Chapter 2 Solid Phase Peptide Synthesis: An Overview
O v e r the last two decades, there has been a rapid progress in the chemistry
of large peptides and Peptide synthesis has proven as an
indispensable tool for the structural elucidation of many recently isolated
natural products having a peptide structure such as hormones, neuropeptides
and antibiotics, which often could be isolated only in minute quantities.
Recent developments in the biotechnology of new proteins as well as
advances in immunology and the development of pharmaceuticals based on
inhibitors and antagonists have led to immense demands for synthetic
peptides. The fields of research in modem peptide chemistry include
synthesis and analysis, isolation and structure determination, conformation
investigations and molecular modeling.
The advances in chemical peptide synthesis over the last fifty years
have made the synthesis of large peptides and proteins a realistic possibility.
Chemical synthesis is probably the most practical way of providing usehl
quantities of material, and in addition, allows the systen~atic variation of
structure necessary for the development of peptides for therapeutic use.2
Analogs of the peptides and modified structures containing specifically
labeled amino acids or non-DNA-encoded amino acids and also peptide
mimetics, are more efficiently made by chemical synthesis. Reports on the
studies connected with the synthetic peptides revealed that they are used to
raise anti-peptide antibodies, to study enzyme substrates and the binding
properties of viral proteins to identify and locate gene products and in NMR
studies of peptide structure.
The earliest modes of peptide bond formation pioneered by
~ ~ r t i u s ~ ~ and ish her'^ at the turn of this century yielded impressive but not
yet practical results. Introduction of the amino protecting benzyloxycabonyl
led to a new era of peptide synthesis. Improvements in the method
of pcptide bond formation, particularly the invention of carbonic acid mixed
anhydridez8 gave a new impetus to peptide synthesis and in 1953, the
methodology reached a degree of sophistication which allowed the
synthesis of a peptide hormone, Oxytocin, by Du Vigneaud and his
associates. From here on, synthesis of peptides progressed by leaps and
bounds. Introduction of di~~clohex~lcarbodiimide~~ a still unsurpassed
coupling reagent had a major impact on the methodology of peptide bond
formation and further refinement was brought about by the development of
active esters.30 Equally important improvements could be noted in the
methods of protection: acid labile blocking groups built on the stability and
thus ready formation of the tertiary butyl m at ion,^' the
tertiarybutyloxycarbonyl group particularly, remain among the tools of
unchallenged importance even after the introduction of base sensitive
blocking in the form of the 9-Fmoc Yet, the most conspicuous
milestone in the history of peptide synthesis is the invention of solid phasc
peptide synthesis by R. B. Merrifield in 1963.~ Through painstaking
meticulous research, Merrifield determined the best conditions for his solid %
phase synthesis. Since his 1963 article appeared, thousands of peptides and - - -
other biological polymers including carbohydrates and nucleic acids have
been synthesized using this method both by him and by others. In 1965,
together with John Stewart, he automated the process, and today
commercial models with microcomputer controls are available. In
recognition for Merrifield's development of methodology for chemical
synthesis on a solid support, he was awarded the 1984 Nobel prize in
chemistry.
The synthesis of peptides is achieved either by the solution phase
or by the solid phase methods. The solution phase method of peptide
synthesis is laborious and time consuming as the intermediate products have
to be removed, purified and characterized before proceeding to the next
coupling step. Insolubility of the intermediate peptide in solvents used for
the synthesis and mechanical losses are other problems associated with this
method. Therefore, a new approach was needed if large amounts of
peptides were required or if larger and more complex peptides were to be
made.
The advances made in peptide chemistry and biology would not
have been possible without the availability of the new methods of peptide
synthesis. The feasibility of this technique was first shown by the synthesis
of the crystalline tetrapeptide, L-leucyl-Lalanyl-glycyl-L-valine.
Numerous developments have been made which widened the scope of the
method.33 There is a greater demand for new strategies, faster synthesis,34
better coupling reagents, protecting groups and especially methods for
simultaneous preparation and analysis of very large number of peptides in a
short time. Stepwise peptide synthesis on polymer supports is regaining
importance due to the recent developments made in protecting group 35,36 37-39 strategy, anchoring techniques and support properties. 40,41
2. 1. Principles of Solid Phase Peptide Synthesis
Solid phase peptide synthesis follows the stepwise assembly of
peptides by consecutive coupling of amino acids. Memfield employed an
insoluble and filterable polymer support such as chloromethylated, 1% <- ~~
~~ - \
DVB-crosslinked polystyrene which functions as the carboxy protecting
group for the C-terminal amino acid. Ailer incorporation of the first amino
acid to the polymer through a benzyl ester linkage, the terminal amino
group is deprotected under conditions which do not cleave the resin-amino
acid ester bond. Then, another Na protected amino acid is coupled to the
amino group of the polymer bound substrate using DCC
ester coupling. The W deblocking and coupling steps are
desired sequence is assembled on the polymer support.
After completion of the synthesis, the peptide is
support. Memfield's strategy used strong acids like TFA, HBr-AcOH for
the cleavage of peptides fiom the polymer. This results in simultaneous Na-
deblocking and deprotection of most of the side chain functionalities to give
the eee peptide which is then purified by suitable procedures. Owing to
standardization of the steps involved, solid phase synthesis can be
automated. The chemical steps involved in the Memfield's synthesis using
chloromethylated polystyrene are outlined in Scheme 2 . 1.
The Essential Advantages Associated with SPPS
The reactions can be driven to completion using excess soluble low
molecular weight reagents and final products are obtained in good yield.
The excess reagents can be easily separated from the polymer-bound
peptide by simple filtration. As a result, the laborious and cumbersome
purification of intermediate peptide is avoided and this results in a
tremendous saving of time. Since it is possible to cany out all reactions in a
single reaction vessel, manipulations and attendant losses involved in the
repeated transfer of materials can be avoided. After synthesis, the spent
resin can be recycled as such or with some chemical modifications. So the
process is economical. The polymeric support should be insoluble, rigid
and capable of funtionalization to a relatively high degree. The functional
groups should undergo a straight forward reaction with reagents and must
be free of any side reactions. The support should swell in suitable solvents
and should be physicochemically compatible with the bound substrate,
reagents and solvents used, for effective reactions to occur. There are no
solubility problems encountered when adding one amino acid per cycle. In '
this respect, solid phase peptide method appears - to be more suitable for i.,nuo.,
protein synthesis. 1 . / I
The Limitations Associated with the Solid Phase Synthesis
Physicochemical incompatibility of the growing peptide chain with
the polymer support, non-equivalence of functional groups attached to the
polymer support, racemisation leading to optically impure products and
formation of error peptides from deletion and truncated sequences.
(a) First amino acid attachment I Cesium salt method
RI Boc HN~coo-CHI
H
(b) Deprotection & Neutralisation (i) TFA 30% (ii) DlEA 5%
2 H N T C O O - C H 2 a H
(c) Coupling (Active ester) DCC/HOBt
I R2
BOC H N ~ C O O H H
R2 R1 Boc HN~CO-HNtC00-CH*
H H
(d) Elongation of chain Repeat steps (b) and (c) n times
Rn-I R2 i Boo N H V O - ( N H t C O . J t C O - H N r C O O - C H 2 a
H H H H
(e) Cleavage
I TFA/thioanisole
Scheme 2. 1. General protocol for the assembly of amino acids by solid phase peptide synthesis.
2. 2. Insoluble Supports and Anchoring Linkages in SPPS
Most recent work on the chemical modification of polymers has
centered on the introduction and modification of various functionalities on
polystyrene. Usually, 1-2 % DVB-crosslinked polystyrene has been used
successfully in SPPS. The ideal resin with optimum swelling and stability
was found to be 1% crosslinked polystyrene. Polystyrene, chloromethylated
and ring lithiated polystyrene are used in the chemical modification of
styrene polymers as they provide a method of attaching a wide variety of
both electrophilic and nucleophilic species. In addition, a number of other
supports incorporating functional groups like phenacyl, hydrazyl,
acylsulfonyl, benzhydryl, aminomethyl, etc. have been used in S P P S . ~ ~
Newer resins have been developed with different aims such as
improving resin-peptide bond stability, solvent-resin product compatibility,
support loading, coupling efficiency, cleavage of finished resin-peptide
bond and synthesis of protected peptide fragments including peptide esters,
amides or hydrazides. Functionalized resins incorporating safety catch
device^,^' pellicularised resins based on silica,44 polyoxyethylene-
polystyrene graft copolymeric support (POE-PS);~ polyacrylate-DVB
copolymer,46 polyamide-kieselguhr support:7 isocyano resin:' Rink resin,4y
5[4 (9-Fmoc) amino methyl 3,5-dimethoxylphenoxy] valeric acid (PAL)
resin,jO 2-chloro trityl chloro resin," carboxylamide terminal (CAT) resin,"
tertiary alcohol re~in, '~ 2-methoxy-4-benzyloxy benzylalcohol resin,54 4-
nitrobenzophenone resin,55 4-[2, 4-dimethoxy phenyl (amino) methyl]
methyl resins6 and acid labile 9-Xanthenyl resin57 have been developed for
SPPS.
Recently, more supports were developed for multiple peptide
synthesis. In Houghten's tea bag method, PS-DVB (1%) in polypropylene
mesh packets were used as supports.58 In multi-pin synthesis technology,
amino Iimctionalized polyoxyethylene rods are employed as supports.59 An
inexpensive procedure recently developed by Frank et al uses a sheet of
cellulose paper as support.60 In multicolurnn methods, macrosorb-SPR resin
was used?' Multiple peptide synthesis on acid-labile handle derivatised
polyethylene supports has been developed.62 Multipin peptide synthesis at
the micromole scale using 2-hydroxyethyl methacrylate grafted
polyethylene supports have been reported?' Multiple column peptide
synthesis employing Fmoc-amino acid -0-Dhbt or -P@ esters in continuos
flow version of the polyamide method on small packed columns of
Kieselguhr supported resin in a reaction block of Teflon has been
reported.64 An automated multiple peptide synthesis method to synthesis,
cleave and purify several peptides simultaneously in a single batch has been
developed. The technique is based on the synthesis of multiple peptides on
a single solid phase support and is easily adapted to manual or to automated
methods.65
Fmoc SPPS using ~erloza" beaded cellulose has been reported.
Fmoc-amino acids were anchored to amino propyl Perloza beaded cellulose
via the TFA labile Coxymethyl phenoxyacetyl (HMPA) linker. Using '*K"7
Fmoc-aminoacyl-4-oxymethyl phenoxy acetyl-2,4-dichlorophenyl esters, all
20 amino acids were anchored at substitution levels ranging from 0.37-0.65
r n r n ~ l l ~ m . ~ ~ Continuos flow synthesis of peptides using a polyacrylamide
gel resin ( ~ x ~ a n s i n ~ ) has been proved to be ~onvenient.~' The hydrophilic
support beaded cellulose (Perloza) can be used for peptide synthesis with
modified Fmoc and Boc protocols.68 Beaded, hydrophilic, crosslinked
aminoallcjl polydimethyl acrylamide supports have been used for the
assembly of peptides using standard Boc or Fmoc chemistry in automated
equipment. Manual SPPS on resins with high loading capacity requiring
small volumes of solvents have been described.69 NHS-activated Pharmacia
HiTrap Sepharose was modified with 1,3-diaminopropane to give an amino
hctionalized support for S P P S . ~ ~
Some of the recent developments in the field of polymer-supported
peptide synthesis are new hydrophilic matrices for the synthesis of small
peptides by either batch or continuos flow methods7' and [2-(2-nitrophenyl
ethyl)] (NPE) resin7' for the synthesis of protected peptides and
oligonucleotides. Bis-2-acrylamidoprop-1-yl polyethyleneglycol crosslinked
dimethylacrylamide (PEGA) has been introduced as a hydrophilic,
incompatible and flexible support in peptide synthesis.73 Recently, a new
method for preparation of high capacity PEGA resins with well defined
loading of functional groups has been described for continuos flow
synthesis by Meldal and co- worker^.'^
Although the earlier solid phase chemistry was very usehl for
making small peptides and even small proteins, it was clear that there was a
need for improvement in several areas. Several tailor made anchoring
linkages have also been introduced between the first amino acid and the 75,76 polymer support to improve the synthetic procedures of SPPS. These
include phenylacetamido (PAM), p-methyl benzhydrylamide (MBHA), p-
alkoxy benzylester systems, etc. The introduction of multidetachable
anchors in SPPS making use of chemoselectively removable protecting
groups provide maximum flexibility and adaptability to the methods used
for removing the peptide from the polymer supports.77
By introducing a photolabile anchoring group between the resin
and the growing peptide chain, the finished peptide can be cleaved under
mild conditions by photolysis.78 A photolabile o-nitro benzhydrylamino
polystyrene support (NBHA-resin) has been used for solid phase synthesis
and C-terminal amidation of peptides.79 1-chloromethyl-2-nitro TTEGDA-
crosslinked polystyrene resin has been used as a photosensitive solid
support for preparation of fully protected peptides.80 Development of new
photolabile protecting groups like Menpoc (a-methyl nitropiperonyl
oxycarbonyl) and Menvoc (a-methyl nitro veratryloxycarbonyl) were
reported in the 13" American peptide symposium.8' Polymer-supported
solid phase synthetic procedures have been reported for the synthesis of C-
terminal peptide arnides using a new photolytically cleavable a-methyl
phenyl arnido anchoring linkage between the support and growing peptide.82
In Fmoc methodology, all resins are substituted with a linker such
as 4-hydroxymethyl phenoxy acetic acid (HMF'A) requiring attachment of
the first amino acid as an ester. This step can lead to low substitution,
racemisation or dipeptide formation. An efficient catalyst is DMAP. A
good alternative is the use of 2,6-dichloro benzoylchloride in DMF.'~ This
method can be used for the attachment of the first amino acid onto
hydroxymethylated resins. 5, 9-(9-Fmoc amino xanthen-2-oxy) valeric acid
(XAL) has been introduced as an acid labile handle for Fmoc-based peptide
amide synthesis.84 Other acid labile anchoring linkages used in SPPS are
dimethoxyacido labile linker ( D A L ) ~ ~ and 3-methoxy-4-hydroxymethyl
phenoxyacetic acid.86 Barany and Merrifield have reviewed on the recent q
developments on handles and supports for S P P S ~ An oxidation labile [ -- < C '
phenyl hydrazide group has been recently reported as a linker for solid
phase peptide synthesis.88 PAL handle is usehl for the synthesis of
dipeptide C-terminal amides using Fmoc/t-Bu strategy.50 Mc.lnnes and co-
workers have developed an efficient linker for SPPS based on
dibenzocyclohepta-l,4-diene system.89
Barter and co-workers have demonstrated the synthesis and
application of a novel silicon linker offering highly efficient cleavage to
small molecule libraries bearing no memory of resin atta~hment.~' 2-(NM
tert.butyloxycarbonyI-5-methyl-imidazol-4yl)-2-hydroxyacetic acid, a
safety catch linker recently developed by Hoffman and Frank allows direct
release of peptide acids into aqueous buffer after Fmoc solid phase
synthesis.9' A new procedure using dihydropyran-2-carboxylic acid as a
bifunctional linker for the synthesis of peptide alcohols has been
de~cribed.~' Linkers based on the Dde [I-(4,4-Dimethyl-2,6-
dioxocyclohexylidene) ethyl] primary mine protecting strategy have been
developed and their utility demonstrated in the solid phase synthesis of a
naturally occurring spider toxin. The linkers are stable to both acid and
base conditions and cleaved either by 2% vlv hydrazine hydrate or by
transamination with a volatile primary alkylamine in a variety of organic
solvents.
Some new polymeric supports based on polyacrylamide and
polystyrene have been developed for solid phase peptide synthesis.
Polyacrylamide supports include N,N'-methylene - bis - acrylarnide
(NNMBA), tetraethyleneglycol diacrylate (TTEGDA), triethyleneglycol
dimethacrylate (TEGDMA) and divinylbenzene (DVB)-crosslinked
polystyrene supports.'3s93 Polystyrene supports include TTEGDA,
TEGDMA and 1,6-hexanediol diacrylate (HD0DA)-crosslinked supports.93
These new resins were found superior to PS-DVB in terms of stability and
solvation properties. This resulted in the increased use of these resins in
solid phase peptide synthesis. Here, we have used polystyrene crosslinked
with tetraethyleneglycol diacrylate (IITEGDA) as the polymer support,
which can be easily prepared and functionalized. The polymer has high
mechanical stability, good solvation properties and suitable reactivity
characteristics, thus favoring speed and completion of all reactions during
synthesis.
2. 3. Use of Fmoc Groups in Peptide Chemistry
Carpino and Han described the use of Fmoc group for the
protection of amino function, which provides a simple, rapid and efficient
alternative to the common use of Boc-amino acids.32 It is also an
exceptionally mild procedure, avoiding both the repetitive trifluoroacetic
acid treatment in each cycle and the harsh liquid HF cleavage of the product
from the solid support. It offers the following advantages:
quantitative and rapid p-protective group cleavable by mild
nonhydrolytic base treatment.
use of tertiary butyl type side chain protection, which is known to be
completely stable to base.
cleavage of completed target peptide from resin by mild acidolysis . facile UV monitoring of both coupling and Nu-deprotection steps, thus
opening up the possibility of spectroscopically monitoring the peptide
synthesis.
Frnoc chemistry can be enhanced by combining it with HBTU
activation, a combination known as Fast Moc chemistry.94 Fmoc polyamide
solid phase synthesis was designed by Sheppard et a1 to overcome some of I > * , , A
4 the problems associated with SPPS using Boc chemistry. The new
polyamide support has a polarity similar to peptides and swells much better
than polystyrene in the solvents commonly used in peptide synthesis, eg:
NMP, DMF etc. A recent development in the polyamide method has been
the introduction of continuos flow synthesis which has advantages over the
batch synthesis recommended by Merrifield. Bayer et al described the first
continuos flow synthesis on chemically modified silica gel supports.95
Frank and Gausepohl employed loosely packed cartridges of different C'
polymeric gels for the continuos flow synthesis.96 Atherton and Sheppard
et al polymerised polydimethyl acrylamide gels on Kieselguhr and
successfully used the material obtained for continuos flow synthesis using 97 an automated synthesizer.-
2.4. Solvents in SPPS
Numerous developments in the area of SPPS have widened the
scope of the method, but one problem remained substantially unresolved,
i.e., the occurrence of aggregation within the peptide-resin matrix.98 It was
suggested that the incomplete solvation of peptide-resin complex might be a
source of difficulty in solid phase synthesis.99 Such incomplete solvation
was thought to encourage inter or intra chain association leading to more
compact structures within the complex compared with the freely solvated
state.
Dimethylsulfoxide has proved to be a superior reaction medium for
SPPS in aggregating systems and a solution to the difficult sequence
problem.100 The influence of resin swelling was recognized at the very
beginning of SPPS. HCI-dioxane, a powerful swelling solvent was found to
give good deprotection of Boc groups whereas HCI-AcOH, which did not / c
swell the resin, did not."' - Mixed solvent systems may optimize peptide- -- - 7
resin solvation by combining relatively polar and non-polar solvents.'02
Several mixed solvent systems used successfully in solid phase peptide
synthesis include trifluoroethanol (TFE)-DCM,"' dimethylsulfoxide
(DMSO)-DCM,"~ p-dioxane-~MF,'05 urea-DM~,"~ DMSO-NMP,'" and
1, I, 1,3,3,3-hexafluoro-2-propanol (HFIP)-DCM.'" An improved solid
phase synthesis of a difficult-sequence peptide using hexafluoro-2-propanol
has been suggested.
Recently, theory incorporating solvent electron donor and acceptor
numbers have been used to create mixed-solvent systems that minimize the
intermolecular 0-sheet formation.'09 Strong electron donor solvents such as
hexamethylphosphonic triamide (HMPA) or DMSO are mixed with DMA,
DMF or NMP."~ The partial substitution or complete replacement of tBu-
based side chain protecting groups for carboxyl, hydroxyl and amino side
chains by more polar groups would also aid peptide-resin so~vation."~ The
use of solvent mixtures containing both a polar and non-polar component,
such as 35% 1'HF-NMP or 20% TFE-DCM is recommended to alleviate the
problem of side chain-induced resin collapse.'"
Chaotropic salts have been shown to inhibit interchain P-sheet
aggregates and hence improve peptide-resin solvation and coupling
efficiencies. ' I 2 0.4 M NaC104, KSCN or LiBr was helpful for several
couplings in DCM-DMF (1:l) during Boc SPPS of Rnase I-13-MBHA-
PS."' The use of LiBr in anhydrous THF was reported to be extremely
effective in disrupting P-sheet structure in resin bound peptides."4 The use
of hexafluoro acetone trihydrate as a structure stabilizer for peptides have
been reported."5
2. 5. New Methods of Protection and Deprotection in SPPS
N-2-(2,4-dinitro phenyl) ethoxy carbonyl, 2-chloro-3-indenyl
methyloxycarbonyl (CLIMOC) and Benz (F) inden-3-yl methyloxy
carbonyl (BIMOC) are some of the base labile protecting groups similar to
9-fluorenyl methyloxy carbonyl group suitable for solid phase synthesis."6
Another newly introduced base labile a-amino protecting group is 2-(4-nitro
phenyl) sulphonyl ethoxy carbonyl (Nsc)."~
Several modifications to the widely used tertiarybutyloxycarbonyl
(Boc) group have been made. 118,119 4-methyl sulphenyl benzyloxycarbonyl
(Msz) group is a new protecting group removed by reductive acidolysis.
Acid labile monomethoxy trityl and dimethoxy trityl were used as amino
protecting groups in SPPS. Several new side chain protecting groups for
amino acids such as S-phenylacetamidomethyl (Phacm) group for
cysteine,120 2-(4-acetyl-2-nitrophenyl) ethyl group for the y carboxyl of
aspartic acid,I2' Nm -cyclohexyloxycarbonyl group (HOC) '~~ and N" -
allyloxycarbonyl group (Aloc) for tryptophan,'23 2,4-dinitrophenyl group
(Dnp) for hydroxyl function of t y r ~ s i n e , ' ~ ~ p-(methyl sulphinyl) benzyl
group for serine,12' 2,2,4,6,7-pentamethyl dihydrobenzofuran-5-sulfonyl
group (Pbf) for ~ ~ i n i n e ' ~ ~ and 2-adamantyl oxycarbonyl group (2-Adoc)
for &-amino group of sine'^^ have been introduced. For imidazole group
of histidine, W (1-adamantyloxy methyl) group (W-1-Adom) removed by
TFA, W-t-butoxymethyl group removed by mild acidolysis and W-
benzyloxymethyl group cleaved rapidly and cleanly by HBr in TFA or by
catalytic hydrogenolysis has been reported. 128-130 The new mild acid labile
protecting groups for the guanidino function of N-Fmoc-L-Arginine, 10,ll-
dihydro-5H dibenzo [a, dl cyclohepten-5-yl, 2-methoxy-l0,l I-dihydro-5H-
dibenzo [a, dl cyclohepten-5-yl and 5 H-dibenzo [a, dl cyclohepten-5-yl
groups in SPPS has been reported. 13'
New protecting groups like 2-(1-adamanty1)-propanol-2-esters
(Adp) removable under mild acid and 2-bromoethyl and 2-
iodoethyl esters deprotected by samarium diiodide found application in
SPPS . '~~ The use of 9-fluorenyl methylesters for protection of carboxyl
group has been proposed.'34 The advantage is selective deprotection under
mild conditions using secondary mines without racemization. The use of
2-phenyl isopropyl esters as carboxyl terminus protecting groups have been I .
reported in the fast synthesis of peptide fragments."' Robles and co-
workers have reportkd a new base labile carboxyl protecting group [2-(4-
acetyl-2-nitrophenyl) ethyl] ( A n ~ e ) . ' ~ ~
Tetrafluoroboric acid has been employed as a useful deprotecting
reagent in Fmoc-based SPPS.'~' A newly developed reagent for the
deprotection of t-butyloxycarbonyl and No-benzyloxycarbonyl
is iodotrichlorosilane. A report from the 22"d European peptide
symposium (1992) is about the optimized deprotection procedure for
peptides containing Arg (Mtr), Cys (Acm), Trp and Met residues.'40 A new
stepwise deprotection method using reductive acidolysis followed by
fluoide - ion in SPPS has been found to minimize aspartimide formation in
peptides containing an Asp-Gly sequence.I4' Sodium borohydride in
presence of palladium (0) catalyst could be used for effective removal of N-
ailyl oxycarbonyl (Alloc) protecting Recently, Wensbo has
described the selective removal of N-Boc protecting group using silica gel 143 at low pressure. The microwave assisted silica gel deprotection of N-Boc
derivatives have been reported by Vaquero and c o - ~ o r k e r s . ' ~ ~
The use of Fmoc-N-(2-hydroxy-4-methoxybenzyl) amino acids in
peptide synthesis has been reported.'45 The use of 2-methoxyethylester as a
protecting group in peptide and glycopeptide synthesis has been reported.
The selective removal of ME esters by lipases was achieved under mild
conditions (pH 7, 37OC) leaving all other linkages including peptide bonds
and other ester protecting groups ~naf fec ted . '~~
2. 6. Coupling Reagents in SPPS
The nature of acylating agent, protected amino acid activated
species and solvation of the growing peptide chain decide the efficiency of
coupling reactions. The introduction of DCC as a reagent for peptide bond
formation was a major event in the history of peptide synthesis. Other
carbodiimides utilized in SPPS are diisopropyl carbodiimide (DIPCDI) and
di-t-butyl ethylcarbodiimide. 147,148 Recently, benzotriazol-1-yl-oxy-tris
(dimethylamino) phosphonium hexafluoro phosphate (BOP) found
application in the synthesis of fairly complex pep tide^.'^^ Novel activating
agents like benzotriazolyloxy tri (pyrro1idine)-phosphonium
hexafluorophosphate (P~BOP),"' Bis[4-(2,2-dimethyl 1,3-dioxolyl)
methyl]-carbodiimide (BDDC),'" bromo tris (pyrro1idino)-phosphonium
hexa fluoro phosphate (P~B~oP)"* have been introduced.
Spccific sequences which contain dificult couplings during SPPS
can bc drivcn to completion using 2-(1H-bcnzotriazol- I -yl) 1,1,3,3-
tetramethyl ammonium hexafluoro phosphate (HBTU) or 2-(1H-
benzotriazol-1 -yl) 1,1,3,3-tetramethyl uronium tetrafluoroborate (TBTU)
mediated coupling. 153-155 Enhancement of peptide couplings was
recommended by a combination of 4-dimethylaminopyridine (DMAF')-
dicyclohexyl carbodiimide (DCC) in the case of hindered amino acid
residues.Is6 Simultaneous use of 1-HOBt and copper (11) chloride as
additives for racemisation free and efficient peptide synthesis by the
carbodiirnide method has been reported. Recently, l-Hydroxy-7-
azabenzotriazole (HOAt) has been described as a superior peptide coupling
additive, which enhances couplings yields in solution by about 6-32
times.Is7 The uronium and phosphonium salts of HOAt is also used. 3-
Dimethyl phosphinothioyl-2(H)-oxazolone (MPTO) was introduced as a
promising new reagent for racemisation eee couplings.'s8 Some of the
other newly introduced coupling reagents are N-cyclohexyl-N-isopropyl
carbodiimide (cIc),"~ 2-(benzotriazol-1 -yl) oxy- 1,3-dimethyl
imidazolidinium hexafluoro phosphate (BOI),'~' Tris (pyrrolidino)
phosphonium reagent,l6I Toppip U [2-(2-0x0-l(2H)pyridyl)-1,1,3,3-
bispentamethylene uronium tetrafluorob~rate]'~~ and FDP
(pentafluorophenyl diphenyl phosphate).163 Some of the newly developed
activating agents in peptide synthesis are 1-a-Naphthalene sulfonyloxy
benzotriazole ( N S B ~ ) , ' ~ 6-Nitro-1-a-naphthalene sulfonyloxy
benz~triazole,'~~ di-ter-butyl pyrocarbonate in presence of pyridine and
ammonium hydrogen ~arbona te , '~~ and tetramethylfluoroformadinium
hexafluorophosphate for solution and solid phase synthesis.167 Coupling
methods for the safe incorporation of cysteine with minimal racemisation in
9-Fmoc SPPS include BOP (or HBTU or HATU)/HOBt (or H0At)EMP
(4:4:4) without preactivation in CH2C12-DMF (1:1), DIPCDI/HOBt (or
HOAt) (4:4) with Smin. preactivation and preformed pentafluoro
phenylesters in CH2CI2-DMF (1 : 1).'6s A novel algorithm for the coupling
control in SPPS has been described. The control scheme relies on a feed-
forward artificial neutral network algorithm which can predict the final
yield of the reaction within its initial 5 min. by analyzing the conductivity
signal profile.169
Goodman and co-workers have described the utility of a-aminoacid
N-carboxyanhydrides protected with the three urethane protecting groups
most commonly used for peptide synthesis, 9-Fmoc, Boc and Cbz, as highly
effective reagents in peptide synthesis in both solid phase and in solution.'70
Fmoc-amino acid fluorides, whether as the stable isolated species or as
intermediates generated in situ, represent convenient inexpensive reagents
for peptide coupling.17' Rapid coupling occurs even for sterically hindered
amino acids. In the case of preformed acid fluorides, coupling does not
require the presence of base, thus precluding the incursion of any base-
catalyzed side reactions, including loss of configuration at the carboxyl
group undergoing reaction. The coupling of Fmoc-amino acid chlorides can
be mediated by the potassium salt of 1-HOBt and 1-HOAt. 172.173 Coupling
is fast and racemisation free.
2. 7. Cleavage of the Peptide-Resin Bond
On completion of the chemical synthesis of the peptide chain, the
final step requires removal from the solid phase support and liberation of
the protected side-chains of the trihctional amino acids. The most popular
reagent for cleavage of peptides from Boc-based resins is anhydrous HF.
But, this necessitates the use of expensive HF resistant fumehoods and
cleavage apparatus.
More recently, an improvement over the conventional strong acid
SN1 deprotection process has been developed by Tam and co -~orke r s . ' ~~
This is a two step procedure that incorporates an SN2 process as an initial
step followed by an SN1 step to remove the more resistant pretecting
groups. This two step procedure has been named the low-high HF
deprotection procedure. Over the past few years, the use of other strong
acids such as TFMSA (trifluoromethanesulfonic acid) or TMSOTf
(trimethyl silyl trifluoroacetate) have begun to appear in the literature as
alternatives to HF cleavage of PAM and MBHA resins. Dilute HBr in TFA
containing pentamethylbenzene and thioanisole was used in the cleavage
and deprotection of peptides on MBHA-resin.
A new two-step deprotection/cleavage procedure for Boc-based
SPPS is reported.'75 First, the protective groups are removed from 4-
(oxymethy1)-phenylacetamidomethyl (PAM) resin attached peptide with the
weak hard acid, trimethylsilylbromide-thioanisole~TFA. In the second step,
the peptide is cleaved flom the resin with a stronger hard acid such as
trimethylsilyl trifluoromethane sulfonate in TFA or with HF. The hard acid
deprotection with 1M trirnethylsilyl trifluoromethanesulfonate in TFA has
been applied for the porcine peptide w."~
In addition to acid cleavage, several resin types such as oxime can
be cleaved using a variety of different methods to yield peptide hydrazides
and analogs of protected fragments. Some resins like Br-Wang and the Br-
PPOA resins are even cleavable by light. Cleavage of peptide from
benzylester type linkers by 2-dimethyl arninoethanol or N,N-diethyl
hydroxylamine yield sidechain protected peptide acids.177 Reductive
cleavage of peptide-resin ester bond in classical Memfield resins give
peptide-alcohols when treated with lithium borohydride in THF.'~*
2. 8. Purification and Characterization of Cleaved Peptides
Since Memfield's breakthrough in peptide chemistry, advances in
instrumentation, chemistry and computer technology have simplified
peptide synthesis and have made purification and characterization easier.
Automated peptide synthesizers eliminate time consuming manual
operations and minimize contact with caustic chemicals.
Solid phase peptide synthesis has proved to be an established
procedure only after the introduction of efficient HPLC. Semipreparative or
preparative HPLC, usually on reverse phase is necessary to achieve purities
of > 95%.179 Depending on the peptide size and hydrophobicity, packings
of C18, C8 or C4 are recommended. Newer techniques such as reverse
phase flash chromatography and perfusion chromatography may become
very important in the future. 180,181
Mass spectroscopy, especially in the FAB mode is a very usehl
technique for the structure elucidation of peptide The
introduction of electrospray ionization (ESI) and matrix assisted laser
desorption ionization (MALDI) has enabled the intact ionization of large
biomolecules for mass spectral analysis. 183,184 High resolution 2D NMR
spectroscopy has become one of the important tools in the elucidation of
three dimensional solution structure of fair sized biomolecules. Some recent
reviews put an insight into structure determination of proteins by three- and
four-dimensional NMR spectroscopy. 185,186 Bandekar studied the IR and
Raman spectroscopic results on amide bands in proteins, peptides and
polypeptides. Copenhagen has described the use of near-1R.Fourier
Transform Rarnan spectroscopy as a new method for monitoring the
secondary structure of the peptide chain during solid phase peptide
synthesis. 187,188 Some more examples of conformational analysis of
peptides come kom the laboratories of Throntan, Narita, Baldwin and
Ponnuswamy. 189-192 The use of molar ellipticity at 222 nm and
deconvolution of the experimental CD curves for secondary structure
estimation is well established. 193*194 Among the interesting new additions to
the technique of solid phase synthesis, the incorporation of internal
standards such as norleucine seem to have a lasting value, as it allows
monitoring of the progress of a synthesis by simple means. 195,196
2. 9. S S Bridging in Peptides
Disulfide bridges formed between cysteine residues constitute an
important structural determinant in peptide and protein s t ruct~res . '~~ One
need only mention the role of Actinomycin D in the study of protein
biosynthesis and of Valinomycin in the study of membranes. The artificial
introduction of extra disulfide bridges into peptides or proteins allows the
creation of conformational constraints that can improve biological activity
or confer therm~stability.'~~
Starting with the pioneering work of du Vigneaud on Oxytocin, the
challenge to reproduce and engineer increasingly complex arrays of
disulfide bridges as found in natural peptides and proteins has stimulated
the efforts and ingenuities of many peptide chemists.199 Within the peptide
field, there are a number of native molecules containing disulfide bonds
including Somatostatin, Endothelin and Calcitonin. Many of the peptide
toxins from snakes and scorpions possess multiple disulfides. Following
these examples, disulfide cystine linkages have been incorporated into a
large number of synthetic peptide sequences with the aim of reducing the
conformational freedom of the native molecules. For example, cysteine
residues have been incorporated in place of non-essential residues in the
peptide Opiate enkephalins, RGD sequences and Somatostatin. 200,201 In
1982, Schiller and co-workers used a D-C~S', L-C~S' cyclization to
incorporate conformational constraint into Leu-Enkephalin and
~ ~ n o r p h i n . ~ ~ ~
A number of model peptides containing disulfide linkages have
been examined using NMR and X-ray dimaction. These studies have
mainly focused on the ability to induce p-turns and P-sheets?'' P-hairpin
mimics constrained by disulfide bridges have been explored in solution?04
Cystine containing cyclic and acyclic bicystine peptides have been reported
to represent excellent models of antiparallel P-sheets. The use of disulfide
linkages across helices to stabilize helical bundles is reported by several 205,206 groups. In TASP approach of protein designing, disulfides have been
used as conformational constraints of templates.207 More recently, the
importance of small disulfide loops in the functional active site in the
redox-proteins, thioredoxins and glutaredoxins has been recognized?08
Disulfide bridges are important structural motifs in natural and
engineered peptides and proteins. The preparation of disulfide-containing
peptides hinges on reliable chemistry to form disulfide bonds. Usually, a
linear sequence is assembled by solid phase method and then protecting
groups as well as the anchoring linkage is cleaved. There follows oxidation
in dilute solution to minimize unwanted dimerisation and oligomerisation.
The alternative of carrying out deprotection and oxidation of the cysteines
while the peptide chain remains anchored to the polymer support is of
obvious interest and has received considerable recent attraction. 209,210 Such
an approach takes advantage of pseudo-dilution, which is a kinetic
phenomena expected to favor facile intramolecular processes.2'l
The disulfide bond forming reaction is a key step in the synthesis of
cystine-containing peptides. Usually, air oxidation or iodine oxidation has
been employed for this reaction.212 However, the former is time-consuming
under highly diluted conditions and the latter needs particularly controlled
conditions. Methyl trichlorosilane or tetrachlorosilane in trifluoroacetic
acid (TFA) in the presence of sulfoxide, can cleave various S-protecting
groups of cysteine to form cystine directly?'3 A mild and highly efficient
method for intramolecular disulfide bond formation in peptides mediated by
charcoal has been developed. Completion of charcoal-assisted catalysis of
disulfide bond formation took less than 6 h, testing a series of peptides with
ring sizes varying from 2 to 17 amino
Cyclic peptides have become powerful tools in the hands of
biochemists and have served as the means whereby great
made in the study of a number of biochemical processes. .$ ,. . .
2.10. Synthesis of Hydrophobic Peptides
In 1959, K a m a n n suggested that
defined as the tendency of non-polar side chains to pack together in the
interior of the protein molecule where they can avoid contact with water,
might be important for the stabilization of the native structure of
It is now widely assumed that hydrophobicity is the major factor in
maintaining the specific structure of native The hydrophilic-
hydrophobic balance can be estimated theoretically from the calculated
hydrophobicity values and experimentally £rom the retention times in
reverse phase high performance chromatography.
The hydrophobicities of the individual amino acid side chains have
been measured experimentally in a variety of ways, using the free amino
acids, amino acids with the amino and carboxyl groups blocked and side
chain analogues with the backbone replaced by a hydrogen atom and using
a variety of non-polar solvents including ethanol, octanol, cyclohexane and
di~xane.~" Synthesis of hydrophobic peptides is a difficult process because
of the non-polar side chains and due to the coiling tendency of the
peptides.2's Peptides with substantial hydrophobic character also tend to
aggregate with increasing concentrati~n.~'~
Hydrophobic proteins are the vital components of every living cell.
Often associated with cell membranes, their functions vary from ion or
molecule transport to cell recognition to signal transduction. Owing to their
limited solubility in aqueous solvents, structural analysis by conventional
techniques has been problematic. Mass spectrometry has become a
valuable tool for the peptide and protein analysis. The development of mass
spectroscopy for the analysis of hydrophobic peptides and proteins provide
an extremely valuable tool in structural studies of this important class of
proteins.
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