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SOLID PHASE PEPTIDE SYNTHESIS - Pharmaceutical-Networking ...
SOLID
PHASE
PEPTIDE
SYNTHESIS
SOLID PHASE PEPTIDE SYNTHESIS - Pharmaceutical-Networking ...
Solid-Phase Peptide Synthesis

TIPS AND TRICKS FOR
SOLID PHASE PEPTIDE SYNTHESIS
FROM THE EXPERTS AT BACHEM
      Table of Contents

                  List of Abbreviations                                                          05
                  Foreword                                                                       08
       I          Introduction                                                                   08
       1.         Historical Background                                                          08
       2.         Fmoc or Boc?                                                                   10
       3.         Equipment                                                                      10
       3.1.       Manual Synthesis                                                               10
       3.2.       “Quasi Continuous Flow”                                                        11
       3.3.       Fully Automated SPPS                                                           12
       II         Fmoc Based SPPS                                                                12
       1.         Resins                                                                         12
       1.1.       General Remarks                                                                12
       1.2.       General Handling of the Resins                                                 13
       1.3.       Resins Available from Bachem                                                   14
       1.3.1      Resins for the Synthesis of Peptide Acids                                      14
                  Wang resin and preloaded Wang resins
                  DHPP-resin and Fmoc-Pro-DHPP-resin
                  PDDM-resin
       1.3.2.     Resins for the Synthesis of Peptide Amides                                     15
                  Tricyclic amide linker resin
                  Rink amide resin
                  4,4’-Dialkoxybenzhydrylamine resin
                  Other TFA-labile amide resins
       1.3.3.     Resins for the Synthesis of Fully Protected Peptide Fragments                  15
                  SASRIN and preloaded SASRIN resins
                  2-Chlorotrityl chloride resin and preloaded 2-chlorotrityl resins
                  Xanthenyl linker resin (for the synthesis of fully protected peptide amides)
                  PDDM-resin
       1.3.4.     Resins for the Synthesis of Peptide Alcohols                                   16
                  SASRIN
                  PDDM-resin
                  2-Chlorotrityl chloride resin
                  3,4-Dihydro-2H-pyran-2-ylmethoxymethyl resin (Ellman’s dihydropyrane resin)
                  Further resins

2
SOLID PHASE PEPTIDE SYNTHESIS - Pharmaceutical-Networking ...
1.4.     Linkers                                                                      20
2.       The Fmoc Group                                                               22
2.1.     General Remarks                                                              22
2.2.     Cleavage Procedures                                                          22
3.       Fmoc Amino Acid Derivatives                                                  23
3.1.     Side-Chain Protecting Groups                                                 23
3.2.     Side-Chain Protection Schemes                                                24
3.3.     Protection of Cys During Fmoc SPPS of Peptides Containing Disulfide Bridges   26
3.3.1.   Peptides Containing a Single Disulfide Bridge                                 27
3.3.2.   Peptides Containing Two Disulfide Bridges                                     27
3.3.3.   Peptides Containing Three Disulfide Bridges                                   28
3.3.4.   Simultaneous Formation of Disulfide Bridges                                   28
4.       Coupling Reagents and Methods                                                29
4.1.     General Remarks                                                              29
4.2.     Activation Methods                                                           30
4.2.1.   Carbodiimides – Carbodiimide/HOBt                                            30
4.2.2.   Activation by Phosphonium and Uronium/Aminium Salts                          31
4.2.3.   Fmoc Amino Acid Active Esters                                                31
4.2.4.   Fmoc Amino Acid Fluorides and Chlorides                                      31
4.3.     Monitoring of Coupling and Deblocking                                        32
4.3.1.   Kaiser Test                                                                  32
4.3.2.   TNBS Test                                                                    32
4.3.3.   Acetaldehyde/Chloranil Test                                                  33
4.3.4.   Bromophenol Blue Test                                                        33
4.3.5.   Cleavage of Samples                                                          33
4.4.     Capping                                                                      34
4.5.     Aggregation/ Difficult Sequences                                              34
5.       Cleavage from the Resin                                                      35
5.1.     Simultaneous Cleavage from the Resin/Side-Chain Deprotection                 35
5.2.     Mix Your Own Cocktail                                                        36
5.3.     Cleavage of Protected Peptide Fragments                                      36
6.       Side Reactions in Fmoc SPPS                                                  36
6.1.     Diketopiperazine Formation                                                   36
6.2.     Aspartimide Formation                                                        37
6.3.     Transfer of Pmc to Trp During TFA Cleavage                                   37
6.4.     3-(1-Piperidinyl)alanine Formation                                           37
6.5.     Incomplete Fmoc Cleavage                                                     39
6.6.     Guanidinylation of Free Amino Moieties During Coupling                       39
6.7.     Side Reactions of Methionine                                                 39
6.8.     N-O Shift                                                                    39
7.       Standard Fmoc Cycle                                                          39
8.       References                                                                   41

                                                                                           3
Solid-Phase Peptide Synthesis

       III        Boc Based SPPS                               46
       1.         Resins                                       46
       1.1.       Resins for the Synthesis of Peptide Acids    46
                  Chloromethyl polystyrene (Merrifield resin)
                  PAM-resin
       1.2.       Resins for the Synthesis of Peptide Amides   46
                  BHA-resin
                  MBHA-resin
       1.3.       Further Resins                               47
                  4-Formyl-phenoxymethyl polystyrene
       2.         The Boc Group                                47
       2.1.       General Remarks                              47
       2.2.       Deprotection                                 48
       2.3.       Neutralization                               49
       3.         Boc Amino Acid Derivatives                   49
       4.         Coupling Reagents and Methods                51
       5.         Cleavage from the Resin                      51
       5.1.       HF                                           51
       5.2.       TFMSA                                        52
       5.3.       TMSOTf                                       52
       5.4.       HBr/TFA                                      52
       6.         Side Reactions in Boc SPPS                   52
       6.1.       Diketopiperazine Formation                   53
       6.2.       Aspartimide Formation                        53
       6.3.       Homoserine Lactone Formation                 53
       6.4.       N-O Shift                                    53
       6.5.       Side Reactions Involving Glu                 53
       6.6.       Asp-Pro Cleavage                             53
       7.         Standard Boc Cycle                           53
       8.         References                                   55

4
List of Abbreviations
Protecting Groups and Active Esters

Acm           Acetamidomethyl
Adpoc         2-(1’-Adamantyl)-2-propyloxycarbonyl
Aloc          Allyloxycarbonyl
Boc           tert. Butyloxycarbonyl
Bom           Benzyloxymethyl
2-BrZ         2-Bromobenzyloxycarbonyl
tBu           tert. Butyl
Bzl           Benzyl
2-ClZ         2-Chlorobenzyloxycarbonyl
Dde           1-(4,4-Dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl
2,6-diClBzl   2,6-Dichlorobenzyl
Dmb           2,4-Dimethoxybenzyl
Dnp           2,4-Dinitrophenyl
Fm            9-Fluorenylmethyl
Fmoc          9-Fluorenylmethyloxycarbonyl
For           Formyl
Hmb           2-Hydroxy-4-methoxybenzyl
MBzl          4-Methylbenzyl
Mmt           4-Methoxytrityl
Mob           4-Methoxybenzyl
Mtr           4-Methoxy-2,3,6-trimethylphenylsulfonyl
Mtt           4-Methyltrityl
Npys          3-Nitro-2-pyridylsulfenyl
OAll          Allyl ester
OtBu          tert. Butyl ester
OBt           3-Hydroxy-1,2,3-benzotriazole ester
OcHex         Cyclohexyl ester
OcPen         Cyclopentyl ester
ODhbt         3-Hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine ester
ODmab         4-{-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl]amino}benzyl ester
OFm           9-Fluorenylmethyl ester
OMpe          3-Methylpent-3-yl ester
OPfp          Pentafluorophenyl ester
OPp           2-Phenylisopropyl ester
OSu           Hydroxysuccinimide ester
Pbf           2,2,4,6,7-Pentamethyldihydrobenzofurane-5-sulfonyl
Pmc           2,2,5,7,8-Pentamethylchroman-6-sulfonyl
StBu          tert. Butylthio
Tfa           Trifluoroacetyl
Tmob          2,4,6-Trimethoxybenzyl
Trt           Trityl
Tos           p-Toluenesulfonyl
Xan           9-Xanthydryl
Z             Benzyloxycarbonyl
                                                                                                5
Solid-Phase Peptide Synthesis

      Reagents

       BTFFH         Bis(tetramethylene)fluoroformamidinium hexafluorophosphate
       BOP           Benzotriazolyloxytris(dimethylamino)phosphonium hexafluorophosphate
       DBU           Diazabicyclo[5.4.0]undec-7-ene
       DCC           Dicyclohexylcarbodiimide
       DEBPT         3-(Diethoxy-phosphoryloxy)-3H-benzo [d][1,2,3] triazin-4-one
       DIC           Diisopropylcarbodiimide
       DTE           Dithioerythritol
       DIPEA         Diisopropylethylamine
       DMAP          N,N-Dimethylaminopyridine
       EDT           Ethanedithiol
       HATU          O-(7-Azabenzotriazolyl)-tetramethyluronium hexafluorophosphate*
       HBTU          (Benzotriazole-1-yl) tetramethyluronium hexafluorophosphate*
       HOAt          1-Hydroxy-7-aza-benzotriazole
       HOBt          1-Hydroxybenzotriazole
       PyBOP         (Benzotriazol-1-yl)oxy-tris-pyrrolidino-phosphonium hexafluorophosphate
       TATU          (7-Azabenzotriazolyl) tetramethyluronium tetrafluoroborate*
       TBTU          (Benzotriazolyl) tetramethyluronium tetrafluoroborate*
       TEA           Triethylamine
       TFA           Trfluoroacetic acid
       TFMSA         Trifluoromethanesulfonic acid
       TES           Triethylsilane
       TFFH          Tetramethylfluoroformamidinium hexafluorophosphate
       TIS           Triisopropylsilane
       TMSBr         Trimethylsilyl bromide
       TMSCl         Trimethylsilyl chloride
       TMSOTf        Trimethylsilyl trifluoromethanesulfonate
       TNBS          2,4,6-Trinitrobenzenesulfonic acid

      Resins

       BHA           Benzhydrylamine
       DHPP          4-(1’,1’-Dimethyl-1’-hydroxypropyl)phenoxyacetyl alanyl aminomethylpolystyrene
       MBHA          4-Methylbenzhydrylamine
       PAM           Phenylacetamidomethyl
       PDDM          Polymeric diphenyldiazomethane

      * cf. I. Abdelmoty, F. Albericio, L.A. Carpino, B.M. Foxman, and S.A. Kates, Lett. Pept. Sci. 1 (1994) 57.

6
Solvents

AcOH       Acetic acid
DCM        Dichloromethane
DMA        N,N-Dimethylacetamide
DMF        N,N-Dimethylformamide
DMSO       Dimethyl sulfoxide
HFIP       Hexafluoroisopropanol
IPA        Isopropanol
MTBE       Methyl tert. butyl ether
NMP        N-Methylpyrrolidone
TFE        Trifluoroethanol

Miscellaneous

AA         Amino Acid
DKP        Diketopiperazine
FTIR       Fourier Transformed Infra Red
HPLC       High Performance Liquid Chromatography
MALDI-MS   Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry
MAS-NMR    Magic Angle Spinning Nuclear Magnetic Resonance
MS         Mass Spectrometry
SPOS       Solid Phase Organic Synthesis
SPPS       Solid Phase Peptide Synthesis
TLC        Thin Layer Chromatography

                                                                           7
Solid-Phase Peptide Synthesis

      FOREWORD
      This publication is a practical vademecum       • Methods in Enzymology 289,
      in which Bachem’s chemists involved in          Solid Phase Peptide Synthesis,
      solid phase synthesis for many years have       (G.B. Fields Ed)
      gathered their knowledge and experience         Academic Press 1997.
      in SPPS.                                        • Chemical Approaches to the Synthesis of
      The idea is to discuss the variables of solid   Peptides and Proteins,
      phase synthesis and to present the choices,     (P. Lloyd-Williams, F. Albericio, E. Giralt Eds),
      advantages and drawbacks of each one            CRC Press 1997.
      enabling an optimal selection for an “easy”     • Fmoc Solid Phase Peptide Synthesis, A
      and successful synthesis.                       Practical Approach,
      The procedures described in this brochure       (W.C. Chan, P.D. White Eds),
      are routinely used but we can’t guarantee       Oxford University Press 2000.
      that they can be applied in all cases. When     • Solid Phase Synthesis, A Practical Guide,
      in doubt it is strongly recommended to per-     (S.F. Kates, F. Albericio Eds),
      form feasibility experiments before using       Marcel Dekker 2000.
      the bulk of the material.                       • Houben-Weyl E22a, Synthesis of Peptides
      During the last years, several books have       and Peptidomimetics
      been published in which SPPS is a major         (M. Goodman, Editor-in- chief; A. Felix, L.
      topic. We want to cite them apart from the      Moroder, C. Toniolo, Eds),
      literature references.                          Thieme 2002, p.665ff.

      I INTRODUCTION

      1. Historical Background                        As the growing chain is bound to an
                                                      insoluble support the excess of reagents
      Solid Phase Peptide Synthesis (SPPS) can        and soluble by-products can be removed
      be defined as a process in which a peptide       by simple filtration. Washing steps with
      anchored by its C-terminus to an insoluble      appropriate solvents ensure the complete
      polymer is assembled by the successive ad-      removal of cleavage agents after the de-
      dition of the protected amino acids consti-     protection step as well as the elimination
      tuting its sequence.                            of excesses of reagents and by-products
      Each amino acid addition is referred to as a    resulting from the coupling step.
      cycle consisting of:                            For a general scheme of SPPS see Fig. 1
                                                      on p. 10. Table 1 gives an overview of im-
       a) cleavage of the Nα-protecting group         portant developments during the history of
       b) washing steps                               SPPS.
       c) coupling of a protected amino acid
       d) washing steps

8
Table 1. 50 Years of history - A choice of key dates.

 Year     Authors                   Development
 1963     Merrifield                 Development of SPPS [1], insoluble carrier: crosslinked polystyrene;
                                    Nα-protecting group: Boc
 1967     Sakakibara                HF-cleavage [2]
 1970     Pietta & Marshall         Introduction of BHA-resin for the synthesis of peptide amides [3],
                                    MBHA-resin: Matsueda & Stewart 1981 [4]
 1970     Carpino & Han             Fmoc, a base labile Nα-protecting group [5]
 1973     Wang                      Development of p-alkoxybenzyl alcohol resin (Wang resin) [6], cleav-
                                    age: TFA; Nα-protection: Bpoc
 1976     Burgus & Rivier           Application of preparative reversed phase HPLC for the purification
                                    of peptides prepared by Boc SPPS [7]
 1977     Barany et al.             The concept of “orthogonal” protection schemes [8]
 1978     Meienhofer et al.         Fmoc/tButyl strategy. Carrier: p-alkoxybenzyl alcohol resin;
                                    Nα-protection: Fmoc; side-chain protection: TFA- labile, e.g. Boc, tBu;
                                    final cleavage: TFA [9]
 1985     Houghten and others       Simultaneous parallel peptide synthesis, synthesis of peptide
                                    libraries (T-bags, pins, etc.) [10,11]
 1985     Rapp and others           Polystyrene-polyethylene glycol grafts e.g. TentaGel [12]
 1987     Rink and others           Introduction 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 al.            Development 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 al.             2-Chlorotritylchloride 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     Combinatorial Chemistry; Solid Phase Organic Synthesis (for rapid
          and others                synthesis of libraries of small molecules [19-22])
 1995     Mutter et al.             Pseudoproline dipeptides [23]
 2002     Gogoll and others         Microwave-accelerated SPPS [24]
 2003     White and others          Fmoc SPPS of long peptides (100 AA) [25]

Although in general acidolytic cleavage from            whereas allyl-based anchors [29] are re-
the resin is the method of choice to release            sistant towards the cleavage conditions of
the peptide at the end of the synthesis,                Boc as well as Fmoc protecting groups. The
a broad range of resins susceptible to be               so-called “safety-catch linkers” are per-
cleaved by nucleophiles such as the “Kaiser             fectly compatible with both Boc and Fmoc
oxime resin” [26] and the p-carboxybenzyl               chemistries. Only after an activation step
alcohol linker [27] or by photolysis [28] has           they are highly sensitive towards nucleo-
gained popularity.                                      philes e.g. the sulfonamide linker [30] or
Quite often, these moieties are not com-                4-hydrazinobenzoic acid [31].
patible with the conditions of Fmoc SPPS,

                                                                                                              9
Solid-Phase Peptide Synthesis

                                                           HX      linker         P

                                                                       coupling of
                                                                       TPG-AA1(PG1)-OH
                                                       PG1

                                          TPG          AA1         linker         P

                                                                       cleavage of TPG
                                                      PG1

                                                H     AA1         linker          P
                                    removed during
                                    the final cleavage
                                                                       further coupling and
                                                                       deprotection steps
                                  PG3      PG2        PG1                                                  Fig. 1.
                                                                                                           General scheme of
                     H    AA4     AA3      AA2        AA1         linker          P                        SPPS.
                                                                                                           X = O, NH
                     not all amino acids                               final                                AA = Amino Acid
                     require side-chain protection                     cleavage                            PG = Protecting Group
                                                                                                           P = Polymer Support
                                    H    AA4         AA3        AA2     AA1   XH desired                   TPG = Temporary Pro-
                                                                                 peptide                   tecting Group

      2. Boc or Fmoc?                                           mation of disulfide bridges, derivatization of
                                                                side chains, etc ).
      The choice of an adequate combination of                  The main characteristics of the two general
      protecting groups/solid support is the first               approaches are outlined in Table 2.
      step on the way to achieve a successful
      synthesis. For standard SPPS this choice
      is generally limited to a Boc/benzyl or a                 3. Equipment
      Fmoc/tBu based scheme. During the first 15
      years of SPPS, the Boc group has been used                3.1. Manual Synthesis
      almost exclusively.                                       The “classical” reactor for SPPS merely
       Even if this technique permitted remark-                 consists of a cylindrical vessel with a fritted
      able synthetic achievements [32,33] the                   disc and a removable lid equipped with a
      introduction of a new type of protecting                  mechanical stirrer. Shakers have already
      group has offered more flexibility for the                 been used by Merrifield, for a popular
      modification of the peptide chain and/or                   model see the photograph on p. 74 in [34].
      more specificity in the cleavage of the Nα-                The resin may also be stirred by bubbling
      versus the side-chain protecting groups.                  nitrogen through, however more elaborate
      The combination Fmoc/tBu has met these                    equipment is required. For rapid small scale
      requirements and broadened the scope of                   synthesis a small fritted glass funnel is
      SPPS. Moreover, the development of new                    sufficient. Oxygen and moisture need not
      resin derivatives has allowed the cleavage                be strictly excluded, but the cleavage of the
      of fully protected sequences which can be                 Nα protecting group should be performed
      further coupled in SPPS or in a classical                 under a hood as to avoid exposure to piperi-
      solution process.                                         dine (Fmoc cleavage) or TFA (Boc cleavage).
      In addition, a variety of selectively cleavable           The swelling of the resin has to be taken
      protecting groups offers new perspectives                 into consideration in the choice of the
      for “on-resin” modification (cyclization, for-             reactor size. Normally, the volume of the

10
Table 2. Fmoc/tBu or Boc/Bzl?

Topic                              Fmoc/tBu                                     Boc/Bzl
Use                                Routine synthesis                            Requires special equipment
     α                                        1)
N /side chain protection           orthogonal                                   both acid labile
TFA treatment                      final cleavage                                repetitive cleavage
HF treatment                       none                                         final cleavage
Automation                         yes                                          yes
Scale                              any scale, including final cleavage           HF cleavage: limited scale
Monitoring:                        UV-absorption, chromophores:                 quantitative ninhydrin test: cumber-
Nα-deblocking,                     Fmoc, dibenzofulvene-piperidine adduct       some
completion of coupling
Synthetic steps                    deblock, wash, couple, wash                  additional neutralization step
Avoidance of DKP                   circumvention tedious;                       change of coupling protocol: con-
formation                          synthesis on 2-chlorotrityl resin:           comitant coupling/neutralization
                                   suppression of DKP formation
Final cleavage                     in SPPS vessel                               special equipment required
Especially                         acid sensitive peptides & derivates, e.g.    base labile peptides;“difficult
recommended for                    O-glycosylated or sulfated peptides          sequences”, aggregation impeded by
                                                                                repetitive TFA treatment
1)
     For a definition see II.3.1.

swollen peptide resin will slowly increase                 Solvents are filtered off by slight suction, or,
during chain elongation. When synthesizing                 more gently, by applying inert gas pressure.
a medium-sized peptide (20–30 AA) using                    In Fmoc/tBu based SPPS the vessel may
Fmoc SPPS, a 100–150 ml reactor will suf-                  also be used for the final cleavage or for the
fice for ca. 10 g of resin. The swelling will be            cleavage of fully protected peptides from
more important in Boc SPPS mostly during                   very acid-labile resins such as SASRIN.
the TFA deprotection step; a 250 ml reactor                Using a manual synthesizer may be more
would be recommended for the above-                        cumbersome than employing a fully
mentioned synthesis. Vessels for small-                    automated one, but any parameter can
scale SPPS are depicted in Fig 2a (p.12),                  be changed at any time. A more thorough
Fig. 2b (p. 17) shows a large-scale reactor.               monitoring is possible as samples for
 At the beginning of each coupling cycle,                  analysis can be removed at each stage of
deblocking or washing step the resin and                   the synthesis.
the solution have to be mixed thoroughly,
followed by slow stirring or shaking for the               3.2. “Quasi Continuous Flow”
remaining process. All the beads have to                   In this approach the solid support is packed
be suspended in the liquid for thorough                    into a column and the reagents and sol-
washing, efficient coupling, and complete                   vents are delivered by a pump. The resins
deblocking. It is important to watch for                   used in this technique must be able to with-
beads sticking to the wall of the vessel                   stand considerable pressure and, at the
especially during the coupling and rinse                   same time, keep a constant volume while
them from the wall with a small amount of                  changing solvents. The standard polysty-
solvent if necessary. “Sticking beads” may                 rene-based resin is not suitable for that
become a problem when stirring too vigor-                  purpose as the volume of the beads mark-
ously. Silylation of the glassware improves                edly depends on the solvent. Nevertheless,
the surface hydrophobicity and prevents                    continuous-flow synthesizers taking into
the beads from sticking to the wall of the                 account the shortcomings of swollen poly-
vessel.                                                    styrene have been developed [35,36].

                                                                                                                       11
Solid-Phase Peptide Synthesis

       This type of synthesizer is best used for      [45] or “consumed” [46] (the Fmoc amino
       Fmoc-based protocols. The Boc protocols        acid derivative) and concomitantly released
       generate ionic species during the Boc cleav-   (HOBt or HOAt) during coupling. Monitoring
       age, which cause considerable changes in       via changes of conductivity [46] allows the
       swelling due to electrostatic forces.          monitoring on a real-time basis and end
       A synthesizer has been developed in which      point value can be given to determine the
       swelling is monitored, considering that dur-   completion of the coupling reaction.
       ing Fmoc-SPPS, volume changes in a given
       solvent can only be caused by the growing
       peptide chain [37].
       Composite material made from a rigid sup-      II FMOC-BASED SPPS
       port such as Kieselguhr particles [38] or
       large pore crosslinked polystyrene [39] in     1. Resins
       which dimethylacrylamide [40] has been
       polymerized are used for continuous flow        1.1. General Remarks
       synthesis.                                     All resins marketed by Bachem are obtained
       Poly(ethylene glycol)-based supports such      from beaded polystyrene crosslinked with
       as TentaGel or PEGA have been introduced       1% divinylbenzene (a mixture of the meta
       for batch as well as continuous flow synthe-    and the para isomer). This degree of cross-
       sis [41–43]. For a review of recent develop-   linking is optimal for SPPS. A higher level
       ments in this field see [12].                   of crosslinking would reduce the swelling
                                                      whereas a decrease would cause a consid-
       3.3. Fully Automated SPPS                      erable loss of mechanical stability in the
       A variety of fully automated synthesizers      swollen state.
       for batchwise and continuous flow SPPS is       The carrier resins for SPPS are obtained
       commercially available [44]. Fig. 2a shows     from this polymer or from the chlorometh-
       the reaction vessels of such a machine         ylated material. In the second case, the
       allowing parallel small-scale syntheses. In    available load is restricted by the degree of
       the meantime, fully automated synthesizers     chloromethylation. The average bead size is
       employing microwave irradiation for accel-     adjusted by the conditions of polymeriza-
       erating the synthetic steps were success-      tion. Bachem offers the most popular size
       fully introduced to the market [24].           distribution 200–400 mesh (average diam-
       Fmoc/tBu SPPS permits automatic moni-          eter 38–75 μm). A variety of resin derivatives
       toring and adequate adjustment of de-          is also available as large beads: 100–200
       protection and coupling times in order to      mesh (average diameter 75–150 μm). With
       achieve complete conversions. The monitor-     such resins, reaction times may have to be
       ing relies on strong chromophores which        prolonged due to limited diffusion towards
       are either released during deprotection        the interior of the beads.

 Fig. 2a.
 Fully auto-
 mated reactor
 for parallel
 small-scale
 SPPS, reac-
 tion vessels.

12
The load of the resins is adapted to the          As mentioned above, the coupling rate is
needs of routine SPPS: 0.7–1 meq/g before         controlled by the diffusion of the activated
the loading of the first Fmoc amino acid.          species into the swollen bead, i.e. the larger
Loads may be deliberately reduced, e.g., for      the bead the slower the coupling. Thus, the
side-chain cyclization, for the synthesis of      coupling can’t be accelerated by vigorous
long peptide chains (above 30–40 residues),       stirring. Slight stirring or shaking is suf-
or for the preparation of sequences pre-          ficient to support the diffusion of reagents
senting intrinsic difficulties. Resins having      into the beads.
a particularly high load can be prepared by       Sudden shrinkages should alarm the op-
Bachem on request.                                erator. This phenomenon is caused by the
                                                  aggregation of the peptide chain which will
1.2. General Handling of the Resin                impede the continuation of the synthesis.
The term “Solid phase” peptide synthesis          Complete coupling reaction and deblocking
is actually misleading, gel-phase synthe-         will be difficult to attain due to the steric
sis would be more appropriate [47]. The           hindrance created by the aggregation. A
swelling, i.e. the solvation of the polystyrene   range of methods to improve the efficiency
chains and the functionalized moieties            of these key steps will be dealt with later on.
including the growing peptide, remains            As the peptide resin may be deliberately
essential for successful SPPS and even            swollen or shrinked, washes with shrinking
more so in SPOS. The swelling volumes             solvents such as IPA accelerate the removal
of polystyrene-based resins in the most           of excesses of reagents, and time must then
important solvents have been determined           be allowed for proper swelling, e.g. in DMF.
by Santini [48]. The complete swelling of the     The peptide resin should not be shrinked
dry resin may take up to 1 hour [48].             when the peptide may aggregate, which is
Unmodified crosslinked polystyrene and             rather difficult to predict [50] and during
chloromethylated polystyrene swell very           the first cycle of a synthesis. On the other
well in apolar solvents such as toluene,          hand, when shrinking the resin with MTBE
dioxane and DCM, moderately in DMF and            before coupling the maximum concentra-
poorly, if at all in alcohols and water. The      tion of coupling reagents is attained. For
swelling behaviour of derivatized polysty-        fragment coupling it has even been recom-
rene depends on the load and the polarity         mended to treat the dried resin with a solu-
of the functional groups. These moieties          tion of the activated fragment [51].
are usually rather polar: amides, alcohols,       A WASH represents a short treatment
amines, esters, ethers, etc., and improve the     (1–5 min, depending on the amount) of the
interactions with polar solvents whereas no       peptide resin with a solvent under gentle
“additional polarity” is gained when working      stirring. The swollen resin may be inspected
with the “purely aromatic” 2-chlorotrityl         under a microscope. Regular round spheres
chloride resin. So, after the loading with an     should be observed, but not neccesar-
Fmoc amino acid, the Fmoc group is split          ily smooth surfaces. Torn beads and fines
off with piperidine/DMF (1:4) considerably        result from inappropriate treatment of the
slower from the 2-chlorotrityl resin than         resin and will clog frits. Mechanical stress
from the Wang resin derivative.                   has to be minimized as swollen beads are
SPPS relies on proper swelling in polar           rather susceptible to abrasion. So they
solvents as polar aprotic solvents facilitate     should not be stirred with a magnetic bar
coupling (see II. 4); good swelling means         (except for cleavage, as the carrier is nor-
good accessibility of coupling sites and          mally not recovered). Reaction vessels and
thus, a smooth reaction (even though a few        stirrer blades have to be designed to mini-
exceptions to this rule have been observed        mize shearing forces. As already mentioned,
[49]). Concurrently with the peptide elonga-      a slight stirring will suffice, and the system
tion, swelling in DMF normally increases.         has to be thoroughly mixed only when start-
But it should be kept in mind especially          ing a washing step or a reaction. Vigorous
when synthesizing long peptides that swell-       suction and suction to dryness will unnec-
ing also means dilution of coupling sites         essarily stress the peptide-resin. Applying
and reagents. A slow and steady increase of       inert gas pressure to remove the solvent is
their excess will compensate for this effect.     a gentle alternative. The inert atmosphere

                                                                                                    13
Solid-Phase Peptide Synthesis

      may be beneficial, though inertization is not     of Fmoc-Pro-OH (yielding a modified tert.
      an esential requirement of SPPS.                 butyl ester) is impeded as well. Bachem
      The load of the carrier resin is determined      offers the preloaded Fmoc-Pro-DHPP resin
      by elemental analysis (N, Cl) and/or by cou-     (D-1830).
      pling an Fmoc amino acid and determining
      the resulting conversion (see below). Resins     Diphenyldiazomethane resin (PDDM-resin)
      may also be characterized, e.g., by FTIR-        (D-2230)
      spectroscopy. The growing interest in SPOS       PDDM-resin, i.e. diphenyldiazomethane
      led to renewed interest in the method and        resin D-2230, readily reacts with carboxylic
      refined instrumentation for the character-        acids in DCM [57,58]. The rate correlates
      ization of solid samples [52]. MAS-NMR can       with the acidity of the substrate. Nitrogen,
      also been carried out if the resin is properly   the only by-product, is evolved concomi-
      swollen [53].                                    tantly. As the incoming amino acid is not
      Requirements of storage depend on the            activated racemization is suppressed; as a
      nature of the resin: PDDM-resin and photo-       result, PDDM-resin lends itself especially
      labile resins have to be protected from light,   for the synthesis of peptides containing a
      2-chlorotritylchloride resin is sensitive to     C-terminal Cys or His.
      humidity; in most cases the resins have to       Due to the simple and reliable linkage pro-
      be stored in the deep-freezer.                   tocol PDDM-resin is especially recommend-
                                                       ed for the anchoring of expensive amino
      1.3. Resins Available from Bachem                acids. The excess of derivative can also be
                                                       easily recovered as no coupling reagent is
      1.3.1. Resins for the synthesis of peptide       used. Bulky amino acids such as Fmoc-Aib
      acids                                            and peptide fragments react readily with
      Wang resin and preloaded Wang resins             PDDM-resin. The acids don’t have to be pure
      (D-1250 (200–400 mesh) and D-2115                but they must not be contaminated by other
      (100–200 mesh))                                  acids of comparable strength or stronger;
      Wang resin, i.e. p-alkoxybenzyl alcohol          on the other hand, selective alkylation of
      resin, may be termed the standard resin for      the more acidic carboxyl group may be at-
      Fmoc/tBu SPPS of “peptide acids”. The tert.      tained. PDDM-resin reacts preferentially
      butyl type side-chain protection is concomi-     with the α-carboxyl group of Fmoc-Glu-OH
      tantly removed during acidolytic cleavage        in DCM/DMF (3:1), the γ-carboxyl can be
      from this resin.                                 modified otherwise (M. Mergler, unpub-
      The esterification of Wang resin as well          lished results). Loading of PDDM-resin is
      as of other resins bearing hydroxyl groups       best performed in DCM but small amounts
      with Fmoc amino acids is a crucial step in       of other solvents such as DMF, THF, or
      SPPS. It is more difficult than it may seem       dioxane may be added to improve solubil-
      considering that high conversion and, espe-      ity. In contrast to standard esterification
      cially, minimal racemization are desired. We     procedures where conversion can’t be easily
      therefore recommend the use of preloaded         followed, alkylation with PDDM-resin can by
      resins. Bachem offers a broad range of           monitored visually due to the color change
      Fmoc L- and D-amino acids coupled to             of the resin. The deeply violet resin turns
      Wang resin. If the resin derivative you need     yellowish while nitrogen evolves. After dis-
      is not yet available please ask for a quota-     coloration, shaking or stirring is continued
      tion. In any case, Bachem guarantees high        for 4 to 6 hours and the resin is carefully
      loading and minimal racemization.                washed with DCM.
                                                       Moreover, PDDM-resin is the resin of choice
      DHPP Resin and Fmoc-Pro-DHPP resin               for the side-chain anchoring of Fmoc-Asp
      DHPP-resin, i.e. 4-(1’,1’-dimethyl-1’-hy-        and Fmoc-Glu derivatives, especially when
      droxypropyl) phenoxyacetyl alanyl amino-         the standard esterification of Wang resin or
      methyl polystyrene, has been developed           SASRIN (e.g., with DCC/DMAP) has proven
      especially for the synthesis of peptides         difficult. When anchoring Fmoc-Asp-NHR
      containing a C-terminal proline [54,55,56].      via the β-carboxyl functionality to PDDM-
      The bulkiness of the linker prevents diketo-     resin, losses due to aspartimide formation
      piperazine formation, but the esterification      can’t occur.

14
Peptides can be cleaved from PDDM-              amides from this resin requires harsher
resin under the same conditions as from         conditions, e.g. a treatment with 95% TFA
Wang resin, even though 2–5% TFA/DCM            and scavengers at 35°C for 2 hours. Peptide
is sufficiently strong to promote cleavage.      amides containing a C-terminal Gly may be
Fully protected peptide fragments may be        split off under milder conditions.
obtained if Tyr(tBu), Lys(Boc), or His(Trt)
are not present. Thus, for safe synthesis of    Other TFA-labile amide resins
fully protected peptide fragments, SASRIN       Bachem also offers the well-established
or 2-chlorotritylchloride resin should be       “PAL” resin 4-alkoxy-2,6-dimethoxybenzyl-
preferred.                                      amine resin [14] (D-2125). Cleavage from
                                                PAL resin requires a lower concentration of
1.3.2. Resins for the synthesis of peptide      TFA than cleavage from the resins de-
amides                                          scribed above.
A special functionality must be introduced      Additionally, peptides containing C-terminal
on the resin to allow the release of the pep-   Asn or Gln derivatives may be obtained by
tide as an amide.                               side-chain linkage of the corresponding
These linkers possess an amino function to      Fmoc-Asp or Fmoc-Glu derivative followed
which the C-terminal amino acid is coupled      by SPPS, acidolytic cleavage yields the C-
and present an electronic structure such        terminal Asn or Gln [63].
that the final acid treatment splits off the     The aldehyde resins D-2570 and D-2575
peptide as an amide.                            may be used for backbone (-CO-NH-)
                                                anchoring of peptides or SPPS of peptide
Tricyclic amide linker resin                    N-alkylamides [64]. The appropriate educt
(D-2200)                                        resins are obtained via reductive amination
The 5-Fmoc-amino-10,11-dihydro-5H-              of D-2570/D-2575. D-2570 should be pre-
dibenzo[a,d]cycloheptenyl-2-oxyacetyl           ferred for Fmoc-SPPS, as the final cleavage
linker based on the dibenzosuberyl protect-     can be performed with TFA under standard
ing group of Pless [59] has been developed      conditions.
by Ramage [60] to enable smooth cleavage
of peptide amides with concomitant side-        1.3.3. Resins for the synthesis of fully pro-
chain deprotection. The linker is coupled to    tected peptide fragments
MBHA-resin modified with DL-norleucine.
The final cleavage is performed using stan-      SASRIN and preloaded SASRIN resins
dard cocktails (see II. 5.)                     (D-1295 (200–400 mesh) and D-2440
                                                (100–200 mesh))
“Rink amide” resins                             SASRIN (Super Acid-Sensitive ResIN) cor-
( D-1675 and D-2080)                            responds to 2-methoxy-4-alkoxy-benzyl
Rink amide AM resin (or Knorr resin)            alcohol resin [17].
D-1675 is obtained by the attachment            As already discussed before, we recom-
of the linker Fmoc-2,4-dimethoxy-4’-            mend the use of SASRIN preloaded with the
(carboxymethyloxy)-benzhydrylamine to           desired Fmoc amino acid. Fully protected
aminomethyl resin [61].                         peptide fragments are obtained by cleavage
The ether derivative 4-(2’,4’-dimethoxy-        with 0.5 to 1% TFA in DCM or by treatment
phenyl-Fmoc-aminomethyl) phenoxymethyl          with HFIP/DCM (1:4) [65]. Fully protected
polystyrene as originally described by Rink     peptide hydrazides can be obtained con-
[13] is also available from Bachem (D-2080).    veniently by Fmoc SPPS employing the
Peptide amides are split off from these res-    SASRIN-derivative D-2285. The derivative
ins by 95% aqueous TFA; scavengers being        D-2550 allows the synthesis of fully pro-
added if necessary.                             tected peptide hydroxamic acids.

4,4’-Dialkoxybenzhydrylamine resin
(D-1600)
N-Fmoc-4-Methoxy-4’-(-carbonylpropyloxy)
benzhydrylamine is linked to H-Ala-amino-
methyl resin [15]. The cleavage of peptide

                                                                                                15
Solid-Phase Peptide Synthesis

      Detailed cleavage protocols and compre-           ides allowing very mild cleavage conditions
      hensive information concerning the use            [16]. The 4-(9-Fmoc-aminoxanthen-3-yloxy)
      of SASRIN are contained in our brochure           butyryl linker coupled to MBHA resin [68]
      SASRIN – a review of its manifold applica-        yields fully protected peptide amides after
      tions which is available free of charge upon      repetitive short treatments of the pep-
      request. It can also be downloaded from our       tide resin with 1% TFA/DCM. The resin will
      homepage at www. bachem.com.                      turn yellow during acidolytic cleavage. The
                                                        cleavage protocol and work-up procedures
      2-Chlorotrityl chloride resin and preloaded       described in the SASRIN brochure can also
      2-chlorotrityl resins                             be applied to this product. The N-terminus
      (D-1955 (200–400 mesh) and D-2930                 may be deprotected before cleavage. Traces
      (100–200 mesh))                                   of carboxylic acids have to be carefully
      2-Chlorotrityl resin [18] is somewhat more        removed if the product is to be subjected to
      acid-labile than SASRIN.                          fragment coupling.
      Loading of 2-chlorotritylchloride resin is        Fully protected fragments obtained from
      achieved by treatment with the triethylam-        SASRIN or 2-chlorotrityl resin can be
      monium salt of the desired Fmoc amino             coupled in solution to amidated fragments
      acid, thus, concomitant racemization is           cleaved from the title resin, providing a way
      minimized. To proceed with SPPS, Fmoc has         for the synthesis of peptide amides by a
      to be split off, but the first deprotection with   convergent approach [69, 70].
      piperidine/DMF takes longer than usual (2 x
      30 minutes).                                      PDDM-resin
      On the other hand, Fmoc-AA-2-chlorotrityl         For the synthesis of protected peptide frag-
      resins are not stable, the Fmoc amino acid        ments on PDDM-resin see p. 14.
      is slowly cleaved upon storage, whereas the
      H-AA-2-chlorotrityl resins can be stored.         1.3.4. Resins for the synthesis of peptide
      Bachem offers a broad range of preloaded          alcohols
      H-AA-2-chlorotrityl resins, resins with           Bachem offers a range of resins which can
      standard substitution as well as the cor-         also be used for the synthesis of (fully pro-
      responding low-load (LL) derivatives. The         tected) peptide alcohols and thiols.
      resin is especially suitable for the synthesis
      of fully protected peptides containing a          SASRIN
      C-terminal Cys or Pro. In case of C-terminal      The alcohol is generated by reductive cleav-
      Pro, diketopiperazines can’t be formed            age. A detailed procedure is described in the
      when proceeding with the SPPS due to the          SASRIN-brochure.
      steric hindrance of the trityl moiety.
      Moreover, 2-chlorotrityl resin is the opti-       PDDM-resin
      mal carrier for the synthesis of peptides         This resin readily alkylates Fmoc amino
      containing a C-terminal tryptophan as the         alcohols in the presence of a catalytic
      bulkiness of the chlorotrityl group prevents      amount of BF3· Et2O in DCM [71]. High loads
      the alkylation of the indole moiety, and thus,    can be obtained rapidly under these mild
      irreversible binding of peptide during final       conditions provided that the alcohol is suf-
      cleavage.                                         ficiently soluble in DCM. Satisfactory loads
      Rapid cleavage of fully protected peptide         have been obtained as well with secondary
      fragments is attained by treatment with           alcohols. The resulting benzhydryl ether
      0.5–1% TFA in DCM or HFIP/DCM (1:4 or 3:7)        can be cleaved by repetitive short treat-
      [66]; further cleavage mixtures have been         ments with 1–2% TFA/DCM. This simple and
      described by Barlos et al. [18].                  efficient approach is our preferred method
                                                        for the preparation of protected peptide
      Xanthenyl linker resin                            alcohols.
      (for the synthesis of fully protected peptide     Thiols are alkylated by PDDM under the
      amides)                                           same conditions with equally good results.
      (D-2040)                                          The resulting thioethers can be cleaved with
      Xanthenyl resin (or Sieber resin) has been        TFA/phenol (9:1) [72] or 95% TFA.
      developed for the synthesis of peptide am-

16
2-Chlorotrityl chloride resin                   Further resins
This resin reacts with Fmoc amino alcohols      For the preparation of C-terminally modi-
in the presence of pyridine (or DIPEA/DMAP)     fied peptides, the side-chain hydroxyl
in DCM/DMF. The reaction is slower than the     groups of Ser and Thr can be alkylated with
alkylation with the PDDM-resin [73,74]. The     PDDM-resin or 2-chlorotrityl chloride resin
resulting ethers can be cleaved by mild acid.   [74] or acetalated by Ellman’s resin.
Thiols react more readily with the resin.       The reaction is less favorable with Thr due
                                                to the steric hindrance of the amino acid
3,4-Dihydro-2H-pyran-2-ylmethoxymethyl          side-chain.
resin                                           Cys and cysteamine derivatives have
(Ellman’s dihydropyrane resin )                 been obtained from SASRIN (D-2165 and
(D-2530)                                        D-2170), PDDM [72] and chlorotrityl resin.
This resin has been conceived especially for
the anchoring of alcohols [75].                 Table 3 gives an overview of the resins avail-
It reacts with Fmoc amino alcohols in           able from Bachem.
dichloroethane to form acetals. Acetalation
and mild cleavage via transacetalation are
catalyzed by strong acids such as benzene-
sulfonic acid.

                                                                                                 Fig. 2b.
                                                                                                 Large-scale
                                                                                                 reactor
                                                                                                 for SPPS
                                                                                                 allowing
                                                                                                 to produce
                                                                                                 kilograms of
                                                                                                 peptide.

                                                                                                                17
Solid-Phase Peptide Synthesis

      Table 3. Resins for Fmoc-SPPS

       Product No.       Name                                          Structure
       D-1250            Wang resin
       (200-400 mesh)    (4-Alkoxybenzyl alcohol resin)
       D-2115
       (100–200 mesh)
       D-1295         SASRINTM resin
       (200-400 mesh) (2-Methoxy-4-alkoxybenzyl alcohol resin)
       D-2440
       (100–200 mesh)
       D-1600         Fmoc-4-methoxy-4’-(γ-carboxypropyloxy)-
       (200–400 mesh) benzhydrylamine linked to Alanyl- aminomethyl
                      resin

       D-1675         Fmoc-2,4-dimethoxy-4’-(carboxy-methyloxy)-
       (200–400 mesh) benzhydrylamine linked to aminomethyl-resin
                      (Rink amide AM resin)

       D-1830         Fmoc-Pro-DHPP-resin
       (200–400 mesh)

       D-1965         2-Chlorotrityl chloride resin
       (200–400 mesh)
       D-2930
       (100-200 mesh)

       D-2040            Xanthenyl linker resin
       (200-400 mesh)    (Sieber resin, 4-[9-Fmoc-amino-xanthen-
                         3-yloxy]-butyryl)-4-methyl-benzhydrylamide
                         resin)

       D-2080            4-(2’,4’-Dimethoxyphenyl-Fmoc-aminomethyl)-
       (200-400 mesh)    phenoxymethyl-polystyrene resin
                         (Rink resin)

       D-2125         PAL resin
       (200–400 mesh) (4-Alkoxy-2,6-dimethoxybenzylamine resin)

       D-2165         Fmoc-cysteamine-SASRINTM
       (200–400 mesh) (Fmoc-2-aminoethanethiol-SASRINTM)

18
Table 3. Resins for Fmoc-SPPS (continued)

Product No.       Name                                            Structure
                                   TM
D-2170         Fmoc-Cys(SASRIN )-OH
(200–400 mesh)

D-2200            Tricyclic amide linker resin (200–400 mesh)
                  (Ramage resin, 5-Fmoc-amino-10,11-di-
                  hydro-5H-dibenzo [a,d]cycloheptenyl-2-oxyace-
                  tyl-DL-Nle- 4-methyl-benzhydrylamide resin)

D-2230         PDDM-resin
(200–400 mesh)

D-2285            SASRINTM-carbazate
(200-400 mesh)

D-2415            Hydroxylamine-Wang-resin
(200-400 mesh)

D-2530         3,4-Dihydro-2H-pyran-2-ylmethoxymethyl resin
(200–400 mesh) (Ellman resin)

D-2550         O-Alkyl hydroxylamino-SASRINTM
(200–400 mesh)

D-2560            4-(Fmoc-hydrazino)-benzoyl aminomethyl resin
(200-400 mesh)

D-2570         4-Formyl-3-methoxy-phenyloxymethyl polysty-
(200–400 mesh) rene resin

D-2575            4-Formyl-phenyloxymethyl polystyrene resin
                  (200-400 mesh)
                  (4-Alkoxybenzaldehyde resin)

                                                                              19
Solid-Phase Peptide Synthesis

                    1.4. Linkers                                    Q-1550
                    Linkers are bifunctional molecules anchor-      4-Hydroxymethyl-3-methoxy-phenoxy-
                    ing the growing peptide to the insoluble        acetic acid, somewhat less acid-labile
                    carrier. Linkers may be coupled to any car-     than SASRIN [76,77,79].
                    rier suitable for SPPS, an important option
                    if alternatives to polystyrene-based resins     Q-1190
                    have to be considered.                          4-Hydroxymethyl-phenoxyacetic acid
                    The C-terminal Fmoc amino acid may be           (HMP linker), a “Wang equivalent” [78].
                    coupled to the linker yielding the so-called
                    handle which can be purified before loading      Q-2545
                    the polymer. High loads regardless of the       4-(Fmoc-hydrazino)-benzoic acid, acid-
                    bulkiness of the amino acid are obtained by     base stable linker which can yield various
                    coupling these handles.                         esters/amides upon the cleavage from
                    The linkers available from Bachem are           the resin requiring a Cu(II) catalyst and a
                    presented in Table 4. Some of them are          nucleophile [31].
                    better suited for use in solid phase organic
                    synthesis.                                      Q-2345
                                                                    4(4-(1-hydroxyethyl)-2-methoxy-5-nitro-
                    Q-1755                                          phenoxy)-butyric acid, a photolabile linker
                    (4-(3-hydroxy-3-methyl-butyl)-phenoxy)-         releasing the peptides as carboxylic acids
                    acetic acid, a “tert.butyl-equivalent” anchor   [80].
                    especially suitable for the synthesis of
                    peptides with a C-terminal Pro since DKP        Q-2745
                    formation is suppressed by its bulkiness        Fmoc-Suberol (5-Fmoc-amino-2-carboxy-
                    [54 - 56].                                      methoxy-10,11-dihydro-5H-dibenzo[a,d]
                                                                    cycloheptene), the Ramage linker for the
                    Q-1660                                          synthesis of peptide amides [60].
                    Fmoc-2,4-dimethoxy-4’-(carboxy-
                    methyloxy)-benzhydrylamine
                    (Rink Amide Linker), for the synthesis of
                    peptide amides [61].

                    B-1750
                    Fmoc-4-methoxy-4’-(-carboxypropyloxy)-
                    benzhydrylamine, for the synthesis of
                    peptide amides [15].

                    Q-1095
                    4-Formyl-3-methoxy-phenoxyacetic acid,
                    may be reduced to the alcohol before or
                    after coupling [64,76,77]; it can also be
                    used as a formyl anchor e.g. for reductive
                    amination [64].

                    Q-2290
                    2-Hydroxy-5-dibenzosuberone, reacts with
                    resins carrying chloromethyl groups.

                    Q-1185
                    4-Hydroxymethylbenzoic acid (HMBA), an
                    anchor especially suited for cleavage with
                    nucleophiles, thus Boc should be preferred
                    for Nα-protection [78].

20
Table 4. Linkers for Fmoc SPPS

 Product No.    Name                                    Structure
 B-1750         Fmoc-4-methoxy-4’-(γ-carboxy-
                propyloxy)-benzhydrylamine

 Q-1095         4-Formyl-3-methoxy-phenoxyacetic
                acid

 Q-1185         4-Hydroxymethyl-benzoic acid
                (HMBA)

 Q-1190         4-Hydroxymethyl-phenoxyacetic acid

 Q-1550         4-Hydroxymethyl-3-methoxy-phenoxy-
                acetic acid

 Q-1660         Fmoc-2,4-dimethoxy-4’-
                (carboxymethyloxy)- benzhydrylamine
                (Rink linker)

 Q-1755         (4-[3-Hydroxy-3-methyl-butyl]-phe-
                noxy)- acetic acid

 Q-2290         2-Hydroxy-5-dibenzosuberone

 Q-2345         4-(4’-[1-Hydroxyethyl]-2’-methoxy-5’-
                nitrophenoxy) butyric acid

 Q-2545         4-(Fmoc-hydrazino)-benzoic acid

 Q-2745         Fmoc-Suberol (5-Fmoc-amino-
                2-carboxymethoxy-10,11-dihydro-5H-
                dibenzo[a,d]cycloheptene
                (Ramage linker)

                                                                    21
Solid-Phase Peptide Synthesis

                    2. The Fmoc Group

                    2.1. General Remarks
                    Due to the development of strategies based
                    on orthogonal protection, Fmoc has become
                    the most important base-labile N-protect-
                    ing group. The main stability features of the
                    Fmoc group are summarized below:

                     Stability   Fmoc is acid-stable, withstands cleavage of Boc/tBu (TFA) and Z/Bzl (HF). Fmoc
                                 is stable under the cleavage conditions of Aloc/OAll (Pd°).
                     Limited     Limited stability towards tertiary amines such as DIPEA, pyridine [81]; the rela-
                     stability   tive stability depends on base concentration, solvent and temperature. Stability
                                 towards hydrogenolysis is controversial [82] and should be evaluated for each
                                 individual case.
                     Lability    Lability towards bases, especially secondary amines
                                 [81] (piperidine > diethylamine). Fortunately the Fmoc group is less labile towards
                                 primary amines, including the amino group of the amino acid involved in the
                                 coupling reaction.
                                 Premature Fmoc cleavage may nevertheless occur during very slow couplings.
                                 N-silylation of the coupling site prevents this side reaction, it can accelerate the
                                 coupling [83].

                    The Fmoc group is removed via base-             By-products generated by the repetitive
                    induced β-elimination (see Fig. 3). As          treatment with base have been described.
                    a result dibenzofulvene and carbon              Aspartimide formation is the best docu-
                    dioxide are split off. Secondary bases          mented side-reaction (see II.6.2). Epimer-
                    such as piperidine add to the former            ization and subsequent piperidide forma-
                    molecule whereas bases such as DBU              tion have been detected, even though the
                    don’t react with the dibenzofulvene.            bulky tert. butyl group impedes reactions
                    Hence, it has to be removed rapidly             involving the β-carboxy group. Aggregation
                    from the peptide resin or scavenged by          during chain elongation interferes with the
                    a secondary amine such as piperidine            couplings as well as with the Fmoc cleav-
                    to avoid irreversible attachment to the         ages. If an incomplete deblocking occurs or
                    liberated amino group. Since both cleav-        is suspected, more active cleavage reagents
                    age products are strong chromophores            should be tested (see below).
                    the deblocking can be monitored by UV
                    spectroscopy.                                   2.2. Cleavage Procedures
                                                                    Usually, Fmoc is split off by a short treat-
                                                                    ment (3 to 5 minutes) with piperidine/DMF
                                                                    (1:4). In general this treatment is repeated
                                                                    and slightly prolonged (7 to 10 minutes). Un-
                                                                    der those conditions complete deblocking is
                                                                    attained in most cases. Thus, deviations are
                                                                    restricted to cases of sluggish cleavage (see
                                                                    difficult couplings, II.4.5.) or base-sensitive
                                                                    sequences.
                                                                    Harsher alternatives to piperidine/DMF
                                                                    have been developed as well as milder
                                                                    cleavage reagents [84]. In case of sluggish
                                                                    deblocking, even slight variations of the
                                                                    reagent may considerably accelerate the
               Fig. 3. Removal of the Fmoc group with               cleavage, e.g.:
               piperidine

22
• 1 to 5% DBU/DMF, more reactive than           pected load, for a load of ca. 0.5 meq/g
piperidine [85], for glycopeptides [86],        20 mg is sufficient).
• 20% piperidine and 1–5% DBU in DMF, for       Piperidine/DMF (1:4) is added to the mark,
difficult deblockings,                           beads sticking to the neck have to be care-
• morpholine/DMF (1:1), milder than piperi-     fully rinsed off.
dine for highly sensitive glycopeptides [87],   The mixture is shaken thoroughly and left to
• piperidine/DMF (1:4) at 45°C, for “difficult   settle for 25 to 30 min.
sequences” [88],                                The resin is filtered off and the absor-
• acceleration by microwave treatment [24],     bance of the filtrate is measured at 301 nm
• 0.1 M HOBt in piperidine/DMF (1:4), sup-      (ε=7800).
pression of DKP and aspartimide formation
[89],                                           Recommended Standard Procedure
• Bu4N+F– in DMF and other tetraalkylam-        Determination of the Completion of Fmoc
monium fluorides [90] (not recommended),         Cleavage
• 2% HOBt, 2% hexamethyleneimine, 25%           The sample is washed and dried as de-
N-methylpyrrolidine in DMSO/NMP 1:1, mild       scribed above. The sample is cleaved with
cleavage conditions keeping thioesters          the appropriate reagent.
intact [91] and reducing aspartimide forma-     Super acid-sensitive resins: the sample is
tion [92].                                      thoroughly washed with DCM and treated
Whichever cleavage reagent is preferred,        with 1% TFA/DCM (ca. 5 min) or HFIP/DCM
it has to be washed out very carefully after    (1:4) (15 min - 1 hr).
Fmoc removal and the last washing must          The resulting solution can be applied directly
be neutral.                                     to a TLC plate, which should be dried in
When synthesizing large peptides the dura-      vacuo before development. Fmoc is
tion of Fmoc cleavage should be gradually       readily detected at 254 nm, but Pmc, Pbf,
increased. For safe removal of the de-          Mtt, and Trt are strong chromophores as
blocking reagent the resin may have to be       well.
washed more often.                              For further analysis e.g. by HPLC, the peptide
                                                may be isolated and deprotected with TFA as
Recommended Standard Procedure                  described below.
Fmoc Cleavage                                   Other resins (and large peptides, as TLC may
Prewash with DMF (2x)                           show ambiguous results): treat with 95% aq
Treat with piperidine/DMF (1:4), 5 and 10       TFA containing 5% EDT (or TIS) for at least
min, 10 mL of reagent/g peptide-resin.          1 hr.
Wash alternately with DMF and IPA until         Filter off the resin and precipitate the pep-
neutral pH.                                     tide with MTBE.
                                                Minute amounts of Fmoc peptide can be
As mentioned above, the generation and          detected by HPLC, TLC or MS.
disappearance of Fmoc based
chromophores allows the monitoring of
the synthesis. Furthermore, samples may         3. Fmoc Amino Acid Derivates
be taken to determine the load of Fmoc
peptide. The completion of the deprotection     3.1. Side-Chain Protecting Groups
reaction may be checked by cleaving             Fmoc/tBu probably represents the most
samples and analyzing the obtained pep-         popular “orthogonal” combination of pro-
tide.                                           tecting groups. The term “orthogonal” was
                                                coined by Barany and Merrifield in 1977
Recommended Standard Procedure                  to designate “classes of protecting groups
Determination of Load                           which are removed by differing chemical
A sample of peptide-resin is washed 4x DMF,     mechanisms. Therefore they can be re-
5x IPA and 2x MeOH or ether,                    moved in any order and in the presence of
and dried to constant weight.                   the other classes. Orthogonal protection
10 to 20 mg of dried resin are weighted ex-     schemes allow for milder overall reaction
actly into a 100 ml measuring flask              conditions as well as the synthesis of par-
(the amount of resin depends on the ex-         tially protected peptides” [8].

                                                                                                 23
Solid-Phase Peptide Synthesis

                                                                                                           Table 5.
                                                                                                           Orthogonality of
                                                                                                           protecting groups.
                                                                                                           X, Y, Z: protecting
                                                                                                           groups
                                                                                                           A = O, yields peptide
                                                                                                           acid
                                                                                                           A = NH, yields pep-
                                                                                                           tide amide.

       Strategic choices
       A–Linker cleaved by strong acid (Wang, Ramage)
       Side-chain protecting groups                  Options
       X, Y, Z cleaved by strong acid                No cleavage specificity, standard synthesis.
                                                     Final cleavage from the resin provides deprotected
                                                     peptide.
       X, Y cleaved by strong acid (tBu, Boc, ...)   Partial and specific on-resin deprotection and modi-
       Z orthogonal (Allyl, ...)                     fication of the peptide chain.
       cleaved by weak acid (Trt, Mtt, ...)
       A–Linker cleaved by weak acid (SASRIN, 2-Chlorotrityl, Xanthenyl)
       Side-chain protecting groups                  Options
       X, Y, Z cleaved by strong acid or orthogo-    Cleavage from the resin provides fully protected pep-
       nal                                           tide for future chain elongation (convergent synthe-
                                                     sis) or chain modification in solution.
       X, Z cleaved by strong acid                   Partial and specific on-resin deprotection and modi-
       Y orthogonal                                  fication of the peptide chain.
                                                     Cleavage of the modified peptide from the resin
                                                     with or without concomitant removal of side- chain
                                                     protection.

      The combination Fmoc/tBu is truly orthogo-         acid is linked is a protecting group as well.
      nal whereas Boc/ Bzl is not, at least not un-      When conceiving the synthesis of complex
      der the conditions of SPPS as they both are        or “modified” peptides (e.g. side-chain
      cleaved by acids. As Boc can be selectively        cyclized peptides) the tactics of synthesis,
      removed in the presence of Z/Bzl, the              that is, the combination of side-chain pro-
      combination has been termed “quasi-                tecting groups and type of resin has to be
      orthogonal”.                                       considered thoroughly. A few combinations
      A brochure Orthogonaly of Protecting Groups        are presented in Table 5.
      proposing combinations of selectively              For example, side-chain cyclization, usually
      cleavable side-chain protecting groups for         via amide bond (e.g. for increasing the rigid-
      use in Fmoc-SPPS is also available from            ity of a peptide and thus stabilizing desired
      Bachem and can be downloaded from our              conformations) has become an important
      homepage.                                          structural element in designing peptide
                                                         analogues. Appropriate synthetic strategies
      3.2. Side-Chain Protection Schemes                 relying on (quasi) orthogonal side-chain
      As mentioned earlier the choice of orthogo-        protecting groups have been conceived,
      nal protecting groups has broadened the            cyclization may be achieved either “on-
      scope of the Fmoc/tBu based SPPS. The              resin” or following cleavage of the (partially)
      solid support to which the C-terminal amino        protected linear precursor from the resin.

24
Table 6. Side-chain protected Fmoc derivatives of proteinogenic amino acids.

 Amino       Protecting      Cleavage Conditions       Remarks
 Acid        Group
 Arg         Pmc             95% aq TFA                standard
             Pbf             95% aq TFA                standard, slightly more acid labile than Pmc
             Mtr             95% aq TFA (35°C)         more acid stable than Pmc
 Asn/Gln     Trt             95% aq TFA                standard, more stable to acidolysis than Mtt
             Mtt             95% aq TFA                standard
             Xan             95% aq TFA                standard
 Asp/Glu     OtBu            95% aq TFA                standard
             OMpe            95% aq TFA                Asp: suppression of aspartimide formation
             OPp             1% TFA/DCM                on-resin modification
             OBzl            H2/Pd or HF               acid sensitive peptides, SASRIN then H2/Pd, rarely used
             OAll            Pd(PPh3)4                 orthogonal to Fmoc/tBu/resin linkage
             ODmab           2% N2H4·H2O/DMF           quasi orthogonal to Fmoc, acid stable
 Cys                                                   see II.3.3.
 His         Trt             95% aq TFA                standard
             Mtt             95% aq TFA                standard
 Met         O               NH4I/Me2S                 rarely used (see II.6.7.)
 Lys         Boc             95% aq TFA                standard
             Aloc            Pd(PPh3)4                 orthogonal to Fmoc/tBu/resin linkage
             Adpoc           1% TFA/DCM                on-resin modification
             Mtt             1% TFA/DCM                on-resin modification
             Dde             2% N2H4·H2O/DMF           cf. Dmab
             ivDde           2% N2H4·H2O/DMF           improved stability towards piperidine
             Z               H2/Pd or HF               rarely used
             Fmoc            20% piperidine/DMF        multiple antigenic peptides, dendrimers
 Ser/Thr/    tBu             95% aq TFA                standard
 Tyr         Trt             1% TFA/DCM                on-resin modification
             Bzl             H2/Pd or HF               rarely used
 Trp         Boc             95% aq TFA then aq        standard
                             AcOH

The combinations All/Aloc and Dmab/Dde              Arginine
have become very popular for side-chain             Pmc and Pbf are mostly used for the protection
cyclization (via an amide bond) of resin            of the guanidino function of Arg. The cleavage
bound peptides.                                     is accelerated in the presence of thiols in the
If treatment with 1% TFA/DCM is kept suffi-          cocktail (see II.5.1.). The cleavage of the Mtr
ciently short, Mtt (or Adpoc) in combination        group requires prolonged reaction time or reac-
with OPp may be employed in combination             tion at elevated temperature which can lead to
with a Wang type resin [93,94] as well.             undesired side reactions [95].
For peptides synthesized on SASRIN, side-
chain modifications of the partially protect-        Asparagine and Glutamine
ed peptide are performed in solution after          The Trt and Mtt protected amino acids are
cleavage from the resin.                            perfectly suited for Fmoc based SPPS. The
                                                    protecting groups are efficient in impeding the
A choice of side-chain protected Fmoc amino         dehydration of the side chain carboxamide
acids is presented in Table 6. For further infor-   during the activation step. In addition these
mation on these and other derivatives please go     protecting groups increase the solubility of the
to our online shop at www.bachem.com                poorly soluble Fmoc-Asn-OH and Fmoc-Gln-

                                                                                                                 25
Solid-Phase Peptide Synthesis

      OH. As Trt is removed rather sluggishly from an       protecting groups exist for syntheses in which
      N-terminal Asn, Mtt carboxamide protection            on-resin derivatization is desired. Among them
      should be preferred in this position [96].            the most commonly used are Aloc, cleaved
                                                            by nucleophiles in the presence of Pd. Adpoc
      Aspartic Acid and Glutamic Acid                       and Mtt are more acid labile than Boc and are
      In routine Fmoc SPPS the side-chain is protect-       cleaved by repeated treatment with 1–2% TFA/
      ed as the tert. butyl ester. This protecting group    DCM. The combination of the various Asp/Glu
      is stable under the conditions of SPPS and is         and Lys/Orn protecting groups has enabled the
      readily removed during the final TFA cleavage.         synthesis of complex molecules benefitting
      The OMpe derivative is less prone to base-            from the “multidimensional orthogonality” [102].
      catalyzed aspartimide formation [97]. OPp, OAll,
      or ODmab-esters are used when an additional           Serine, Threonine and Tyrosine
      level of orthogonality is required, for on-resin      The tert. butyl ethers possess the qualities of
      cyclization for example. These esters can be          good protecting groups and are widely used. Trt
      cleaved specifically in the presence of other          can be used for the protection of Ser and Thr if
      protecting groups such as Boc, OtBu, Fmoc.            on-resin derivatization is required. Thr and Tyr
                                                            may also be coupled with unprotected side-
      Cysteine                                              chain functionalities .
      See also II.3.3.
      Cys derivatives are notorious for base-               Tryptophan
      catalyzed racemization during activation              Trp has been used without protection, however
      and coupling [98]. Considerable amounts               the indole nucleus can then be alkylated in the
      of D-Cys epimer are obtained when cou-                final TFA cleavage. For that reason we strongly
      pling Cys(Trt) derivatives in the presence of         advise coupling indole-protected tryptophan.
      bases. Cys(Acm) derivatives show a lower              Fmoc-Trp(Boc) has become the standard
      tendency to racemize. They tolerate weak              derivative for incorporating the amino acid . Dur-
      bases as collidine. Attempted syntheses               ing the final TFA cleavage the Boc group yields
      of peptides containing several disulfide               isobutylene and leaves the N(indole)-carboxy
      bridges following standard Fmoc protocols             moiety which prevents alkylation of the indole
      may have failed for this reason. The extent           nucleus. This intermediate is decarboxylated
      of this side-reaction can be reduced by               during a subsequent treatment with diluted
      using weak bases as collidine in combina-             AcOH.
      tion with uronium/aminium or phophonium
      reagents or, more effectively, by coupling            3.3. Protection of Cys During Fmoc SPPS of
      in the absence of bases, e.g. with carbodi-           Peptides Containing Disulfide bonds
      imides and HOBt (or HOAt). Racemization               We offers a brochure on cysteine
      is further impeded by using less polar sol-           and further mercapto amino acids including a
      vents for the coupling.                               compilation of our offer of derivatives of these
                                                            compounds which can be downloaded from our
      Histidine                                             web page.
      Trt or Mtt are the most common protecting             Cys has always required particular attention
      groups for the protection of the imidazole ring of    in peptide chemistry. Protection of the highly
      His. Due to steric hindrance tritylation occurs       reactive side chain thiol function during peptide
      exclusively at the Nτ (1-position). Trt and Mtt are   synthesis is mandatory, and peptides contain-
      stable under the conditions of SPPS but they          ing free cysteines have to be protected from
      don’t prevent catalysis of racemization during        random oxidation (for a choice of often-used
      activation by the imidazole moiety (free Nπ) [99].    S-protecting groups see Table 7). In most cases,
      Fortunately, a coupling protocol minimizing this      the liberated sulfhydryl moieties are oxidized
      side reaction has been described. See 4.2.2.          to generate intra-or intermolecular disulfide
      [100, 101].                                           bonds selectively. By choosing appropriate
                                                            protecting groups, the disulfide bridges may be
      Lysine                                                formed at various stages of the synthesis: on-
      In routine Fmoc SPPS, Boc is used for the pro-        resin as well as in solution.
      tection of the amino function. It is cleaved dur-     S-Protection has to be chosen according to the
      ing the final TFA cleavage. Special orthogonal         synthetic strategy. Furthermore, removal of

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