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`!"i£7€3{3J
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`
`139.)
`;..!?f'J.'3<’2~§<i-‘_-3.‘)
`
`-
`
`May/June 1993
`
`The Journal of Peptide Application, Synthesis and Analysis. Vol. 6, N0. 3
`
`
`
`Page 1 of 12
`
`ILMN EXHIBIT 1039
`
`

`
`Peptide Research
`
`STAFF
`
`Francis W. Eaton, Jr.
`Publisher
`
`J .E. Gilchrist
`Associate Publisher
`
`J ama Ellingboe
`Scientific Editor
`Susan Starratt
`Advertising, Manager
`Esta Warshofsky
`Asst. Advertising Manager
`Thomas J. Stevens
`Assistant Sc ientilic Editor
`
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`Editorial Assistant
`Sandra Lamont
`Production Manager
`Karen Shulman
`Circulation Manager
`
`Copy:-ight'“’ I993 Eaton Publishing Co.
`Ptep1i‘r.‘r- Re.t'r'w':-l'r [ISSN 1{}4[i—S'.I'D4) is pub-
`lished six times a year by Eaton Publishing
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`
`CONTENTS
`
`Review
`
`Synthetic Peptides and Titmor Cell Metastasis
`G.B. Fields... . . . .
`
`Short Communications
`
`Synthesis of Arginine Aldehydes for
`the Preparation of Pse"d°P9P“d93
`G. Guicbard. J.P. Briand and M. Friedc
`. . . . . .
`.
`.
`. . . . . . .
`.
`.
`. . . . . . . .
`.
`.
`.
`. . . . . . _
`A Gentle Method for Linking Tris to Arnino Acids
`and Peptides
`R.G. Whittaker. P.J. Hayes and VJ. Bender
`. . .
`.
`.
`. . .
`
`,
`
`. 121
`
`Research Reports
`
`A. Kassarjian. V. Sehellenberger and C.W. Turck . . . .
`
`.
`
`.
`
`.
`
`.
`
`. . . . . .
`
`.
`
`.
`
`.
`
`.
`
`. . _
`
`129
`
`M.K. Luidens. C.S. Aks. Q —l. Zhu. T.F. Smith. R. MacColl and J. Figgc. . . . ..
`Pegglated Peptides I: Solid-Phase Synthesis of N”-Pegylatcd Peptides
`Using Fmoc Strategy
`. . . . .. 140
`. . . . . .
`.
`. . . . . .
`. . . . . . . . . . . .
`.
`.
`. . . . .
`.
`.
`Y.—A.Lu and A.M.Felix . . . .
`b u. UNCA
`A Comparative Synthesis of the [t>'I‘ip"] LH-RH Analo
`y
`e
`g
`Method in Solution and by Solid Phase
`. . . , .
`.
`. . . . . .
`.
`. .
`P.A. Swain. B.L. Anderson, M. Goodman and WD. Fuller . . . .
`Solid-Phase Synthesis and Biological Activity of the Parallel Dimer of
`Deamtno-Oxytocin
`. . . . . . . . . . . . . . . . . _
`.
`.
`.
`.
`M-C Mumiolt. M. Lebl. J. Slaninovai and G. Barany . .
`Peptide-Encoding for Structure Detennination of Nonsequeneeable
`P0l5'mers Within Libraries Synthesized and Tested on Solid-Phase
`Supports
`V. Nikolaiev, A. Stierandovai. V. K1'Cl"ll‘iiik. B. Seligmann. K.S. Lam. S.E. Salmon
`andM.Lehl . .
`.
`.
`. . . .
`. .
`.
`.
`.
`. . . .
`. .
`. . . . . .
`. .
`. .
`. . .
`. . .
`.
`. . . . . . . .
`. .
`. . . . ..t6t
`
`134
`
`, _ 147
`
`_ . [55
`
`_
`
`_
`
`_
`
`Calendar of Events ................... ... . ....................... .. 172
`
`Information for Authors
`
`Purpose and Scope
`Peptirtc Re.s'carc!i is an interdisciplittaryjoumal for rapid publication
`of articles describing original research on peptides in biochemistry,
`molecular biology. chemistry. cell biology. endocrinology. pharma-
`cology. immunology , neuroscience, septtralion scienceand newdevel-
`opments in the sy nthcsis, purification attdcharactcrizat ion ofpcptides.
`Article Formats
`
`Research Papers t4—6 typeset pages) focus on original research.
`techniques and applteations.The required format is that oftltestandard
`scientific paper: TI1l6.r‘\bSlr=lCl. Introduction. Materials and Methods.
`Results. Discussion and References.
`
`Short Communications t l -3 typeset pages} provide for papers shoner
`Ihan the usual Research Paper. Authors need not adhere strictly to
`the explicit fonnat oi‘ the Research Paper.
`Reviews (6-8 typeset pages) or Mini-Reviews (2 pages) are usually
`written by invitation. but unsolicited papers are welcomed.
`Peptide Product Forum (2-4 typeset pages) is intended for the
`description of applications of new research instruments or products.
`
`Vol. 6 No. 3 (I993)
`
`Papers are peer reviewed for interest. utility and scieittitig merit.
`Unsubstantiated claims are not pemtntcd.
`
`Review, Acceptance and Publication
`A rrtanuscripl
`is submitted with the understanding that it. or the
`essence of its content. has not been published and is not being
`considered for publication elsewhere. All authors are presumed to
`have had significant roles in the reported research. have agreed to
`be listed as authors and have reviewed and approved the paper. It is
`also understood that responsibility for the oontent ol‘ the paper is
`shared by all authors in accordance with their roles in carrying out
`the research and preparing the manuscript.
`
`Papers will be reviewed promptly and anony mottsly by qualified
`independent referees. All papers are privileged communications that
`are not to be released to the press or public during the review process.
`Pttblictttion should he expected within foormonths ollinal approval.
`Detailed instructions for authors can be found in the Jan/Feb isstte or
`can be obtained front the Scientific Editor, Peptide Research. I54 E.
`Central St.. Natick. MAUI 7'6ll(Tcl.‘. 503—o55—s2s2; Fax: 5()8w655-99 I 0).
`
`Pcotidc Rose‘
`
`'
`
`
`
`Page 2 of 12
`
`

`
`Peptide Research
`
`Peptide-Encoding for Structure
`Determination of Nonsequence-
`able Polymers Within Libraries
`Synthesized and Tested on
`Solid-Phase Supports
`
`mixtures (rt : number of residues in the
`sequence) with another position de-
`fined in each group, serial approaches
`cart be readily applied to the generation
`and screening of libraries of norrpep—
`tide or nonsequcnccablc compounds
`without modified methods
`for
`se-
`
`quence determination.
`The parallel approach, exenrplified
`either by phage display screening (20,
`for review see 22) or by the Sclcctidc
`technology (15), is based on sirnultarte-
`ous screening of millions of trniquc
`structures (prepared by simple modifi-
`cation (5.15) of the solid-phase peptide
`synthesis principle (18)) separ'atcd in
`space but contained in the same reac-
`tion vessel. After identifying and iso-
`lating the active particle, it is necessary
`to deterrnine the structur'e of the mo]-
`
`ecule responsible for the given activity.
`The application of nnnpcptide struc-
`tures for library building is possible
`using the Selcctide technology. but is
`limited by the ability to determine the
`structure of the active molecule, which
`is usually available in a limited amount
`(the amount present in one particle of
`the carrier).
`The rationale for the construction
`and screening of norrpcptide libraries is
`obvious. The goal of most drug-discov-
`cry programs is the development of a
`specific ligand for a macromolecular
`target. The ability to mimic peptide-
`protein interaction with nonpeptidc
`structures was demortsttated by several
`authors (7,8,l9,25~27). Therefore,
`it
`would be desirable to construct and
`screen libraries based on building
`blocks capable of all major types of in-
`teractions (ionic, hydrogen bonding,
`hydrophobic, charge—transfer, chclation.
`aromatic, etc.). and assuming a wide
`variety of tertiary structures,
`in addi-
`tion to those created by peptide bonds.
`Coding for the structure of an active
`molcctrle is not itself a novel concept.
`The phage-display technique uses the
`nucleic acids of the phage to code for
`the sequence of the interacting peptide.
`Sequencing of nucleic acids attached
`
`INTRODUCTION
`
`identifying new
`The process of
`structures that interact with pharmaco-
`logically
`important macromolecular
`targets has been significantly acceler-
`ated by the introduction of so-called
`“library techniques" (4,6.9,l5,20; for re-
`views see 10,22,241). These approaches
`are designed to generate and readily
`screen a multiplicity of structures that
`interact with a given acceptor (recep-
`tor, enzyme, antibody). There are in
`principle two approaches to scrcenirtg
`the libraries: serial and parallei. In the
`serial approach [Geyscn ct at. (6) and
`Houghten er al. (9)]. a multiplicity of
`setrti-random libraries is generated and
`tested. These libraries consist of groups
`of peptide mixtures with one or two
`defined positions. Tire most active of
`these groups in a given hioassay is se-
`lected for an iterative process of syn-
`thesis and screening, during which all
`of the positions of the active sequence
`are successively defined. Sincc step-
`wise sequence—rcvealing is inherent to
`this approach. as weli as to the concept
`of positional scanning libraries (23).
`which consists of it groups of peptide
`
`V. Nikolaiev, A. Stierandova,
`V. Krcltnak, B. Seiigmann,
`K.S. Lam‘, S.E. Salmon‘
`and M. Lebl
`
`Selecride Corporation and
`‘Arizona Cancer Center
`
`ABS'l'RAC'l‘
`
`A rrreritod of irrdirccriy (iererrrrirrirrg the
`.s'!rt£C.t‘IU’c' of nortpepririic or rr0itseqrrerrce-
`able crJtnprJrrrtrIs firm‘
`itrtve been syrm’rc-
`sized rm irtdit't'rt'trc.=i pm‘ticies of .t'(2iirr' sup-
`port
`is de.t‘crii)ed. The recittriqrre portraits
`rite pcrrrrilel .-.'ynr:'te.tr'.r of ct corrrpotrrrrt rim!
`is not st.-sccpribie to Edrrrcm rIegr'rtdarr'mr
`f('.g., N-terrrrirttti-biricircri peptide). or‘ one
`rmrrtaitritrg r'orrrp(:rrern's
`ritrrr
`crnttrot‘ be
`tclerrrified by wrrirro crcici seqtmrciirg.
`ro-
`gerlrer with (I corre.\'por:a'irr.g "codirrg" pep-
`tide. Each corrplirrg .i‘fep in the rr.rsemb1'y of
`tire
`rrorrseqrrertceablr:
`cornporrrrd is foi-
`iotveri by the c'r)rrpt'irrg 0_,t'rtrr_rtmirrrJ rrciri to
`ct
`rt'r',I_',icr'enr crrrrrchmeirt site (attire saute car'-
`rfer prrrricie. whereby the runiiro (tciri rur-
`ctirrbigtrotrfly ct.-de.r'.fi)r tire pr'evi()trs!'y corr-
`pl'ed‘t')rri!'dirrg bfrick of tire n0n.icqrrerrr:e—
`able coarporurrt’. The rarinrrrric is to enable
`the seqtterrce derenrrizrrrtiarr of rt birrhogicrriiy
`active cotr1porrrrr." rim! iras beran irierrrrjfic-cl
`through the .screerrr'rrg of ar r’r’bn-try of non-
`egrrrenc'errt')r’e
`t'rmt_rJorrrrr1.t‘.
`by fr'ar-tsirrriirg
`the .\‘er_rtrerIce of its "crJriitr_r.;" _rJepriri'e‘, deter-
`mirrert‘ by Edrrrrrrr
`riegrztrirtrirrrt,
`into the
`smrcrrrrr: of the rrc.'ive crmrprrrurri. The
`redrttique frrciiirrrrex the cortsrr-rtcrjpri and
`screettirtg of rrorrpepridic lr'i2mrr'e.v for the
`tii.§'c.‘0ver‘y qf
`r'mpr.u'r(rnr pirarmcrcctrricai
`r.-rmrpotrrrc.-'5.
`
`/'tbi;r'evirrrioir.t': The oI1e—letteI' notatiott of amino acid I‘esitit.Ie$ is used in this manuscript for
`description of arnirrn acid residues and peptide sequences. Dcrivati vcs of amino acids are represented
`by thI'ec-letter codes. Other ztbbrevialions used: Boc, i('!'t‘-lI)11l5«’l0X.}'Ci1Ii)(ltl}-'l; BOP, bcn‘.r.otri:JI.olyl—
`oxy-trisdinretlrylarrrino—phospltoniurn lrexrti"|uor’opl1osplratc; Cl-IA. cyclohcxylarnirre: DC}-IA. tiicy—
`clohexylamtnc: DCM. dichloromcthanc; Dd:r.. 3,5—r.iitnetlroxy—dimethylbcnzylmtycarbtirtyl; DIC,
`I-|iiS0[3I'UPy|CaI'b0diilttit-lc: DIFA. diisopropyletlrylaminc; DMF, dinrethylforrnatnide; DMPU,
`|,3—
`dimcthyl-3.-4,5,6-tctmiIydr0—2(IH)-pyrimidinonc tdirrrclhyiptopyIerrctarea); Fmoc, [luorcny1mcllr-
`oxycarbonyl; HOBI. N-hydrrrxyhcnzotriazole: HPLC. high pressure liquid cltI'ornatugrap|ry'.
`PyBrOP. bronto—tt'is-pyrroiidirtophosplrnrriurn hertafluoroplrosphate: SCAL. satiety-catch amide
`linker; TFA. triliuomacctic acid; Tfa. triI‘luoroacelyl.
`
`eljenngt
`This material was co ied
`
`
`
`Page 3 of 12
`
`

`
`directly to peptide molecules was sug-
`gested by Brenner and Lerner (2) for
`determination of a structure of a pep-
`tide in a library. llowcver, successful
`¢o-synthesis of nucleic acids and pep-
`tides requires novel chemistry which
`has not yet been described. [The use of
`peptides to code for rtonnatural amino
`acid-containing peptides in peptide
`libraries in solution was published (1 1)
`while this manuscript was in prepara-
`tion.] Methods available for protein se-
`quencing require
`only
`femtomolar
`quantities and are therefore well suited
`for the technology employing screen-
`ing and sequencing of pure peptides
`synthesized on the polythene beads
`(15). There are no other types of poly-
`meric molecules besides DNA and
`peptides for which techniques of struc-
`ture determination have been well de-
`veloped. Biophysical
`techniques of
`molecular structure determination are
`significantly less sensitive (MS) andlor
`Eess powerful (NMR, IR, x-ray). and
`typically much slower and more labor
`intensive.
`
`MATERIALS AND METHODS
`
`Instrumentation
`
`Fast atom bombardment (FAB) mass
`spectroscopic measurements were car-
`ried out on a ZAB EQ spectrometer
`(VG Analytical Ltd, Manchester, UK).
`‘H NMR spectra were obtained on a
`General Electric QE 300 instniment
`(Fullerton, CA). Sequencing by Edman
`degradation was perfonned on an AB[
`4778 protein sequencer (Applied Bio-
`sy stems. Foster City, CA) and a Porton
`P1 3010 instrument
`(Porton Instru-
`ments, Tareana, CA). Both analytical
`and preparative HPLC were carried out
`on a Millipore 625 LC system (Bed-
`forri, MA) with -.1 Millipore 490E Pro-
`grammable Multiwavclength Detector
`using Vydae Peptide and Protein C18
`analytical
`(4.6 X 250 tom, 5 pm,
`1
`ml/min) and preparative (I0 X 250 mm,
`10 ton, 3 mllmin) columns (The Sepa-
`rations Group, Hesperia, CA}. respec-
`tively. Analyses of mixtures released
`from one bead were performed on an
`Ultrttfttst Microprotein
`Analyzer
`(Michrom l3i0Resources, Pleasanton,
`CA) usinia Reliasil C18 column (5
`pm, 300
`,
`1 X 150 mm). All spectra
`are reported in ppm relative to tetra-
`methylsiiane (6) using either CDCI3 or
`_/G2
`
`CD3SOCD3 as solvents. UV/V IS ab-
`sorption spectra were recorded on a
`Hewlett Packard HP 8452A Diode-
`
`Array spectrophotometer (Palo Alto,
`CA) using a |—cm quartz cuvette. Amino
`acid analyses were carried out on a D-
`500 system (Durrum, Palo Alto, CA) .
`
`General Procedures
`
`synthesis was per-
`Solid—phase
`formed manually in polypropylene
`syringes as described by Krcltriak and
`Vagner
`(13). Syntheses were per-
`formed on TentaGel (TG) resin (Rapp
`Polymere, Tubingen, Germany) (130
`or 80 pm, 0.23 mmol/g) modified with
`SCAL handle (21) (safety—catch amide
`linker) or with all appropriate linker.
`F'moc—protecting groups were cleaved
`with 50% piperidineJDMF for l X 10
`min. Boc groups were cleaved for 20
`min with 30% TFAIDCM containing
`3% anisole. Ddz groups were cleaved
`for 30 min with 2% TFAIDCM. After
`
`Boc cleavage, a solution of DIEAI
`DCM (10%) was used for neutraliza-
`tion. A mixture of BOP? HOBHDIEA
`
`(1: l :2 eq) in DMF was used for the ac-
`tivation of both Nt1—Fmoc and Boc
`
`amino acids. The completeness of each
`condensation reaction (15-40 h) was
`checked by the ninhydrin test or by the
`chlora nil test in the cases of coupling to
`secondary amino groups. The coupling
`protocol included washing with DMF
`(6-8 times) [followed by washing with
`DCM in the case of Boc—protected
`amino acids] between coupling and de-
`protection, and between deprotection
`and coupling. The SCAL linker was re-
`duced by 20% (EtO)2P(S)SH in DMPU
`for 2 h. Final cleavage was done by
`95% TFA-5% water mixture.
`
`Materials
`
`Commercial-grade solvents were
`used without further purification. Pro-
`tected amino acids were obtained from
`
`Bachem (Torrance, CA), Advanced
`ChemTech (Louisville, KY) or Propep—
`tide (Vert-le-Petit, France).
`
`Models of a Peptide Library
`Encoded by a Peptide Sequence
`
`Boc-Lys(Fmoc)—0H was coupled as
`the first amino acid to SCAL-TG. the
`NE-Fmoc group was deprotccted and
`Fmoc-Lys(Fmoc)-OH was coupled to
`the side chain of the lirst lysine. The
`
`Nu- and NE-Fmoc groups of lysine
`were cleaved and the resin was divided
`into three parts. Frnoc-Ala-OH. Fmoc-
`Phc—OH and Fmoc-Val-Oll,
`respec-
`tively, were each coupled to one por-
`tion of the resin. The corresponding
`Boc amino acids (Gly, Tyr and Lea —
`Boc—Tyr—0H was used with the unpro-
`tected hydroxyl group) were coupled in
`the next step to the ot—amino group of
`lysine after Boc deprotection, while the
`“Fmoe branch" remained protecte-,c1_
`After completion of Boc amino acid
`condensations, all three portions of the
`resin were combined, and the “Fmoc
`branch“ was deprotected. The follow-
`ing randomization was performed ex-
`actly the same way as the first one after
`the splitting of the resin into three
`equal portions. After randomization of
`three positions (coupling of three dif-
`ferent amino acids in each position),
`the resin was divided into smaller pans
`and treated differently.
`
`I. Cleavage of both N—ferrm'm;[
`FrJ10c— om.’ Boc-prorecfing gmrrps
`
`Two completely deprotectcd beads
`were sepzuately submitted for sequence
`analyses. Correct “cotnplementary"
`amino acids were found in all three
`cycles in the expected ratio 2:1. Results
`(values in pmol): Isr bead: lst cycle: V
`251, L 146; 2nd cycle: V 244, L 147;
`3rd cycle: V 245. L ll9. 2nd bead.‘ lst
`cycle: A l02, G 39; 2nd cycle: V 121,
`L 59: 3rd cycle: F 125, Y 50.
`Part of the resin (about 100 mg) was
`treated with 20% diethyldithiop|tos-
`phate in DMPU (2 X 1 h shaking) to re-
`duce the SCAL handle. The mixture of
`peptides was cleaved from the reduced
`SCAL with TFA/H20 (‘)5:5) for I h,
`The cleavage mixture was concentrated
`in vacno and precipitated with Et2O_
`The precipitate was collected by cen-
`trifugation and dried. The mixture of
`peptides was
`dissolved
`in
`0.1%
`Tl’-‘Ari-I20 and analyzed by HPLC_
`First, a faster gradient 0%-100% of
`acetonitrile and 0.1% TFA in 100 min
`was run for orientation. A slow gradi-
`ent 0%—50% of acetonitrile and 0.1%
`TFA over 200 min revealed that the ex-
`
`pected 27 peaks were present. Several
`additional minor peaks were identified,
`the formation of which was attributed
`to the use of side—chain unprotected
`tyrosine during the synthesis. Because
`of possible losses of some hydrophobic
`sequences during the ether precipita-
`tion,
`the
`second cleavage of
`the
`
`
`
`Page 4 of 12
`
`

`
`
`
`mixture avoided this step. The cleav-
`age mixture of TFA and water was di-
`luted by additional water, concentrated
`on an evacuated centrifuge and lyopliil—
`ized. HPLC evaluation of the mixture
`demonstrated
`an
`equimolar
`repre-
`sentation of all expected peaks.
`
`2. Deprorecrimi of the N-rcrnifiirrl
`Frrinr: group and rrcerylrrtirui of the
`“Fmoc f)l"(H".lCfl ”
`
`The free N-terminal amino groups
`were acetylated with a 0.3 M solution
`of N—acetylimidazole in DMF for 20
`min (ninhydrin test negative). N-termj-
`rial Boc groups on the other branch
`were deprotected after
`acetylation.
`Three randomly chosen beads were so-
`quenced and provided the following
`readings (values in pmol): lst cycle: Y
`213 (bead 1), G 161 (bead 2), Y 201
`(bead 3); 2nd cycle: L 165 (1), Y 166
`(2), Y 205 (3); 3rd cycle: Y I88 (I), L
`128 (2). G 162 (3). The readings were
`not contaminated by the amino acids
`present in the acetylated arm.
`A part of
`the aeetylated beads
`(about 100 mg) was treated the same
`way as described above (precipitation
`of the mixture by ethyl ether and/or
`evaporation of cleavage mixture and
`lyophilization)
`to reduce the handle
`and cleave the acetyiated peptides.
`HPLC analysis under the same condi-
`tions has shown that during ether pre-
`cipitation a significant proportion of
`the library was lost due to its solubility
`in ether. Evaporated and lyophilized
`sample provided the same number of
`peaks ol'approximate1y the same pat-
`tem as in the case of unprotected li-
`brary, with retention times shifted to
`higher values.
`
`3. Repl'rtceni.eirt‘ of the Boc-prot‘ccr-
`fttg grrmp by the Tfrr group
`
`The trifluoroacetyl group was intro-
`duced as at protecting group instead of
`the Boc group at the N-terminiis in or-
`der to pcrlnit stepwise sequencing ex-
`periments. First.
`the N-terminal Boc
`group was cleaved from a resin sample
`(50 mg) while the “Prime branch” was
`left protected. The free amino groups
`were protected with lrifluoroaeetyl by
`treatrnerlt with 10 equivalents (0.14
`mmol, '31 ill) of tiilluroacetic acid an-
`hydridc in dichloromethane (0.5 ml) in
`the presence of D11-EA (0.16 mmol, 23
`],l,l). The reaction was complete after 20
`min (ninhydrin test negative). After the
`trifluoroacetylation, the Fmoc group on
`
`the other brancl
`;
`and
`three beads weri: Vfgr
`se-
`moc
`qucncing. The sequence of the 1::
`- d
`branch was determined sup ort
`for the Seqllencing Was lremorved fbe
`the sequencer, and thg support bcatlflfm
`sembly was treated with a 0 2 M
`mm (if NEIQH (24 h- 200C). dried and
`submitted for an additional three cycles
`of sequencing The appmprime Se_
`4
`.
`.,
`.
`0
`quences predicted from the sequeneinc
`Eiffhe rm“ ifhfllch were obtained. 15:‘
`mid (values in pino1): 1. F (735), 2. V
`(6431 3- A (337); after TFA removal:
`I. Y (207), 2. L (187). 3. G (76), se-
`quence FVAIYLG; 2nd bend:
`1. A
`(2 '53» 2- A (230). 3. F (I93); after TPA
`removal: I. G (88), 2, C, (35), 3_ Y (80),
`Sequence AAF/GGY; 3rd bcrid:
`1. F
`(63): 2- F (57), 3. V (41); after TFA re.
`moval: I. Y (15), 2_ Y (12), 3_ L (4% Sc_
`quence FFWYYL.
`
`4' Clm"’a!€(5
`berrrf
`
`(if 1! pepflde front one
`
`Several beads of the resin coiitain-
`_
`1'13 WHY deprotected sequences on the
`rctluecd SCAL handle (set: point 1)
`“(Cm Plflccd Separately into small glass
`vials andgtreated overnight with 30 pl
`of neat TFA. Aliquots (3 til) were with-
`drawn and diluted with H30 to the (cm
`Volume of 20 ill and analyzed by HPLC
`on a mrcrobore 1-IPLC (Michrom appa-
`m_”’3)_ (gmdlflnl; 5%—60% of acetoni-
`tn_le in 0.1% TFA in water over 20
`min). C_alcul-ation based on the average
`extinction coefficient of peptides; at
`215 nm have shown that about 100.
`200 1311301 of peptide was released from
`one polymeric bead.
`'
`
`i\‘l0n-peptide Screening Libraries
`Isncoded by a Peptide Structure
`
`‘ Couplings of amino acids were per-
`formed by a manual method using
`standard protocol at room temperature:
`protected amino acid (3 eq) in DMF
`was mixed with DIC (3 eq) (or DIC and
`H03! I3 eq each] and the resin). and
`coupling was followed by analytical
`tests. Symmetric anhydiides were used
`where specified.
`linker and Boc-
`Fmoc-SCA1.
`Lys(Fmoc) were coupled first
`to the
`resin (TentaGel S NH2, 1 g) using DIC
`and HOBL After cleavage of the Frnoc
`group, Fmoc-Trp was coupled and
`Fmoc deprotected. The peptide—resin
`was divided into three equal portions,
`and three different bromoacids (one in
`
`each reaction vessel, 3 eq each) were
`coupled using DIC in DMF (3 eq). The
`three acids were bromoacetic, ot-b1'0-
`movaleric and ot—bromo-p—toluic acid-
`The coupling of the last acid was re-
`peated because of its low reactivity, US’
`ing a 6-fold excess of both acid and
`DIC. Boc protection of the ot—a1riin0
`group of Lys was removed by TFA.
`and the first coding sequence Boc-pro-
`tected amino acids (Gly, Ala. Lctl)
`were coupled by DIC. Coding amino
`acids were chosen according to the
`molecular weight of the nonpcptidc
`building blocks, so that
`the lightest
`block (bromoacetic acid, in this case)
`was coded by Gly;
`the heaviest one
`(bromotoluic acid) was coded by bill;
`and medium-weight one (brotnovalcric
`acid) by Ala.
`The three resin parts were pooled
`together, washed thoroughly with DCM
`and deprotected by TFAIDCM in
`preparation for the coupling ol' the next
`coding amino acids. After deprotec—
`tion,
`the resin was divided again into
`three portions. Couplings of Boc-pro-
`tected amino acids (again Gly, Ala and
`Lea) were performed as usual by
`means of DIC. Two parts of the resin
`were treated with 2—M solutions of
`amines (benzylamine and l-amino—4-
`methylpiperazine) in DMF overnight.
`The third part was treated with a 2-M
`solution of tluorenylmethyloxycarbonyl-
`2-aniinoethanethiol, and after comple-
`tion of the reaction the Fmoc group was
`removed. Coding of amines was based
`again on their molecular weights.
`The resin was pooled together once
`more, mixed and divided into three
`parts for the final couplings. Carboxylic
`acids (cycloliexylacetic acid, phenyloxy—
`acetic acid and 4—pyridylthioacetic
`acid) were coupled to amines obtained
`(primary and secondary) by DIC, and
`the coupling reactions were repeated
`twice using pre—formed symmetrical
`anhydridcs in 3-fold to 5-fold excess.
`After obtaining a negative chloranil
`test.
`the three batches of resin were
`treated separately by TFA, neutralized,
`and the
`last coding Boc—protected
`amino acids were coupled using DIC
`and I-1OBt. Coding of the last carbnx—
`ylic acids was based on the satire
`scheme as before. Finally, all the resin
`was pooled together.
`Two “unnatural” libraries have been
`completed using this general approach.
`The only difference between those two
`is the location of the SCAL linker. In
`
`".._‘X/I3
`
`Page 5 of12
`
`
`
`Page 5 of 12
`
`

`
`the first library, the SCAL linker is
`attached to the NE of Lys attached
`directly to the resin, and therefore Tip
`amide was the last amino acid in all the
`compounds of this library. The coding
`peptides remained on the resin beads
`after cleavage. The synthesis of the
`second library started with attaching
`the SCAL linker to the resin, and the
`last amino acid in all the compounds
`was Lys. All the compounds released
`from this library included also their
`coding sequence peptides.
`Beads from the first library (exclu-
`sive, A) were treated with the reducing
`agent and individual beads were picked
`up for separate cleavage and sequence
`analyses. Five beads were studied.
`After cleavage of the nonpeptide pan,
`the beads were successfully sequenced
`(see Table 1) and the strticture of the
`nonpeptide compound could be de-
`duced.
`Solutions
`containing
`the
`cleaved compounds were analyzed on a
`micro—HPLC system.
`A sample (800 mg) of the second
`library (inclusive, B) was treated with
`95% TFA after reduction of the SCAI.
`linker, freeze-dried, dissolved in water,
`and separated on a semi-preparative
`HPLC column yielding 44 peaks using
`a gradient of 0'?/w60% acctonitrile in
`0.1% TFA in water over 200 min. (The
`higher—than—predicted number of peaks
`observed in the HPLC trace can be ex-
`
`plained by synthetic problems experi-
`enced in the solid-phase nonpeptide
`couplings. This conclusion is supported
`by the analysis of products obtained in
`the synthesis of individual components
`of this model
`libraiy.) The fractions
`representing the different peaks were
`lyophilized, and several peaks were
`analyzed by FAB MS and sequencing
`to show the correspondence between
`the structure predicted from the amino
`acid coding sequence and the molecu-
`lar weight of the construct. Examples
`of the results: Pccik 4: RT 25.31 min,
`sequencing: 1. Leu (364 pmol), 2. Gly
`(139), 3. Gly (422); FAB MS - 827.0
`(building
`block combination
`169);
`Peak 8: RT 28.69 min, sequencing:
`1. Gly (261), 2. Leu (176), 3. Ala (225);
`FAB MS - 770.2 (building block com-
`bination 257 we block 7); Peak 13:
`RT 31.47 min,
`sequencing:
`1. Leu
`(792), 2. Leu (551), 3. Gly (128); FAB
`MS - 921.0 (building block combina-
`tion l59); Peak 14: RT 32.27 min,
`sequencing: 1. Leu (7930), 2. Gly (1810),
`3. Ala (1763); FAB MS — 883.0 (build-
`=:/A 4}
`
`Table 1. Structures Contained on Randomly Selected Be-ads from a Library of Nonpcptide
`Structures
`
`Bead Building Block M.W.
`No.
`Combination
`(mIz}
`
`Amino Acid Detected (pmol)
`
`1st cycle
`
`2nd cycle
`
`3rd cycle
`
`ing block combination 269); Peak 15:
`RT 32.77 min, sequencing: 1. Leu (784),
`2. Ala (447), 3. Ala (360); FAB MS —
`776.2 (building block combination 249
`w/o block 9); Peak 1'6: RT 33.16 min,
`sequencing: 1. Leu (1286), 2. Ala (918),
`3. Ala (688); FAB MS — 776.2 (building
`block combination 249 w/o block 9);
`Peak 17: RT 33.51 min, sequencing:
`1. Leu (298), 2. Leu (280), 3. Ala (202);
`FAB MS - 826.2 (building block coin-
`bination 259 we block 9); Peak 19:
`RT 34.80 min, sequencing: 1. Leu (641).
`2. Gly (412), 3. Ala (460); FAB MS -
`883.1 (building block combination 269);
`Peak 20: RT 36.66 min, sequencing:
`1. Leu (150), 2. Leu (119), 3. Leu (80);
`FAB MS - 902.2 (building block com-
`bination 359 we block 9); Peak 26:
`RT 41.77 min, sequencing: 1. Gly (39),
`2. Gly (38), 3. Gly (23); FAB MS - 744.1
`(building block combination
`167);
`Peak 3]: RT 48.86 min, sequencing:
`1. Ala (180), 2. Gly (98), 3. Ala ([06);
`FAB MS - 824.0 (building block com-
`bination 268); Park 32: RT 49.46 min,
`sequencing: 1. Leu (234), 2. Leu (320),
`3. Ala (277); FAB MS - 826.1 (building
`block combination 259 we block 9);
`Peak 33: RT 5070 min, sequencing:
`1. Gly (152), 2. Gly (120), 3. Ala (94);
`FAB MS — 800.1 (building block com-
`’ bination 267).
`
`Synthesis of Representative
`Compounds from the Nonpeptide
`Library
`
`A component of the exclusive
`library A, compound 1, was synthesized
`on 0.23 g of Knorr
`resin (Bachem,
`Biibendorf, Switzerland) (0.5 meqlg).
`Fmoc—Tip was coupled first according
`to the general protocol, using DIC and
`HOBI. After deprotection of the amino
`group, ct-bromoacetie acid (50 mg)
`was cotiplcd using DIC (50i1l) in DMF
`
`(0.5 ml). Benzylamine (100 ill) was
`dissolved in 0.5 ml of DMSO and the
`bromoresin was treated with this solu-
`tion ovemight. The final carboxylic
`acid, 4-pyridylthioacetic acid (80 mg),
`was dissolved in 0.85 ml of DMPU and
`preactivated with DIC (80 (Ll) and
`HOBt (80 mg), and coupled to the airti-
`noresin for 10 h. The coupling was re-
`peated using PyBrOl’ and DIEA for ac-
`tivation. Compound ] was cleaved by
`95% TFA. After cleavage, TFA was
`evaporated in vcicuo and the residue
`was dissolved in 30% aqueous acetoni-
`trile, and lyophilizcd. The product ob-
`tained afteI' drying was redissolved in
`neat acetonitrile and precipitated by
`ether. This operation was repeated twice
`and an almost white precipitate was ob-
`tained. The product showed two peaks
`on RP HPLC from which the second
`one gave correct n'iass—spectrum. Yield
`after purification on semi-preparative
`RP HPLC —-
`18 mg. Formula:
`C27H2"iN5O3S; MS expected 4 501.6;
`MS found — 502.2 (M+H)+. ‘H l\_lMR
`data (DMSO-d6): 10.804 d (1H, N'"H);
`8.49 d (2H, pyridyl C2H and CGH); 8.35
`d (1H, NH); 7.62 Ll (2H, pyridyl C3H
`and C511); 6.9-7.7 mm (B21 and Tip
`aromatic protons); 4.59 m (1H, Tip
`C°‘H); 3.75-4.65 m (aliphatic pfiotons);
`3.19 dd and 2.91 dd (2H, Trp C H).
`The second component ofthe exclu-
`sive library, compound 11, was synthe-
`sized according to the same scheme as
`compound I, using ot-bicrnovaleric acid
`(40 til), 4-methyl-1-aminopiperazin
`(100 pl) and cyclohexylacetic acid (80
`mg)
`as building blocks. Formula:
`C29H44N5O3', MS expected —-— 524.7;
`MS found 2 525.3 (M+H)+, 558.2
`(M+Na)+ and 573.2 (M+i<)*.
`The third component of the exclu-
`sive library, compound III, was synthe-
`sized according to a scheme similar to
`compound I, using ot-bromo-p-toluic
`
`
`
`Page 6 of 12
`
`

`
`
`
`H-[3Ala—Gly-[3Ala—G]y—TG; 2. Fmoc
`cleavage;
`3. Coupling of
`Frnoc—
`Lys(Fmoc); 4. B00 cleavage; 5. The
`resin was divided into 9 parts and the
`following Drl7.—protected amino acids
`were coupled: A,D,l,K,M,N,S,T,V; 6.
`Fmoc cleavage; 7. Coupling of nine
`Fmoc-protected amino acids: Y,G,F,l,,
`I-l,P,Q,R,E (Y was coupled to that part
`of resin that had already attached A,
`etc.)', 8. Resin combined and Dd:
`cleaved; 9. Repeat steps 5-7; 10. Fmoc
`cleavage; ll. Coupling of nine Fmoc-
`protected amino acids: Y,G,F,L,H,P,
`Q,R,E;
`l2. Repeat steps
`l0—l I: 13.
`Fmoc cleavage; 14. Side-chain protect-
`ing groups and Ddz removed by mix-
`ture K ( I 2).
`One head was submitted to four cy-
`cles of Edman degradation: Is: r-ya-lg:
`Arg (64), He (67); 2nd cycle: Gly (45),
`Tl1r' (14); 3m’ cycle: Phe (42); 4th cycle:
`Arg (35).
`Ile was coupled Dd?.-pto-
`tected and found in the first cycle. It
`coded for Phe, which was detected in
`the third cycle. In the second cycle Thr
`was detected as the amino acid that had
`
`been coupled Ddz protected. Arg, coded
`by Thr, was accordingly found in the
`fourth cycle of sequencing.
`
`Screening Protocol of the Library
`
`The peptide library was screened
`according to the published procedure
`(14). The peptide beads were first
`mixed with incrementally increasing
`double-distilled water to remove the
`DMF. After extensive washing with
`PBS (137 mM NaCl, 2.7 rnM KC], 4.3
`mM Na2l'lPO4. 1.4 rnM KH2P041 PH
`7.2), the beads were coated with 0.05%
`gelatin (wt/vol) to block any nonspe-
`cific binding. The beads were then
`incubated with a l:l000O0 dilution of
`streplavidin—alkaline phosphatase at 2
`rngfml
`(Pierce, Rockford,
`IL)
`in 2X
`PBSi'Tweenlgclalin (2>< PBS, {J.l%
`Tween—20 (volfvol) and 0.05% gelatin
`(wt/vol). The beads were then thor-
`oughly washed with TBS (137 mM
`NaCl, 2.7 1nM KC], 25 mM Tris base,
`pH 7.4) and the standard substrate 5—
`brorno—4—ehioro—3—irtdoly1
`phosphate
`was added. The beads. together with
`the substrate, were then transferred to
`petri dishes for color development.
`After 30 min to l h, the colored beads
`were collected with the aid of at rnicro-
`
`piper, washed with 6 M guanidine
`hydrochloride, pH 1.0, and subjected to
`sequencing as described (14,15).
`
`J-dcé;
`
`acid (120 mg), fluorcny|1ncthyl0xycar—
`bonyl—2—aminoethanethiol (280 mg)
`(deprotection after coupling with
`piperitlinfDMF) and phenoxyacetic
`acid (80 mg) as building blocks. For-
`mula: C29H3tJN404S. MS expected —-
`530.6; MS found — 553.0 (M+Na)*.
`
`Model Library Synthesis:
`
`Lr'brcr.r'y.' XXXX—L_t>s{XXXX)-L)tr(ZZJ-
`BAla-Gly—[5Ala-Gly-TG
`
`The library was synthesized accord-
`ing to the follow