woau) INTELLECl1JAL PROPERTY ORGANIZATION
`International Bureau
`
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(51) lntemational Patent Classification 6 =
`1
`C07K /04
`
`(11) International Publication Number:
`
`W0 98ll1l25
`
`(43) Intenultlonal Publication Date:
`
`I9 March I998 (l9.03.98)
`
`(21) Intemational Application Number:
`
`PCT/DK97/00375
`
`(22) International Filing Date:
`
`9 September I997 (09.09.97)
`
`(30) Priority Data:
`097l/96
`
`9 September 1996 (09.09.96)
`
`DK
`
`(7l)(72) Applicants and Inventors: HOLM, Ame [DK/DK]; Mar-
`grethevej I9, DK-2840 I-Iolte (DK). LARSEN, Bjarne, Due
`[DK/DK]; Arildsgard S, DK-2700 Bronshraj (DK).
`
`(81) Designated States: AL, AM, AT, AU, AZ, BA, BB, BG, BR,
`BY, CA, CH, CN, CU, CZ, DE, DK, EE. ES, FI. GB, GE,
`GH, HU, ID, IL. IS, JP. KE, KG, KP, KR. KZ, LC, LK,
`LR. LS. LT, LU, LV. MD, MG, MK, MN, MW, MX, NO,
`NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR,
`Tl‘, UA. UG, US, UZ, VN, YU. ZW, ARIPO patent (GH,
`KE, LS, MW, SD, SZ, UG, ZW), Eurasian patent (AM, AZ,
`BY, KG, KZ, MD, RU. TJ, TM), European patent (AT, BE,
`CH. DE. DK. ES, FI, FR, GB, GR. IE, IT, LU, MC, NL,
`PT, SE), OAPI patent (BF, BJ, CF, CG. CI, CM, GA, GN.
`ML, MR, NE, SN, TD, TG).
`
`(74) Agent: HOFMAN-BANG & BOUTARD LEHMAN &. REE Published
`A/S; Hans Belckevolds Alle 7, DK-2900 Hellernp (DK).
`With international search reporl.
`
`(54) Title:
`
`IMPROVED SOLID-PHASE PEPTIDE SYNTHESIS AND AGENT FOR USE IN SUCH SYNTHESIS
`
`(57) Abstract
`
`Peptides X-AA;-AA2...AA.,-Y, wherein AA is an L- or D-amino acid residue, X is hydrogen or an amino protective group, Y is OH,
`NH; or an amino acid sequence comprising from 3 to 9 amino acid residues and n is an integer greater than 2, are prepared by solid phase
`synthesis, preferably using Fmoc-chemistry. the improvement consisting in that the C-terminal part attached to the solid phase comprises a
`pre-sequence comprising from 3 to 9. preferably from S to 7, amino acid residues independently selected from native L-amino acids having
`a side chain functionality which is suitably protected during the coupling steps and having a propensity factor Pa > 0.57 and a propensity
`factor P/3 > l.l0, preferably Lys and/or Glu. or the corresponding D-amino acids and said pre-sequence is optionally cleaved from the
`formed peptide.
`
`SANOFI-AVENTIS Exhibit 1028 - Page i
`IPR for Patent No. 8,951 ,962
`
`

`
`FOR THE PURPOSES OF INFORMATION ONLY
`
`Codes used to identify States party to the PCf on the front pages of pamphlets publishing international applications under the PCf.
`
`AL
`AM
`AT
`AU
`AZ
`BA
`88
`BE
`BF
`BG
`BJ
`BR
`BY
`CA
`CF
`CG
`CH
`Cl
`CM
`CN
`cu
`cz
`DE
`DK
`EE
`
`Albania
`Annenia
`Austria
`Australia
`Azerbaijan
`Bosnia and Herzegovina
`Barbados
`Belgium
`Burkina Faso
`Bulgaria
`Benin
`Brazil
`Belarus
`Canada
`Central African Republic
`Congo
`Switzerland
`COte d'lvoire
`Cameroon
`China
`Cuba
`Czech Republic
`Gennany
`Denmark
`Estonia
`
`ES
`FI
`FR
`GA
`GB
`GE
`GH
`GN
`GR
`HU
`IE
`IL
`IS
`IT
`JP
`KE
`KG
`KP
`
`KR
`KZ
`LC
`Ll
`LK
`LR
`
`Spain
`Finland
`France
`Gabon
`United Kingdom
`Georgia
`Ghana
`Guinea
`Greece
`Hungary
`Ireland
`Israel
`Iceland
`Italy
`Japan
`Kenya
`Kyrgyzstan
`Democratic People's
`Republic of Korea
`Republic of Korea
`Kazakstan
`Saint Lucia
`Liechtenstein
`Sri Lanka
`Liberia
`
`LS
`I.T
`LU
`LV
`MC
`MD
`MG
`MK
`
`ML
`MN
`MR
`MW
`MX
`NE
`NL
`NO
`NZ
`PL
`PT
`RO
`RU
`SD
`SE
`SG
`
`Lesotho
`Lithuania
`Luxembourg
`Latvia
`Monaco
`Republic of Moldova
`Madagascar
`The former Yugoslav
`Republic of Macedonia
`Mali
`Mongolia
`Mauritania
`Malawi
`Mexico
`Niger
`Netherlands
`Norway
`New Zealand
`Poland
`Ponugal
`Romania
`Russian Federation
`Sudan
`Sweden
`Singapore
`
`Sl
`SK
`SN
`sz
`TD
`TG
`TJ
`TM
`TR
`TT
`UA
`UG
`us
`uz
`VN
`YU
`zw
`
`Slovenia
`Slovakia
`Senegal
`Swaziland
`Chad
`Togo
`Tajikistan
`Turkmenistan
`Turkey
`Trinidad and Tobago
`Ukraine
`Uganda
`United States of America
`Uzbekistan
`Viet Narn
`Yugoslavia
`Zimbabwe
`
`SANOFI-AVENTIS Exhibit 1028 - Page ii
`
`IPR for Patent No. 8,951,962
`
`

`
`W098/11125
`
`PCT/DK97/00375
`
`1
`
`Improved solid-phase peptide synthesis and agent for
`
`use in such synthesis.
`
`Background of the invention
`
`1. Technical Field
`
`5
`
`The present invention relates to an improved process
`for the production of peptides by solid-phase synthe(cid:173)
`sis. The invention also relates to an agent which is
`useful in solid-phase peptide synthesis.
`
`2. Background Art
`
`10
`
`15
`
`Solid-phase peptide synthesis (SPPS) is a highly suc(cid:173)
`cessful method introduced by Merrifield in 1963 (Ref.
`1). Numerous peptides have been synthesized with this
`technique since then. An excellent review of the cur(cid:173)
`rent chemical synthesis of peptides and proteins is
`provided by S.B.H. Kent
`(Ref. 2) which is
`incorpo(cid:173)
`rated herein by reference.
`
`In practice,
`tide chains
`
`two strategies
`by solid-phase
`
`for the assembly of pep(cid:173)
`synthesis are used, viz.
`
`the stepwise solid-phase synthesis, and solid-phase
`fragment condensation.
`
`20
`
`In stepwise
`the C-terminal amino acid in the
`SPPS,
`form of an
`N-a-protected,
`if
`necessary side-chain
`protected reactive derivative
`is covalently coupled
`either directly or by means of a suitable linker to a
`" so 1 i d ··
`support ,
`e . g .
`a po 1 ym eric res in , which
`is
`swollen
`in an organic solvent. The N-a-protective
`group is removed, and the subsequent protected amino
`acids are added in a stepwise fashion.
`
`2 5
`
`the des ired peptide chain length has been ob-
`When
`tained, the side-chain protective groups are removed,
`
`30
`
`SANOFI-AVENTIS Exhibit 1028 - Page 1
`
`IPR for Patent No. 8,951,962
`
`

`
`W098/11125
`
`')
`L
`
`PCT/DK97/00375
`
`the resin, which
`from
`is cleaved
`the peptide
`and
`might be done in separate steps or at the same time.
`
`In solid-phase fragment condensation the target se(cid:173)
`quence is as sembled by consecutive condensation of
`fragments on a solid support using protected frag(cid:173)
`ments prepared by stepwise SPPS.
`
`5
`
`Over the years, two coupling strategies have been de(cid:173)
`
`veloped based on the use of different N-a-protective
`groups and matching side-chain protective groups.
`
`a
`
`10 Merrifield used tert.butyloxycarbonyl (Boc) as the N(cid:173)
`protective
`group, while
`9-fluorenylmethyloxy(cid:173)
`carbonyl
`(Fmoc) was
`introduced by Carpino and Han
`(Ref. 12) .
`
`15
`
`The operations involved in one cycle of chain exten-
`sian in stepwise SPPC using Boc- and Fmoc-chemistries
`are illustrated in the reaction-schemes below (taken
`from Ref. 2).
`
`SANOFI-AVENTIS Exhibit 1028 - Page 2
`
`IPR for Patent No. 8,951,962
`
`

`
`~ ---1
`~
`(!5 g
`
`Ul
`
`w
`
`Couple
`
`O~prolec 1
`
`Rl':)it~
`
`l
`--------~ rm0c A.A -0-Bzi-0-CH
`
`"
`
`-RlSIN
`
`2
`
`Dewole<l
`
`CONH-CH
`
`2
`
`-M,-00zi(H
`
`1
`
`Tlo-0-•NH
`
`.----~ Boc -M,.-00ziCH2CONH -0~2-RE :,1N
`
`~
`loC
`0
`~
`
`Ul
`
`""" """ """ N
`
`B.
`
`A.
`
`f"moc -M., _1-M,. -0-Bzi-0-CH2-R[S.N
`
`8oc-M,_1-M., -08ziCH2CONH-CH2 -R[SIH
`
`-M., -0 -Bzl-0-CH l-R(SIN
`
`2
`
`NH
`
`f mocM-OBJI 1
`
`HOSt
`
`Couple
`
`Neut'o""
`
`BocM--OH
`
`NH2--M.,-00ziCH2CONH-CH2-R(SIN
`
`(BocM)20 ) 1
`l
`J
`
`SANOFI-AVENTIS Exhibit 1028 - Page 3
`
`IPR for Patent No. 8,951,962
`
`

`
`wo 98/11125
`
`4
`
`PCT/DK97/00375
`
`The N-a-Boc-protected peptide coupled to a PAM-resin
`
`is N-a-deprotected with trifluoro-acetic acid
`
`(TFA).
`
`The resulting amine salt is washed and neutralized
`with a tertiary amine. The subsequent peptide bond is
`
`5
`
`formed by reaction with an activated Boc-amino acid,
`
`e.g. a symmetric anhydride. Generally, the side-chain
`
`protection is benzy 1-based, and
`
`the deprotection is
`
`made with HF or a sulphonic acid.
`
`The N-a-Fmoc protected peptide coupled to a resin is
`
`10 N-et.-deprotected by treatment with a secondary amine,
`
`normally piperidine, in an organic solvent, e.g. N,N(cid:173)
`
`dimethyl
`
`formamide
`
`(DMF) or dichloromethane
`
`(DCM).
`
`the neutral peptide resin is reacted
`After washing,
`with an activated Fmoc-arnino acid, e.g. a
`hydroxy-
`
`15
`
`benzotriazole active ester.
`
`The side-chain protection is tert. butyl, trityl and
`
`arylsulfonyl based, and for deprotection of the side(cid:173)
`
`chains, preferably TFA is used.
`
`While the Boc- and Fmoc-strategies are used for es-
`
`20
`
`sentially all current practical peptide synthesis,
`
`other N-a protective groups
`(Steward & Young, Ref .13).
`
`have
`
`been proposed
`
`Boc
`
`forms an ac id-1 abi le urethane group, and other
`
`proposals of this category are biphenylisopropyloxy-
`
`2 5
`
`c arbony 1
`
`( Bpoc) ,
`
`3, 5 -dimethoxypheny 1 isopropy loxy(cid:173)
`
`carbonyl
`
`( Ddz), phenylisopropyloxycarbonyl
`
`(Poe) and
`
`2, 3, 5-tetrarnethylbenzyloxycarboxyl
`
`( Tmz).
`
`Other
`
`types of N-o. protecting groups available in(cid:173)
`
`clude nitrophenylsulfenyl (Nps) which can be removed
`
`30
`
`by either very dilute anhydrous acid, e.g. HCl, or by
`
`nucleophilic
`
`attack,
`
`e.g. with methyl-3-nitro-4-
`
`SANOFI-AVENTIS Exhibit 1028 - Page 4
`
`IPR for Patent No. 8,951,962
`
`

`
`W098/11125
`
`5
`
`PCT/DK97100375
`
`( Dts), which
`mercapto benzoate. Also di thiasucc inyl
`is removable by nucleophilic attack, might be used.
`
`5
`
`SPPS has the genera 1 advantage that it lends itself
`to fully automated or semi-automated chain assembly
`chemistry. A system for SPPS under low pressure con(cid:173)
`tinuous
`flow conditions was developed by Dryland &
`Sheppard (Ref. 7) and was further refined, see Cam(cid:173)
`eron, Meldal & Sheppard
`(Ref.
`14) Holm & Meldal
`(Ref.15), and WO 90/02605.
`
`10 While SPPS has now developed to be a corners tone in
`protein and peptide synthesis, certain problems still
`remain
`to be so 1 ved. Since some of
`these problems
`might well be related to
`the peptide structure,
`a
`brief discussion is deemed proper.
`
`20
`
`15 Empirical predictions of protein conformations have
`been made by Chou & Fasman (Ref. 6). It is well-known
`that protein architectures may be described in terms
`of primary, secondary, tertiary and quaternary struc(cid:173)
`ture. The primary structure refers to the amino acid
`sequence of the protein. The secondary structure is
`the 1 o c a 1 spatia 1 organization o f
`the p o 1 ym e r back(cid:173)
`bone without consideration of the side-chain confor(cid:173)
`mation. As
`examples of
`secondary structures, a(cid:173)
`helixes, n-sheets and ~-turns, which are chain rever-
`sal regions consisting of tetrapeptides can be men(cid:173)
`tioned. The tertiary structure is the arrangement of
`all the atoms in space, including disulphide bridges
`and side-chain positions,
`so
`that all short-
`and
`long-range interactions are considered.
`
`25
`
`30
`
`The term quaternary structure may be used to denote
`the interaction between subunits of the protein, e.g.
`the a- and 0-chains of hemoglobins.
`
`SANOFI-AVENTIS Exhibit 1028 - Page 5
`
`IPR for Patent No. 8,951,962
`
`

`
`W098/1112S
`
`6
`
`PCT/DK97/0037S
`
`5
`
`10
`
`15
`
`Following a discussion of earlier attempts to corre(cid:173)
`late protein secondary structure with amino acid corn(cid:173)
`positions, where e.g. Ser, Thr, Val, Ile and Cys were
`classified as "helix breakers·· and Ala, Leu and Glu
`as "helix formers .. , while hydrophobic residues were
`"P-formers··,
`and proline
`classified as
`strong
`to-
`"P-
`residues
`as
`amino acid
`gether with
`charged
`breakers"", Chou & Fasman made a statistical analysis
`of 29 proteins with known X-ray structure in order to
`establish prediction rules for u_- and fl-regions.
`
`Based on these studies they determined so-called pro(cid:173)
`pensity factors Pu., Pf\ and Pt which are conforma(cid:173)
`tional parameters expressing the positional prefer(cid:173)
`ences as a-helix, f)-sheet and f)-turn, respectively,
`for the natural L-amina acids
`forming part of pro(cid:173)
`teins.
`
`For the sake of convenience, the Pa and PB are listed
`below. One-letter abbreviations
`for
`the
`individual
`amino acids are given in parenthesis.
`
`SANOFI-AVENTIS Exhibit 1028 - Page 6
`
`IPR for Patent No. 8,951,962
`
`

`
`wo 98/11125
`
`7
`
`PCT/DK97/00375
`
`Pa
`
`Glu
`
`( E) 1.51
`
`Val
`
`Met
`
`( M) 1.45
`
`Ile
`
`Ala (A) 1. 4 2
`
`Tyr
`
`5
`
`Leu ( L) 1. 21
`
`Phe
`
`Lys
`
`( K) 1. 16
`
`Trp
`
`Phe
`
`( F ) 1. 13
`
`Leu
`
`G1n
`
`( Q) 1. 11
`
`Cyr
`
`Trp ( w) 1. 08
`
`Thr
`
`10
`
`Ile ( I ) 1. 08
`
`Gln
`
`Val
`
`( v) 1. 06
`
`Met
`
`Asp (D) 1. 01
`
`Arg
`
`p r~
`
`1. 7 0
`
`1. 60
`
`1. 4 7
`
`1. 38
`
`1. 37
`
`1. 30
`
`1. 19
`
`1. 19
`
`1.10
`
`1.05
`
`0.93
`
`His (H) 1.00
`
`Asn
`
`0.89
`
`Arg
`
`( R) 0.98
`
`His
`
`0.87
`
`15
`
`Thr ( T) 0.83
`
`Ala
`
`0.83
`
`Ser ( s) 0.77
`
`Ser
`
`0.75
`
`Cys (c) 0.70
`
`G1y
`
`0.75
`
`Tyr ( y) 0.69
`
`Lys
`
`0.74
`
`Asn ( N) 0.67
`
`Pro
`
`0.55
`
`20
`
`Pro ( p) 0.57
`
`Asp
`
`0.54
`
`Gly (G) 0.57
`
`Glu
`
`0.37
`
`SANOFI-AVENTIS Exhibit 1028 - Page 7
`
`IPR for Patent No. 8,951,962
`
`

`
`wo 98/11125
`
`8
`
`PCT/DK97/00375
`
`Generally speaking, values below 1. 00
`
`indicate that
`
`the amino acid in question must be regarded as unfa(cid:173)
`
`vourable for the particular secondary structure.
`
`As an example, the hydrophobic acids (e.g. Val,
`
`I le,
`
`5
`
`Leu) are strong n-sheet formers, while
`
`the charged
`
`amino acids (e.g. Glu, Asp, His) are [1-sheet break-
`
`ers.
`
`In the a-helix structure, the spiral configuration of
`
`the peptide
`
`is held rigidly
`
`in place by hydrogen
`
`10
`
`bonds between the hydrogen atom attached to the ni(cid:173)
`
`trogen atom in one repeating unit
`
`H
`
`0
`II
`- C - N -)
`
`(C
`
`and the oxygen atom attached to a carbon atom three
`
`15
`
`units along the chain.
`
`If a polypeptide is brought
`
`the a.(cid:173)
`helix can be made to unwind to form a random coil, by
`
`into solution,
`
`adjustment of the pH. The transition from u.-helix to
`
`random coil occurs within a narrow pH. Since the hy-
`drogen bonds are all equivalent in bond strength in
`
`20
`
`the a-helix,
`
`they tend to let go all at once. The
`
`change can also be induced by heat.
`
`The P-sheet structure consists of fully extended pep(cid:173)
`
`tide chains in which hydrogen bonds link the hydrogen
`
`25
`
`atoms on one chain to the oxygen atoms in the adjoin(cid:173)
`
`ing chain. Thus hydrogen bonds do not contribute to
`the internal organization of the chain as they do in
`
`the a-helix, but only bond chain to chain. Adjacent
`
`chains may be parallel or antiparallel.
`
`SANOFI-AVENTIS Exhibit 1028 - Page 8
`
`IPR for Patent No. 8,951,962
`
`

`
`W098/lll25
`
`9
`
`PCT/DK97/00375
`
`0-turns are frequently observed in these parts of a
`peptide chain which connect antiparallel chains in a
`
`O-sheet structure.
`
`In a P-turn,
`
`the CO-
`
`and NH-groups
`
`from amino acid
`
`5
`
`No. n
`
`in the peptide chain form hydrogen bond to the
`
`corresponding groups in amino acid No. n+4.
`
`f\-sheet constitute strongly var iat ing
`a-helix and
`parts of the peptide conformation of proteins (from 0
`to 80 %), and the remaining parts of the proteins are
`folded in other structures. In most proteins sections
`
`10
`
`the peptide chains appear as
`of
`''random coi 1 s ··.
`
`irregularly folded
`
`Turning now to the general problems
`
`still prevailing
`
`in connection with SPPS, S.B.H. Kent
`
`(Ref. 2) high-
`
`15
`
`lights the synthesis of "difficult sequences
`
`Obviously,
`
`the whole rationale of SPPS is
`
`based
`
`on a
`
`complete N-a-deprotect ion prior to each
`
`of the
`
`cou-
`
`pling steps involved.
`
`20
`
`By the same token, ideally all of the N-a-deprotected
`amino groups should be coupled to the reactive amino
`acid derivative according
`to
`the des ired sequence,
`
`i.e. a complete aminoacylation should take place.
`
`Kent states that the most serious potential problem
`
`25
`
`in stepwise SPPS is incomplete peptide bond formation
`giving rise to peptides with one or more amino acids
`missing (deletions), but with properties similar to
`the target sequence.
`
`Such incomplete couplings are more prevalent in some
`
`term "difficult
`sequences than in others, hence the
`sequences .. , and are apparently also more predominant
`
`30
`
`in Fmoc-chemistry than in Soc-chemistry.
`
`SANOFI-AVENTIS Exhibit 1028 - Page 9
`
`IPR for Patent No. 8,951,962
`
`

`
`W098/lll25
`
`10
`
`PCT/DK97/00375
`
`recognized "difficult sequences" have
`A number of
`been studied by scientists including the present in(cid:173)
`ventors.
`
`During SPPS of homo-oligopeptides containing leucine
`or alanine using the Fmoc-strategy, ineffective N-a(cid:173)
`deprotection with piperidine in a sequence dependent
`manner (Refs. 3 and 4) was observed. Investigations
`showed that this phenomenon was associated with sub(cid:173)
`sequent slow or
`incomplete amino acid coupling and
`evidence for f3-sheet aggregation of the growing pep(cid:173)
`tide chain was presented as a cause for the difficult
`couplings
`and
`incomplete Fmoc-deprotections. This
`evidence was based
`on general physical-chemical ob(cid:173)
`servations (Ref.
`4)
`and on a detailed Raman Near In-
`frared Spectroscopic study
`(Ref. 5).
`'rhe physical(cid:173)
`chemical observations referred to may be summarized
`as follows:
`In case of synthesis of H-(Ala)n-Lys-OH
`on a polyamide polymerized kieselguhr matrix
`( PepSyn
`K), no problems were observed until n
`5, but ap-
`proximately 20 -
`25% Fmoc-protected peptide was still
`present after standard deprotection with piperidine
`(20% piperidine in DMF) with n
`6. When continuing
`the synthesis to n = 10 a relatively complicated mix(cid:173)
`ture was obtained comprising the target peptide (n ~
`10) as well as deletion peptides corresponding to n ~
`6, 7, 8, and 9 and deletion pept ides with the Fmoc
`group still attached
`the N-terminal where n
`to
`6 ' 7 ' 8 ' and 9 respectively (Fig. 1 ) This mixture was
`identified by FAB MS after hplc separation of
`the
`single components. Failure sequences or partial de-
`protection with n ~ 2 -
`5 or incomplete deprotection
`of the target peptide (n
`10) was not observed, thus
`confining
`the problems
`to a given stretch of
`the
`homo-oligopeptide chain. This type of difficult ami-
`noacylations and incomplete deprotections can there-
`
`~
`
`5
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`10
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`15
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`20
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`25
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`30
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`SANOFI-AVENTIS Exhibit 1028 - Page 10
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`PCTIDK97/00375
`
`fore be referred to non-random in contrast to random
`difficulties which are due only
`to
`common steric
`problems
`(Ref. 2) . Although experimental conditions
`sol(cid:173)
`were varied, e.g. resin-type, deprotection time,
`vents, addition of chaotropes, problems still
`per(cid:173)
`sisted although heating to 50 °C had an optimal
`ef-
`feet on incomplete Fmoc-deprotection (Ref. 4).
`
`It is a most characteristic feature of the homo-oligo
`alanine chain, that the incomplete Fmoc-deprotection
`steps are followed by strikingly slow acylations with
`the next amino acid in the sequence. Thus effective
`acylation times for each of the first six alanines
`are less than 60 min, while complete acylation with
`Ala7, Alas, Alag, Ala1o is 26, 28, 30 and 7 hours,
`respectively (Fig. 2).
`
`Kent (Ref. 2) proposes a number of solutions to the
`problem related to sequence-dependent coupling diffi(cid:173)
`culties, viz.
`the use of heat
`in the coupling step
`and a quantitative conversion of residual unreacted
`resin-bound peptide chains to terminated species in a
`"capping" procedure.
`
`Summary of the invention
`
`The object of the present invention is to provide an
`improved SPPS according to which peptides, which are
`recognized as or prove to be "difficult sequences ,
`can be synthesized in high yield and purity.
`
`A further object of the invention
`is to provide an
`improved SPPS which provides
`for
`reduced coupling
`times, not only for difficult sequences, but also for
`otherwise uncomplicated sequences where it is desir(cid:173)
`able to reduce the normally long coupling times.
`
`5
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`10
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`15
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`25
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`30
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`SANOFI-AVENTIS Exhibit 1028 - Page 11
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`

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`WO 98/11125
`
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`
`PCT/DK97/0037S
`
`A still further object of the invention is to provide
`an agent or a kit for use in SPPS, whereby the above(cid:173)
`mentioned process improvements are obtained.
`
`The manner in which these and other objects and ad-
`vantages of
`the
`invention
`is achieved will appear
`more fully from the following description and accom(cid:173)
`panying drawings showing hplc diagrams
`for various
`peptides obtained by solid phase synthesis.
`
`Brief description of the drawings
`
`Fig. 1 is an hplc of crude H-Ala 1o-Lys-OH showing a
`substantial amount of deletion peptides (peak
`1,
`2,
`and
`4)
`and
`incompletely
`Fmoc(cid:173)
`3
`deprotected peptides (peak 6, 7, 8 and 9) be(cid:173)
`sides the target peptide (peak 5).
`
`Fig. 2 is a diagram showing sequential coupling times
`for each of the alanines in the synthesis of
`H-Ala1o-Lys-OH.
`
`Fig. 3
`
`is an hplc of H-Ala1o-Lys3-0H showing the tar(cid:173)
`get peptide. No deletion peptides are ob-
`served.
`
`Fig. 4 is an hplc of H-Ala 20 -Lys 3-0H showing deletion
`peptides (peak 1, 2, 3, 4 and 5) besides the
`target peptide (peak 6).
`
`5
`
`10
`
`15
`
`20
`
`Fig. 5 is an hplc of H-Ala 10 -Lys6-0H. No deletion
`peptides are observed.
`
`25
`
`Fig. 6
`
`is an hplc of H-Ala 2 o-Lys6-0H. No deletion
`peptides are observed.
`
`Fig. 7 is an hplc of H-VQAAIDYING-OH, Acyl Carrier
`Protein (ACP) 65-74). The target peptide (peak
`
`SANOFI-AVENTIS Exhibit 1028 - Page 12
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`
`

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`3) is accompanied with deletion peptides (peak
`2 is the des-Val peptide).
`
`Fig. 8
`
`is an hplc of H-VQAAIDYING-K6-0H, Acyl Carrier
`Protein ( ACP) 6 5-7 4 coupled to the Lys ( Boc) 6-
`pre-sequence with only a minor amount of des(cid:173)
`Val peptide (peak 1).
`
`Fig. 9 is an hplc of H-Ala 10 -Lys-OH prepared by in(cid:173)
`troduction of an HMPA linker. Several deletion
`peptides are observed
`(from Ala 1 o-Lys(tBOC)(cid:173)
`HMPA-(Lys(tBoc))6-resin).
`
`Fig.10 is a similar hplc of H-Ala 10 -Lys-OH prepared
`by using an MMa-linker showing a significant
`reduction the amount of deletion peptides com(cid:173)
`pared
`to
`Fig.
`9
`(from
`Alalo-Lys-MMa(cid:173)
`(Lys(tBoc) )6-resin).
`
`Detailed disclosure of the invention
`
`To investigate the problems described above with ref(cid:173)
`erence to Ref. 3 and 4 further the present inventors
`addressed themselves to the question to which degree
`the ~-sheet formation of the homo oligo-alanine chain
`may be affected by inclusion of a shorter peptide se(cid:173)
`quence,
`a pre-sequence,
`in
`the chain at
`the C(cid:173)
`t e rm in us . This question is ass o c i ate d with the fact
`that protein structures and polypeptide sequences may
`have stretches of well defined structures dependent
`on the amino acids in the sequence and of preceding
`amino acids. As mentioned earlier Chou and Fasmans
`
`(Ref. 6) on protein structures have
`investigations
`led to recognition of classes of amino acids which
`are defined as predominantly a-helix
`inducing, ~­
`sheet or random coil inducing. Although it a priori
`may be assumed that similar predictions may apply for
`
`5
`
`10
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`15
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`20
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`30
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`SANOFI-AVENTIS Exhibit 1028 - Page 13
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`
`

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`wo 98/11125
`
`14
`
`PCT/DK97/00375
`
`homo oligo alanines or leucines it was pointed out,
`however,
`in Ref. 4 that Chou and Fasman rules do not
`predict homo oligo alanine or leucine peptides as ~­
`sheet forming chains. Furthermore, Chou and Fasman' s
`rules cannot be expected to apply during peptide syn(cid:173)
`thesis on a resin and do clearly not apply when side(cid:173)
`chain protected amino acids are part of the synthe(cid:173)
`sis.
`
`the synthesis of H-(Ala)n-
`the basic studies
`In
`(Lys}m-OH, where
`(Lys)m represent
`the pre-sequence
`with m equal to 1, 3 and 6 was investigated. A con(cid:173)
`tinuous-flow version of
`the polyamide solid-phase
`method (Ref. 7) on a fully automated peptide synthe(cid:173)
`sizer developed was used as previously described
`(Ref. 14} with DMF as solvent and 3 times excess of
`Fmoc-alanine and Fmoc-lysine(tBoc)-pfp esters,
`re(cid:173)
`spectively and standard Fmoc-deprotection with 20%
`piperidine in DMF for 10 minutes. Coupling times were
`monitored with Dhbt -OH which is deprotona ted to the
`yellow Dbht-o-
`anion when un-acylated amino groups
`are still present and disappearance of
`the yellow
`colour marks
`the end-point of
`the synthesis. After
`cleavage of the peptide from the resin with 95% aque(cid:173)
`ous TFA,
`the product was washed with ether and ana-
`lyzed by hplc.
`'l'he results with m
`== 1 are described
`above (Fig. 1). In case of m = 3, it is seen from the
`hplc trace shown in Fig. 3 that the synthesis may be
`continued to Ala1o without detectable amounts of de(cid:173)
`letion peptides or incomplete Fmoc-deprotection. How-
`ever, when continuing
`the synthesis
`to Ala20
`the
`chromatogram (Fig. 4) shows the presence of deletion
`peptides. The results are even more striking with H(cid:173)
`(Ala)n-(Lys)6-0H where products without detectable
`deletion peptides are obtained with both Ala1o (Fig.
`5) and Ala20
`(Fig. 6). Furthermore, coupling
`times
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`SANOFI-AVENTIS Exhibit 1028 - Page 14
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`

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`W09S/11125
`
`15
`
`PCTIDK97/00375
`
`are drastically reduced from up to 30 hours to stan(cid:173)
`dard coupling times
`(< 2 hours) in the single steps.
`The H-Ala2o-OH sequence has previously been attempted
`synthesized by the Soc-methodology but high levels of
`deletion and insertion peptides were obtained (Ref.
`8). Clearly the pre-sequence Lys6, which under
`the
`prevailing synthesis conditions
`is fully protected
`with the tBoc-group, has a most definitive and fa(cid:173)
`vourable effect on the structure of the growing pep-
`tide chain eliminating the otherwise very severe syn(cid:173)
`thetic problems due
`to incomplete deprotections and
`extremely slow couplings.
`
`In accordance with these surprising findings the pre(cid:173)
`sent invention is based on
`the
`incorporation of a
`particular pre-sequence
`in the C-terminal part at(cid:173)
`tached to the solid support.
`
`fundamental breach with the prior art at(cid:173)
`This is a
`deal with difficult sequences, where
`the
`tempts
`to
`on the reaction conditions and the nature
`focus was
`of the solid support.
`
`As further discussed below the C-terminal sequence by
`which the desired peptide is attached to the solid
`support might also include suitable linkers in order
`to provide for e.g. better attachment or cleavage
`conditions.
`
`Thus in a first aspect the present invention relates
`to a process for the production of peptides
`
`5
`
`10
`
`15
`
`20
`
`25
`
`X-AA 1 -AAz
`
`.
`
`30
`
`wherein AA is an L- or D-amino acid residue, X is hy-
`drogen or an amino protective group, Y is OH, NHz or
`an amino acid sequence comprising from 3 to 9 amino
`
`SANOFI-AVENTIS Exhibit 1028 - Page 15
`
`IPR for Patent No. 8,951,962
`
`

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`wo 98/11125
`
`16
`
`PCT/DK97100375
`
`acid residues and n is an integer greater than 2 by
`phase
`synthesis wherein
`the C-terminal amino
`solid
`acid
`in the
`form of an N-a-protected, if necessary
`chain
`protected reactive derivative is coupled
`side
`solid
`support or a polymer optionally by means
`to a
`of a linker,
`subsequently N-a-deprotected, whereafter
`the subsequent amino acids forming
`the peptide se(cid:173)
`quence are stepwise coupled or coupled as a peptide
`fragment in the form of suitably protected reactive
`derivatives or fragments, wherein the N-c(-protective
`group is removed following formation of the desired
`from
`peptide and
`the peptide is cleaved
`the solid
`in that the C-
`r a c
`support, c h a
`t e
`i
`z e d
`r
`terminal part attached to the support or polymer com-
`prises a pre-sequence comprising from 3 to 9, pref(cid:173)
`erably from 5 to 7 amino acid residues independently
`selected
`from native L-amino acids having
`a
`side
`chain functionality which
`is protected during
`the
`coupling steps and having a propensity factor Pa >
`0,57 and a propensity factor PIJ > 1,10 or the corre(cid:173)
`sponding 0-amino acids and said pre-sequence is op(cid:173)
`tionally cleaved from the formed peptide.
`
`5
`
`10
`
`15
`
`20
`
`L-amino acids meeting the above-mentioned limits for
`the propensity factors Pa and Pf5 are Lys, Glu, Asp,
`25 Ser, His, Asn, Arg, Met and Gln.
`
`These amino acids all have a side chain functionality
`selected from a carboxy, carboxamido, amino, hydroxy,
`guanidino, sulphide or imidazole group.
`
`30
`
`in the pre-sequence
`Presently preferred amino acids
`and
`combinations
`and Glu
`thereof, e.g.
`are Lys
`(Glu)q(Lys)p, where p + q is 3 to 9 , preferably 6 to
`9 1 and the order of Lys and Glu is arbitrarily cho-
`sen.
`
`SANOFI-AVENTIS Exhibit 1028 - Page 16
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`
`PCT/DK97/00375
`
`5
`
`10
`
`15
`
`20
`
`25
`
`the amino acids or peptide
`The N-u. amino group of
`fragments used in each coupling step should be suita(cid:173)
`bly protected during
`the coupling. The protective
`
`group may be Fmoc or Boc or any other suitable pro-
`those described above with refer-
`tective group, e.g.
`ence to Ref. 13 and
`18. The presently preferred N-a
`protective group is Fmoc.
`
`It is important that the side chain functionality in
`the pre-sequence
`is suitably protected during
`the
`
`coupling steps. Such protective groups are well-known
`to a person skilled
`in
`the art and
`the preferred
`groups are listed in claims 7-12.
`
`Without wishing to be bound by any particular theory,
`it is assumed that the physical-chemical properties
`of the protected side-chain of the pre-sequence exem(cid:173)
`plified by lysine are responsible for
`the observed
`"structural assisted peptide synthesis" (SAPS) by re(cid:173)
`ducing or eliminating B-sheet formation in the poly
`alanine sequence.
`
`In case of other homo-oligo pre-sequences it has been
`observed that (Glu(tBu) )6, as well as the mixed se(cid:173)
`quence
`( Glu ( tBu) Lys ( tBoc)) 3
`induces
`a
`favourable
`structure in the poly-alanine chain affording prod(cid:173)
`ucts without deletion peptides.
`
`a more genera 1 phe(cid:173)
`is
`To investigate whether SAPS
`nomenon or if it is confined
`only
`to homo-oligo-
`peptides such as
`the poly alanine
`sequence,
`some
`mixed
`sequences
`reputedly known
`as
`difficult se-
`
`quences were investigated.
`
`30
`
`The synthesis of H-VQAAIDYING-OH, Acyl Carrier Pro(cid:173)
`tein (ACP) 65 - 74, is a well known difficult synthe(cid:173)
`sis which has been used as a model reaction in a num-
`
`SANOFI-AVENTIS Exhibit 1028 - Page 17
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`
`

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`
`1 8
`
`PCTJDK97/00375
`
`ber of cases (Ref. 9). Deletion peptides are observed
`in standard synthesis with a des-Val peptide (peak 2)
`as a prominent by-product (see example 7 and figure
`
`7 ) .
`
`5
`
`In accordance with
`
`the
`
`invention
`
`the sequence H(cid:173)
`
`10
`
`15
`
`20
`
`25
`
`the pre(cid:173)
`synthesized using
`VQAAIDYING-K6-0H was
`sequence (Lys(tBoc))6 attached at the C-terminus on a
`pepsyn K. The synthesis proceeded to give a product
`with the correct molecular weight in
`high purity and
`a significantly
`reduced amount of
`des-Val peptide
`(see example 8 and figure 8).
`
`to be H(cid:173)
`reported
`is
`sequence
`Another difficult
`VNVNVQVQVD-OH which has been synthesized on a variety
`of flow resins (Rapp polymer, PEG-PS, Pepsyn K, PEGA
`1900/300, PEGA 800/130 and PEGA 300/130) in all cases
`accompanied by considerable glutamine preview already
`from Vall except when using a PEGA 1900/300 resin
`(Ref. 10). In accordance with the invention the syn(cid:173)
`thesis of H-VNVNVQVQVDK6-0H proceeded to give a prod-
`uct with the correct molecular weight. Deletion pep(cid:173)
`tides were not detected in the spectrum (see example
`
`9) .
`
`In order to verify another important aspect of the
`present
`invention, viz.
`the reduced coupling
`times
`obtained by
`introducing a pre-sequence at
`the C(cid:173)
`terminal part of desired peptide the coupling times
`of
`the
`individual amino acids
`in
`the synthesis of
`enkephalin, H-Tyr-Gly-Gly-Phe-Leu-OII, have been moni(cid:173)
`tored with and without the pre-sequence (Lys(tBoc))6
`
`30
`
`as described in example 15·
`
`The results of the measurements demonstrate that the
`coupling times
`in this otherwise uncomplicated syn-
`
`SANOFI-AVENTIS Exhibit 1028 - Page 18
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`
`

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`PCT/DK97/00375
`
`thesis proceeds e f feet i vely in combination with the
`
`chosen pre-sequence.
`
`In
`
`the above described cases
`
`the peptide sequences
`
`have been obtained with a hexa
`
`lysine pre-sequence
`
`5 which for certain purposes may be acceptable or even
`
`of advantage. Thus,
`
`a P-sheet
`
`forming sequence may
`
`cause severe solubility problems, but in case of H(cid:173)
`
`Ala2Q(Lys)6-0H the peptide turned out to be soluble
`
`in aqueous solutions while H-Ala 1 o -Lys -OH very rap-
`
`10
`
`idly precipitated
`from TFA solutions when diluted
`with water. In case of (Glu) 6 the solubility provided
`by the pre-sequence and the multi tude of carboxylic
`
`acid groups may be utilized for e.g. ELISA, where
`
`site-d