,37 the Boc group version of compound 1, as an idealchoice.

    Linker 1 was synthesized based on literature precedents3840

    in a three-step procedure (Scheme 1). 4-Hydroxybenzophe-none was alkylated with R-bromoacetic acid and, afterreduction of the ketone intermediate, displacement with9-fluorenylmethylcarbamate (Fmoc-NH2) provided protectedlinker 1.

    Linker 1 was used in conjunction with our previouslydescribed multicomponent MS-coding tag,41 which is com-posed of the amino acids arginine, 4-bromophenylala-nine, and 2,2-ethylenedioxybis(ethylamine)monosuccinamide(Ebes).42 The rationalization for each of these componentsis (i) Arg provides a protonation site to help generate a strongMS signal, (ii) Phe(4-Br) generates a distinctive isotopic

    Figure 2. Mass spectrometry-based sequencing with the PAD synthesis method.

    Figure 3. Global transformations of resin-bound peptides togenerate peptidomimetic libraries from libraries. Reagents andconditions: (i) BH3THF; (ii) Li-tBuO, R-X; (iii) RNCX.

    Scheme 1. Synthesis of Cleavable Linker 1a

    a Reagents and conditions: (i) Br-CH2-CO2H, K2CO3, acetone, reflux,8 h; (ii) NaBH4, EtOHiPrOHH2O (2.5:2.5:1.0), 0 C, 1 h then 0 to 20C, 1 h; (iii) Fmoc-NH2, AcOH, TsOH, 20 C, 12 h.

    334 Journal of Combinatorial Chemistry, 2008 Vol. 10, No. 2 Kappel et al.

  • pattern allowing easy identification of relevant peaks, and(iii) Ebes is a hydrophilic spacer, which facilitates postcleavage extraction and provides additional mass so the peaksof interest will be pushed beyond the matrix region of thespectrum. The coding tag was assembled as illustrated inScheme 2. Linker 1 was coupled to Tentagel resin, andfollowing Fmoc deprotection, the coding tag elements werecoupled in sequential order to give resin 2. A single bead of2 was treated with hydrogen fluoride (HF) gas to release thecoding tag, which was easily detected by MALDI-TOF MS(Figure 4A). In anticipation of library synthesis, the codingtag was treated with diborane to generate polyamine resin3. Single-bead HF cleavage revealed the reduced coding tag(Figure 4B).

    After the establishment of a single-bead HF cleavageprotocol using linker 1, a series of model-encoded peptidesincorporating the above system was synthesized as a test ofour ability to observe peptide ladders cleaved from single

    Tentagel beads (Scheme 3). Resin 2 was acylated with Fmoc-Phe-OH and, after Fmoc deblocking, vigorously shaken inwater then reacted with 0.4 equiv of Fmoc-OSuc to generatethe bilayer resin 4 in which the inner-layer Phe wasunprotected. A series of Fmoc-protected amino acids werecoupled to resin 4, followed by Fmoc deblocking, to givethe dipeptide ladder resins 5a-q. A single bead from resins5a-q was treated with HF, and the crude cleavage productswere analyzed by MALDI-MS. The results are summarizedin Table 1. In each case, the expected masses of 7a-q wereeasily observed in the respective spectrums. Figure 5 showsa representative example, in which the Ala-Phe (7a) andPhe (6) ladders were cleaved from resin 5a.

    To preview the global transformation chemistry weplanned to use, resin 5a was chosen as model system andwas reacted under diborane reduction conditions to providepolyamine resin 8 (Scheme 4). Treatment of 8 with anisolethen HF to cleave the polyamine ladders 9 and 10 revealedonly a small signal for the Phe polyamine 9, in addition toan unknown impurity with a m/z of 698.5 (Figure 6A).Because an amine-resin linkage is more stable and thusharder to cleave than the corresponding amide linkage it wasproposed to extend the time of HF exposure of 7 to improvethe cleavage yield and MS signaling. However, the sameresult was obtained when extending the cleavage time to 7h.

    Alternatively, treatment of 8 with acetic anhydride andthen anisole/HF was attempted to provide poly-N-acylamines11 and 12. The MS spectrum shows both of the expectedproducts at 960.5 and 1059.6 m/z respectively (Figure 6B).Also present are two peaks (918.5 and 1017.6 m/z), whichare 42 mass units less than 11 and 12, respectively, indicatingeither incomplete acetylation or incomplete reduction.

    OBOC library 13 (Scheme 5) was prepared to test thecleavage/decoding method on a completely random library.The synthesis used the same procedure as that for 5a-q except

    Scheme 2. Synthesis and Reduction of the Mass Spectrometry Coding Tag on Tentagel Beadsa

    a Reagents and conditions: (i) 1, DIC, HOBt, DMF; (ii) 25% piperidine/DMF; (iii) Boc-Arg(Tos)-OH, DIC, HOBt, DMF; (iv) TFADCM (1:1), 30 min;(v) Boc-Phe(4-Br)-OH, DIC, HOBt, DMF; (vi) N-Fmoc-2,2-ethylenedioxy-bis(ethylamine) monosuccinamide, DIC, HOBt, DMF; (vii) B(OH)3, B(OCH3)3,1 M BH3THF, 65 C, 72 h; (viii) piperidine, 65 C, 16 h.

    Scheme 3. Synthesis and Cleavage of a Model ParallelDipeptide Librarya

    a Reagents and conditions: (i) Fmoc-Phe-OH, DIC, HOBt, DMF; (ii)25% piperidine-DMF; (iii) swell in H2O, 5 h; (iv) Fmoc-OSu (0.4 equiv),DIEA (0.8 equiv), DCMEt2O (55:45), 30 min; (v) Fmoc-Aa-OH, DIC,HOBt, DMF; (vi) anisole vapors then HF, 20 C, 2 h.

    MS Encoding of OBOC Libraries from Libraries Journal of Combinatorial Chemistry, 2008 Vol. 10, No. 2 335

  • both Aa1 and Aa2 were randomized with 19 proteinogenic aminoacids (Cys was not used) to give a dipeptide library with 361permutations. After the library was synthesized, 20 beads werepicked and subjected to the HF cleavage procedure. Followingextraction with ACNH2O (1:1; 20 L), the crude residuegenerated from each of the beads was analyzed by MALDI-TOF MS. Table 2 summarizes the results of the MS data. Eachof the beads, with one exception (Table 2, entry 1), was decodedunambiguously by matching the spectrum peaks with theexpected masses of the potential products.

    Sublibraries of 13 were prepared (Scheme 6) using thesame modifications used to make Ala-Phe dipeptide mi-metics 10 and 12. Exhaustive diborane reduction generatedsublibrary 14, which in turn was used to make the poly-N-acetylamine sublibrary 15. For each of the sublibraries, 20beads were chosen for cleavage/decoding.

    The MS data for the 20 polyamine, sublibrary 14 beadsare illustrated in Table 3. Nine of the beads were fullydecoded, and eight of the beads were nonsequenceable. Inaddition, three of the beads were partially decoded so thatone of the amino acids could be determined. Althoughnonideal, this reduces the potential pool of products from361 to 19 and thus makes resynthesis a practical possibility.As mentioned in regards to the model Ala-Phe-based

    polyamine model 7, difficulty in analyzing these beads maybe a result of the additional stability afforded to the aminoproduct-linker bond. To improve the reliability of decodingpolyamine beads, single bea...


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