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SOLID PHASE PEPTIDE SYNTHESIS
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
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
3Solid-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
4List 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
5Solid-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.
6Solvents
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
7Solid-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
8Table 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,
9Solid-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
10Table 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].
11Solid-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.
12The 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
13Solid-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.
14Peptides 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
15Solid-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-
162-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.
17Solid-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)
18Table 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)
19Solid-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].
20Table 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)
21Solid-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].
23Solid-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.
24Table 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-
25Solid-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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