METHODS IN AMOLECULARBIQLOCY
`
`VölumeZ98
`
`Pepide
`Syuliesis
`andpplicatians
`
`Edied by
`ohu Howl
`
`KHUMANAWBNASS
`
`MPI EXHIBIT 1058 PAGE 1
`
`

`

`Peptide Synthesis.and Applications
`
`MPI EXHIBIT 1058 PAGE 2
`
`

`

`METHODS IN MOLECULAR BIOLOGYt#
`
`Peptide Synthesis
`and Applications
`
`Edited by
`John Howl
`Research Institute in Healthcare Science,
`School of Applied Sciences, University of Wolverhampton,
`Wolverhampton, UK
`
`HUMANA PRESS
`
`TOTOWA, NEWJERSEY
`
`MPI EXHIBIT 1058 PAGE 3
`
`

`

`O 2005 Humana Press Inc.
`999 Riverview Drive, Suite 208
`Totowa, New Jersey 07512
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`www.humanapress.com
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`All rightsreserved. No part of this book may be reproduced, stored in a retrieval system or
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`PrintedintheUnitedStatesof America. 10 9
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`Library ofCongress Cataloging-in-Publication Data
`
`Peptidesynthesis and applications / edited by John Howl.
`
`P. ; cm. -
`
`(Methods in molecular biology; 298)
`
`Includes bibliographical references and index.
`
`ISBN 1-58829-317-3 (alk. paper)
`
`1.
`
`Peptides-Synthesis--Laboratory manuals.
`
`(DNLM: 1. Peptide Synthesis--Laboratory Manuals. QU 25 P4246 2005]
`
`I.
`
`Howl, John. II. Series: Methods in molecular biology (Clifton, N.J.) ; v.
`298.
`
`QD431.25.S93P47 2005
`
`547.756--dc22
`
`2004020037
`
`MPI EXHIBIT 1058 PAGE 4
`
`

`

`Contents
`
`Preface
`
`Contributors
`
`PART I: COMMON STRATEGIES
`
`1
`
`Fundamentals of Modern Peptide Synthesis
`Muriel Amblard, Jean-Alain Fehrentz, Jean Martinez,
`and Gilles Subra ..
`2 Chimerism: A Strategy to Expand the Utility
`and Applications of Peptides
`JohnHowl .....
`
`PART I1: SYNTHETIC METHODOLOGIES AND APPLICATIONS
`
`3 Modification of Peptides and Other Drugs Using
`Lipoamino Acids and Sugars
`Joanne T. Blanchfield and Istvan Toth..
`
`4 Synthesis of Linear, Branched, and Cyclic Peptide Chimera
`Gábor Mezö and Ferenc Hudecz...
`
`6
`
`5 Synthesis of Cell-Penetrating Peptides for Cargo Delivery
`Margus Pooga and Ülo Langel....
`Incorporation of the Phosphotyrosyl Mimetic
`4(Phosphonodifluoromethylphenylalanine (F,Pmp)
`Into Signal Transduction-Directed Peptides
`Zhu-Jun Yao, Kyeong Lee, and Terrence R. Burke, Jr.
`
`7 Expressed Protein Ligation for Protein Semisynthesis
`and Engineering
`Zuzana Machova and Annette G. Beck-Sickinger....
`
`8 Cellular Delivery of Peptide Nucleic Acid
`by Cell-Penetrating Peptides
`Kalle Kilk and Ülo Langel...
`9 Quenched Fluorescent Substrate-Based Peptidase Assays
`Rebecca A. Lew, Nathalie Tochon-Danguy,
`Catherine A. Hamilton, Karen M. Stewart,
`Marie-Isabel Aguilar, and A. lan Smith .....
`
`vii
`
`.V
`...ix
`
`ooooness3
`
`****.*.... 25
`
`45
`
`..
`
`..... 63
`
`*. 77
`
`91
`
`eeeeeeeeo
`
`105
`
`131
`
`143
`
`MPI EXHIBIT 1058 PAGE 5
`
`

`

`Vii
`
`Content
`
`10 A Convenient Method for the Synthesis of Cyclic Peptide I :A.
`braries
`Gregory T. Bourne, Jonathon L. Nielson, Justin F. Coughlan.
`Paul Darwen, Marc R. Campitelli, Douglas A. Horton,
`Andreas Rhümann, Stephen G. Love, Tran I. Tran,
`and Mark L. Smythe.
`
`0000eeeeesesess,
`*****1$1
`
`11
`
`High-Throughput Peptide Synthesis
`Michal Lebl and John Hachmann
`
`12 Backbone Amide Linker Strategies for the Solid-Phase Synthesis
`of C-Terminal Modified Peptides
`Jordi Alsina, Steven A. Kates, George Barany,
`andFernando Albericio...
`
`eeeee
`
`19%
`
`13 Synthesis of Peptide Bioconjugates
`FerencHudecz ....
`
`PART III: PRACTICALGUIDES
`
`eeeeeeeeeeeses,
`
`20
`
`14 Protein ldentification by Mas Spectrometric Analyses ofPeptides
`Ashley Martin ...
`15 Manual Solid-Phase Synthesis of Glutathione Analogs:
`A Laboratory-Based Short Course
`UrselSoomets, Mihkel Zilmer, and Ülo Langel...
`Index...
`
`•ee0eeeeeesssss
`
`22
`
`24
`
`250
`
`MPI EXHIBIT 1058 PAGE 6
`
`

`

`1 F
`
`undamentals of Modern Peptide Synthesis
`
`Muriel Amblard, Jean-Alain Fehrentz, Jean Martinez, and Gilles Subra
`
`Summary
`The purpose of this chapter is to delineate strategic considerations and provide
`practical procedures to enable non-experts to synthesize peptides with a reasona-
`ble chance of success. This chapter focuses on Fmoc chemistry, which is now the
`most commonly employed strategy for solid phase peptide synthesis (SPPS). Proto-
`cols for the synthesis of
`fully deprotected peptides are presented, together with a
`review of linkers and supports currently employed for SPPS. The principles and the
`different steps of SPPS (anchoring. deprotection, coupling reaction, and cleavage)
`are all discussed, along with their possible side reactions.
`
`Key Words: Solid phase peptide synthesis; side reaction coupling; anchoring;
`deprotection; cleavage;
`linker.
`
`1.
`
`Introduction
`
`Nowadays, "peptide synthesis" includes a large range of techniques and pro-
`cedures that enable the preparation of materials ranging from small peptides to
`large proteins. The pioneering work of Bruce Merrifield (1), which introduced
`solid phase peptide synthesis (SPPS), dramatically changed the strategy of pep-
`tide synthesis and simplified the tedious and demanding steps of purification
`associated with solution phase synthesis. Moreover, Merrifield's SPPS also per-
`mitted the development of automation and the extensive range of robotic instru-
`mentation now available. After defining a synthesis strategy and programming
`the amino acid sequence of peptides, machines can automatically perform all
`the synthesis steps required to prepare multiple peptide samples. SPPS has now
`become the method of choice to produce peptides, though solution phase syn-
`thesis can still be useful
`for large-scale production of a given peptide.
`
`From: Methods in Molecular Biology, vol. 298: Peptide Synthesis and Applications
`Edited by: ). Howl O Humana Press Inc., Totowa, NJ
`
`3
`
`MPI EXHIBIT 1058 PAGE 7
`
`

`

`4
`
`Amblard etal.
`
`frit
`
`to vacuum
`
`Fig. 1. Basic equipment
`
`for SPPS.
`
`2. Materials
`
`(Fig. 1).
`1. Reaction vessel
`2. Polytetrafluoroethylene (PTFE) stick (15 cm length, 0.6-0.8 cmdiameter).
`3. Rotor.
`4. Filtration flask.
`5. Porous frit.
`6. Lyophilizer.
`7. HPLC equipped with reverse phase Cis column.
`8. pH-Indicating paper.
`9. Solvents
`(N,N-dimethylformamide
`(DCM)
`in wash bottles.
`(DIPEA).
`10. Diisopropylethylamide
`11. Piperidine solution in DMF (20:80).
`12. Kaiser test solutions (ninhydrin, pyridine, phenol) (see Note 1).
`13. Fmoc-amino-acids with protected side-chains (see Table 1).
`14. Trifluoroacetic acid (TFA).
`15. Triisopropylsilane (TIS).
`16.
`tert-butyl methyl ether (MTBE).
`
`(DMFJ, methanol
`
`methank
`[MeOH],dichloromedti
`
`3. Methods
`
`3.1. Principles of SPPS
`
`Steps, the useofas
`As peptide synthesis involves numerous repetitive steps, the
`support has obvious advantages. With such a system a large excess
`
`MPI EXHIBIT 1058 PAGE 8
`
`

`

`Modern Peptide Synthesis
`
`Table 1
`Proteinogenic Amino Acids
`
`5
`
`Side-chain
`H-
`CH3-
`(CH)),CH-
`(CH))CHCH,-
`CH,CH,(CH,)CH-
`HOOC-CH-
`H,NOC-CH,-
`HOOC-CH,CH,-
`H,NOC-CH,CH,-
`HN-CH,CH,CH,CH;-
`
`NH-CHCH;CH
`
`H,N
`
`CH,
`HO-CH,-
`CH-CH(OH)-
`
`-CHy
`
`HO CH
`ÇH-
`
`HS-CH;-
`CH,-S-CH,CHạ-
`
`One-letter code
`
`G A V L D N Q K R
`
`H
`
`S T
`
`Y
`
`W
`
`C M P
`
`H,N OH
`HR
`
`Amino acid
`Glycine
`Alanine
`Valine
`Leucine
`Isoleucine
`Aspartic acid
`Asparagine
`Glutamic acid
`Glutamine
`Lysine
`Arginine
`
`Histidine
`
`Serine
`Threonine
`Phenylalanine
`
`Tyrosine
`
`Tryptophan
`
`Cysteine
`Methionine
`Proline
`
`Three-letter code
`Gly
`Ala
`Val
`Leu
`Ile
`Asp
`Asn
`Glu
`Gln
`Lys
`Arg
`
`His
`
`Ser
`Thr
`Phe
`
`Tyr
`
`Trp
`
`Cys
`Met
`Pro
`
`Note: All the 20 DNA-encoded or proteinogenic a-amino acids are of L stereochemistry.
`
`high concentration can drive coupling reactions to completion. Excess reagents
`and side products can be separated from the growing and insoluble peptide sim-
`ply by filtration and washings, and all the synthesis steps can be performed in the
`Samevessel without any transfer of material.
`The principles of SPPS are illustrated in Fig. 2. The N-protected C-terminal
`amino acid residue is anchored via its carboxyl group to a hydroxyl (or chloro)
`
`MPI EXHIBIT 1058 PAGE 9
`
`

`

`Amblardeta.
`
`OH +
`
`Linker
`
`Loading of theresin
`
`N
`
`Linker
`
`P
`
`HN
`
`Removal of thetemporary
`Na protectinggroup
`
`Linker
`
`P
`
`permanent side-chain protecting group
`
`temporaryurethane Na protecting group
`
`solid support
`
`activating group
`
`NH or O
`
`6
`
`P T
`
`A X
`
`SPPS cycles
`
`Coupling of thenext
`activated amino acid
`
`Linker
`
`n cydes
`
`Linker
`
`Linker
`
`HN
`T
`
`FAn+2 H
`
`Rx
`Px
`
`Pn:2
`
`HN
`
`n
`
`Fig. 2. Principles of SPPS.
`
`MPI EXHIBIT 1058 PAGE 10
`
`

`

`Modern Peptide Synthesis
`
`7
`
`or amino resin to yield respectively an ester or amide linked peptide that will
`ultimately produce a C-terminal acid or a C-terminal amide peptide. After load-
`ing the first amino acid, the desired peptide sequence is assembled in a linear
`fashion from the C-terminus to the N-terminus (the C→ Nstrategy) by repeti-
`tive cycles of N deprotection and amino acid couplingreactions.
`Side-chain functional groups of amino acids must be masked with permanent
`protecting groups (P,) that are stable in the reaction conditions used during pep-
`tide elongation. The a-amino group is protected by a temporary protecting group
`(T) that is usually a urethane derivative. The temporary protecting group (T) can
`be easily removed under mild conditions that preserve peptide integrity and
`reduce the rate of epimerization, which can occur via 5(4H)-Oxazolone forma-
`tion of the activated amino acid during the coupling step (2,3) as indicated in
`Fig. 3. The protective role of urethanes against epimerization also explains the
`predominanceof the C→N strategy.
`After coupling, the excess of reactants is removed by filtration and washings.
`The temporary N-terminal protecting group is removed allowing the addition
`of the next N-urethane protected amino acid residue by activation of its a-car-
`boxylic acid. This process (deprotection/coupling)
`is repeated until the desired
`sequence is obtained.
`In a final step, the peptide is released from the resin and
`the side-chain protecting groups (P) concomitantly removed.
`
`3.2. Fmo/tBu SPPS
`
`In SPPS, two main strategies are used: the Boc/Bzl and the FmoctBu approaches
`for T/P, protecting groups. The former strategy is based on the graduated acid
`lability of the side-chain protecting groups. In this approach, the Boc group is
`removed by neat TFA or TFA in dichloromethane, and side-chain protecting
`groups and peptide-resin linkages are removed at the end of the synthesis by
`treatment with a strong acid such as anhydrous hydrofluoric acid (HF). While
`this method allows efficient syntheses of large peptides and small proteins, the
`use of highly toxic HF and the need for special polytetrafluoroethylene-lined
`apparatus limit the applicability of this approach to specialists only. Moreover,
`theuse of strongly acidic conditions can produce deleterious changes in the struc-
`tural integrity of peptides containing fragile sequences.
`The Fmoc/Bu method (4) is based on an orthogonal protecting group strat-
`egy. This approach uses the base-labile N-Fmoc group for protection of the a-
`amino function, acid-labile side-chain protecting groups and acid-labile linkers
`that constitute the C-terminal amino acid protecting group. This latter strategy has
`the advantage that
`temporary and permanent orthogonal protections are removed
`by different mechanisms, allowing the use of milder acidic conditions for final de-
`protection and cleavage of the peptide from the resin. For all thesereasons, Fmoc-
`basedSPPS is now the method of choice for the routine synthesis of peptides.
`
`MPI EXHIBIT 1058 PAGE 11
`
`

`

`8
`
`Amblard etal.
`
`RO
`
`H
`
`Base
`
`RO
`R,
`
`RO
`
`RO
`
`Base
`
`ROʻ
`
`RNH2
`
`RO,
`
`NHR,
`
`R1
`
`Fig. 3. Epimerization by oxazolone formation.
`
`3.3. Solid Supports
`
`The matrix polymer and the linker can characterize solid supports (5).Often
`the term "resin" is improperly used in place of the linker system, ignoringthe
`fact that the matrix polymer is as important
`in supported chemistry asthesolu-
`tion phase (6). As hundreds of different
`resins are commercially available,somt
`of them carrying the same linkers, special care should be taken to properlychoose
`the most suitable linker for the synthesis.
`
`3.3.1. Matrix Polymers
`
`commonly usedtor
`resins are most
`Cross-linked polystyrene (PS) -based
`(corresponding t01
`routine SPPS. Beads of 200 to 400 mesh size
`distribution
`diameter of about 50 um) and a loading of 0.5 to 0.8 mmol/g present goodchar
`acteristics for polymer swelling in solvents such as DMF and DCM,diffusto
`of reactants into the polymer matrix, and accessibility
`of
`linker sitesburncu
`into the bead. For larger peptides (more than 25 amino acids) or more diit
`sequences,a lower loading is required (0.1-0.2 mmol/g).
`vlene
`Cross-linked polyamide (PA)-based resins and composite PS-polyethy)e
`ohys
`glycol (PEG)-based resins are much more hydrophilic supports exhibitingp
`levels
`ical properties different
`from PS resins at microscopic and macroscopicle
`aler-
`(7). These supports, often with a lower
`loading capacity, may representa
`uences
`native to standard cross-linked PS resins for the synthesis of difficultseque
`and large peptides.
`
`MPI EXHIBIT 1058 PAGE 12
`
`

`

`Modern Peptide Synthesis
`
`Table 2
`Type of SPPS Reaction Vessels
`
`9
`
`length
`
`Vessel
`(cm)
`
`Vessel diameter
`(cm)
`
`Max. resin weight
`(g)
`
`Working volume
`(mL)
`
`5
`
`11
`15
`
`2
`2.6
`3.4
`
`0.5
`2
`
`10
`40
`90
`
`3.4. Resin Handling
`
`3.4.1. SPPS Reaction Vessels
`
`SPPS can be performed in classical glass reaction vessels that can be made by
`glassblowers or purchased from manufacturers (Fig. 1). Alternatively, syringes
`equipped with PTFE or glass frits may also be used.
`Reaction vessel size should be in relation to the amount of resin used, accord-
`ing to Table 2.
`
`3.4.2. Solvents
`
`As 99% of coupling sites are not at the surface but inside the resin beads,
`swelling of beads carrying the growing peptide chain is essential for the opti-
`mal permeation of activated N-protected amino acids within the polymer matrix,
`thus improving coupling yields. Before starting the solid phase synthesis, the
`resin has to be swollen in an adequate solvent such as DCM or DMF for 20 to
`30 min (Protocol 1). For cross-linked polystyrene beads used in SPPS, DCM
`presents optimal swelling properties. For coupling steps, polar aprotic solvents
`such as DMF or N-methylpyrrolidone
`(NMP) are preferred to improve solubil-
`ity of reactants. Alcohols and water are not adequate solvents for PS resins (see
`Note 2). Nevertheless, methanol or isopropanol can be used during the washing
`steps (Protocol 2) to shrink PS resin beads. This shrinking will efficiently remove
`reactants in excess. After such treatment, PS beads should be swollen in DCM
`or DME.
`
`3.4.3. Stirring and Mixing
`
`It is not necessary to agitate the reaction vessel vigorously, as diffusion phe-
`nomena dictate the kinetic reaction in SPPS. Moreover, most types of resin beads
`used for peptide synthesis are fragile, so magnetic stirring is not recommended.
`An old rotary evaporator rotor can be used for stirring during coupling and depro-
`tection steps or alternately any apparatus enabling smooth agitation by rocking
`
`MPI EXHIBIT 1058 PAGE 13
`
`

`

`10
`
`Amblard et al.
`
`or vortexing is appropriate. As beads usually stick to glass, the importantcon-
`dition is that all surfaces of the reactor must be in contact with thereaction
`mixture during stirTing.
`
`3.4.4. Washing
`
`to remove soluble side products and theexcessof
`Washing steps are essential
`reactants used during coupling and deprotection steps. Filling the reactorwith
`solvent contained in a wash bottle and emptying it under vacuum is anappro-
`priate and simple method.
`If necessary, stirring and mixing of the resininthe
`washing solvent can be performed with a PTFE stick.
`
`PROTOCOL 1. RESIN SWELLING
`
`1. Place the dry resin in the appropriate reaction vessel (see Subheading 3.4.1.).
`2. Fill the reactor with DCM until all
`resin beads are immersed. Resinsuspension
`can be gently mixed with a PTFE stick.
`3. Leave for 20 to 30 min.
`4. Remove DCM by filtration under vacuum.
`
`PROTOCOL 2. STANDARD WASHING PROCEDURES
`
`1. Fill the reaction vessel with DMF.
`filtration.
`2. Leave for 10 s and remove the solvent by
`3. Carefully wash the screw cap and the edge of the reactor with DMF.
`4. Repeat steps 1 and 2 twice with DME.
`5. Repeat steps 1 and 2 with MeOH.
`6. Repeat steps 1 and 2 with DCM.
`7. Repeat steps 1 and 2 with DMF.
`
`3.5. Linkers and Resins for Fmoc-Based SPPS
`
`d b
`
`loading) of theN-protected
`The first step of SPPS is the anchoring (or
`ond
`terminal amino acid residue to the solid support via an ester or an amidebo
`depending on the C-terminal
`functional group of target peptide(respectve
`nthe
`acid or amide). Most of the linkers are commercially available anchoredont
`different matrices (PS, PA, PEG-PS). The bead (ball) symbol used in thetolo
`ing paragraph is generic and does not refer to a particular matrix.
`
`3.5.1. Peptide Amides
`
`resins
`For the synthesis of C-terminal peptide amides, the commonly useuand
`are I to 3 (Fig. 4)
`(8-l0). These resins are compatible with Fmoc chemisty
`esidue,
`final TFA cleavage. For attachment of the first urethane N-protectedresu
`standard peptide coupling procedures (Protocol 5) can be used. Theseresl
`usually supplied Fmoc-protected and should be deprotected beforeinco
`
`MPI EXHIBIT 1058 PAGE 14
`
`

`

`Modern Peptide Synthesis
`
`11
`
`OMe NHạ
`
`OMe
`
`H,N
`Me0
`
`1: Rink am ide resin
`
`2: Pal resin
`
`3: Sieber resin
`
`HO
`
`Fig. 4. Resins for peptide amide synthesis.oOMe
`
`Me0
`
`HO
`
`4: X = H, Wang resin
`5: X = OMe, Sasrin resin
`
`6: HMPB resin
`
`7:X=H, Tritylchorideresin
`8: X =Cl 2- Chlorotrityl choride resin
`
`Fig. 5. Resins for peptide acid synthesis.
`
`tion of the first residue. With bulky C-terminal amino acids, a double coupling
`step can be necessary.
`
`3.5.2. Peptide Acids
`
`The anchoring of an amino acid to the solid support by esterification is often
`more difficult, and even hazardous,
`for some residues and can lead to epimer-
`ization, dipeptide formation, and low substitution. Thus, we recommend the
`purchase of resins preloaded with the first C-terminal N-protected amino acid;
`these are commercially available from various manufacturers.
`Commonly used resins in Fmoc/tBu strategy for the synthesis of C-terminal
`peptide acid are reported in Fig. 5 (11-14). Anchoring reactions must be per-
`formed in an anhydrous medium and amino acids containing water should be
`dried before use.
`
`3.5.3. Hydroxymethyl-Based Resins
`
`For hydroxymethyl-based resins 4 to 6, formation of the ester linkage is easier
`with unhindered resins such as Wang resin 4 compared with resins possessing
`withdrawing methoxy groups 5 and 6. The most commonly used esterification
`process is the symmetrical anhydride method (Protocol 3). Determination of
`the loading can be performed by Fmoc release measurement (see Note 3). In the
`case of difficult anchoring,
`the esterification step can be repeated with fresh
`reactants. Arginine derivatives can need three esterification steps to achieve
`
`MPI EXHIBIT 1058 PAGE 15
`
`

`

`12
`
`Amblard et al.
`
`corect loading. After anchoring, unreacted resin-bound hydroxyl groupsshould
`be capped by benzoic anhydride or acetic anhydride (see Protocol 3, step8).
`
`PROTOCOL 3. ATTACHMENT TO HYDROXYMETHYL-BASED RESIN
`
`1. Place the resin in the appropriate SPPS reactor.
`2. Swell
`the resin as described in Protocol 1.
`is placedinadry,
`3. The desired Fmoc amino acid (10 eq relative to resin substitution)
`round-bottom flask with a magnetic stirrer and dissolved in dry DCM at 0°C(3mU
`mmol). Some drops of dry DMF may be useful
`to achieve completedissolution.
`4. Add DIC (5 eq) and stir the mixture for 10 min at 0°C.
`If a precipitate isobserved,
`add DMF until dissolution and stir for 10 min longer.
`5. Add the solution to the hydroxyImethyl
`resin.
`6. Dissolve
`dimethylaminopyridine
`(DMAP)
`(0.1-1 eq) in DMF and add thesolu-
`tion to the reaction mixture.
`7. After 1 h stirring, wash the resin with DMF (three times) and finally withDCM.
`8. Dry the resin in vacuo for 18 h before
`performing Fmoc release measurementon
`a sample (see Note 3). When the loading is less than 70% repeat theesterification
`step.
`9. When the desired substitution is achieved, cap the remaining hydroxyl groupsby
`adding benzoic or acetic anhydride (5 eq) and pyridine (1 eq) in DMF totheresin
`(previously swelled) and stir for 30 min.
`10. Wash the resin (Protocol 2) and start classical elongation with N-protectedamino
`acid after Fmoc deprotection (Protocol 7).
`
`3.5.4. Trityl-Based Resins
`
`Trityl-based resins are highly acid-labile. The steric hindrance of thelinker
`prevents diketopiperazine formation and the resins are recommendedfor o
`and Gly C-terminal peptides. Extremely mild acidolysis conditionsenablete
`cleavage of protected peptide segments from the resin. These resins arecommer
`cially available as their chloride or alcohol precursors. The trityl chlorideresl
`is extremely moisture-sensitive, so reagents and glassware should becareruly
`dried before use to avoid hydrolysis into the alcohol
`form.
`It isnecessary
`tivate
`activate the trityl alcohol precursor and it is highly recommended toreacti
`ofthe
`the chloride just before use (see Note 4). After activation, attachmento
`pres
`first residue occurs by reaction with the Fmoc amino acid derivative inthe
`isfiee
`ence of a base. This reaction does not
`involve an activated species, soit1S
`from epimerization. Special precautions should be taken for Cys andHIs
`dues that are particularly sensitive to epimerization during activation (Tabe
`
`PROTOCOL 4. ATTACHMENT TO TRITYL-BASED RESIN
`
`1. Place 1 gof
`vessel.
`
`trityl-based resin (1.0-2.0 mmol chloride/g resin) in anSPPSI
`
`action
`
`MPI EXHIBIT 1058 PAGE 16
`
`

`

`Modern Peptide Synthesis
`
`13
`
`the resin as described in Protocol 1.
`2. Swell
`3. Add a solution of 3 eq of Fmoc amino acid and 7.5 eq of DIPEA in dry DCM
`(10 mL/g resin). When a lower substitution resin is desired, reduce the amount of
`amino acid.
`4. Stir the mixture for 30 to 60 min at room temperature.
`5. Wash the resin with DMF (two to three times).
`6 Add 10 mlL of a mixture of DCM/MeOH/DIPEA (80:15:5) to cap any remaining
`reactive chloride group.
`7. Mix for 15 min and filter.
`8. Wash the resin three times with DMF and DCM. After drying in vacuo, the substi-
`tution can be measured from Fmoc release (see Note 3).
`
`.6 Side-Chain Protecting Groups
`
`limit the description of side-chain protecting groups (5) to those that
`We will
`have been found most effective for the preparation of a large number of classi-
`cal peptide sequences in Fmoc SPPS and are commercially available from most
`of the protected amino acid providers. For routine synthesis, TFA-labile protect-
`ing groups are usually used. However,
`for selective modifications of a partic-
`ular residue on the solid support (e.g., side-chain cyclized peptides, biotinylated
`peptides), special orthogonal protecting groups are needed (15). Some of the
`most commonly used side-chain protecting groups are reported in Table 3.
`
`3.7. Coupling Reaction
`
`The most simple and rapid procedure for the stepwise introduction of N-pro-
`tected amino acids in SPPS is the in situ carboxylic function activation (Proto-
`col 5). A large excess of the activated amino acid is used (typically 2-10 times
`excess compared to the resin functionality, which is provided by the manufac-
`turer or empirically determined; see Note 3). This excess allows a high concen-
`tration of reactants (typically 60-200 mM) to ensure effective diffusion. The
`time required for a complete acylation reaction depends on the nature of the
`activated species, the peptide sequence that is already linked to the resin, and
`the concentration of reagents. This last parameter must be as high as possible
`and is in connection with the volume of the reaction vessel and the resin substi-
`tution (Table 2). The preferred coupling reagents for in situ activation are ben-
`zotriazol-1-yl-oxytripyrrolidino phosphonium hexafluorophosphate (PYBOP)
`(16) for phosphonium-based activation and O-(benzotriazol-1-yl)-1,1,3,3-
`tetramethyluronium tetrafluoroborate (TBTU) (17) or N-[1H-benzotriazol-1-yl)
`(dimeth-ylamino)methylene}-N-methyl-methanaminium hexafluorophosphate
`N-oxide (HBTU) (18) for aminium/uronium-based activation (Fig. 6). These
`coupling reagents convert N-protected amino acids into their corresponding OBt
`esters. A tertiary amine (generally disopropylethylamine)
`is required to produce
`
`MPI EXHIBIT 1058 PAGE 17
`
`

`

`14
`
`Amblardet l
`
`Table 3
`Side-Chain Protecting Groups in Fmoc-Based SPPS
`
`Protecting
`groups
`
`Pmc
`Pbf
`
`Removal
`condition
`
`95% TFA
`95% TFA
`
`Remarks and side reactions
`
`Presence of thiols may
`accelerate the cleavage.
`
`OtBu
`OAI|4
`
`95% TFA
`Pd(Ph;P)4/PhSiH,
`
`Aspartimide formation
`(see Subheading 12.2.).
`
`Amino acid
`and side-chain
`functionality
`
`Arg
`
`HN NH2
`
`Asp/Glu
`
`OH
`Asn/Gln
`
`NH2
`
`Cys
`nSH
`
`His
`
`Trt
`
`95% TFA
`
`Trt
`
`95% TFA
`
`Trt (NHr)
`Mta
`
`TFA
`1% TFA
`
`Protections avoiddehydration
`of the carboxamideside-chain
`during
`activation and helpto
`solubilize
`Fmoc-Asn-OH and
`Fmoc-GIn-OH.
`PyrGlu formation forN-terminal
`glutamine peptides
`(see Note 5).
`High level of epimerizationcan
`occur during activation
`(see Note 6).
`Participate in the folding of
`peptides and proteins by
`disulfide bridge formation
`(see Subheading 3.11.).
`
`Even with protection ofthe
`imidazole ring,problems
`of epimerization canoccr
`during activation.
`
`Trp can be usedunprotecleuo
`However, ifArg(Pme)
`Arg(Pbf)
`is
`esent inthecan
`Sequence, side-reaction
`Occur.
`
`Lys
`mNH2
`
`Ser/Thr/Tyr
`mOH
`
`Trp
`
`Boc
`Mtta
`Aloca
`tBu
`Trte
`
`Boc
`
`TFA
`1% TFA
`Pd(Ph,P)4JPHSİH,
`TFA
`1% TFA
`
`TFA
`
`"Protection used for on-resin derivatization.
`
`MPI EXHIBIT 1058 PAGE 18
`
`

`

`Modern Peptide Synthesis
`
`15
`
`(H,C),N O_N(CH)l2
`
`(H,0),N N(CH)2
`
`PF
`
`PYBOP
`
`BE
`
`O_
`TBTU
`
`HATU
`
`(HsC)N
`
`N(CH)2
`
`PE
`
`PPF,
`
`PYAOP
`HBTU
`Fig. 6. Phosphonium and uronium/aminium coupling reagents.
`
`Me
`Me-Nt
`
`MeN
`Me
`
`-HNa
`
`Fig. 7. N-terminal
`
`tetramethylguanidinated peptides.
`
`the carboxylate of N-protected amino acids which reacts with coupling reagents.
`More recently,
`N-[1H-benzotriazol-1-yl(dimethylamino)methylene]-N-methyl-
`methanaminium hexafluorophosphate (HATU)
`(19) and 7-azabenzotriazol-
`lyl-oxytris(pyrrolidino) hosphonium hexafluorophosphate (PYAOP) (20).
`that generate OAt esters, have been reported to be more efficient and to reduce
`epimerization.
`Uronium/aminium-based reagents (HBTU, HATU, TBTU) are thought to mech-
`anistically function in a similar way to their phosphonium analogs but unlike them,
`hey can irreversibly block the free N-terminal amino function of the peptide-
`Dy Tormingtetramethylguanidiniumderivatives(Fig. 7) (21). To avoid
`s side-reaction, it is recommended to generate the carboxylate of the amino
`acid before adding these coupling reagents at a slightly reduced equivalent com
`pared to the amino acid.
`
`MPI EXHIBIT 1058 PAGE 19
`
`

`

`16
`
`Amblard et al.
`
`PROTOCOL 5. STANDARDCOUPLING PROCEDURE WITH HBTU
`
`1. After Fmoc deprotection and washings (or properly conditioning andswellingwa
`the resin is dry), wash the resin once with DME.
`(usually 3 eq).
`2. Add the N-a Fmoc-protected
`amino acid as a powder
`3. Fill the SPPS reaction vessel (at least 2/3 of volume) with DMF and stir withaPIFE
`stick for 10-20 s.
`slight excess, 3.5-4 eq) and stir with aPIFE
`(usually
`4. Add DIPEA to the vessel
`stick until complete dissolution of the N-protected amino acid occurs.Otherteri:
`ary amines can be used such as N-methyl morpholine or triethylamine.
`5. Add HBTU (2.9 eq compared with 3 eq of amino acid), screw the cap, andstirfor
`30 min. For difficult sequences it is recommended to preactivate theN-protected
`amino acid before coupling to the free N"-amino function to limit theguanylaia
`side-reaction.
`6. Remove the coupling solution by filtration.
`7. Wash the resin properly according to Protocol 2.
`8. Perform qualitative monitoring of the coupling reaction using acolorimetrictes
`(see Note 1).
`
`test willreved
`In some cases coupling is not complete and a colorimetric
`the presence of free amino groups. On these occasions a double couplingwih
`fresh reagents must be performed. When
`acylation is incomplete after aseconl
`coupling, a capping procedure through acetic anhydride (Protocol 6)can
`performed to stop the elongation of these less-reactive amino groups.When
`colorimetric test still reveals the presence of free amine functions aftercapply
`aggregation can be suspected.
`
`PROTOCOL 6. CAPPING
`
`the resin in DCM.
`1. Swell
`2. Remove the DCM by filtration.
`3. Fill the SPPS reactor (at least 2/3 of volume) with a 50/50 DCM/aceticanhyu
`solution (in case of trityl
`linker, see Note 7) and mix for 3 min.
`4. Remove the capping solution by filtration and repeat step 3 for 7 min.
`5. Wash three times with DCM.
`etrice
`6. Check the disappearance of free amino groups by a convenient colorimeui
`(see Note 1) and repeat the operation if necessary.
`
`3.8. Difficult Sequences and Aggregation
`goW
`When N-deprotection reactions and amino acid coupling stepsdoo
`shoull
`completion or proceed in low yields, repeated or prolonged reactiontine
`overcomethese problems. Nevertheless, this is not always sufficient.Te
`of this failure is thought to be self-association of the peptide chainby nk
`bond formation leading to aggregation. This aggregation results in
`
`MPI EXHIBIT 1058 PAGE 20
`
`

`

`Modern Peptide Synthesis
`
`17
`
`Fmoc,
`
`OH
`
`Me0
`O-Fmoc
`Fig. 8. Fmoc-(FmocHmb) amino acid.
`
`solvation of the peptide-resin and inaccessibility of the reagents to the N-termi-
`nal amino group. This phenomenon is sequence-dependent with particular pro-
`pensity for sequences containing high proportions of hydrophobic residues and
`can start from the fifth coupled residue. Two general methods can be used to
`disrupt the formation of secondary structures. The first consists of changing the
`peptide environment by adding chaotropic salts such as potassium thiocyanate
`or lithium chloride at a 0.4 M concentration, detergent solvents at 1% (v/v), or
`solvents such as DMSO,
`trifluoroethanol, or hexafluoroisopropanol
`to the reac-
`tion medium (22-28). The second approach involves structural changes in the
`peptidebackbone itself by the introduction of protecting groups to selected amide
`bonds. This is usually achieved by using the 2-hydroxy-4-methoxybenzyl
`(Hmb)
`derivative of glycine. Other amino acids can also be incorporated as their N,O-
`bis(Fmoc)-N-(Hmb) derivatives approximately every 6-7 residues (Fig. 8) (29,
`30). The incorporation of (Hmb) amino acids can be performed through their
`corresponding pentafluorophenyl esters, which are commercially available, or
`by standard coupling methods. For optimal N-acylation of the terminal Hmb
`residue that can be slow,
`it is recommended to use powerful coupling methods
`suchas symmetrical anhydrides or preformed acid fluorides in DCM. The 0-
`Fmoc protection of the Hmb derivatives is cleaved under piperidine treatment
`andthe Hmb group in the final TFA cleavage. For Cys-, Ser-, and Thr-contain-
`ing peptides their pseudoproline derivatives, also known to disrupt aggregation,
`can also be used (31-33).
`
`3.9. Fmoc Deprotection
`
`Removal of the temporary Fmoc protecting group from the N-terminus of the
`peptidyl-resin is normally achieved by short treatment with 20% piperidine in
`DMF (Protocol 7). The reaction is generally complete within 10 min, but can
`be longer in some cases. For safe removal a 20-min deprotection time is recom-
`mended. The deprotection results in formation of a dibenzofulvene-piperidine
`adduct that strongly absorbs in the UV range (see Note 3). Fmoc removal can
`be monitored by UV spectroscopy. This is a common procedure with automatic
`
`MPI EXHIBIT 1058 PAGE 21
`
`

`

`18
`
`Amblard etal.
`
`synthesizers but not in standard manual SPPS. When incompletedeprotection
`is suspected, the use of 20% piperidine
`containing 1 to 5% DBU in DMF isteç.
`ommended. However, DBU can promote
`aspartimide
`formation,
`thus itsusg
`should be avoided in Asp- or