Solid Phase Peptide Synthesis - Bachem

1 Solid Phase Peptide SYNTHESISS olid- Phase Peptide Synthesis2 TIPS AND TRICKS FOR Solid Phase Peptide SYNTHESISFROM THE EXPERTS AT BACHEMList of or Boc? Quasi Continuous Flow Automated SPPS12 IIFmoc Based Handling of the Available from for the Synthesis of Peptide Acids14 Wang resin and preloaded Wang resinsDHPP-resin and for the Synthesis of Peptide Amides15 Tricyclic amide linker resinRink amide resin4,4 -Dialkoxybenzhydrylamine resinOther TFA-labile amide for the Synthesis of Fully Protected Peptide Fragments15 SASRIN and preloaded SASRIN resins2-Chlorotrityl chloride resin and preloaded 2-chlorotrityl resinsXanthenyl linker resin (for the Synthesis of fully protected Peptide amides) for the Synthesis of Peptide Alcohols16 SASRINPDDM-resin2-Chlorotrityl chloride resin3,4-Dihydro-2H-pyran-2-ylmethoxymet hyl resin (Ellman s dihydropyrane resin)Further resinsTable of Fmoc Amino Acid Protecting Protection of Cys During Fmoc SPPS of peptides Containing Disulfi de peptides Containing a Single Disulfi de Containing Two Disulfi de Containing Three Disulfi de Formation of Disulfi de Reagents and by Phosphonium and Uronium/Aminium Amino Acid Active Amino Acid Fluorides and of Coupling and Blue of Diffi cult from the Cleavage from the Resin/Side-Chain Your Own of Protected Peptide Reactions in Fmoc of Pmc to Trp During TFA (1-Piperidinyl)alanine Fmoc of Free Amino Moieties During Reactions of Fmoc Peptide Synthesis4 IIIBoc Based for the Synthesis of Peptide Acids46 Chloromethyl polystyrene (Merrifi eld resin)

2 For the Synthesis of Peptide Resins474-Formyl-phenoxymethyl Boc Amino Acid Reagents and from the Reactions in Boc Lactone Reactions Involving Boc Groups and Active EstersAcmAcetamidomethylAdpoc2-(1 -Adamantyl)-2-propyloxycarbonylAlocAllyl oxycarbonylBoctert. ButyloxycarbonylBomBenzyloxymethyl2-BrZ2 -BromobenzyloxycarbonyltButert. ButylBzlBenzyl2-ClZ 2-ChlorobenzyloxycarbonylDde1-(4,4-Dimet hyl-2,6-dioxocyclohexylidene)-3-methylbu tyl2,6-diClBzl2,6-DichlorobenzylDmb2,4-D imethoxybenzylDnp2,4-DinitrophenylFm9-Fl uorenylmethylFmoc9-Fluorenylmethyloxycar bonylForFormylHmb2-Hydroxy-4-methoxybenz ylMBzl4-MethylbenzylMmt4-MethoxytritylMo b4-MethoxybenzylMtr4-Methoxy-2,3,6-trime thylphenylsulfonylMtt4-MethyltritylNpys3 -Nitro-2-pyridylsulfenylOAllAllyl esterOtButert. Butyl esterOBt3-Hydroxy-1,2,3-benzotriazole esterOcHexCyclohexyl esterOcPenCyclopentyl esterODhbt3-Hydroxy-4-oxo-3,4-dihydro-1, 2,3-benzotriazine esterODmab4-{-[1-(4,4-dimethyl-2,6-dioxo cyclohexylidene)-3-methylbutyl]amino}ben zyl esterOFm9-Fluorenylmethyl esterOMpe3-Methylpent-3-yl esterOPfpPentafl uorophenyl esterOPp2-Phenylisopropyl esterOSuHydroxysuccinimide esterPbf2,2,4,6,7-Pentamethyldihydrobenz ofurane-5-sulfonylPmc2,2,5,7,8-Pentameth ylchroman-6-sulfonylStButert.

3 ButylthioTfaTrifl uoroacetylTmob2,4,6-TrimethoxybenzylTrtT ritylTosp-ToluenesulfonylXan9-Xanthydryl ZBenzyloxycarbonylList of AbbreviationsSolid- Phase Peptide Synthesis6 ReagentsBTFFHBis(tetramethylene)fl uoroformamidinium hexafl uorophosphateBOPB enzotriazolyloxytris(dimethylamino)phosp honium hexafl uorophosphateDBUD iazabicyclo[ ]undec-7-eneDCCD icyclohexylcarbodiimideDEBPT3-(Diethoxy- phosphoryloxy)-3H-benzo [d][1,2,3] triazin-4-oneDICD iisopropylcarbodiimideDTED ithioerythritolDIPEAD iisopropylethylamineDMAPN,N-Dimethylamin opyridineEDTE thanedithiolHATUO-(7-Azabenzotriazolyl)- tetramethyluronium hexafl uorophosphate* HBTU(Benzotriazole-1-yl) tetramethyluronium hexafl uorophosphate*HOAt1-Hydroxy-7-aza-benzot riazoleHOBt1-HydroxybenzotriazolePyBOP(B enzotriazol-1-yl)oxy-tris-pyrrolidino-ph osphonium hexafl uorophosphateTATU(7-Azabenzotriazolyl) tetramethyluronium tetrafl uoroborate*TBTU(Benzotriazolyl) tetramethyluronium tetrafl uoroborate*TEAT riethylamineTFATrfl uoroacetic acidTFMSAT rifl uoromethanesulfonic acidTEST riethylsilaneTFFHT etramethylfl uoroformamidinium hexafl uorophosphateTIST riisopropylsilaneTMSBrTrimethylsilyl bromideTMSClTrimethylsilyl chlorideTMSOTfTrimethylsilyl trifl uoromethanesulfonateTNBS2,4,6-Trinitrobe nzenesulfonic acidResinsBHAB enzhydrylamineDHPP4-(1 ,1 -Dimethyl-1 -hydroxypropyl)phenoxyacetyl alanyl aminomethylpolystyreneMBHA4-Methylbenzhy drylaminePAMP henylacetamidomethylPDDMP olymeric diphenyldiazomethane* cf.

4 I. Abdelmoty, F. Albericio, Carpino, Foxman, and Kates, Lett. Pept. Sci. 1 (1994) 57. 7 SolventsAcOHAcetic acidDCMD ichloromethaneDMAN,N-DimethylacetamideDM FN,N-DimethylformamideDMSOD imethyl sulfoxideHFIPH exafl uoroisopropanolIPAI sopropanolMTBEM ethyl tert. butyl etherNMPN-MethylpyrrolidoneTFET rifl uoroethanolMiscellaneousAAAmino AcidDKPD iketopiperazineFTIRF ourier Transformed Infra RedHPLCHigh Performance Liquid ChromatographyMALDI-MSMatrix-Assisted Laser Desorption/Ionization Mass SpectrometryMAS-NMRM agic Angle Spinning Nuclear Magnetic ResonanceMSMass SpectrometrySPOSS olid Phase Organic SynthesisSPPSS olid Phase Peptide SynthesisTLCThin Layer ChromatographySolid- Phase Peptide Synthesis8 This publication is a practical vademecum in which Bachem s chemists involved in Solid Phase Synthesis for many years have gathered their knowledge and experience in idea is to discuss the variables of Solid Phase Synthesis and to present the choices, advantages and drawbacks of each one enabling an optimal selection for an easy and successful procedures described in this brochure are routinely used but we can t guarantee that they can be applied in all cases.

5 When in doubt it is strongly recommended to per-form feasibility experiments before using the bulk of the the last years, several books have been published in which SPPS is a major topic. We want to cite them apart from the literature references. Methods in Enzymology 289, Solid Phase Peptide Synthesis ,( Fields Ed) Academic Press 1997. Chemical Approaches to the Synthesis of peptides and Proteins,(P. Lloyd-Williams, F. Albericio, E. Giralt Eds), CRC Press 1997. Fmoc Solid Phase Peptide Synthesis , A Practical Approach, ( Chan, White Eds), Oxford University Press 2000. Solid Phase Synthesis , A Practical Guide,( Kates, F. Albericio Eds), Marcel Dekker 2000. Houben-Weyl E22a, Synthesis of peptides and Peptidomimetics(M. Goodman, Editor-in- chief; A. Felix, L. Moroder, C. Toniolo, Eds), Thieme 2002, Historical BackgroundSolid Phase Peptide Synthesis (SPPS) can be defi ned as a process in which a Peptide anchored by its C-terminus to an insoluble polymer is assembled by the successive ad-dition of the protected amino acids consti-tuting its amino acid addition is referred to as a cycle consisting of: a) cleavage of the N -protecting group b) washing steps c) coupling of a protected amino acid d) washing stepsAs the growing chain is bound to an insoluble support the excess of reagents and soluble by-products can be removed by simple fi ltration.

6 Washing steps with appropriate solvents ensure the complete removal of cleavage agents after the de-protection step as well as the elimination of excesses of reagents and by-products resulting from the coupling step. For a general scheme of SPPS see Fig. 1 on p. 10. Table 1 gives an overview of im-portant developments during the history of INTRODUCTION9 Table 1. 50 Years of history - A choice of key eldDevelopment of SPPS [1], insoluble carrier: crosslinked polystyrene; N -protecting group: Boc1967 SakakibaraHF-cleavage [2]1970 Pietta & MarshallIntroduction of BHA-resin for the Synthesis of Peptide amides [3], MBHA-resin: Matsueda & Stewart 1981 [4]1970 Carpino & HanFmoc, a base labile N -protecting group [5]1973 WangDevelopment of p-alkoxybenzyl alcohol resin (Wang resin) [6], cleav-age: TFA; N -protection: Bpoc1976 Burgus & RivierApplication of preparative reversed Phase HPLC for the purifi cation of peptides prepared by Boc SPPS [7]1977 Barany et concept of orthogonal protection schemes [8]1978 Meienhofer et strategy.

7 Carrier: p-alkoxybenzyl alcohol resin; N -protection: Fmoc; side-chain protection: TFA- labile, Boc, tBu; fi nal cleavage: TFA [9]1985 Houghten and othersSimultaneous parallel Peptide Synthesis , Synthesis of Peptide libraries (T-bags, pins, etc.) [10,11]1985 Rapp and othersPolystyrene-polyethylene glycol grafts TentaGel [12]1987 Rink and othersIntroduction of various TFA-labile linkers for the Fmoc/tBu SPPS of Peptide amides [13 15]1987 Sieber Xanthenyl linker for the Fmoc/tBu SPPS of fully protected Peptide amides, cleavage: 1% TFA/DCM [16]1987 Mergler et of 2-methoxy-4-alkoxybenzyl alcohol resin SASRIN (Super Acid Sensitive ResIN) for the Fmoc/tBu SPPS of fully pro-tected Peptide fragments, cleavage: 1% TFA/DCM [17]1988 Barlos et resin for the Fmoc/tBu SPPS of fully protected Peptide fragments, cleavage: AcOH/TFE/ DCM (1:1:3) or HFIP/DCM (1:4) [18]1993 Hobbs de Witt, Ellman and othersCombinatorial Chemistry; Solid Phase Organic Synthesis (for rapid Synthesis of libraries of small molecules [19-22])1995 Mutter et dipeptides [23]2002 Gogoll and othersMicrowave-accelerated SPPS [24]2003 White and othersFmoc SPPS of long peptides (100 AA) [25]Although in general acidolytic cleavage from the resin is the method of choice to release the Peptide at the end of the Synthesis , a broad range of resins susceptible to be cleaved by nucleophiles such as the Kaiser oxime resin [26] and the p-carboxybenzyl alcohol linker [27] or by photolysis [28] has gained often, these moieties are not com-patible with the conditions of Fmoc SPPS, whereas allyl-based anchors [29] are re-sistant towards the cleavage conditions of Boc as well as Fmoc protecting groups.

8 The so-called safety-catch linkers are per-fectly compatible with both Boc and Fmoc chemistries. Only after an activation step they are highly sensitive towards nucleo-philes the sulfonamide linker [30] or 4-hydrazinobenzoic acid [31]. Solid - Phase Peptide Synthesis102. Boc or Fmoc?The choice of an adequate combination of protecting groups/ Solid support is the fi rst step on the way to achieve a successful Synthesis . For standard SPPS this choice is generally limited to a Boc/benzyl or a Fmoc/tBu based scheme. During the fi rst 15 years of SPPS, the Boc group has been used almost exclusively. Even if this technique permitted remark-able synthetic achievements [32,33] the introduction of a new type of protecting group has offered more fl exibility for the modifi cation of the Peptide chain and/or more specifi city in the cleavage of the N - versus the side-chain protecting combination Fmoc/tBu has met these requirements and broadened the scope of SPPS.

9 Moreover, the development of new resin derivatives has allowed the cleavage of fully protected sequences which can be further coupled in SPPS or in a classical solution addition, a variety of selectively cleavable protecting groups offers new perspectives for on-resin modifi cation (cyclization, for-mation of disulfi de bridges, derivatization of side chains, etc ).The main characteristics of the two general approaches are outlined in Table Manual SynthesisThe classical reactor for SPPS merely consists of a cylindrical vessel with a fritted disc and a removable lid equipped with a mechanical stirrer. Shakers have already been used by Merrifi eld, for a popular model see the photograph on p. 74 in [34]. The resin may also be stirred by bubbling nitrogen through, however more elaborate equipment is required. For rapid small scale Synthesis a small fritted glass funnel is suffi cient. Oxygen and moisture need not be strictly excluded, but the cleavage of the N protecting group should be performed under a hood as to avoid exposure to piperi-dine (Fmoc cleavage) or TFA (Boc cleavage).

10 The swelling of the resin has to be taken into consideration in the choice of the reactor size. Normally, the volume of the PlinkerAA1PG1 TPGHXP linkerAA1PG1 PlinkerHAA1PG1 PlinkerAA2PG2AA3PG3AA4 HAA1AA2AA3AA4 HXHcoupling of TPG-AA1(PG1)-OHcleavage of TPGfurther coupling anddeprotection stepsnot all amino acidsrequire side-chain protectionfi nalcleavageremoved during the fi nal cleavagedesiredpeptideFig. scheme of SPPS. X = O, NH AA = Amino Acid PG = Protecting GroupP = Polymer SupportTPG = Temporary Pro-tecting Group11swollen Peptide resin will slowly increase during chain elongation. When synthesizing a medium-sized Peptide (20 30 AA) using Fmoc SPPS, a 100 150 ml reactor will suf-fi ce for ca. 10 g of resin. The swelling will be more important in Boc SPPS mostly during the TFA deprotection step; a 250 ml reactor would be recommended for the above- mentioned Synthesis . Vessels for small-scale SPPS are depicted in Fig 2a ( ), Fig. 2b (p. 17) shows a large-scale reactor.