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`ILMN EXHIBIT 1018
`
`
`
`Page 1 of 10
`
`ILMN EXHIBIT 1018
`
`

`
`l_lllCl ll(1Lll}ll(1lJ[)lll ucu [)1
`
`.
`
`'
`
`t
`e 1 e
`Protein
`Research
`
`0
`
`Head afliw:
`MUNKSGAARD International Publishers Ltd.
`35 Norre Sogade
`Postbox 2148
`
`or
`
`DK—l0l6 Copenhagen K, Denmark
`Regional ()_[/ice in USA.'
`
`MUNKSGAARD International Publishers Ltd.
`Three Cambridge Center
`Suite 208
`Cambridge, MA 02142
`USA
`
`or with any bookseller.
`
`
`
`_
`Aim and Scope
`The INTERNATIONAL JOURNAL OF PEPTIDE & PROTEIN RESEARCH Wlll
`be of the highest possible scientific and technical standard and will_cover not only
`proteins as such but also peptides and amino acids. The Journal will be open to
`original papers that contribute to the further development of peptide and protein
`research.
`
`Rapid Publication
`This is indeed a large field and there is need for a journal that can publish the latest
`results in the shortest possible time. It is, therefore, a principal aim to keep
`publication time at an absolute minimum, ensuring that articles within the field of‘
`peptide and protein research are gathered quickly in one place and thereby are of
`easy access to all interested in the field. Special attention will be given to the rapid
`publication of Short Communications.
`
`Subscription 1991
`Two volumes are published annually, one issue per month. Subscription price 1991:
`DKK 2700.00 postage included (GBP 253.00, DEM 750.00). USA, Canada and
`Japan 1991: USD 513.00 including postage and air freight.
`Reduced rate for private subscribers 1991: DKK 1460.00 postage included (GBP
`137.00, DEM 40500). USA, Canada and Japan: USD 287.00 including postage and
`air freight.
`Prices are subject to exchange-rate fluctuations.
`
`© 1991 Munksgaard lnteriiational Publishers Ltd. Authorization to photocopy items
`for internal or personal use, or the internal or personal use of specific clients, is
`granted by Munksgaard International Publishers, Ltd. for libraries and other users
`registered with the Copyright Clearance Center (CCC) Transactional Reporting
`Service, provided that the base fee of $02.50 per copy is paid directly to CCC, 27
`Congress Street, Salem, MA 01970. 0367-8377/91/$02.50 + 0.00. All other rights,
`including microfilm, reserved.
`
`Interlzalional Journal of Peptide & P)'()l(!fVI Researo/1 (ISSN 0367-8377) is published
`monthly by Munksgaard International Publishers Ltd, 35 Nerre Sogade, P.O. Box
`2148, DK-1016 Copenhagen K, Denmark. USA subscription price is USD 513.00
`including airspeed delivery. Second class postage paid at Jamaica, NY 11431. USA
`Postmaster I‘or North American subscribers: send address changes to Publications
`Expediting Inc., 200 Meacham Avenue, Elmont, NY 11003. Air freight and mailing in
`the USA by Publications Expediting Inc. Printed in Great Britain at the Alden Press,
`Oxford.
`
`Editor-in-Chief
`Victor J. Hruby, USA
`
`Editorial Board
`R. Acher, France
`P. Balaram, India
`E. Benedetti, Italy
`N. L. Benoiton, Canada
`E. Breslow, USA
`B. Castro, France
`C. Deber, Canada
`W. DeGrado, USA
`R. E. Feeney, USA
`L. Gierasch, USA
`N. Go, Japan
`M. Goodman, USA
`R. Hirschmann, USA
`V. T. Ivanov, USSR
`.1. H. Jones, England
`G. Jung, Germany
`I. Karle, USA
`D. S. Kemp, USA
`H. Kessler, Germany
`K. D. Kopple, USA
`M. Lebl, Czechoslovakia
`M. Marraud, France
`G. Marshall, USA
`R. B. Merrificld, USA
`D. H. Rich, USA
`R. Roechi, Italy
`B. P. Roques, France
`f S. Sakakibara, Japan
`H. A. Scheraga, USA
`P. W. Schiller, Canada
`R. C. Sheppard, England
`J.
`A. Smith, USA
`A.
`F. Spatola, USA
`G.
`I. Tesser, The Netherlands
`G.
`Van Binst, Belgium
`H. Yajima, Japan
`
`This mate-ri 5| w‘as»:n-pied
`at the NLM a rid may be
`Subject US €”c>~i'.in;riglit. Laws
`
`Page 2 of10
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`

`
`A
`
`-
`
`Volume 37, No. 6, June 1991
`Indexed in Current Contents
`
`Original Articles
`
`B.N. Violand, J.S. Ton,
`Determination of the disulfide bond pairings in bovine transforming
`B.I). Vineyard, N.R. Siege/,
`growth factor-at
`‘
`CE. Smith, P.D. Pyla, J.F. Zobe/,
`
`P.C. Toren & E. W. Kolodzicf/'
`
`463
`
`Preparations, solution conformations and molecular structures of
`N,N’-ethylene-bridged dipeptides and their derivatives
`
`Synthesis of S-alkyl and C-terminal analogs of the Sac-r:Iz(1rr)rrzyz'e.v
`cerevisiue a-factor. Influence of temperature on the stability of Fmoc and
`OFm groups toward HF
`
`468
`
`Y. Kojima, Y. Ikeda, E. Kumata,
`J. Maruo, A. Okumom, K. Hirotsu,
`K. S/iibata & /1. Olzsuku
`
`476
`
`C-B. Xue, J.M. Ber/car & I". Naider
`
`General method for rapid synthesis of multieomponent peptide mixtures
`
`487
`
`A. Fur/((1, F. Sebestyén, M. Asgedom
`& G. Dibé
`
`Hydrolysis of /J-casein by gastric proteases. I. Comparison of proteolytic
`action of bovine ehymosin and pepsin A
`
`494
`
`H. Guillou, G. Miranda &
`J-P. Pelissier
`
`R. Schmidt & K. Neubcrt
`502
`Cyclization studies with tetra- and pentapeptidc sequences corresponding
`
`to /I-casomorphins
`
`Infrared spectroscopic discrimination between a- and 3,0-helices in
`globular proteins. Reexamination ofAmide I infrared bands of
`oz-lactalbumin and their assignment to secondary structures
`2-Chlorotrityl chloride resin. Studies on anchoring of Fmoe-amino acids
`and pcplidc Clcavflgc
`
`508
`
`513
`
`S.J. Prertrelski, D.M. Byler &
`M.P. T/iompsrm
`
`K. Barlos, 0, Chalzi, D. Gums &
`G. Stawopoulos
`
`G. Valle, M‘ Cri‘9""l. C. 710I1I'0/0.
`Peptides from chiral C""‘—disubstituted glyeincs. Crystallographic
`5- P0/l'VlC'//i. W-11-I 1>’0c’Sl€’I1.
`characterization of conformation of C’-methyl, C’-isopropy|g|ycine
`[(otMe)Val] in simple derivatives and model peptides
`HE. Sc/Irienltlkct‘. E-M- MW.’/€"‘ &
`
`J. Kamplmis
`G- Y. Xu & CM. Delier
`
`521
`
`528
`
`Conformations of neurotensin in solution and in membrane
`environments studied by 2-D NMR spectroscopy
`Conformation and inhibitory properties of peptides based on the tissue
`kallikrein-aprotinin complex
`
`536 M.S. Des/zpumle, J. Baylmz,
`.I.A. Hcmzilzon & J. Burton
`
`Amatoxins bearing amino and carboxyl groups prepared by selective
`alteration of the aldehyde generated by periodate oxidation of
`
`methylated at-amanitin
`
`J.E. Mullersman & J.F. Preston, 1]]
`
`544
`
`Synthesis and biological evaluation of mouse growth hormone-releasing
`factor
`
`Enhancement of solubility by temporary dimethoxybenzyl-substitution of
`peptide bonds. Towards the synthesis of defined oligomers of alanine and
`of lysyl-glutamyl-glycine
`
`Evidence for a glycoeonjugate form of glutathione S-transferasc pl
`
`Fmoe/solid-phase synthesis of Tyr(P)-containing peptides through t-butyl
`phosphate protection
`
`552
`
`556
`
`E.P. Ilcimer, M. Ahmad,
`T.J. Lllm[)I'().&', A.M. Fe/ix,
`T.R. I)0wns & L./l. Fro/urmn
`
`J. Blaakmeer, T. Tijsse-K/aseiz &
`G.[. Tesser
`
`565
`
`S. Kuzmic/1, L.A. Vamle/-veer &
`K.D. Tau‘
`
`572
`
`J. W. Peric/1 & E.C. Reynolds
`
`'f.r"“ "E.-:'.l' Lil!
`
`Ill.
`
`-Ii.'.ll'
`-lilac
`
`Munksgaard ° Copenhagen
`
`This matxarialwas {spied
`at the NLM and may he
`S‘ul:sje»ct US Copyright Laws.
`
`Page 3 of10
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`Page 3 of 10
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`

`
`Im. J. Peptide Protein Res. 37, 1991, 487-493
`
`General method for rapid synthesis of multicomponent peptide
`mixtures
`
`ARPAD FURKA, FERENC SEBESTYEN, MAMO ASGEDOM* and GABOR DIBO
`
`Department of Organic Chemistry, Eiitvfis Lordnd University, Budapest, Hungary
`
`Received 12 February, accepted for publication 21 November 1990
`
`A method is suggested for the synthesis of multicomponent peptide mixtures. The method is a solid phase
`synthesis modified in order to give a closely equimolar mixture of peptides with predetermined sequences.
`The main point of modification is that before every coupling cycle the resin is divided into equal parts and
`each portion is coupled with a different amino acid. Then the portions are mixed and before the next
`coupling cycle the resin is again distributed into equal portions. The method is illustrated by the synthesis
`of a mixture of 27 tetrapeptides and that of 180 pentapeptides.
`Key W()r((.t‘1 peptide mixtures, synthetic; peptide synthesis, new method; peptide synthesis, solid phase; peptides, electro-
`phoretic identification; peptides, HPLC separation
`
`Due to the outstanding importance of peptides in
`biological processes there is an increasing need for
`synthetic peptides in a variety of applications. Al-
`though the introduction of the solid phase method (1)
`and its automation have considerably speeded up the
`synthetic procedure itself, the one by one synthesis of
`peptides still seems to be slow to comply with the need.
`A possible strategy to improve the productivity of the
`synthetic methods is the simultaneous synthesis oftwo
`or morc—-cven several hundreds of—peptides. Dif-
`ferent methods have been developed to achieve this
`goal. van Rietschoten et al. (2) succeeded in synthesiz-
`ing two peptides on two easily separable resins.
`Gorman (3) constructed a multi-vessel apparatus and
`successfully applied it for the simultaneous synthesis
`of four peptides. The multiple continuous-flow solid
`phase method devised by Krchnak at al.
`(4) made
`possible the synthesis of a decapeptide and its nine
`omission analogs in a single run. Geysen et al. (5)
`synthesized 208 hexapeptides on polyethylene rods
`and tested them without
`removal
`from the solid
`support. In the remarkable method of Houghten (6)
`40-80 peptides were simultaneously synthesized on
`40-80 portions of resin placed in solvent-permeable
`
`Abbreviations follow the recommendations of the IUPAC-lUB
`Joint Commission on Biochemical Nomenclature (European J.
`Birirr/rem. 138, I984, 9-37).
`*l’resent address: PO, Box 62379, Addis Ababa, Ethiopia.
`
`bag5_ Frank & Daring (7) applied paper discs as solid
`support in their synthesis and the coupling operations
`were carried out on 100 discs at a time. Much labor
`can be
`saved by using simultaneous
`synthetic
`methods, which is well demonstrated by H0ughteI1’S
`experiments in which 260 different l3-residue peptides
`were synthesized in less than 4 weeks.
`Further improvement can be expected in the effi-
`ciency of the synthesis if one compromises by using
`peptide mixtures
`instead of
`individual peptides.
`Tjoeng er al. (8) succeeded in synthesizing mixtures of
`four to seven oligopeptides in a single run_. The
`mixture was synthesized on solid support by using, in
`one of the coupling cycles. a mixture of four to seven
`acylating amino acid derivatives.
`By exploiting the additional possibilities inherent in
`the Merrifield method, a new synthetic strategy can be
`introduced assuring, besides a dramatic reduction in
`the number of the coupling cycles, the closely equi-
`molar formation of the components of the peptide
`mixture (9).
`
`The principle of the method. A mixture of a large
`number of peptides, each of them containing the same
`number of residues but different sequences (which can
`be deduced from that of a parent peptide by varying
`amino acids in all or several positions), can be synthe-
`sized in a single run. A normal solid phase synthesis is
`carried out, but before every coupling step the resin is
`
`487
`
`\§33§4
`
`
`
`This material wastopied
`attithe NLM and may be
`in l::—jes:t U13 {Le-pyright Law;
`
`Page 4 of10
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`

`
`A. I‘Lll'K‘c1 (3! al.
`
`divided into equal parts (the number depending on the
`number of amino acids intended to vary in that par-
`ticular position). Each portion is coupled with the
`desired amino acid, and the samples are then mixed.
`The number of the components in the mixture synthe-
`sized this way is given by the product of the numbers
`of the amino acids varied in the different positions.
`
`MATERIALS AND METHODS
`
`Reagents and solvents were products of Fluka AG
`(Buchs, Switzerland). Boc-amino acids were pur-
`chased from Reanal (Budapest, Hungary). Side chain
`protecting groups were: OBzl for Glu and Z for Lys.
`Boc-Ala-resin (0.74 mmol Ala/g) was prepared from
`Bio-Beads S-X1 chloromethyl resin (Bio-Rad Labora-
`tories, Richmond, CA) by Gisin's method (10).
`
`Solidplzase synthesis. The coupling protocol of Gutte
`& Merrifield (ll) was adapted with slight modifica-
`tions: diisopropylcarbodiimide (l2) and l-hydroxy-
`benzotriazole (13) were used;
`the Boe—amino acids
`were added in 100% molar excess; the activated Boc-
`amino acid derivatives were dissolved in dichloro-
`methane—dimethyl formamide mixture (3: 1, v/v). The
`progress of the coupling reaction was followed with
`Kaiser”'s ninhydrin test (14).
`
`Mixing and portioning of the resin samples were per-
`formed on Boc-protected peptidyl resins. The dilferent
`resin samples (ca. 120 mg of each) were suspended in
`l0—l0mL of dimethylformamide and poured into a
`common vessel. The mixture was shaken for 10 min
`and,
`to avoid sedimentation, quickly divided into
`equal volumes.
`
`Cleavage ofpeptides from the resin (50 mg) was carried
`out by trifluoromethanesulfonic acid (15). The reac-
`tion mixture was filtered and washed with trifluo-
`
`roacetic acid into 25 mL dry ether. The mixture was
`allowed to stand overnight at — 20°C, then the preci-
`pitate was collected by filtration, washed twice with
`ether and dried "over KOH, then over P205.
`
`HPLC separation of the peptides was performed on a
`Vydac 2l8TP54 C18 (25 cm X 2.1 mm, i.d.) reversed-
`phase column using a Beckman system (Model 421
`Controller, Model 340 Organizer). Elution was iso-
`eratie at 100% A (0.1 % aqueous trilluoroacetic acid)
`for 5 min; then followed by a linear gradient of 0—20%
`B (90% acetonitrile containing 0.1% trifluoroaeetic
`acid)
`for 20 min, and 20-50% for an additional
`20 min. The flow rate was 1 mL/min. The peptides were
`detected at 214nm (0.5 AUFS).
`
`Sequential degradation was carried out on a gas-phase
`sequencer built at the City of Hope according to the
`method ofHawke et al. (l 6) using the continuous-flow
`488
`
`reactor of Shively et al. (17); the plienylthiohydantoin
`amino acids were identified by using an on-line re-
`versed-phase HPLC system.
`
`Two-dimensional paper electrop/zoresis. The samples of
`the peptide mixtures were applied to :1 30cm band on
`Whatman 3 MM paper (0.3—0.4 mg/cm), together with
`reference markers of taurine and leucine methyl ester
`(50 nmol/em) on both sides. Electrophoresis was ac-
`complished on a horizontal cooled plate apparatus
`(Labor MIM, Hungary) at 32 V/cm. The first run was
`made at pH 6.5 for 2h, using pyridine acetate buffer
`(18). After drying, two side strips were cut out and
`stained with eadrnium-ninhydrin (19). A guide strip of
`2.5 cm (parallel
`to the side strips) was excised and
`sewn to a fresh sheet. Electrophoresis was performed,
`again with markers on the two sides,
`in a perpen-
`dicular direction at pH 2.0 applying ACOH-formic
`acid solution as buffer (20). The peptide map was
`stained and the migration distances then measured.
`For the preparation of the individual peptides, the
`bands were cut out from the first electrophoretogram,
`stitched to new sheets. Each sheet was subjected to
`electrophoresis at pH 2.0 for 2 h. The ninhydrin-posi-
`tive strips were cut out and the peptides were eluted
`from the paper with dilute ACOH, then freeze dried.
`
`RESULTS
`
`Synthesis of a mixture af27 tetrapeptitles. The com-
`ponents of the mixture to be synthesized were de-
`signed to have Ala at
`the C-terminus and,
`in the
`remaining 3 positions, Glu, Phe and Lys were varied.
`
`TABLE l
`
`Peptit1e_s'f(1rtnetl on polymer ([1) mp/2(»'t as result oftlte three coupling
`steps in .s'yntltesi.s n_/'27 tetrape/)tt'tle.r
`
`A-tv
`
`Coupling step I
`FA-p
`
`Coupling step 2
`FEA-p
`FFA-p
`FKA-p
`
`Coupling step 3
`FEEA-p
`FEFA-p
`FEKA-p
`l"l7EA-p
`FFFA-p
`FFKA-p
`FKEA-p
`FKFA-p
`FKKA-p
`
`EA—p
`
`EE/\—p
`EFA-p
`EKA-p
`
`EEEA-p
`EEFA-p
`EEKA-p
`EFEA-p
`EFFA-p
`EFKA—p
`EKEA-p
`EKFA-p
`EKKA-p
`
`KA-p
`
`KEA-p
`KFA-p
`KKA-p
`
`KEEA-p
`KEFA-p
`KEKA-p
`KFEA-p
`KFFA-p
`KFKA-p
`KKEA-p
`KKl"A-p
`KKKA-p
`
`This rnaterialwas el:=:—|JlE{l
`a1.‘theNLM and <'rsa‘-_.r‘l:re
`S‘ul:«jeet:t‘ USQU-wright Law;
`
`Page 5 of10
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`
`LJ_)’llLll\)DlD U1 kJ\4}JLl\J\.4 LIIIALLILUD
`
`The synthesis started from Ala-resin, which was sub-
`mitted to three consecutive coupling steps. The steps
`consisted of the following three operations:
`
`a result, a mixture of 180 pentapeptides was formed:
`
`EGKLP-EFGK-EKP-EFK—A(180)
`
`1. dividing the resin into three portions
`2. coupling the portions (after deprotection) with
`Glu, Phe and Lys, respectively
`3. mixing the portions
`
`Again in the final (No. 4) coupling step, samples were
`removed before mixing the five portions of resin and
`submitted to cleavage separately. In this way the fol-
`lowing simpler mixtures were formed:
`
`The sequences of the peptides formed in the three
`coupling steps are shown in Table 1 where one-letter
`symbols are used for the amino acid residues and p
`denotes the polymer.
`Describing the composition of such multicom-
`ponent peptide mixtures by writing down the sequen-
`ces of their components required a great deal of space.
`Therefore, a shorter notation has been introduced.
`For the synthesized mixture the following formula can
`be used:
`
`EFK-EFK-EFK-A(27)
`
`where the figure in parentheses shows the number of
`peptides in the mixture and the letters separated by
`hyphens indicate the amino acid residues varied in
`different positions of the peptides. A single letter (like
`A) means that
`in all peptides the same residue is
`present at that position.
`In order to facilitate the identification of the synthe-
`sized peptides, a sample was removed from each
`portion of resin before mixing in the final coupling
`step and the cleavage on these tetrapeptide resin mix-
`tures was performed separately. Thus the synthesis
`ended with a mixture of 27 peptides and with three
`simpler mixtures containing only nine components
`each:
`
`E-EFK-EFK-A(9)
`
`F—EFK—EFK-A(9)
`
`K—EFK—EFK-A(9)
`
`Synthesis of a mixture of 180 pentapeptides. The syn-
`thesis again started from Ala-resin and was carried
`through four coupling steps according to Table 2. As
`
`TABLE 2
`
`Number of portions and amim) acids z/sod in coupling operations in
`syntIiesi.\‘ of I80 pentapeptides
`
`Step.
`No.
`
`1
`2
`3
`4
`
`No. of
`portions
`
`3
`3
`4
`5
`
`Coupled amino acids
`
`Glu, Phe, Lys
`Glu, Lys, Pro
`Glu, Phe, Gly, Lys
`Glu, Gly, Lys. Leu, Pro
`
`E-EFGK-EKP-EFK-A(36)
`
`G-EFGK-EKP-EFK-A(36)
`
`K-EFGK-EKP-EFK-A(36)
`
`L-EFGK-EKP-EFK-A(36)
`
`P-EFGK—EKP-EFK-A(36)
`
`Since these 36 components still seemed to be too
`many for easy identification, the course of the synthe-
`sis was checked in the tetrapeptide stage, too. To make
`this possible, the samples were removed from the four
`portions before mixing them in the No. 3 coupling
`step, and again the cleavage was made separately. This
`resulted in four tetrapeptide mixtures containing only
`nine components each:
`
`E—EKP-EFK—A(9)
`
`F-EKP-EFK-A(9)
`
`G-EKP-EFK-A(9)
`
`K-EKP-EFK-A(9)
`
`Identification of the components of the synthetic pep_tia'c
`mixtures. In order to show that the desired peptides
`are really present in the synthetic mixtures the com-
`ponents had to be identified. Two approaches were
`used for this purpose: 1) separation of peptides by
`HPLC, followed by their sequential degradation and
`2) computer-assisted paper electrophoretic identifica-
`tion.
`
`The first approach is demonstrated for the tetrapep-
`tide mixture E-EFK-EFK-A(9). Fig.
`1 shows the
`result of the analytical scale HPLC separation. The
`large differences of peak heights can be attributed to
`the presence of one or two phcnylalanines in some
`peptides. The components corresponding to the nine
`peaks were isolated by a preparative scale HPLC run
`and then submitted to N-terminal stepwise degrada-
`tion. The sequences found (Table 3) proved to be
`exactly those expected (Table 1, coupling step 3, first
`column).
`In addition to separations by HPLC, two-dimen-
`sional paper electrophoresis was also applied and
`proved to be a very useful and quick qualitative
`method for the analysis of the peptide mixtures in any
`stage of the synthesis. In dealing with our synthetic
`mixtures paper electrophoresis has been found to be
`superior to HPLC in one important respect: from the
`
`489
`
`This mate rial was copied
`at.t.h«e N LM and may be
`Subjeezt US Eicypy right Law:
`
`Page 6 of10
`
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`Page 6 of 10
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`

`
`/‘\.
`
`l“LlI’K2i
`
`€’f
`
`(ll.
`
`T/\BL[i 3
`
`AM _a
`
`.S'm[t1('ri¢'o of /11'/2/1‘:/es‘ /.\‘(I/(ll(‘l/ lzr 1lPL(',/rant xrnr/to/it’ mi.\'Iur¢'
`[Li-[JFK-I;'FI\'-A { 9)
`
`Peak number in
`Fig.
`I
`
`l
`Z
`I
`4
`5
`6
`7
`8
`‘)
`
`Sequence
`
`EKKA
`F.KF,.’\
`l‘:l7iK/—\
`l.’l.’IiA
`EFKA
`l7.l'£F/\
`EKF/\
`EFE/\
`TIFFA
`
`position ofa peptide occupied on the two-dirnensional
`clectrophoretic map its amino acid composition can
`be deduced with considerable certainty. This is made
`possible by the close relation existing
`as pointed out
`by Otlbrd (2l)—bctween the mobility ofpeptidcs on
`one side, and their molecular weight and electric
`charge on the other.
`Based on Offord‘s relation a computer program has
`recently been developed (22) for generating predicted
`peptide maps, and these were compared with the ex-
`perimental ones to facilitate assignment of the com-
`ponents of the synthetic mixtures to the spots. This
`comparison also facilitated the detection of missing
`
`0
`
`10"”
`
`"
`
`50‘
`2b
`Tl|VlE(rnm)
`
`1.0 7
`
`so
`
`FIGURE I
`
`tetrapeptide mixture.
`HPLC separatiolt of E-F,FK—EliK—A(9)
`Column: Vydac 2I8Tl’54 (‘l8 (25em x ?_.l mm, i.d.). Solvent A:
`().l% aqueous TF/\. solvent B: 90% acetonitrile containing 0.l”/A)
`Tl‘A. Elution: isocratic at 0% B For 5 min; then linear gradient of
`()—20% B l'or 20min, 20-50% B For 20min. Detection at 2l4nm (0.5
`AUFS). Flow rate
`lmt,/min. Peak I : EKK/\, 2 = EKEA,
`3 = EEKA. 4 : F
`‘EA. 5 : EFK/\, 6 : F.FjF/\. 7 : FZKFA.
`
`8 : El<‘[iA. ‘) = 7]-FA (see Table 3).
`
`490
`
`_ 6
`
`_4-
`
`5
`
`5
`
`,
`
`i
`
`1
`
`'2
`
`L
`
`Q
`
`rt '
`
`L?
`
`r_
`
`)
`
`PEPTIDE
`
`EEEA
`
`EFEA
`EEFA
`
`EFl"A
`
`EKEA
`FZEKA
`
`EKFA
`EFKA
`EKKA
`
`FIGURE 2
`
`6 5/2
`
`— [74/48
`
`- [I5/46
`—— I l5/'46
`
`— 57/46
`
`59/99
`- 5‘)/99
`
`0/96
`0/‘)6
`56/150
`
`No
`
`l
`
`2
`
`3
`
`4
`
`5
`
`6
`
`tetrapcptide
`of Ti-EFK-EFK—A(9)
`Computer-designed map
`mixture. Migration distances of peptides at pH 6.5 and plll are
`given in millimeters. The experimental migration ol’ the reference
`substance l.eu()Me:
`l35mm at pH6 S and I20 mm at pH 2.
`
`peptides (if any) and nonpredicted spots on the ex-
`perimental maps.
`As a response to the input data (number of amino
`acid residues in the peptides. varied amino acids for all
`positions and migration distance on the experimental
`map of LcuOT\/Ie used as a reference substance), the
`computer calculated the number of the expected pep-
`tides. their sequences and expected migration distan-
`ces on the experimental map at both pH 6.5 and pH 2.
`In addition,
`it drew up the expected peptide map.
`making possible the assignment of the peptides to the
`appropriate spots on the map. Taking into account
`that, dtte to incomplete reactions, some deletion pep-
`tides may appear
`in the synthetic mixtures,
`the
`program can also generate such sequences and cal-
`culate their migration positions on the maps.
`The usefulness and reliability of the clectrophoretic
`method is demonstrated with the same tetrapeptide
`mixture used i11 HPLC separations: E-EFK-EFK-
`—A(9). Fig. 2 shows the computer map of the nine
`tetrapeptides and their assignments to the spots. The
`peptides occupy six positions. Three ol‘
`them are
`shown to be doubly occupied by sequence isomers.
`The computer-designed map can be compared to the
`experimental one (Fig. 3). The resemblance is quite
`obvious. All of the six predicted spots are present on
`
`This material was tnpiad
`atthe NL|'-:1 and ma‘-,r be
`E-u bjatt US Copyright Law:
`
`Page 7 of10
`
`
`
`Page 7 of 10
`
`

`
`1'
`
`,. _.
`
`..<’.,‘..-,.g§_»;l,,“_ ‘, _ .
`
`FIGURE 3
`
`D)/HUICSIS O1 ]3CpLl(.lC IIIIXLLIICS
`
`then at pH 2) corresponding to spots I to 6 of Fig. 3.
`Samples 1, 3, and 6 were submitted to stepwise de-
`gradation yielding the expected sequences EEEA,
`EFFA, and EKKA, respectively. Further treatment of
`samples 2, 4, and 5 is exemplified by that of sample 4.
`This sample was combined with a portion of the origi-
`nal synthetic mixture of the nine peptides and then
`submitted to HPLC separation. The result is demon-
`strated by Fig. 4. When comparing this figure to Fig. 1
`two peaks (peaks 2 and 3) are seen to have consider-
`ably increased heights. This means that spot no. 4. of
`Fig. 3 is occupied by two peptides corresponding to
`peaks 2 and 3. The two peptides EKEA and EEKA
`are exactly the predicted ones. Similar analysis of
`samples 2 and 5 also supported the correctness of the
`predictions.
`Fig. 5 shows the two-dimensional map of the 27
`tetrapeptides EFK-EFK—EFK-A. All
`the
`spots
`(except those marked by letters) are expected ones
`and, according to the assignments, they correspond to
`one to six components. To the very faint spots, a and
`b deletion peptides have been assigned:
`
`spot a KEA and EKA
`
`spot b KFA and FKA
`
`Two—dimensional paper electrophorctogram of E-EFK-EFK-A(9)
`telrapeptide mixture.
`
`As to the appearance of deletion peptides, one has
`to take into consideration that in this particular syn-
`
`the experimental map, and there are no unpredicted
`extra spots.
`In order to check the correctness of the assignments,
`the synthetic mixture was separated into six samples
`(using preparative paper electrophoresis at pH 6.5,
`
`’
`
`K!’
`N
`<1:
`
`2
`
`‘]
`
`
`l
`4
`ll l l
`-V_J.
`_
`‘. ‘W
`
`57
`6
`
`5
`
`.
`
`
`
`W e“
`
`3
`
`l
`
`O
`
`i0
`
`FIGURE4
`
`30
`20
`TlME(min)
`
`40
`
`50
`
`FIGURE 5
`
`HPLC separation of the E-BFK-EFK-A(9) synthetic tetrapeptide
`mixture combined with its electrophoretically isolated fraction cor-
`responding to spot 4 on Fig. 3. Experimental details are the same as
`indicated on Fig. l.
`
`Two-dimensional paper electrophoretogram of EFK-EFK-EFK-
`—A(2247) mixture. The faint spots 21 and b have been identified as
`two-sequence isomer pairs of deletion peptides. KEA/EKA and
`KFA/FKA, respectively.
`
`49]
`
`This matzerial wascnpied
`at the NLM a nd may be
`Eu bjact: US Onpvy-right Law:
`
`Page 8 of10
`
`
`
`Page 8 of 10
`
`

`
`1'1. l‘ull\d (:‘l (U.
`
`thesis the same amino acids (Glu, Phe, and Lys) were
`varied in all but the C-terminal position and, as a
`consequence, any deletion peptide could be formed in
`several ways. The number of possible formations was
`calculated by the computer for the four deletion pep-
`tides found: KEA-9, EKA-9, KFA-9, FKA-9. This
`means that the faint a and b spots correspond to very
`low and, in this particular case, probably accumulat-
`ed, quantities of deletion peptides.
`The analysis of the mixture of 180 pentapeptides is
`exemplified by the examination of one of its com-
`ponent mixtures containing 36 peptides (the sample
`was removed before linal mixing). Figs. 6 and 7 show
`the generated and the experimental maps of the
`mixture
`E-EFGK—EKP-EFK-A(36),
`respectively.
`Again the expected spots are seen on the experimental
`map. The number of the assigned components to the
`spots of Fig. 6 varies from 1 to 4.
`Instead of further examinations on the pentapep-
`tide mixtures, more detailed studies were performed in
`the tetrapeptide stage. The electrophoretic maps of the
`four tetrapeptide mixtures each containing nine com-
`ponents (samples removed before mixing in the cou-
`pling step No. 3) were prepared. All the expected spots
`appeared on these experimental maps, leading to the
`conclusion that the synthesis, at least up to this stage,
`has been going well.
`
`DISCUSSION
`
`On the basis of the results of separations made on
`synthetic mixtures by HPLC and paper electrophore-
`sis followed by sequential degradation of the isolated
`components, it may be concluded that the suggested
`method can successfully be used for the preparation of
`multicomponent peptide mixtures. The expected com-
`ponents were really present in the mixtures while by-
`products formed only in low number and quantity. It
`is a characteristic feature of the method that there is a
`good chance for predetermining the approximate
`molar ratio of the peptides in the synthesized mixtures
`
`it!
`
`13- II
`
`LJ Ul
`
`LJ
`
`FIGURE 6
`
`Computer—dcsigned map of the pentapcptidc mixture E—EFGK—
`-EKP-EFK-A(36).
`
`492
`
`
`
`FIGURE 7
`Two-dimensional paper electrophoretogram of the mixture of 36
`pentapeptides (E-EFGK-EKP-EFK-A).
`
`simply by appropriate portioning of the resin before
`the acylation steps. In this respect the present method
`is superior to that ofTjoeng er al. (8) who used a single
`resin and a mixture of acylating reagents in the cou-
`pling stages. Since in all of the described experiments
`only a single amino acid resin (Ala-resin) was used as
`starting material
`it seems worthwhile to note that
`when the C-terminal amino acid is also intended to be
`varied. the synthesis should be started with the appro-
`priate mixture of amino acyl resins.
`The efficiency of the method is remarkable. For
`example the synthesis of I80 pentapeptides by in-
`dependent, one—by—one synthesis (leaving out of the
`consideration coupling of the first amino acid to the
`resin) requires 720 coupling cycles. The synthesis of
`the same 180-component mixture can be accom-
`plished by only I5 coupling cycles. In general, in a
`synthetic mixture of 1' residue peptides where the
`number of amino acids varied in the 1.2.
`. ith posi-
`tion are nl, n2, ..., m‘, the number of the components
`is given by the product of these numbers. while the
`number of the coupling cycles can be expressed by the
`mere sum of them. The time and labor savings in-
`crease rapidly with the number of components of the
`synthetic mixtures. Although the method enables syn-
`thesis of thousands of peptides in one single run, the
`practical number of components is obviously limited.
`An important question that deserves consideration
`is the practical applicability of the peptide mixtures.
`As was shown (see Fig. 1) all of the nine components
`of the E-EFK-EFK-A(9) mixture could be separated
`and isolated by HPLC in a single run. Taking this into
`
`This material was t-up-ia-cl
`atthaNL|'u'| and may be
`Subject USCnpqrright Laws
`
`Page 9 of10
`
`
`
`Page 9 of 10
`
`

`
`—_;
`
`—~—~
`
`~
`
`r—r——~—~ »——~———~-v~
`
`.»t¢'ml. Sm". US 82, 5l3l—
`
`l922—
`
`l’.l.
`
`.l.li.
`
`(1985) Anal.
`
`(I955)
`
`consideration, the synthesis of peptide mixtures com-
`pleted by the separation of the components seems to
`compete favorably witli the one-by-one synthesis fol-
`lowed by one—by—one purification it‘ the mixture is not
`too complex. This assures the immediate applicability
`of the suggested method.
`Extra advantages might be gained in hunting for
`new biologically active peptides by directly submitting
`the simpler mixtures to biological screening. This
`would make it possible to focus further ell'orts only on
`mixtures showing biological activity. Of course the
`authors are aware ol‘ the possibility that in some eases
`the presence 01" an active component can escape detec-
`tion because of being masked by antagonist peptides.
`For this reason determination of the complexity ofthe
`mixtures suitable in these kinds of experiments needs
`further investigations.
`Multieomponent peptide mixtures containing com-
`ponents with predetermined sequence and size also
`oller themselves as useful samples in studies determin-
`ing the factors influencing the retention times in
`HPLC separations.
`
`ACKNOWLEDGMENTS
`
`l. CTsaszz'1r‘ and Mrs. Zs. Cvemes for their
`The authors thank Mrs.
`skillful
`technical assistance. (}.I). wishes to thank Dr. John E.
`Shivcly (at the City olllopc. Duarte, Caliliornia. USA) for making
`it possible to use the facilities of his laboratory as well as Mr.
`Michael Ronk (of the same laboratory) for the N—terminal sequene—
`mg.
`
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`
`I2.
`
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`.I
`l’:77IizIt> I'mIr’iI1 Rz*.s'. 35,
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`9. Furka. /\.. Sebcstycn. Ii. Asgedom. M. & Dibo, G. (1988)
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`PP-
`lt). Huwke. D.H.. Harris. D.C. & Shively.
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`.l.L.. Miller.
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`l8. Ryle,
`/\.P., Sanger. F.. Smith. Ll‘. & Kitai. R.
`Bloc/i<’m.
`.l. 60. 54l—55()
`l9. Heilmann. .l.. Barrollier. J. & Walzke. E. (1957) fInppv»St'rIm'.i'
`7..
`I’/1_i'.s'iu/. (‘/win. 309. 3l9—23U
`20. Atlield, (j.N. & Morris, C..l.O.R. (l‘)(>l) b’I'0z'/1('m.J, 8|. (>0()—
`(il4
`2| Ollbltl. R.E. (1966) Nature (London) 211. 59l~593
`22
`Furka. /i\.. Sebestyen.
`I7‘. & Gulyas. J. (1988) Proc. 2nd lnt.
`Cont‘. Bioehem. Separations, Keszthely. Hungary. pp. 35-42
`
`l. Merrilieltl, R.B. (1963) .1. Am. ('/mu. S01‘. 85. 2l49a2l54
`.~
`van Rietsehoten. J.. Tregear. G.W., Leeman. S.. Powell. D..
`Niall, H. & Potts, J.T. (1975) in l’v/>Iirlu.t‘ /974 (Wolman. Y..
`ed.). pp.
`ll3—l I5. Wiley.