HORMONAL
`PROTEINS AND
`PEPTIDES
`
`Edited by CHOH HAO LI
`
`The Hormone Research Laboratory
`University of California
`San Francisco, California
`
`VOLUME II
`
`ACADEMIC PRESS New York and London
`A Subsidiary of Harcourt Brace Jovanovich, Publishers
`
`1973
`
`MPI EXHIBIT 1039 PAGE 1
`
`MPI EXHIBIT 1039 PAGE 1
`
`

`

`CONTMBUTORS
`
`MiKLOS BODANSZKY
`JOHANNES MEIENHOFER
`J. RAMACHANDRAN
`
`MPI EXHIBIT 1039 PAGE 2
`
`MPI EXHIBIT 1039 PAGE 2
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`

`

`C O P Y R I G HT © 1973, BY A C A D E M IC P R E S S, I N C.
`ALL RIGHTS RESERVED.
`NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR
`TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC
`OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY
`INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT
`PERMISSION IN WRITING FROM THE PUBLISHER.
`
`ACADEMIC PRESS, INC.
`Ill Fifth Avenue, New York, New York 10003
`
`United Kingdom Edition published by
`ACADEMIC PRESS, INC. (LONDON) LTD.
`24/28 Oval Road, London NW1
`
`LIBRARY OF CONGRESS CATALOG CARD NUMBER:
`
`72-88367
`
`PRINTED IN THE UNITED STATES OF AMERICA
`
`MPI EXHIBIT 1039 PAGE 3
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`MPI EXHIBIT 1039 PAGE 3
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`

`

`List of Contributors
`
`Numbers in parentheses indicate the pages on which the authors' contributions begin.
`
`MIKLOS BODANSZKY (29), Department of Chemistry, Case Western Re­
`serve University, Cleveland, Ohio
`JOHANNES MEIENHOFER (45), The Children's Cancer Research Founda­
`tion, and Department of Biological Chemistry, Harvard Medical School,
`Boston, Massachusetts
`J. RAMACHANDRAN (1), The Hormone Research Laboratory, University
`of California, San Francisco, California
`
`MPI EXHIBIT 1039 PAGE 4
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`

`

`3
`
`Peptide Synthesis: A Review of the
`Solid-Phase Method
`
`JOHANNES MEIENHOFER
`
`Introduction
`I.
`Π. The Solid-Phase Concept
`A. Basic Features
`B. Experimental Scheme
`C. Intrinsic Problems
`D. Related Methods
`III. The Chemistry
`A. The Solid Support and Introduction of Functional Groups
`B. Attachment of Amino Acids or Peptides
`C. Protection-Deprotection
`D. Coupling Methods
`E. Removal of the Solid Support
`F. Purification and Characterization
`IV. The Operation
`A. Reaction Vessel and Stirring
`B. Solvents and Reagents
`C. Schedule for Synthesis
`D. Analytic Control
`E. Mechanization and Programming
`V. Applications
`A. Model Peptides
`B. Peptides with Biological Functions
`C. Special Applications of the Solid-Phase Method
`D. Other Polymeric Products
`VI. Comparison and Choice between Solid-Phase and Solution Synthesis
`A. Present State of Solid-Phase Synthesis
`B. Present State of Solution Synthesis
`C. Project-Oriented Choice of Method
`D. Outlook
`References
`
`48
`54
`54
`56
`58
`66
`72
`72
`82
`95
`114
`135
`146
`155
`157
`163
`164
`169
`175
`178
`179
`179
`191
`221
`221
`221
`227
`239
`243
`244
`
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`

`

`46
`
`Abbreviations
`
`JOHANNES MEIENHOFER
`
`The abbreviations used for amino acids and protecting groups and the
`designation of peptides and side-chain linkages follow the rules of the
`IUPAC-IUB Commission on Biochemical Nomenclature, in Biochemistry 5,
`1445 and 2485 (1966). The rules for naming synthetic modifications of
`natural peptides have been described in Biochemistry 6, 362 (1967)· The
`following additional abbreviations have been used:
`
`A. AMINO AND HYDROXY ACIDS
`Aad
`L-2-aminoadipic acid
`L-a-aminobutyric acid
`Abu
`e-aminocaproic acid
`Acp
`L-a-amino-ß-ethylvaleric acid
`Aev
`a-aminoisobutyric acid
`Aib
`3 -amino-4-phenylbuty rie
`Apb
`acid
`3 -amino-3 '-phenylisobuty rie
`acid
`ω-aminoundecanoic acid
`δ-aminovaleric acid
`L-cyclohexylalanine
`L-a-cyclohexylglycine
`cycloleucine, 1-aminocyclo-
`pentanecarboxylic acid
`L-a-cyclopentylglycine
`S-carboxymethylcysteine
`i/YM.y-4-fluoroproline
`
`Apib
`
`Aud
`Avi
`Cha
`Chg
`Cle
`
`Cpg
`cys(CM)
`FPro
`
`Gly*
`Har
`He*
`Lac
`Leu*
`MePro
`Met(O)
`Nie
`Pab
`Phg
`Pic
`Pic
`Pro*
`<Glu
`
`Thi
`Tyr(Me)
`</-Val
`
`["Clglycine
`Λο/770-L-arginine
`P^Jisoleucine
`L-lactic acid
`p4C]leucine
`trans-3 -methy lproline
`methionine sulfoxide
`L-norleucine
`p-aminobenzoic acid
`D-C-phenylglycine
`L-pipecolic acid
`phenyllactic acid
`p'CJproline
`pyroglutamic acid, L-pyr-
`rolidone-2-carboxylic acid
`)3-2-thienyl-D, L-alanine
`O-methyltyrosine
`deuterated L-valine
`
`B. PROTECTING AND ACYLATING GROUPS
`acetyl
`Ac
`Dnboc
`acetamidomethyl
`Acm
`adamantyloxycarbonyl
`Adoc
`alkyl
`Alk
`/e/7-amyloxycarbonyl
`Aoc
`2-benzoylmethylvinyl
`Bmv
`ter/-butyloxycarbonyl
`Boc
`Bpoc
`2- ( p-bipheny ly 1 ) isopropy 1-
`oxycarbonyl
`benzylthiomethyl
`tert-buiy\
`benzoyl
`benzyl
`dimedonyl, 5,5-dimethyl-
`cyclohex-2-en-1 -one-3 -yl
`3,5-dimethoxybenzyloxy-
`carbonyl
`
`Btm
`Bu'
`Bz
`Bzl
`Dim
`
`Dimoz
`
`2,2'-dinitrodiphenylmethyl-
`oxycarbonyl
`2,4-dinitrophenyl
`dansyl, 5-dimethylamino-
`naphthalene-1 -sulf onyl
`diphenylmethyl
`ethylcarbamoyl
`ethyl
`9-fluorenylmethoxycarbonyl
`furfuryloxycarbonyl
`formyl
`4,4'-dimethoxybenzhydryl
`p-methoxybenzyl
`p-methoxybenzyloxycarbonyl
`)3-mercaptopropionyl
`/3-mercaptoundecanoyl
`δ-mercaptovaleryl
`
`Dnp
`Dns
`
`Dpm
`Ec
`Et
`Fmoc
`Foe
`For
`Mbh
`Meb
`Meoz
`Mpr
`Mud
`Mvl
`
`MPI EXHIBIT 1039 PAGE 6
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`

`

`3. SOLID-PHASE PEPTIDE SYNTHESIS
`
`47
`
`Ν02·Ζ
`Nps
`OBu'
`OBzl
`Oct
`OEt
`OMe
`OMtp
`ONb
`ONm
`ONo
`ONp
`OPcp
`
`p-nitrobenzyloxycarbonyl
`o-nitrophenylsulfenyl
`ter/-butyl ester
`benzyl ester
`octanoyl
`ethyl ester
`methyl ester
`methylthiophenyl ester
`p-nitrobenzyl ester
`w-nitrophenyl ester
`o-nitrophenyl ester
`p-nitrophenyl ester
`pentachlorophenyl ester
`
`C. O T H ER ABBREVIATIONS
`ACTH
`adrenocorticotropin
`aryl
`AT
`CHA
`cyclohexylamine
`CDI
`iV,N'-carbonyldiimidazole
`DCCI
`dicyclohexylcarbodiimide
`DCHA
`dicyclohexylamine
`DEAE
`diethylaminoethyl
`DMF
`dimethylformamide
`1,4-divinylbenzene
`DVB
`1 -ethyl-(3-dimethylaminopropyl)
`EDC
`carbodiimide x HC1
`iV-ethoxycarbonyl-2-ethoxy-1,2-
`dihydroquinoline
`ethanol
`acetic acid (AcOH)
`N-hydroxysuccinimide
`mixed anhydride
`
`EtOH
`HOAc
`HOSu
`MA
`
`EEDQ
`
`OPic
`OSu
`Pbu
`Pel
`Pth
`Prot
`Tfa
`Thp
`Tos
`Tri
`Z
`Ztf
`
`4-picolyl ester
`iV-hydroxysuccinimide ester
`phenylbutyryl
`pelargonyl
`phthalyl
`protecting group
`trifluoroacetyl
`tetrahydropyranyl
`p-toluenesulfonyl
`triphenylmethyl
`benzyloxycarbonyl
`2,2,2-trifluorobenzyloxy-
`carbonylaminoethyl
`
`MeOH
`MSH
`NEPIS
`
`NEU
`Resin*
`
`RNase
`SDS
`SE
`SOL
`SPS
`TEA
`TFA
`TMV
`TRF,
`TRH
`
`methanol
`melanotropin
`iV-ethyl-5-phenylisoxazolium-
`3'-sulfonate
`triethylamine
`copolystyrene-2% divinylben·
`zene*
`ribonuclease
`sodium dodecyl sulfate
`sulfoethyl
`solution synthesis
`solid-phase synthesis
`triethylamine
`trifluoroacetic acid
`tobacco mosaic virus
`
`thyrotropin releasing factor
`
`Arrangement of Peptides in the Tables
`
`The model peptides are listed by size. Within each group the system of
`Goodman and Kenner (1957) is followed, i.e., alphabetical order of amino
`acid residues starting from the C-terminal end.
`Biologically active and related peptides have been grouped under (a)
`hormones, (b) kinins, and (c) others. Within these groups the order is al­
`phabetical by (trivial) name. Within each name the order is generally: (1)
`naturally occurring peptide(s), (2) analogs, homologs, derivatives, and (5)
`
`* The terminology which has become established for derivatives of copolystyrene-
`divinyl benzene and for various linkages with amino acids or peptides is used. It is
`not always consistent depending on whether the benzene ring is included in a term or not.
`
`MPI EXHIBIT 1039 PAGE 7
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`

`

`48
`
`JOHANNES MEIENHOFER
`
`partial sequences. Analogs and derivatives have been ordered by degree of
`substitution, over the (first) position number, over alphabetical order by the
`Goodman and Kenner convention.
`The tables do not contain a complete listing of all peptides prepared by
`solid-phase synthesis. Furthermore, an effort has been made to list only the
`largest (final) peptide of any one synthesis. Literature covered until June
`1971.
`The common amino acids are of the L configuration unless otherwise in­
`dicated.
`
`I. Introduction
`
`Several recent reports on chemical syntheses of proteins have attracted
`wide interest and publicity as milestones of biochemical research. As a re­
`sult, increased attention has been paid to peptide synthesis, a specialized
`field of organic chemistry. At the same time the definitive structure identifi­
`cation of some very small isolated peptides, such as the thyrotropin releasing
`hormone (TRH) or the melanotropin-release inhibiting factor, required
`peptide synthesis to come to the aid of analytic investigations. These and
`other comparatively small peptides possess extremely high biological poten­
`cy as hormonal control factors. The biological roles and the chemical struc­
`tures of an increasing number of peptide and protein hormones are being
`elucidated, and it appears that many other important biological functions of
`peptides are still to be determined, e.g., in nerve and brain function. The in­
`creased visibility of peptides has given further impetus to peptide synthesis.
`Several excellent books have been published on the subject which give de­
`scriptions of the historical developments, of the numerous contributions to
`the presently available arsenal of synthetic methods, of the delineation of
`strategies and tactics of peptide synthesis, of the many actual syntheses of
`peptides, and of their chemical and biological properties (Greenstein and
`Winitz, 1961; Schröder and Liibke, 1965, 1966; Bodanszky and Ondetti,
`1966; Jakubke and Jeschkeit, 1969; Law, 1970). These reviews present an
`enormous number of synthetic procedures for what would outwardly appear
`to be a simple matter: the repeated formation of the same chemical bond.
`Experimentally, however, differences in the ease of formation of a given
`peptide bond can be tremendous, depending on the neighboring sequences
`—just as sequence characteristics confer upon peptides specific physical,
`chemical, and biological properties, of the known immense variety. Delight­
`ful as the legion of methods described in above reviews is to the adept, it
`frequently causes no small confusion or irritation to the uninitiated. On the
`other hand, synthetic peptides, in particular those with biological activity,
`have become of great interest to investigators in many branches of science
`
`MPI EXHIBIT 1039 PAGE 8
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`

`

`3. SOLID-PHASE PEPTIDE SYNTHESIS
`
`49
`
`and medicine. However, for a long time well-defined synthetic peptides have
`not been readily available.
`With the development of the solid-phase method during the past decade,
`this situation has changed because an easy and convenient experimental
`procedure for peptide synthesis became available. When Merrifield (1962,
`1963) published his first accounts on the novel approach, he demonstrated
`its practical usefulness by the preparation of the crystalline tetrapeptide, L-
`leucyl-L-alanyl-glycyl-L-valine. The potential of the procedure for rapid syn­
`thesis of biologically active peptides was shown a year later by preparations
`of 100-500 mg amounts of bradykinin (Merrifield, 1964a,b). Solid-phase
`peptide synthesis offered many advantages over conventional solution syn­
`thesis, in particular, simplicity of operation and dramatic savings in time.
`The method appeared to provide the ideal means for peptide and protein
`synthesis. It attracted quickly wide attention and has found numerous appli­
`cations not only by peptide chemists but also, and perhaps even more, by
`biochemists and biologists, pharmacologists, physiologists, endocrinologists,
`
`Table I—Reviews and Reports about Solid-Phase Peptide Synthesis
`
`Merrifield, R. B. (1965)
`
`(1966)
`
`(1967)
`(1968)
`(1969)
`
`(1970)
`Ferriere, N. (1966)
`Halstr0m, J. (1967)
`Arnold, Z. (1968)
`Izdebski, J., and
`Drabarek, S. (1968)
`Okuda,T. (1968)
`Vesa, V. C. (1968)
`Jouzier, E. (1969)
`Stewart, J. M., and
`Young, J. D. (1969)
`
`Shimonishi, Y. (1969)
`
`Losse, G., and
`Neubert, K. (1970a)
`Sheppard, R. C. (1973)
`
`(a) Science 150, 178-185.
`(b) Endeavour 24, 3-7.
`In "Hypotensive Peptides" (E. G. Erdös, N. Back, and
`F. Sicuteri, eds.), pp. 1-13. Springer-Verlag, Berlin
`and New York.
`Recent Progr. Horm. Res. 23, 451-482.
`(b) Sci. Amer. 218, 56-73.
`(a) Advan. Enzymol. 32, 221-296.
`(b) J. Amer. Med. Ass. 210, 1247-1254.
`Annu. Rev. Biochem. 39, 841-866. (With A. Marglin).
`Sci. Progr., Nature (Paris), No. 3372 127 (in French).
`Saetryk Dan. Kemi 48, 59-63 (in Danish).
`Farm. Pol. 24, 371-376 (in Polish).
`
`Wiad. Chem. 22, 35-43 (in Polish).
`Naturwissenschaften 55, 209-211 (in German).
`Usp. Khim, 37, 246-255 (in Russian).
`Prod. Probi. Pharm. 24, 313-321 (in French).
`
`"Solid Phase Peptide Synthesis." Freeman, San Fran­
`cisco, California.
`Tampakushitsu, Kakusan, Koso 13, 651-658 (in Japa­
`nese).
`
`Z. Chem. 10, 48-64 (in German).
`In "Peptides 1971" (H. Nesvadba, ed.). North-Holland
`Pubi., Amsterdam (in press).
`
`MPI EXHIBIT 1039 PAGE 9
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`

`50
`
`JOHANNES MEIENHOFER
`
`and immunologists. Many reviews have been published, as compiled in Ta­
`ble I. A comprehensive description of concept and methodology by Merri-
`field (1969a) and a book describing experimental details and operational
`instructions (Stewart and Young, 1969) are available.
`Soon solid-phase synthesis aroused vocal criticism from many peptide
`chemists who have generally not taken issue with the basic concept of the
`new method but rather with the ill-defined purity of some of the products.
`Unless the incorporation of an amino acid residue is quantitative, some un-
`reacted lower peptide will be retained in the solid-phase- With the combined
`effects of repetition at every step and of accumulation (removal is not pos­
`sible until after completion of the synthesis), complex mixtures of "micro-
`heterogeneous" nature are formed.
`However, investigators in biological or biochemical areas of work have
`been enthusiastic about the method. They have used products prepared by
`solid-phase synthesis in a variety of applied experimental or biological stud­
`ies with apparent satisfactory results.
`The difference in conclusions reached from the use of the method might
`be rationalized from considerations of vastly differing backgrounds and
`objectives. Thus peptide chemists have prepared homogeneous peptides, tra­
`ditionally through purification of intermediates rather than through in­
`tensive purification of final products. They expected too much from
`solid-phase synthesis too early and were disappointed when the products, par­
`ticularly larger peptides, showed heterogeneity which was unacceptable by
`established standards. Biologists, on the other hand, have very likely been un­
`aware of possible pitfalls and by-product formation during chemical syn­
`thesis of peptides. They did, indeed, not expect very much and were, conse­
`quently, delighted with their progeny. By one or another quick and insensitive
`test they relieved themselves of the required proofs of identity and/or puri­
`ty. Biochemists, furthermore, have frequently acquired a solid background
`in fractionation of multicomponent mixtures from natural isolates. This ex­
`perience has allowed them to purify some crude solid-phase synthetic pep­
`tides, at least up to decapeptides, to high degrees of purity. (In favorable
`cases, even pentadeca—or up to eicosapeptides appear to have been ob­
`tained in rather high degrees of purity. )
`The controversy over these and other aspects of solution synthesis versus
`solid-phase synthesis grew at times into rather heated polemics. Two con­
`fronting camps formed, but the lines were not very rigid. Synthetic achieve­
`ments have been made through both approaches. The results have helped to
`assess specific merits as well as limitations of each. A selection of the largest
`peptides which have been prepared during the past 8 years by solid-phase-
`synthesis and by solution synthesis is shown in Table II for comparion.
`To date the largest peptide prepared by solution synthesis is ribonuclease
`
`MPI EXHIBIT 1039 PAGE 10
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`

`

`3. SOLID-PHASE PEPTIDE SYNTHESIS
`
`51
`
`S-protein possessing 104 amino acid residues. Total syntheses of two adren-
`ocorticotropins (human and bovine), of several calcitonins and insulins, and
`of glucagon and secretin have been achieved. Solid-phase synthesis afforded
`proteins prepared according to target sequences for human growth hormone,
`bovine ribonuclease A, an analog of horse heart cytochrome c, ribonuclease
`Ti, and acyl carrier protein.
`It is apparent from this comparison (Table II) of selected examples that
`both solid-phase synthesis and solution synthesis have produced results
`which surpassed expectations of only a few years ago. It is also apparent
`that a stage of development has now been reached where it might be con­
`sidered unwise judgment to rely principally and categorically on one of the
`two approaches alone and to dismiss the other as either useless or unman­
`ageable.
`
`Table Π—Examples of Achievements by Solid-Phase and by Solution Peptide
`Synthesis
`
`Size a
`188 b
`
`124
`
`104
`
`104
`
`74
`
`Solid-Phase Synthesis
`Peptide and Reference
`Human growth hormone
`Li and Yamashiro (1970)
`Ribonuclease A
`Gutte and Merrifield (1969,
`1971)
`Cytochrome c
`Sano and Kurihara (1969)
`Ribonuclease Ti
`Izumiya et al (1971)
`Acyl carrier protein-(1-74)
`Hancock et al (1971)
`Marshall et al (1973)
`Bovine basic trypsin inhibitor
`Nodaeiö/. (1971)
`Izumiya et al (1972)
`Ferredoxin
`Bayer et al (1968a,b)
`Staphylococcal nuclease T- ( 6-47 ) 42
`Ontjes and Anfinsen (1969a)
`Parathyroid hormone-(1-34)
`Potts et al (1971)
`Insulin
`
`58
`
`55
`
`34
`
`30
`21
`
`Marglin and Merrifield (1966)
`
`Solution Synthesis
`Peptide and Reference
`Ribonuclease S-protein
`Hirschmann et al (1969)
`Adrenocorticotropin
`Porcine: Schwyzer and Sieber
`(1963)
`Human: Bajusz et al (1967)
`Ribonuclease Ti-( 12-47)
`Beacham ei a/. (1971)
`Calcitonin
`Porcine: Rittel et al (1968)
`Guttman et al (1968)
`Human: Sieber et al (1968)
`Salmon: Guttmann et al (1969)
`Insulin
`
`Meienhofer et al (1963)
`Katsoyannis et al (1964)
`Kungetal
`(1965)
`Glucagon
`Wünsch (1967)
`Secretin
`Bodanszky et al (1966)
`
`Size α
`104
`
`39
`
`35
`
`32
`
`30
`21
`
`29
`
`27
`
`a Amino acid residues.
`h According to the originally proposed structure (Li et al, 1966; 1969). For a re­
`vised complete structure, see Li and Dixon (1971).
`
`MPI EXHIBIT 1039 PAGE 11
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`

`

`52
`
`JOHANNES MEIENHOFER
`
`It would be misleading to attribute the exponential increase in synthetic
`achievements solely to progress in methodical developments. The decisive
`impetus and direction which modern peptide chemistry received from du
`Vigneaud's first oxytocin synthesis (du Vigneaud et al, 1953) has been am­
`plified and accelerated by a steadily increasing number of areas of applica­
`tions. In addition to such classical motivations as proof of structure through
`synthesis and establishment of correlation between chemical structure and
`biological activity, it has become possible to synthesize peptides with
`changed specificity of action (inhibitors) and to produce peptide hormones
`commercially by total synthesis (oxytocin, modified ACTH fragments). Re­
`cently, peptide synthesis has been used to provide homogeneous prepara­
`tions for conformational analysis (oxytocin, vasopressin, angiotensin), or to
`establish the structure of an active principle (thyrotropin releasing factor,
`TRF) occurring naturally in extremely small amounts. Furthermore, it ap­
`pears as if synthesis might eventually have to provide for rapidly increasing
`needs of peptide therapeutics whose natural supply is presently still ade­
`quate but destined to remain limited (insulin).
`A basic scheme has been followed in peptide synthesis which proceeds
`through three stages, as shown in Fig. 1. It begins with the preparation of
`protected amino acids. The most commonly used amino protecting groups
`are the benzyloxycarbonyl group (Bergmann and Zervas, 1932), the tert-
`butyloxycarbonyl group (Anderson and McGregor, 1957), and modifica­
`tions of these groups. For carboxyl protection methyl, ethyl, benzyl, and
`ter*-butyl esters are most frequently employed. In addition, side-chain pro­
`tection often is required for trifunctional amino acids. The actual peptide
`bond formation (second stage) is accomplished by activation of the carbox­
`yl group in the carboxyl component followed by reaction with the amine
`component to produce a protected peptide. Partial deprotection in the third
`stage affords intermediates for further condensation. Complete deprotection
`at the end of a synthesis provides the desired free peptide.
`Four efficient methods of peptide bond formation have found wide and
`general application during the past decade (compare, for a brief review,
`Meienhofer, 1962): the azide method (Curtius, 1902); the mixed anhy­
`dride method (Wieland and Bernhard, 1951; Boissonnas, 1951; Vaughan,
`1951); the dicyclohexylcarbodiimide method (Sheehan and Hess, 1955);
`and the active ester method (e.g., nitrophenyl esters: Bodanszky, 1955).
`The choice of protecting groups, of condensing agents, and of deprotec­
`tion procedures is regarded as the tactics of peptide synthesis, while the pat­
`tern of assembling all amino acid residues into the desired peptide sequence
`is referred to as the strategy (Bodanszky and Ondetti, 1966). The two prin­
`cipal strategies "fragment condensation" and "incremental (stepwise) chain
`
`MPI EXHIBIT 1039 PAGE 12
`
`MPI EXHIBIT 1039 PAGE 12
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`

`

`3. SOLID-PHASE PEPTIDE SYNTHESIS
`
`53
`
`First stage
`protection
`
`Second stage
`activation and
`peptide bond
`formation
`
`Third stage
`(selective)
`deprotection
`
`,Θ
`Η,Ν—CH—C—O
`
`R' O
`H8N—CH—C—Ou
`
`O
`R
`II
`I
`Z—NH—CH—C—OH
`
`R' O
`I
`II
`RjN—CH—C— Y
`
`Carboxyl component
`
`Amine component
`
`O
`R
`II
`I
`Z—NH—CH—C—X
`
`Amine component
`
`o
`R'
`R
`I
`I
`II
`Z—NH-CH—C—NH—CH—C—Y
`
`O
`R
`II
`I
`Z—NH—CH—C—NH-
`
`R' O
`I
`II
`-CH—C—OH
`
`O
`R
`II
`I
`H2N—CH—C
`
`R' O
`I
`II
`-NH-CH—C—Y
`
`FIG. 1. General scheme of peptide synthesis.
`
`elongation" (Bodanszky and du Vigneaud, 1959; Bodanszky et al, 1960)
`are schematically depicted in Fig. 2. Fragment condensation allows greater
`flexibility in the choice of protecting groups and condensing methods but is
`impeded by the danger of racemization at the a carbon of the C-terminal
`amino acid in the carboxyl component.
`The above fundamentals of peptide synthesis have been briefly reviewed
`because the solid-phase method "depends primarily on the chemistry of con­
`ventional peptide synthesis. The protecting groups and coupling reactions
`are those which have slowly evolved over the past 70 years, and many of the
`new developments in peptide chemistry will idso have application to the new
`method. The principal change from the chemistry of classical syntheses is
`due to the introduction of the solid support . . ." (Merrifield, 1969a).
`Therefore, many of the discussions about the solid-phase method require an
`understanding of the basic scheme and of the strategies in Figs. 1 and 2, re­
`spectively, and of the common methodology of solution synthesis.
`The above mentioned recent trends in peptide chemistry: preparation of
`increasingly larger molecules and the chemical synthesis of proteins, along
`with the great potential of applications that appear to open up with the new­
`ly discovered small pep tides of high biological potency, clearly indicate that
`the role of peptide synthesis as an integral part of biological research in
`
`MPI EXHIBIT 1039 PAGE 13
`
`MPI EXHIBIT 1039 PAGE 13
`
`

`

`54
`
`JOHANNES MEIENHOFER
`
`Ε Ξ Ξ Ε Π Ϊ Ι , Ξ Θ ,Ξ
`CD
`
`- 1 — 1 — [-
`
`li 2! 3| 4j s l e a l e]
`
`Fragment condensation
`
`m
`ED
`! 1
`6
`5 1 ! 1
`4! i ii 1
`!
`!
`|
`!
`|
`3
`j i i i i 1
`hi
`11 i 2 3 4 5 6 ILLI
`Incremental (stepwise)
`chain elongation
`FIG. 2. Strategies of peptide chain assembly.
`
`many areas will rapidly grow in importance. The question remains: which
`approach should be used with advantage? It will be attempted in this review
`to show that certain projects of peptide synthesis can effectively be accom­
`plished by solid-phase synthesis, and others must be carried out by solution
`synthesis. Ideally, the decision should be a matter of judicious choice be­
`tween available alternatives, and the choice should depend on the nature
`and objective of the individual study.
`
`Π. The Solid-Phase Concept
`
`A. BASIC FEATURES
`The basic features of Merrifield's procedure involve phase separation of
`the peptide from reagents, by-products, and side products. The growing
`
`MPI EXHIBIT 1039 PAGE 14
`
`MPI EXHIBIT 1039 PAGE 14
`
`

`

`3. SOLID-PHASE PEPTIDE SYNTHESIS
`
`55
`
`peptide chain is kept covalently attached to an entirely insoluble support
`throughout all stages of the synthesis. The solid phase must be in a form
`which allows rapid filtration for the removal of the liquid phase, containing
`the soluble reagents, by-products, and side products, from the solid phase.
`To be applicable for multistage syntheses of large polypepüdes, 100%
`quantitative incorporation of each amino acid residue must be accom­
`plished. Finally, the solid support should be fully removable from the com­
`pleted peptide without altering or degrading the desired product. Complete
`achievement of the above essentials should provide a procedure for the rap­
`id preparation of homogeneous peptides in high yields.
`The general plan adopted for the practical implementation is shown in
`
`Strategy
`
`Experimental
`operation
`
`Solid-phase
`reactions
`
`Solvating medium
`(solution)
`
`(soi
`JD )'|
`1 Anchor group reagent
`| Derivatizing agent
`
`X-A-( SOI
`
`^
`
`I Prot.-amino acid
`1 Reagent
`1 Swelling solvent
`
`Prot-HNCHRtCQ-A-l SOLID ) j
`
`1 Deprotecting agent
`Cleaved protection
`1 products
`Acid, base, salts
`
`\|
`V
`HaNCHRiCO-A-fsOl
`JD)\
`
`Prot.-amino acid
`| Coupling reagent
`1
`1 By-products
`\
`|prot-NHCHR2CO~HNCHR1CQ-A-( SOLID ) j
`
`Functionalization
`
`Attachment
`
`stage
`nitial
`ingle
`
`VJ .f-i ~
`
`Deprotection
`
`Coupling
`
`stage (eye
`chain elongc
`Repetitiv
`
`s 1
`(SOLID) j
`
`Acid, base, solvent
`
`Peptide (crude)
`
`!
`
`*
`Product
`
`1
`
`1
`
`Removal
`
`Purification
`
`stage
`final
`Single
`
`FIG. 3. A general scheme of solid-phase peptide synthesis. Abbreviations: A, an­
`choring chain between resin and peptide; X, functional group for the attachment of
`the first amino acid residue.
`
`MPI EXHIBIT 1039 PAGE 15
`
`MPI EXHIBIT 1039 PAGE 15
`
`

`

`56
`
`JOHANNES MEIENHOFER
`
`Fig. 3. A suitable functional group, X, is introduced into the solid support
`of choice through an anchoring chain, A. The first (C-terminal) protected
`amino acid is attached to the solid support through reaction with the func­
`tional group X. "Functionalization" of the solid material and "attachment"
`are the first two nonrepetitive steps. For the assembly of the peptide the
`strategy of incremental (stepwise) chain elongation (Fig. 2, bottom) is used,
`which is characterized by the incorporation of one amino acid residue at a
`time starting from the C-terminal end (Bodanszky and du Vigneaud,
`1959). Selective cleavage of the α-protecting group ("deprotection") is fol­
`lowed by the introduction of the second protected amino acid ("coupling").
`Alternating successive repetitions of deprotection and coupling steps, called
`"cycles," effect the actual peptide chain elongation. The final stage of a syn­
`thesis consists in removal of the solid support after cleavage of the covalent
`peptide to polymer bond and in purification of crude products.
`The procedure offers three principal advantages over solution synthesis:
`(7 ) losses are avoided, which occur during isolation and purification of each
`intermediate through recrystallization, reprecipitation, or other fractionation
`procedures; (2) reactions may be driven to completion through the use of
`large excesses of reactants in the liquid phase; and (3) no solubility prob­
`lems arise. Limited solubility or insolubility constitute the most serious ex­
`perimental obstacles for solution synthesis of many peptides, especially
`when they contain more than twenty to thirty amino acid residues. An addi­
`tional favorable feature over solution synthesis is the use of a single reaction
`vessel throughout all repetitive operations.
`The practical advantages of the solid-phase procedure over solution syn­
`thesis are: unprecedented speed, simplicity of operation, and possibility for
`automation.
`
`B. EXPERIMENTAL SCHEME
`For the experimental application of the general plan, discussed above
`(Fig. 3), Merrifield selected protecting groups and reagents of proven use­
`fulness in conventional peptide synthesis. The solid support of choice is
`beaded copolystyrene-2% divinylbenzene which can be chloromethylated.
`The attachment of the first amino acid through a benzyl ester bond is effect­
`ed by refluxing a mixture of the chloromethylated resin and the Na-
`protected amino acid in ethanol in the presence of base (Fig. 4). For the
`protection of the α-amino function, the ter/-butyloxycarbonyl group is
`mostly used. Deprotection is effected with hydrogen chloride in acetic acid
`or dioxane or with anhydrous trifluoroacetic acid (in méthylène chloride)
`leaving the amino groups in protonated form. These are converted into free
`amino groups by treatment with triethylamine in méthylène chloride. The
`
`MPI EXHIBIT 1039 PAGE 16
`
`MPI EXHIBIT 1039 PAGE 16
`
`

`

`3. SOLID-PHASE PEPTIDE SYNTHESIS
`O
`H3C
`Rx O
`H.
`H3C—C—O—C—N—C—C—Ow + C1C
`Ï©
`I
`II
`H H
`H3C
`Boc-amino acid
`
`57
`
`Λ / Resin
`
`Chloromethyl
`resin
`
`EtOH 80°
`
`Rx O
`O
`H3C
`I
`II H.
`II
`3 1
`H3C—C—O-C—N—C —C—OC
`I
`3
`II
`H H
`H3C
`
`Boc-amino acyl resin
`
`HCl-HOAc
`
`or
`TFA-CH2C12
`
`H R, O
`H5
`<=>©
`I
`I
`II
`C l u ^H—N—C—C—O-C
`I
`I
`H H
`
`EtN„-CH2Cl,
`
`Resin
`
`-Resin
`
`R i °
`H
`H2
`I
`II
`I
`ii-
`H — N - C - C - O - C - ^Λ
`H
`Boc-amino acid
`DCCI-CH2C12
`
`Resin
`
`HgC
`H 3C-C—O-
`I
`H3C
`
`R, O
`O
`Ri o
`I
`II
`II
`I
`II
`H.
`■C—N-C-C—N-
`-c—c-o-c
`I
`I
`I
`I
`H H
`H H
`Boc-dipeptide resin
`
`Resin
`
`HBr-TFA
`or HF
`
`Rx O
`H R, O
`I II
`I
`I
`II
`H—N—C—C—N—C —C-
`I
`I
`H
`H H
`Peptide
`
`OH
`
`FIG. 4. Standard experimental scheme for solid-phase peptide synthesis (Merri-
`field, 1964b). Resin, copolystyrene-2% divinylbenzene.
`
`MPI EXHIBIT 1039 PAGE 17
`
`MPI EXHIBIT 1039 PAGE 17
`
`

`

`58
`
`JOHANNES MEIENHOFER
`
`deprotection step thus requires two successive operations. Coupling with the
`next ter/-butyloxycarbonylamino acid is carried out in méthylène chloride
`with the aid of dicyclohexylcarbodiimide (Sheehan and Hess, 1955) using
`fourfold molar excess of reactants* and 2-6 hour reaction periods. Dicy­
`clohexylcarbodiimide is ideally suited because of its high reactivity. As a
`so-called condensing reagent it can be simply added to component mixtures
`and thus allows easy and convenient manipulation. After completing the re­
`quired number of repetitive cycles, the desired peptide is removed from the
`resin. To achieve this, the benzyl ester bond is cleaved by treatment with hy­
`drogen bromide in anhydrous trifluoroacetic acid (Merrifield, 1964a) or, al­
`ternatively, by anhydrous hydrogen fluoride in the presence of anisole (Len-
`ard and Robinson, 1967).
`The above described procedure is often referred to as the standard Merri­
`field procedure (Merrifield, 1964b,c; Marshall and Merrifield, 1965). For
`the great majority of reported solid-phase syntheses this or essentially simi­
`lar procedures have been used. Numerous minor variations have been made
`to optimize the yield and homogeneity. Among the most frequently changed
`parameters are reaction time, solvents for reactions and washings, and re­
`peated reactions. More substantial modifications such as the use of different
`protecting groups or other reagents for deprotection, coupling, or removal of
`the solid support will be discussed later (Sections III,C-E). The most salient
`features of this experimental scheme are its simplicity and its universality. A
`series of simple successive washing operations has replaced those parts of
`solution synthesis which have always placed the highest demands on the ex­
`pertise and specialized experience of the investigator, i.e., (a) separation of
`the peptide intermediate from unreacted starting materials, reagents, by­
`products, undesired side products or artifacts, and (b) purification of isolat­
`ed peptide intermediates after each step. A rather dramatic reduction of
`workload is attained. Moreover, the repetitive nature of successive opera­
`tions allows mechanization through programmers and timers.
`Indeed, Merrifield's motivation in developing the solid-phase method has
`been the belief in "a need for a rapid, quantitative, automatic method for
`synthesis of long-chain peptides" (M