2.1 Introduction
Synthetic hydrophilic polymers find promising applications in
pharmacology, biotec:hnology and chemistry. The biocompatibility,
biodegradability and phzumacolo~cal activity of these polymers depend
much on their hydrclphilic nature. Polymer-supported synthesis has
achieved a major place in polypeptide and oligonucleotide synthesis over
1-6 the past three decades. Peptide synthesis has proven indispensable for
the structural elucidation and activity studies of many naturally isolated
products having a peptide structure such as hormones, neuropeptides,
antibiotics and enzymes, which can be isolated only in very small
quantities. Synthetic peptides find application in all areas of biomedical
research including immunology, neurobiology, pharmacology,
enzymology and molecular biology.7 The chemical synthesis of peptides
with the naturally oc~cuning structure is possible; it was used for the
development of artificial vaccines and potent drugs that can
substitute the conventional drugs having various side effects.
Investigation of structure-activity relationship of biologically active
peptides also demand:; the synthesis of many analogues of a given
peptide.
In the beginning of 20th century, Emil Fischer synthesised the
first peptide in solutior~.~ The general chemical requirements for the
synthesis of peptide involve the blocking of carboy1 group of one
amino acid and the a~mino group of the second amino acid. The
activation of the free carboxyl group resulted the formation of
amide bond between the amino acids and the selective removal of
the protecting groups resulted a dipeptide. The method developed by
Fischer was laborious and time-consuming because the intermediate
peptides have to be rernoved, purified and characterised before the
next coupling step. The major limitation of classical solution phase
synthesis of peptides is the low yield and solubility of the
intermediate peptides with increase in chain length. A new
approach was needed jor the synthesis of larger and more complex
peptides with high purity and yield.
Merrifield introduced the concept of solid phase synthesis to
achieve more e6cien.t synthesis of peptides. In SPPS, the peptide chain
was assembled in a ste:pwise manner while the C-terminal end of the
peptide was anchored to an inert cross-linked polymer support and
the peptide was grown from C-terminal to N-terminal residue.
Menifield demonstrated the feasibility of the idea by synthesising a
model tetrapepude ~~leuc~l-~-alanyl-~l~c~l-lvaline.~ Simultaneously
with Memfield, Letsinger and Komet reported the synthesis of a
dipeptide, L-leucyl-glycine on a "Popcorn polymer support" using a
different chemical strategy. ' O The N-terminal amino acid was
anchored to the polymer support and the peptide was grown from
N-terminal to C-terminal. This technique is not so popular
because the cleavage from the support under mild condition is not
possible and always there is a problem of racemisation. Memfield's
invention formed the basis of a new technique of peptide synthesis,
which has been used till now with several methodological
improvements and refin~ements. The design of polymer support, its
chemistry and applications for SPPS have been extensively
reviewed. 3,) 1b16 These refinements and recent developments in
designing solid supports and various factors involving in solid
phase peptide synthesis are reviewed in this chapter.
2.2 Principles of Memfield Peptide Synthesis
SPPS follows the strategy of the stepwise assembly of peptides
by consecutive coupling of amino acids. The stepwise synthesis is carried
17.18 out from the C-terminal to the N-terminal of the target peptide. This
approach can eliminate the possibiity of racemisation. The stepwise
synthesis using low rn.olecular C-terminal protecting group finds less
application because of its sparing solubility in organic polar
solvents does not permit a homogeneous reaction. Carrying out the
synthesis as a heterogeneous reaction is also not very promising
due to poor filterability of the reaction mixture.
Merrifield method employs an insoluble and filterable
polymeric support such as cross-linked polystyrene that function
as the carboxy-protescting group for the C-terminal amino acid of the
peptide. The target peptide sequence was formed in a stepwise
manner by attaching temporary N,-protected C-terminal amino acid
to the chloromethylated PS-DVB resin. After the removal of N,-
protection, the next N,-protected amino acid is coupled and the
process is repeated until the entire desired peptide is assembled on
the polymer support. Clicyclohexyl carbodiide (DCC) is used as the
coupling agent,lg and ,all the reactions are cmied out under non-
aqueous conditions in organic solvents. The target peptide was
deprotected and cleaved from the polymer matrix by acidolysis with HF
or anhydrous TFA in the presence of suitable scavengers.2@21
Even though highly pure peptides can be synthesised by
classical solution phase method, it has the following shortcomings:
i. The method is slow, tedious and laborious. In order to obtain a
peptide with high purity, the constituent amino acids are
incorporated in a stepwise manner starting from the peptide's
C-terminus end. Afiter each completed amino acid addition, the
intermediate peptide is separated from any remaining reactants
before its characteri~zation, leading to lengthy synthesis time.
ii. The increasing insollubility of the growing peptide chain in the
reaction medium causes problems in both purification and in
the next coupling step results in the termination of peptide
chain elongation.
iii. Methods such as; chromatography and crystallization required
during the synthesis results in considerable reduction of the
overall yield of the peptide.
Solid phase peptide synthesis has the following advantages
over the classical so~lution phase method.
i. The peptide is synrhesised while its C-terminus is covalently
attached to an insoluble polymeric support. This permits the
easy separation of t:he growing peptide from any by-products or
excess unused amino acid components.
ii. The reactions are driven to completion by using an excess of
reactants and reage:nts.
iii. No mechanical loss occurs because the growing peptide is retained
on the polymer in a single reaction vessel throughout the
synthesis.
iv. The final peptide is detached from the polymer support by a
single cleavage ster~ at the end of synthesis. The side chain
protecting groups can also be cleaved in the same reaction in
order to simplify the work-up and the isolation of the final
peptide. The cleavage step does not degrade the assembled
peptide.
v. The physical ope:raltions involved in the synthesis are simple,
rapid, and amenable to automation.
vi. The spent resin can be recycled.
In spite of these aldvantages, Merrifield's solid phase method
has a number of limitations and is extensively reviewed. 16,22-24
They are:
i. Non-compatibility of resin and growing peptide chain.
ii. Lack of stability of peptide-resin linkage under the conditions of
synthesis.
iii. Non-equivalence of functional groups attached to the polymer
support.
iv. Formation of errosr peptides due to truncated and failure
sequences.
v. Peptide conformation changes in macroscopic environments
inside the polymer matrix and also due to peptide resin linkage.
A number of modifications have been introduced to overcome
the difficulties associated with Merrifield SPPS which includes:
i. Development of new supports with high swelling properties
permitting improved solvation of both matrix and growing
peptide chain.
ii. Introduction of multi-detachable anchoring groups improving
the flexibility of synthetic strategy.
iii. Development of n'ewer separation method (eg. preparative and
semi-preparative HPLC) and characterization techniques in
peptide synthesis.
Extensive investigation upon reaction rates and kinetic course in
solid phase synthesis revealed that the reaction sites within the
polymeric matrix are chemically and kinetically not equivalent,
resulting in deviations from linear kinetics. Consequently quantitative
reactions are difficult to obtain. The preparation and accessibility of
insoluble polymer reagents appears to limit a more general application
of the compounds. These inherent difficulties in solid phase synthesis
were mostly overcome by the 'Yiquid-phase procedure" proposed by
Bayer and Mutter, 25-30 which makes use of soluble polymeric
groups. These macromolecular groups determine the physical and
chemical properties of low molecular weight components covalently
attached to the polymer. Such a technique allows the effective and
quantitative removal of reagents. All reactions proceed in
homogeneous solutiorl in analogy to the low molecular weight system.
The reduced operatio:nal simplicity and changes in the crystallization
tendency makes the liquid phase peptide synthesis less versatile.
2.3 The Role of Solid Support
The stability of' the solid support under all conditions of
functionalisation an.d synthesis is the prime requirement in SPPS.
It is believed that the peptides show a lower tendency to aggregate
because of limited ir~termolecular interactions when bound to a
polymer than in real solution especially at low substitutional levels.
SPPS could therefore be favoured for solution condensations when
31-33 solubility problems in LPPS arise. The restricted mobility of the
peptide anchored to the polymer could become a disadvantage of
SPPS. The PS-DVB support has been widely used for the synthesis of
peptides using BOC-chemistry.w Polystyrene resin with 1% PS-DVB
showed optimum swelling and stabiity while 0.5% PS-DVB resin is too
fragile and above 2% PS-DVB resin does not swell effe~tivel~.~ The
extent of swelling in polar organic solvents increases as the number of
amino acid residues incorporated to the resin increases.36 This is
due to the lowering of the network free energy based on the
additional solvation of' the growing peptide chain. 33.37,38
Merrifield techniclue has undergone a series of mcdifications and
improvements because of the physicochemical incompatibility of the
growing peptide chain and the rigid hydrophobic macromolecular
environment created by the PS-DVB network of the support. In order
to optimize the resin structure in SPPS, Sheppard introduced a polar
polydimethyl acrylmnide resin, which is structurally similar to
peptide backbone. 12.39 This helps easy solvation of the peptidyl
resin and thus reduces the steric hindrance during deprotection and
coupling reactions.4043 Cross-linked and funtionalised polydimethyl
acrylamide gel can detained within the pores of fabricated Keiselguhr
which can be used a:s a matrix in continuous flow method. The
support shows effectiv~: swelling in polar solvents but in non-polar
solvents it is very pc~or. The chemical stability of the resin was also
less comparable to that of polystyrene supports.
The mixed PE:G-PS resin, a highly promising class of solid
support, is used successfully in polypeptide synthesis. 44-46
A cross-linked polystyrene-pol~ethyleneglycol graft co-polymer
with a 2-nitrobenzyl ;anchoring group has been used as a solid
support for the stepwise synthesis of peptides.47 Swelling, which is
a sign of good solva1:ion of the resin, is good for polystyrene resins
in non-polar or less polar solvents like DCM, whereas polyacrylamide
resins swell much better in DMF. Mixed PEG-PS polymers show
excellent swelling in common solvents such as THF, acetonitrile and
alcohols.44 The sirnillar polarity of peptides and polyacrylamide
support, both well soluibilised in DMF, makes the support suitable for
SPPS.48 On the basis of solvent-resin interactions, increasing effort
has been made to introduce polyacrylamide where initially
polystyrene supports were used.49
Bis-2-acrylamidoprop- 1 y l polyethyleneglycol cross-linked
dimethyl acrylamide (PEGA) has been introduced as a hydrophilic,
biocompatible and flexible solid flow stable support in peptide
synthesis (1). 50(a,bl
(1)
The first flow stable synthesis resin was obtained by
polymerization of the soft polydimethyl acrylamide gel inside a
solid matrix of sup:porting ~e i se l~uhr . " Small and Sherrington
replaced the irregu1a.r Keiselguhr with more regular rigid 50% cross-
linked polystyrene sponge containing a grafted polydimethyl acrylamide
This technique IwaLs developed for grafting polyethylene glycol
on to 1% cross-linked1 polystyrene,53 which are monodispersed,
spherical and flow stable. Polystyrene grafted to films of
polyethylene has been used for synthesis of peptides under non-
polar conditions (21.~~ Polyhydroxypropyl acrylate coated
polypropylene and coltton has shown some promise as supports
under polar conditions. 55.56 A co-polymer of bis-acrylamido
polyethylene glycol, lV,IV-dimethyl acrylarnide and acryloyl sarcosin
ethyl ester was succe~;sfully employed for the synthesis of peptides. s7(a-b)
The inert polyethy1e:ne glycol cross-linked resins such as
polyoxyethylene-po1yo:rq~propylene (POEPOP) (3) and polyoxyethylene
polystyrene (POEPS) (4) were efficiently used a s flexible and
biocompatible resins in SPPS.~' Cross-linked Ethoxylate Acrylate
Resin (CLEAR)O supp>rf:s developed by Kempe and Barany were also
used successfully m SPFS.~~
Different concepts for multiple peptide synthesis (MPS) or
simultaneous multiple peptide synthesis (SMPS) were developed to
respond the rapidl-y growing demands for a large number of
peptides with conipletely different sequences. The multiple
synthesis methods are mostly applied in hormone and inhibitor
research. For the identification of relevant.
side chains, every am.ino acid of a biologically active peptide can be
systematically substi.tuted, the chain length can be varied, and N-
as well a s C-termini can be rn~dified.~'
In the initial work of SPPS, the various N-terminal modifications such
i as acetylations, biotinylations, succinimidilations or couplings for
/
preparing irnmunogens could be achieved by multiple methods or by
consecutive syntheses. In multiple peptide synthesis it is possible
to simultaneously prepare the same peptide bound to different anchors
and cleave to obtain peptide acid, peptide arnide, alkylated arnide,
hydrazide or a fully protected fragment. The "tea-bag" method proposed
by Houghten belongs )to the oldest strategies of multiple peptide
synthesis." In "tea bag" method, polystyrene in polypropylene mesh
packets were used as supports. Geysen et al. developed the concept of
multi-pin synthesis technology (PIN) and several hundred peptides
can be simultaneously prepared using this procedure.43 Acrylic acid
coated polyethylene rod:; were used as supports in multi-pin synthesis
technology. Valerio el: al. used 2-hydroxy ethyl methacrylate grafted
polyethylene supports in multi-pin peptide synthesis.62 In multi-
column methods Macrosorb-SPR resin was used.63 Frank et al.
proposed an inexpensive procedure for the preparation of polymer
bound peptides in which the first amino acid was coupled to a sheet of
cellulose paper.64 Recently cross-linked enzyme crystals (CLECs) of
thermolysin were also used for peptide ~ynthesis .~ '
The polymer matrix has a significant role in SPPS. The
success of SPPS depends on the physicochemical characteristics of
the peptide bearing support. For effective swelling of the resin and
solvation of the peptjde, the polymer should have an optimum
50,52 hydrophobic-hydrophili.~ balance. The structure-reactivity and
structure-property correlations in cross-linked polymeric systems
helped to design new :supports with mechanical stability and optimum
reactivity. Pillai et al. 'developed a series of polymer supports by
introducing polar cro:ss-linking agents such as triethyleneglycol
dirnethacrylate (TEGYDMA), tetraethyleneglycol diacrylate (TTEGDA),
hexanediol diacrylate (HDODA) and 1,4-butanedioldimethacrylak
(BDODMA) to po1ysQm:ne network. - These resins have optimum hydrophobic-hydrophlic balance and swell like a gel in most of the.
organic solvents usedl in peptide synthesis. E f f ~ c i e n of these supports
was demonstrated b y :synthesising large number of peptides in high
purity and yield. The new member of this series described in this
thesis, is the g1yce:rol dirnethacrylate cross-linked polymethyl
methacrylate (GDMPi-F'MMA). This support showed very high swelling
characteristics in various solvents, an effective hydrophobic-
hydrophilic balance and is successfully used for the synthesis of
pep tide^.'^^ The optimum reactivities of these newly developed
resins are due to the greater chain mobility of the cross-linker in
solvents that enable effective interaction between the reactants
and resin bound functional groups.
2.4 Resin-peptide Linkages
Polypeptide synthesis by solid phase technique is more
effective when a specific combination of the linker/handle-resin
was used. These linkers can help the cleavage of the peptide from
the support under specific selected conditions that enable the
peptide free from side reactions. The covalent linkage between the
growing peptide chain and the polymer support is one of the
factors that determine the purity of the peptide.' The design and
development of a :series of bifunctional linkers (handles) have
facilitated and extendled enormously the scope and application of
SPPS approach for Ithe preparation of large peptide through a
convergent strategy7' and also hybrids of peptides with other
72 74 biomolecules containmg labile structures. One end of the bi-
functional spacers is attached to a smoothly cleavable protecting
group and the other end allows coupling to a previously
functionalised support. The linkages are easily formed and stable
to repeated cycles of acylation and deprotection steps.
The initial procedure of SPPS was based on temporary Boc
and permanent Bzl- protection (Boc/Bzl) with benzyl ester linkage of
the peptide to the resin. The HF cleavage of the peptide resulted in
concomitant loss of all protecting groups, thus offering no access to
protected peptide fragments. The protected peptides can be obtained
from Memifield resin by trans-esterification, ammonolysis or
hydrogenation depending on the stability of side chain protection.7541
Recently Fmoc/ t-Bu based protection schemes have become
increasingly popular with the development of a great number of
handles or linker-resiLns and cleavage of the protected peptide from
the resin is achieved under milder conditions by a great variety of
reagents.
Table 2.1
-
No.
-
1
-
2
-.
3
-
4
- -.
5
6
-
7
8
Some common linkers utilised in SPPS -- - - v- I
Structure Cleavage Reagent
0 0 Acetoxybenzyl (HMPAIPBA)lw TFA
0 0
0 0 II Methoxy Dilute
aceto~ybenzyl '~~ T F A
o cH,C Methoxyalkoxy benzyl Dilute R-C-(X ii ( S A S R I N ) ~ ~ ~ TFA
Hydroxymethyl phenyl acetamidomethy l (PAM)'''
0 C I I , l 0 Hydmxymethyl
dimethoxy Dilute phenoxyvaleryl T F A (HAL)"'
TFA
Alkoxy dimethoxy benzhydryl i ink)"^
4-benzyloxy- 4',4" dimethoxy hitylamine (BDMTA)"~
Benzhydrylamine ( B H A ) " ~
Methyl benzhydylamine (MBHA)"'
Methoxybenz hydrylamine (MeOBHA)"'
Dilute TFA
Dilute TFA
Dilute TFA
TFA
Aminomethyl dimethoxy
Dimethoxy b e ~ ~ z h ~ d r y l " ~
Amino xanthyloxy valeryl (XAL)"
Nitro henyl ethyl 1 4
TFA
TFA
Dilute TFA
Dilute TFA
* Cleavage reagent for the peptide
1
2.5 Protecting Groups
Of the various amino protecting groups t-butyloxycarbonyl
(Boc)8l,s2 and fluore:n~~lmethoxycarbonyl ( F m o ~ ) ~ ~ - ~ 5 are widely used.
Boc group can be cleaved under acidic conditions and Fmoc group
under basic conditi.ons, hence they are considered as orthogonal
protecting groups.86 For the removal of Boc group 30% TFA in
DCM us used. Though neat TFA is usually used for reducing the
time required for the synthesis of long peptides, studies show that
incomplete removal of Boc group may happen due to the
22
23
Fluorenyl methy 1"'
~ l l y l e s t e r ~ ~ ~
~ i t r o b e n z ~ l ' ~ '
Nibobenz h y d ~ ~ l a m i n e ' ~ ~
0 II
R g - c - - w w R-C-O(H\ -
I t /
~~~
U II E'
R-C-WH, ,+I= n~-C:-NHctl
~~ --
0
Piperidine
Pd(O)
hv
hv
II
25
-
~-
. ~p
insufficient swelling o:i the resin irk TFA.87 Earlier 4 N HC1-dioxane
was used for the cleprotection.88 However this reagent was not
sufficient to give complete removal of Boc group. TFA in DCM
provides an excellenl: medium for the maximum solvation of
peptidyl resin.8"
Fmoc group can be easily and rapidly removed by using a
secondary arnine (20% piperidine in DMF). Unlike Boc method, the
deprotection step in F~noc strategy avoids the neutralization stage,
thus reducing the time for synthesis. Besides this, Fmoc group
permits the easy monitoring of the progress of the synthesis
spectrophotometrically and hence Fmoc amino acids are widely
used in continuous flow synthesis.90 However, recent studies
reveal the fact that Frnoc group induces P-sheet conformation,
hence in solid phase peptide synthesis, using Fmoc protecting
strategy incomplete removal of Fmoc group is frequently notedg'.
The side chain of Boc amino acids are protected by benzyl based
groups. Boc/Bzl groups are removed during the cleavage step of
peptide from the support. In Fmoc strategy, the side chains are
protected by t-butyl groups which are stable in basic conditions,
but: are very sensitive to acid treatments.
Table 2.2
Commonly used W-protecting groups
Cleavage conditions No Name and Structure
1
2,
3
4
5
6
Benzylo:cycarbonyl (2)Iz9
c)- ,,,- @, ? -
~~
HBdHOAc, HBrITFA, Catalytic hydrogenolysis or Liquid HF
4-rneth0x~t1en.z~lox~carbonyl~~~
H3C f \ CH2-f3C- - ~-
Terr-Butylox)~carbonyl (BOC)"'
FH3 ? CH,-C-0-C-
I CH3
2-(4-Biphenyl)-iso ro oxycarbonyl ( ~ p o c ) P32.83
0
-
RSHITENDCM at 25' C for 5 min.
TFA
-
EIF or TFMSA
,4cidolysis, Catalytic hydrogenation
Dilute TFA
.-
12
13
14
15
16
--- ._.-___~--- 143, 144
Dithiosucc'inoyl ( U ~ S )
4 s. L-nN- 0
~
~- Diphenylphosphinyl ( ~ p p ) ' ~ ~
(2 / ( + + p + O
--
p-~iolueneaulphonyl ( T O S ~ ~ ) ' ~ ~
(-'-Jm2-. CH:,-
- ~~ ~ . -__ - - Allyloxycarbonyl(~lloc)'~~
I? CH2= CH-CH2-0-C--
~
148, 149 4-Methoxybem:yloxycarbonyl (Moz)
f H~cD--( \ CH2-(PC-
1 I I
3 -2 _ . _ ~-
2.6 Coupling Reagents
The formation of amide bond is the key step in peptide
synthesis. The reagent used for coupling should be able to form
the peptide bond in mild conditions, and the reaction should be
fast. They should retain the optical integrity of the peptide during
Thiolysis
Photolysis
Base labile
18
19
.
3-Nitro-2-pyridinesulfenyl (~pys) '~ '
02N
(>s-NH
- - 6-Nitrover:atryloxycarbonyl
(Nvoc)l? I . 1'2
WH3 /
P (XZH; CHZ-0-C-
.. . - - 2-[4-(methyls1llfonyl)phenyl~~lf0nyl]
ethoxycarbonyl ( M ~ C ) ' ~ '
rr,cxo, R {Ss ,,-
synthesis. Some of the recently introduced coupling reagents are
given in Table 2.3.
In spite of all th.ese achievements, dicyclohexyl carbodiirnide
(DCC) is still the widely used coupling reagent.22 The mechanism of
the reaction of DCC with carboxyclic acid via the formation of very
reactive 0-acyl isoure:a intermediate has been proposed by Khorana.g2
DCC when used with excess carboxylic acid make symmetrical
anhydride which art: capable of attacking the nucleophile.93
Recently, DCC active esters94-g6 of amino acids have been
widely used in peptide synthesis. As these are stable at room
temperature, they can, be prepared and stored for long time for the
direct reaction in coupling. Among these, pentaflurophenyl esters
of amino acids are very popular.97
Miyazawa et. al used Cu(I1) chloride for suppressing the
racemisation in I)CC assisted coupling reaction.98 I-Hydroxy
benzotriazole (HOE3t) is used as coupling additive which can
prevent racemisation.99 HOBt is used either in conjunction with
DCC or as active ester or built into a stand alone reagent in the
form of phosphoniurnl00~'01 or u r o n i ~ m . ~ ~ ~ ~ ~ ~ ~ The presence of
tertiary amine, e.g diisopropylethyl amine (DIEA), has been
reported1o4 to increase the reaction rate and product yield and are
comparable with other rapid coupling method using (I-H, 1,2,3-
benzotriaw1)- 1-yloxy tris(dimethy1amino) phosphonium hexafluoro
phosphate (BOP) or 2-.(3,4-dihydro-40x0- 1,2,3-benzotriazin-3yl)-
1,1,3,3-tetramethyl-uror~ium tetrafluoroborate (TDBTU) and no
racemisation is noted. lo4
Comparative stucly of various coupling agents to couple
hindered peptides shours that urethane protected amino acid N-
carboxy anhydride (UNCA) and bromo-tris-(pyrro1idino)-
phosphonium hexafluol-ophosphate (PyBrop) are excellent reagent
compared to 2-(1-13 Benzotriazol- 1- 1-yl) 1,1,3,3-tetramethyl-
uronium hexafluoro-phosphate (HBTU), pentafluorophenyl esters
or mixed anhydrides.10"
Table 2.3
Coupling reagents used in peptide synthesis
Reagent Structure
!
1
2
3
4
6
7
N,N'-Dicyclohexylca~rbodiimide (DCC)"~
- ----
N-Hydroxysuccinimide ( H O S U ) ' ~ ~
--
1-Hydroxybenzotriazole ( H O B ~ ) " ~
(Benzotriazol-l -ylo:~y) tris(dimethy1amino) phosphonium hexafluorophosphate BOP)'^'
-
I-0x0-2-hydrox!idihydrobenzotriazene ( H o ~ h b t ) ' ~ '
- - --
7-Aza-I-hydroxybenzotriazole ( H O A ~ ) ' ~ ~
. . . - __ __--___
4-Am-1-hydroxq.benzotriazole
(~-HoA~) '"
.. - - .. -
0
HO-N?J
0
N I OH
QJN% N' I 0 0 PF, I
\ ,pe / N I N,
N / \
dxxoH aN,,N .
N f OH
02 \ OH
N,N'-Diiso ro ylcarbodiimide k'. 2 (DIPCDI)' ' '
1-Ethyl-3-(3'-dimeth laminopropyl) carbodiimide (EDC) x3,,64
tris dimet thy la mi no:^ phosphonium hexafluorophosphate (AOP)"~
tris (pynolidino) phosphonium hexafluorophosphate ( P ~ A O P ) ' ~ ~
N-methylmethaminiurn hexafluorophosphate N-oxide (HBTU)'~'
0 CI
01 H-N w N = c = N - - /
I
I 0 0 PF, I
\ ,pP / N I N,
N /
1-(1-Pyrrolidinyl-IH-1,2,3-triazolo [4,5-b] pyridin-yl methylene) pyrrolidinium hexaflulorophosphate N-oxide (HAP~u)"'
1,2,3-benzotriazin-3-:{I)-l,1,3,3- tetrameth luroniurn hexafluorophoshate (HDTU)lYZ
Tetramethylfluorofonmamidiniurn hexafluorophospha.te (TFFH)
173.174
Pentatluorophenol ( H O P ~ P ) ' ~ ~
Urethane protected amino acid N- carboxy anydride (UNCA) 177-179
2-(3,4-Dihydro-4-0x0-
1,2,3-benzotriazin-3:yl)-l,I ,3,3- tetramethyluronium tetrafluoroborate ( T D B T U ) ' ~ ~
tetramethyluronium tetrafluoroborate (TPTU)I*I
2-(5-Norborene-2,3-dicarboxamido) 1,1,3,3-tetramethyluronium tetrafluoroborate ( ' T ~ I T U ) ' ~ ~
Fmoc amino acid halide 182-188
Tetrabutylammoniuni hydroxide1 p-toluenesulphonyl chloride189
I -(P-Naphthalene: s l~ l honvloxy)- benzotriazole (NSBt) 790 .
Bromo-tris(dimethy1amine) phosphorium hexduorophosphate (B~oP)"'
--
Bromo-tris-byrrolidino) phosphorium hexafluorophosphal:e ( P ~ B ~ O P ) ' ~ ~
Benzotriazolylox.y-bis-pyrrolidino- carbonium hexafluorophosphate ( B B C ) ' ~ ~
p~ ~
Bib (Boc) amino acid f lu~r ide"~
- - - -- - --
4 N~trophenol (ONp) 197 19X
-- - --- --
Q-I N-N
199-200 N-NHCHC,,
0-(Benzotriazol-1 -o!il)- 38 1 ,I ,3,3-pentameth:yleneuronium
hexafluorophosphate (HBPipU) 202,203
0
0-(7-Azobenzotriaz:oI-1-yl)- [TN'i Q PF6
41 1,3-dimethyl- 1.3..trimethyleneuronium N N'
Me hexafluorophosplhate (HAM'TU)'~'
0
0-(7-Azabenzotriazol- 1 yl)- PF6
F4 ? Me
2-(Benzotr~azol- 1 -yl)oxy- 42 1,3-dimethylimidaz.olidin~um
hexafluorophosphate (901)'"~ M e
- - - - - -- ---
40 1,3-dimethyl-l,3-di~nethyluronium hexafluorophosphate (HAMDU)'~'
00 '
2-(1 H-Benzotriazol-1-yl)-1,1,3,3- tetrameth luronium tetrafluoroborate J (TBTU)' '
2.7 Purification and Characterisation of Peptides
A s a general colasideration, homogeneity and correct covalent
structure are the basic goals of the synthetic endeavour and are the
two factors that pervade any and all consideration of the state of
the synthetic product. Even with optimised SPPS, there will be
typically small amounts of deletion peptides and a family of
peptides with chemical modifications due to the side reactions in
the final deprotection.
The evaluatiorl of the homogeneity and covalent structure of the
peptides are prime :importance and the most commonly used
techniques are analytical H P K , capillaryzone electrophoresis (CE),
amino acid analysis, 1JV spectroscopy, sequence analysis and mass
spectrometry. For the homogeneity and structural determination, each
technique makes its, own specialised contribution to the evaluation
process by providing unique information and generally they all are used
to some extent in a complimentary fashion (Figure 2.6).
Homgenay Covalent Stmcture
\
Amino acid UV
Figure 2. 6 ?he relative contribution of analytical t~ools toward the goal of assessing homogeneity and covalent structure
The developmer~t of HPLC in the late seventies revolutionised the
synthetic peptide chemistq by the adequate, rapid and sensitive
analybcal and punfica.tic)n modes of crude synthetic p r o d u c t s . ~ 5 , ~ It
also flouted one of the veiy early-applied peptide purification method-the
gel filtration method. Because amino acids are the fundamental units of
peptides the chromatographic behaviour of a particular peptide will be
determined by the number and properties (polarity, charge potential) of
the residue side chains it contains. In general the major modes of HPLC
employed in peptide separation take advantage of difference in peptide
size (slze-exclusion HPLC), net charge (ion exchange HPLC) or
hydrophobicity (reve:rse phase HPLC). Within this modes mobile
phase condition may be manipulated to maximise the separation
potential of a particultu :HPLC column. The reverse phase HPLC is the
most important mode of chromatographic technique for synthetic
peptide purification. 'Th.e alkyl derivatised (C8 and C18) silica based
packings are the most frequently employed packing support for
reverse phase HPLC separation of peptides. HPLC has proved
extremely versatile for the purification of a single peptide from the
kind of complex peptide mixtures encountered following solid
phase peptide synthesis, where impurities are closely related to
the peptide of interest: (deletion, terminated, chemically modified
peptides), perhaps missing only one amino acid residue and hence
may be difficult to :separate. The HPLC of very hydrophobic
peptides often pose special problems due to their limited solubility
and tendency to aggr'egate and some times it may be absorbed
irreversibly to some reverse phase sorbents.m7 Despite these, the
contaminants frequemt).y encountered in solid phase, including side
chain protecting groups, coupling reagents, cleavage reagents and
scavengers are often difficult to separate from the desired product
peptide during HPLC on Cs and C18 columns.M8
Either andpcal HPLC or CE is normally done as the frst level
of assessment. Capill,~)r electrophoresis is a high resolution, sensitive,
rapid and quantitative electrophoretic separation technique used for
the characterisatior~ of synthetic peptides. The speed and high
resolution capabilities of CE have found ready application in the area
of synthetic peptide c:hemistry. Its one major role has been to analyse
the products of automated peptide synthesis were both deletion and
incompletely deprotected peptides are commonly present. Separation
of peptides by CE occurs as a result of two interacting forces,
electrophoretic migration and electroosmotic flow. In fact, CE can be
an excellent analytical tool for any synthetic peptide manipulation
that results in a change in the net charge or shape of the molecule.
Amino acid analysis quantifies the amount of peptide and
determines whether the requisite amino acids are present a t
reasonable rati0s.m However it is less reliable for certain amino acids
such as cysteine, methionine and tryptophan and the
compositional accuracy decrease as the peptide gets larger.
Sequence analysis, particularly ,the gas phase sequencing and
analysis of the poly1ne:r bound peptides have made tremendous
opportunity to SPPS. UV spectroscopy monitors the integrity of the
aromatic amino acids particularly tryptophan and is very useful in
peptide purification. But the amount of information that can be
obtained about the covalent state of the peptide is very limited.
The recent developments in the field of mass spectrometry,
such as the introduct~orl of desorption ionisation technique and time
of flight mass measurements have made the ionisation and accurate
mass analysis of peptides and proteins feasible at the precise
juncture. In this c1a.s~ the first emerged one was the fast atom
bombardment mass spectrometry (FAB MS).210-211 The FAB MS has
found a particular niche in the mass analysis of biological
macromolecules up to 4 KDa. A valuable application of FAB MS has
been in the verification of the molecular mass of synthetic peptides
produced by automated synthesis.212 Moreover it has the ability to
determine precisely the molecular mass of the peptide with less
than 1Da. Thus amino acid insertions, deletions or chemical
modifications to the peptides can readily be discerned. FAB
MS/MS allows the elucidation of the primary structure of the
target peptide, even1 tiom a mixture and also permits the rapid
identification of synthetic side products.213 So FAB M S and FAB
MS/MS aid in the unec~uivocal characterisation of synthetic peptides.
The second one, electrospray ionisation mass spectrometry (ESI
MS) is an easy and rapid method for the verification of proper peptide
synthesis and for the identification of most: synthetic peptide
byproducts.214-215 Com~pared to other available mass spectrometric
techniques ESI MS offers the advantage of relatively low cost,
convenient automation and ease of interfacing on-line with liquid
chromatography (LC) or capillary electrophoresis. In addition the
multiple charges on most peptides enhance the abiity of ESI MS to
provide structure elucidation or conformation by fragment analysis. In
this series the most a.dvanced electrospray time of flight instrument will
give better perfomances.216
The matrix assisted laser desorption ionisation mass
spectrometry, an outgrowth of direct laser desorption ionisation
spectroscope (LD-MS)I~''~ pioneered by Hillenkamp and Chait is highly
useful in the characterisation of peptides and protein.218 With the
advent of time of flight mass analyzers, MALDI MS extends from
the low molecular weight range to the very high molecular weight
range upto 200 KDa and greater.21g The least complex instrument,
a typical linear mc~de MALDI TOF M S system is suitable for
monitoring most problems encountered in solid phase peptide
synthesis laboratories.
For a primary in:formation of the conformational state, CD,220
ORD221 a d vibrationsll spectrosCopic techniquesm2w were used
extensively. In the characterisation scenario of synthetic peptides, the
most informative of the analysis array, three dimensional (3D)
structural analysis using one dimensional (ID) and two dimensional
(2D) nuclear magnetic resonance (NMR) spectros~opy,224~~~~ and
X ray crystallography .2"6
2.8 Problems Associated with SPPS
The reactions in heterogeneous media are not like any true
solution type reactions. Hence unless all the different stages in a
multi-step synthetic procedure proceed unequivocally and
quantitatively, we cannot expect pure product at the end of the
synthesis. Even if we consider the chances of other side reactions
such as racemisations during coupling, side reactions associated
with individual ami:no acids, branching of peptide chain from the
side chain of a trifunctional amino acid, and the problems of
detachment of peptilde from the support, the yield of peptide after
10 or 15 residue inco~rporation will be too low.. The accumulation
of significant amount. of deletion peptide are well-documented.41
The inherent probllerns which limit the yield of peptides have
identified and these are attributed to the polymer effect or self
aggregating tendency of the peptide bound to the support via.
P-sheet formation or c:ombination of both.227
It was believed that polymer support does 'not have any
influence on the attached peptide chain. However studies show
that the polymer has a significant effect on the growing peptide. In
SPPS methodology, the growing peptide chain is covalently attached
to the insoluble support. The support imbibes organic solvent, and
comes highly swollen gel. Studies indicate that the interior of
peptide-resin is in a highly mobile environment comparable at a
molecular level to free solution, except the fact that diffusion is
restricted, and the homogenities within the swollen resin are
averaged out in NMR time scale.228
The rate of incorporation of a particular amino acid has been
found to decrease with increasing peptide chain The slow
reactivity has been att.ributed to the steric effect of polymer at
various functional sites. Narita and co-workers studied the
influence of peptide chain length in the coupling efficiencies of
amino acid with terminal amino acid of the oligopeptide bound to
soluble polystyrene, copoly(styrene- 1% DVB), and copoly (styrene-
2% DVB).z31,232 They noted little influence of peptide chain length
upto 10 residue amino acid length in the case of soluble and 1%
crosslinked polystyrente. However coupling efficiencies was lowered
with peptide chain length in the case of 2% crosslinked
polystyrene. This may be due the decreased solvation of polymers
in the higher crosslinked system. Free permeation of reactants and
solvents into the polymer support is an essential condition for
effective gel phase reaction. The decrease in coupling yield in SPPS
is believed to result from the restricted permeation of carboxyl
component into resin matrix. Another reason is that functional groups
that are placed near the crosslinks experience steric effects and are
inaccessible to the incoming reagent. Morawetz carried out extensive
investigations on the reactivity of functional groups attached to
polymer s ~ p p o r t . ~ ~ ~ , ~ ~ ~ He pointed out that introduction of
crosslinks in polymer support changes the local polarity of the
system; however the degree of crosslinking did not exert any
marked influence on reactivity. Ford et al. using NMR pulse-
gradient spin echo (PGSE) method showed that all reactive sites in
polystyrene gels are not equally rea~tive.*~5 The SPPS kinetics reveal
that the reactive sites for the first 90% reaction are effectively
homogeneous, but substantial reduction in rate was observed for the
final 10%. Crosslinkinj; of polymer causes the final stages of the
reaction to be extremely slow.
Solvation studies on PS matrix reveal that polystyrene is
hydrophobic and is compatible with hydrophobic solvents, but as
the peptide chain length is increased the amide bond change the
polarity of the peptidyl r e ~ i n . ~ 3 ~ However Sarin et a1.237 has
demonstrated that polymer and peptide chains mutually enhance
one another's solvation, and this is a supporting evidence for Flor's
view.238
Studies from this laboratory have shown that optimum
hydrophobic-hydroph:ilic balance is required for the polymer
support for effecting the solid phase reactions s u ~ c e s s f u l l ~ ~ - ~ ~ . In
this optimum conditions, the polymer will be compatible with the
peptide chain even if the length of peptide is increased or the
polarity of solvent i:; changed.
Bayerr et a]. introduced PEG as a support in order to avoid
matrix effect and the heterogeneous nature of cross-linked
system.23" PEG ouring to its good solubility in many organic
solvents can solubilise even otherwise insoluble peptides. An
advantage of PEG based liquid phase method is the possibility of
carrying out a better analytical control of the reaction, since
spectroscopic techniques like NMR, IR and CD can routinely be
applied which will help to study the changes in conformation of
peptide during ~ynt.hesis.6~.240 The problems involved in the
process of separation of PEG peptide from reaction media and
insolubility problem in the case of larger peptides bound to PEG
hinder the wide spread use of this technique.
The introduction of PEG grafted crosslinked polystyrene
(PEG-PS) was an attempt to overcome the above problems. There
are two forms of P'EG-PS. The anionic polymerization of ethylene
oxide to attach the PEG chain of controlled length on crosslinked
polystyrene bead containing OH group. This resin is marketed a s
tentagel r e ~ i n . ~ ' . ~ ' Preformed polythyleneglycol has been attached
to polystyrene bead ito obtain PS-PEG. The PEG-PS. in contrast to
PS offer better solvation in protic solvents. In PEG, since not
crosslinked, the reaction sites are highly accessible resulting in
greater reactivity.6' The main limitations of PEG-PS in comparison
to PS are higher cost, reduced loading level, and significant
mechanical instability.
Atherton and Stleppard have investigated on the origin of
the steric hindrance in polymer supported peptide synthesis.241
Based on the concepts of polymer-peptide structures they could
explain the polymer effects on the reactivity in polymer-supported
reactions.
2.10 Aggregation of Peptide
Besides the polymer effect described above, another important
factor which is considered as a general source of problem in peptide
synthesis is the insolubility (in classical peptide synthesis) or
aggregation (in SFJR3) of hydrophobic peptide due to freely
interpenetrating c'oils. Internal aggregation of peptide chains
bound to crosslinked polymer brings extra crosslinking to the
system, reduces the swelling behaviour of polymer support,
eventually leading to the inaccessibility of the reagents and
solvents to the reactive groups in the matrix. The onset of
aggregation in solid phase peptide synthesis can be characterized
by:
> A series of reproducible incomplete amino acylations (0.5-15%).242
P Recoupling or capping provides little or no improvement.235.242
P Occurs irrespective of resin type or ~trategy.242~243
> Difficulq increases with high resin loading.2421 N4
P Aggravated by stericdly hindered amino acid."Z
> Associated with specific sequence^.^^^.^^^
The internal aggregation has been shown to be antiparallel P-
sheet structure by 1TIR.245 Studies by Narita have shown that
si@cant solubility o:f protected hydrophobic peptide was noted when
Aib residues were incorporated into peptide ~ h a i n s . ~ 4 6 - ~ ~ ~ Although
the use of Aib in peptide synthesis finds limited application, the above
studies corroborate the idea that insolubility of protected hydrophobic
peptide segment is due to 0-sheet formation, since a-helix promoting
and 0-sheet disrupting property of Aib is well known.249 The view is
supported by the R,arnan, FTIR and CD studies of protein
aggregates."O-25"
References
1. Jones, J. "The Chemical Synthesis of Peptides"; Clarendon
Press: Oxford,, 1991.
2. Atherton, E.; Sheppard, R.C. "Solid Phase Peptide Synthesis:
A Practical Approach"; IRL Press at Oxford Press: Oxford,
1989.
3. Barany, G.; Kneib-Cordonier, N.; Mullen, D. G. Int. J. Peptide
Proteh Res. 1987, 30,305.
4. Bodanszky, M. "Principles of Peptide Synthesis"; Springer
Verlag: New lrol-k, 1984.
5 . Barany, G.; M:emfield, R. B. in "The Peptides: Analysis,
Synthesis arid Biology"; Vol:2; Gross, E., Meienhofer, J.
Eds.; Academic: Press: New York, 1980, 1-228.
6. Birr, C. "Aspects of Merrifield Synthesis"; Springer Verlag:
Berlin, 1978.
7. Galpin, I . J. "Amino Acids, Peptides and Proteins"; Vo1.16,
The Royal Society of Chemistry, Burlington House, London,
1985. 272-341.
8. Fischer, E.; Fourneau, E. Ber. Dtsch. Chem. Ges. 1901, 34,
2668.
9. Merrifield, R. E3. J. Am. Chem. SOC. 1963, 85, 2149.
10. Letsinger, R. I,.; Kornet, J . J. Am. Chem. Soc. 1963, 85,
3045.
Carpino, L. A,; Han, G. Y. J. Am. Chem. Soc. 1970, 92,
5748.
Atherton, E.; Gait, M. J.; Sheppard, R. C.; William, B. J.
Bioorg. Chem. 1979, 8, 351.
Hodge, P.; Sherrington, D. C. "Polymer Supported Reactions
in Organic Synthesis"; Eds. Wiley, New York, 1980.
Mathur, N . K.; :Narang, C. K.; Williams, R. E. "Polymers as
Aids in Organic Chemistry"; Academic Press, New York,
1980, 53-75.
Pillai, V. N. R.; Mutter, M. "New Perspectives in Current
Chemistry"; Vol. 106, Springer Verlag, Berlin, 1982, 120- 175.
Pillai, V. N. R.; Mutter, M. Top. Curr. Chem. 1982, 106, 119.
Bodanszky, M.; Ondetti, M. A. "Peptide Synthesis, Interscience";
New York. 1966, 162.
Hoffmann, ti.; Katsoyannis, P. G. in "The Proteins", Vol. 1, 2
Neurath, Eds. ; Academic Press, New York, 1963.
Stewart, J . M.; Young, ,I. D. "Solid Phase Peptide Synthesis";
2 Eds., Pierce Chemical Co., Rockford, IL, 1984.
Sheehan, J. C:.; Hess, G. P. J. Am. Chem. Soc. 1955, 77,
1067.
Leanard, J.; R~obinson. J. Am. Chem. Soc. 1967, 89, 181.
Wunsch, E. Angew. Chem. Int. Ed. Engl. 1971, 10, 786.
Erickson, B. W.; Merrifield, R. B. "The Proteins" 3 Edn.,
Neurath, H., I-Iil.1, P. R . , Boedar, C. L. Academic Press, New
York, 1976.
Bayer, E. Angeur. Chem. Int. Ed. Engl. 1991, 30, 113.
Mutter, M. ; Bayer, E. Angew. Chern. Int. Ed. Eng1.1974, 86,
101.
Bayer, E.; Mutter, M.; Polster, J.; Uhmann, R. in "Peptides
1974" Wolman, Y. Eds. J.Wiley, New York, 1974, 129.
Mutter, M.; Nagenmaier, H.; Bayer, E. Angew. Chem. Int.
Ed. EngI. 197'1, 10, 8 1 1 .
Bayer, E.; Mutter, M. Nature, 1972, 273, 512.
Mutter, M.; U'hmann, R.; Bayer, E.; Leibeig, S. Ann. Chem.
1975,901.
Mutter, M , Bayer, E "The Peptides- Analysis, Synthesis and
Biology" Gross, E., Meienhofer, J. Eds., Academic Press
New York, Vo1.2 1980, 285.
Hendrix, J . C.; Lansbury, P. T. in "Peptides: Chemistry and
Biology, Proc. of the 12th Am. Pep. Sym.", Smith, J. A.,
River, J. E. Eds., ESCOM: Leiden, 1992, 129.
Weber, U . ; An'dre, M.; Hoppe-Seylers, 2. Physiol. Chem.
1975 356, 701 1.
Sarin, V. K.; Kent, S. B. H.; Merrifield, R. B. J. Am. Chern.
Soc. 1980, 1132, 5463.
34. Stewart, J. M. IMacromol. Sci. Chem. 1976, 10, 254.
Merrifield, R. El. Science 1986, 232, 341.
Hancock, W.S.; Prescott, D. J.; Vagelos, P. R.; Marshall, G. R.. J.
Org. Chem. 1973, 38,774.
Flory, P. J. MacromoIecules.1974, 12, 110.
Tsay, H. M.; Ullman, R. Macromolecules 1988, 21, 2963.
Sheppard, R. (2. in "Peptides 1971, Proc. of the llth Europ.
Pept. Syrnp.", IVesvadba, H. Eds., North Holland: Amsterdam,
1973, 11 1.
Sheppard, R. (2. Sci. Tool.1986, 33, 9.
Kent, S.; Clai-k,, L. I . "Synthetic Peptides in Biology and
Medicine" Alitalo, K.; Partanen, P.; Veheri, A. Eds., Elsevier
Science Publis,hers, Amsterdam, 1985, 29- 57.
Atherton, E.; Sheppard, R. C. "Solid Phase Peptide Synthesis-
A Practical Approach using the Fmoc-Polyamide Technique"
IRL Press, Oxford, 1984.
Geyseri, H. M.;. Meloen, R. H.; Barteling, S. J. Roc. Natl. Acad.
Sci LISA, 1984,81, 3998.
Barany, G.; Albericio, F., in "Peptides 1990, Proc. of the 2 lSt
Europ. Pept. Symp.", r a t E.; Andrew, D. Eds., ESCOM,
Leiden, 1991, 1.39.
Bayer, E.; Dejngler, M.; Hemmasi, B. Int. J. Peptide Protein
Res. 1985, 25, lL78.
Barany, G.; PLlbericio, F.; Sole, N. A.; Griffin, G.W.; Kates,
S.A.; Hudson, I). in "Peptides. 1992, Proc. 22nd Europ. Pept.
Syrnp.", Schneider, C. H.; Eberle, A. N. Eds., ESCOM, Leiden,
1993,267.
Pillai, V. N. F:.; Renil, M.; Haridasan, V. K. Indian J. Chem.
1991, 30B, 205.
Atherton, E.; Cameron, L. R.; Sheppard, R. C. Tetrahedron
1988, 44. 843.
Findeis, M. A,; Kaiser, E. T. J. Org. Chenl. 1989, 54, 3478.
(a) Meldal, M. Tetrahedron Lett. 1992, 33, 3077.
(b) Auzanneau, F. I . ; Meldal, M.; Bock, K. J. Peptide Sci.
1995, 1, 31.
Atherton, E.; Brown, E.; Sheppard, R. C.; Rosever, A. J.
Chem. Soc. Chem. Commun. 1981, 1151.
Small, P W.; Sherrington, D. C. J. Chem. Soc. Chem.
Commun. 1989, 1589
Rapp, W. ; Z:ha.ng, L.; Habich, R.; Bayer, E. in "Peptides,
Proc. of 20" ECur. Pep. Symp.", Jung, G. Ed., Walter de
Gruyter and Cos., Berlin., 1989, 195.
Berg, R.; Amdal, K.; Pedersen, W. B.; Holm, A,; Tam, J. P.;
Merrifield, R. B. J. Am. Chem. Soc. 1989, 111, 824.
Daniels, S. El.; Bematowicz, M. S.; Coull, J. M.; Koster, H.
Tetrahedron Lett. 1989, 30, 4345.
Eichler, J . ; Beinert, A,; Stiernadova, A,; Lebel, M. Peptide
Research 1991, 4, 296.
a , ) Renil, M.; IVleldal, M. Tetrahedron Lett. 1995, 36, 4647.
b) Renil, M.; F'erreras, M.; Delaisse, J. M.; Foged, N. T.;
Meldal, M.. J! Peptide Sci. 1998, 4, 195.
Renil, M.; Melclal, M. Tetrahedron Lett. 1996, 34, 6185.
Kempe, M.; Barany, G. J: Am. Chem. Soc. 1996, 118,7083.
Beck-Siclunger, A. G.; Jung, G.; Gaida, W.; Koppen, H.;
Schorrenberg, (2. ; Lang, R. Eur. J. Biochem. 1990, 176, 458.
Houghten, R. Pi. Proc. Natl. Acad. Sci. USA. 1985, 82, 5131.
Valerio, R. M.; Bary, A. M.; Campbell, R. A.; Dipasquale, A.;
Margellis, C.; Fcodda, S. J.; Geysen, H. M.; Maeji, N. J. Int. J.
Peptide Pr0tei.n Res. 1993, 42, 1.
Holm, A.; Meld.al, M. "Peptide 1988" Jung, G.; Bayer, E. Eds.,
Walter de Gruyter and Co., Berlin. 1989, 208-210.
Frank, R.; Guler, S.; Karuse, S.; Lindenmaier, W. in
"Peptides, 1990, Proc. 21s' Eur. Pept. Symp.", Giralt, E.;
Andreu, D., Eds., ESCOM, Leiden, 1991.
Prescichetti, R. A,; St. Clair, N . L.; Griffith, J. P.; Navia, M. A.;
Margolin, A. L. J. Am. Chem. Soc. 1995, 11 7, 2732.
Chandy, M. C.; Pillai, V. N. R. Indian J. Chem. 1997, 36B, 303.
Renil, M.; Pillai, V. N. R. J. Appl. PoIym. Sci. 1996, 61, 1585.
Kurnar, K . S.; Pil:lai, V. N. R. Tetrahedron 1999, 55, 10437.
Varkey, . J . T.; F'illai, V. N. R. J. Peptide Res. 1998, 51, 49.
Roice, M.; Kurnar, K. S.; Pillai, V. N. R. MacromoIecuIes 1999,
32. 8807
Williams, P. L.; Albericio, F.; Giralt, E. Tetrahedron 1993,
49, 11045.
Kunz, H.; Dombo, B. Angew Chem. ht. Ed. Eng. 1988,27,7 1 1.
Kates, S. A.; de la Torre, B. G.; Eritja, R.; Albericio, F. Tetrahedron
Lett . 1994, 35; 1033.
De la Torre, B'. G.; Avino, A.; Tarrason, G.; Piulats, J.;
Albeicio, F.; Erilja, R. Tetrahedron Lett. 1994, 35, 2733.
Barton, M. A.; krnieux, R. U.; Savoie, J. Y. J. Am. Chem. Soc.
1973, 95, 450 1.
Seebach, D.; Thaler, A.; Blaser, D.; KO, Y. S. Helv. Chim.
Acta. 1991, 74, 1102.
Anantharamaiah, G. M.; Sivanandaiah, K. M. Zndian J. Chem.,
1978, 16B, 79'7.
Larsson, L. E.; Melin, P.; Ragnarsson, U. ht . J. Peptide Protein
Res. 1976, 8, 39.
Liungqvist, A,;; F'olkers, K. Acta Chem. Scand., Sen. B. 1984,
38, 375.
Schlatter, J . M.; Mazur, R. H.; Goodmonson, 0. Tetrahedron
1978, 34, 28513.
Jones, D. A.; Tetrahedron Lett. 1977, 2853.
Anderson, G.W.; Mc Greger, A.C. J. Am. Chem. Soc., 1957,
79 , 6180-6183.
83. Carpino, L.A.; Han, G.Y. J.Org. Chem.1972, 37(22) 3404-
3409.
Atherton, E. ; Fox, H.; Harkiss, D.; Logan, C. J.; Sheppard, R.C.;
William B.J. J. Chem. Soc., Commun. 1978, 537-539.
Carpino, L.; Han. G.Y. J. Am. Chem. Soc. 1970, 92(19),
5748-5749.
Change, C. D.; :Meienhofer, J. Int. J. Peptide and Protein
Res. 1978, 11, 246-249.
Blondlelle, S. E:.; Houghten, R. 2. Int. J. Peptide Protein Res.
1993, 4 1, 522-527.
Steward, J . M.; Wooley, D.W. Nature. 1965, 206, 619-620.
Kent, S.B.H.; In Peptides: Structure and Function,
Proceedings of 9th Amencan Peptide Symposium; Deber, CM;
Hruby, V.J.; k:opple, K., Eds.; Pierce Chemical Company:
Rockford, IL, 1985, 407-414.
Meienhofer, J.; Waki, Heimer, E.P.; Lambros, T.J.;
Makofske, R.C.; Change, C.D. Int. J. Peptie Protein Res.
1979, 13, 35-42.
Larsen, B.B.; Holm, B. Int J. Peptide Protein Res. 1994, 43, 1-9.
Khorana, H.G. Chem. Rev. 1953., 53, 145
Rich, D.H. ; Singh, J. In The Peptides; Water, R; Meienhofer,
J., Eds.; Acade,mic: New York, 1979; Vol. 1 , 242.
Selbioda, M . Pohsh J. Chem. 1994,68, 957-961
Gross, E.; Meienhofer The Peptides: Analysis, Synthesis and
Biology; < I . , Eds; Academic New York, 1979: Vol. 1.
96. Atherton,E.; S:heppard, R.C. In Solid Phase Peptide
Synthesis: A Practical Approach; Rickwood, D.; Hames,
B.D., Eds.; IRL: Oxford University, 1984; 75-85.
97. Kisfaludy, L; CLerpini, M.Q.; B.; Kavocs, J. In Peptides:
Proceedings of gth European Peptide Symposium;
Beyermann, I-I.C.; Van De Linde, A.; Van Den Brink, A.;
Massen, W., E:ds.; North Holland: Amsterdam, 1967; 25-27.
98. Miyazawa, T.; Otomatsu, T.; Fukui, Y.; Yamada, T.; Kuwata, S.
Int. J. Peptide Protein Res. 1992. 39, 237-244.
99. Koning, W.; Geiger, R. Chem. Ber. 1970, 103, 788.
100. Castro, B.; Dormoy, J. R.; Evin, G.; Selve, C. Tetrahedron
Lett. 1975, 121, ('919), 22.
101. Coste, Le-Nguyen, D.; Castro, B. Tetrahedron. Lett. 1990,
31. 205.
102. Kiso, Y., Fujivgara, T.; Nishitani, A.; Akaji, K. Int. J. Peptide
Protein Res. 40, 1992, 308.
103. Knorr, R.; Trzeciak, A,; Bannwaith, W.; Giessen, D. Tetrahedron
Lett. 1992, 32, 647.
104. Bayermann, M.; Henklein, P.; Klose, A,; Sohr, R.; Bienert, M.
Int. J. Peptide Protein Res. 1991, 37, 252-256.
105. Ramage, R.; Eiarron, C. A,; Bielecki, S.; Holden, R.; Thomas,
D. W. Tetrahedron 1992, 48, 499.
106. Merrifield R.B. J. Am. Chem. Soc., 1963, 85, 2 149.
107. Mitchell A. R.; Kent S. B. H., Engelhard M., Merrifield R. B.
J. Org. Chem., 1978, 43, 2845.
108. Wang S. S. J. .Am. Chem. Soc., 1973,95, 1327
109. Sheppard R. C.; Williams B. J. Int. J. Peptide Protein Res.,
1982, 20, 451.
110. Albericio F.; Barany G. Int. J. Peptide Protein Res., 1985,
26, 92.
11 1. Mergler M. ; Tanner R.; Gosteli J . ; Grogg P. Tetrahedron
Lett., 1988, 20, 4005.
1 12. Albericio F.; Barany G. Tetrahedron Lett., 1991, 32, 10 15.
113. Rink H. Tetraliedron Lett., 1987, 28, 3787
114. Eleftheriou S.; Gatos D.; Panagopoulos A.; Stathopoulos S.;
Barlos K. Tetmhedron Lett., 1999, 40, 2825.
115. Shao J.; Voelter W. 'Peptides: Chemistly, Structure and
Bjolod (Hodge~; R. S., Smith J. A., Eds.), ESCOM, Leiden,
The Netherlands, 1994, 149
116. Pietla P. G.; Marshall G. R. Chem. Commun., 1970, 650.
117. Matsueda G. R.; Stewart J. M. Peptides, 1981, 2, 45.
1 18. Sluber W. ; Knolle J.; Breipohl G. Int. J. Peptide Protein Res.,
1989, 34, 215.
119. Penke B.: Nyerges L. 'Peptides 1988' (Jung G., Bayer E.,
Eds.), Walter De Gruyter & Co., Berlin, 1989, 142.
120. Albericio F.; Bcrany G. Int. J. Peptide Protein Res., 1987,
30, 206.
12 1. Sieber P. Tetrahedron Lett., 1987, 28, 2 107.
122. Rich D. H.; Gunvara S. K. J. Am. Chem. Soc., 1975, 97,
1575.
123. Wang S. S. J. Org. Chem., 1976, 41, 3258.
124. Eritja R.; Robles J.; Fernandez -Forner D.; Albericio F.;
Giralt E.; Pedroso E. Tetrahedron Lett., 1991, 32, 15 1 1.
125. Mutter M.; Bellof D. Helv. Chim. Acta, 1984, 67, 2009.
126. Kunz H.; Dombo B. Angew. Chem. Int., 1988, 27, 71 1.
127. Holmes C. P.; Jones D. G.; Frederick B. T.; Dong L. -C. 14th
Am. Pept. Syrnp., Columbus, Ohio, 1995, 004.
128. Ajayagosh A,; Pillai V. N. R. Tetrahedron Lett., 1995, 36, 777.
129. Bergmann M.; Zervas L. Ber. Dtsch. Chem. Gen., 1932, 65,
1192.
130. Mc Kay S.C.; Aylbertson N. F. J. Am. Chem. Soc., 1957, 79,
4686.
131. Anderson G. 'W..; Mc Gregov A. C. J. Am. Chem. Soc., 1987,
79, 6180.
132. Wang S. S.; Merrifield R. B. Int. J. Peptide Protein Res.,
1969, 1, 235.
133. Kemp D. S.; Fotouhi N.; Boyd J. G.; Carey R. I.; Ashton C.;
Hoare J. Int. .J. Peptide Protein Res., 1988, 3 1 , 359.
Birr C.; Lochinger W.; Stahnke G.; Lang P. Justus Liebigs Ann.
Chem., 1972,743, 162.
Voss C.; Birr C . Z. Physiol. Chem., 1981, 362, 717.
Hass W. L.; Krumkalns E. V.; Gerzon K. J. Am. Chem. Soc.,
1966,88, 1988.
Voelter W.; Kalbacher H.; Beni C.; Heinzel W.; Muller J.
'Chemistry of Peptides and Proteins' (Voelter W., Bayer E.,
Ovchinnikov J!. A,, Wunsch E., Eds.), Walter de Gruyter &
Co., Berlin, 1987, 2, 103.
Shao J.; Shekhani M. S.; Krauss S.; Grubler G.; Voelter W.
Tetrahedron Lett., 1991, 32, 345.
Carpino L. A. .4c.c. Chem. Res., 1987, 20, 401.
Woodward R. E3.; Heusler K.; Gosteli J.; Naegeli P.; Oppolzer W.;
Ramage R.; Ranganathan S.; Vorbruggen H. J. Am. Chem.
SOC., 1966, 88, 852.
Carpino L. A,; Han C. Y. J. Am. Chem. Soc., 1970, 92, 5748.
Zervas I,.; Borovas D.; Gazis E. J. Am. Chem. Soc., 1963,
85, 3660.
Barany G.; Albe~icio F. J. Am. Chem. Soc., 1985, 107, 4936.
Zalipsky S.; Albericio F.; Somezynska U.; Barany G. Int. J.
Peptide Proteih Res., 1987, 30, 740.
Kenner G. W.; Moore G. A,; Ramage R. Tetrahedron Lett.,
1976. 3623.
146. Schonheimer I
147. Stevens C. M.; Watanabe R. J. Am. Chem. Soc., 1950,72,725.
148. Wang S. S.; Chen S. T.; Wang K. T.; Merrifield R. B. Znt. J.
Peptide Proteir~ Res., 1987, 30, 662.
149. Chen S. T.; Weng C. S.; Wang K. T. J. Chin. Chern. Soc.,
1987, 34, 1 17.
150. Matsueda R.; Walter R. Int J. Peptide Protein Res., 1980,
16. 392.
151. Patchornik A,; Amit B.; Woodward R. B. J. Am. Chem. Soc.,
1970, 92, 6333.
152. Foder S . P. A, ; Read J L.; Pirrung M. C.; Stryer L.; Lu A. T.;
Solas D. Science, 1991, 251, 767.
153. Schielen W. J. G.; Adams H. P. H. M.; Nieuwenhuizen W.;
Tesser G. 1. ,ht. J. Peptide Protein Res., 1991, 37, 341.
154. Sheehan J. C.; Hess G P. J Am. Chem. SOC, 1955,77,1067.
155. Weygand F.; Ragnarsson U. 2. Naturfomch B., 1966, 21, 1141.
156. Koning W.; Geiger R. Chem. Ber., 1972, 105, 2872
157. Sheppard 6I.C:. Innovations and Perspectives in Solid Phase
Synthesis: 13q7tides Polpptides, OLigonucfeotides (Epton r.,
Ed.) Intercept Ltd., Andover, UK, 1992, 213.
158. Koning W.; Geiger R. Chem. Ber., 1973, 106, 3626.
159. Carpino L. A. J. Am. Chem. Soc., 1993, 1 15, 4397.
160. Carpino L.A.; Imazuml H.; Foxman B. M.; Vela M. J.;
Henklein P.; El--Faham A,; Klose. J.; Bienert M. Organic
Lett., 2000, 2 , 2253.
16l. Sarantakis D.; Teichman J.; Lein E. I,.; Fenichel R. L.
Biochem. Biophys. Res. Commun., 1976, 73, 336.
162. Hudson D.; Kain D.; Ng D. 'Peptide Chemistry 1985, (Kiso Y. ,
Ed.), Protein Res. Foundation, Osaka, Japan, 1986, p 413.
163. Nozaki S. CheznistryLett., 1997, 1
164. Nozaki S. J. Pepride Res., 1999, 54, 162.
165. Carpino L. A.; El-Faham A.; Minor C. A,; Albercio F. J.
Chem. Soc. Chern. Commun., 1994, 201.
166. Kates S. A.; Iliekmann E.; Al-Faham A.; Herman L. W.;
Lonescu D.; M[c Guinness B. F.; Triolo S. A.; Albericio F.;
Carpino L. A. 'Tet:hnique.s in Protein Chemistry' (Marshak D. R.,
Ed.), Academic .Press, New York, 1986, 515.
167. Dourtoglou V.:. (3ross B.; Lambropoulou V.; Znoudrou C.
Synthesis, 1984, 572.
168. Carpino L. A,; El- Faham A.; Truran G.'Triolo S. A.; Shroff H.;
Griffin G. W. ; Minor C. A,; Kates S. A,; Albericio F. 'Peptides'
(Hodges R S., Smith ,J. A., Eds.), ESCOM, Leiden, The
Netherlands, 1994, 126
169. Chen S. 0.; Xu J. C. Tetrahedron Lett., 1992, 33, 647
170. Coste J.; Frerot E.; Jouin P. Tetrahedon Lett., 1991,32, 1967.
171. Abdelmoty I.; Albericio F.; Carpino L. A.; Foxman B. M.;
Kates S. A. Lett,. Peptide Sci., 1994, 1, 52.
172. Carpino L. A,; El-Faham A,; Albericio F. J.L)rgChem., 1995, 60,
3561.
173. Carpino L. A.; El--Faham. A. J. Am. Chem. Soc., 1995, 117,5401
174. Nozaki S. J. f'eptide Res., 1999, 54, 162.
175. Triolo S.A.; Lonescu D.; Wenschuh H.; Sole N.; El-Faham A.;
Carpino L.A.; Kates S.A. Peptides 1996 (Epton R., Ramage,
R., Eds.), Mayflower Scientific Limited., Birmingham, 1998,
839.
1'76. Kisfaludy L.; Schon I . Synthesis, 1983, 325.
177. Hudson D. Peptide Res., 1990, 3, 51.
1'78. Beesley T. E.; Veber D. F.; Schoenewuldt ; Barkemeyer E. F.;
Palevda H. ; Jacob W. J. Jr.; Hirschmarin R. J. Am. Chem.
SOC., 1966, 88, 3163.
179. Fuller W.D.. Cohen M.P.; Shabankareh M.; Blair R.K.;
Goodman M. J: Am. Chem. Soc., 1990, 112, 7414.
180. Xue C.B.; Maider F. J. Org. Chem., 1993,38,315.
18 1. Knorr R.; Trzeuak A.; Bannwarth W.; Gillessen D. Tehhedron
Lett., 1989, 30, 1927.
182. Knorr R ; Trzeuak A.; Bannwarth W.; Gillessen D. 'peptides.,
1990', ESCOM, Leiden, The Netherlands, 1991, 62.
183. Carpino L. A.; Cohen B. J . ; Stephens K. E.; Sadat -Aalace S.
Y.; Tien J. -H.; Langridge D. C. J. Org. Chem., 1986, 51,
3732.
184. Carpino L. A.; Chao H. G.; Beyermann M. ; Bienert M. J. Org.
Chem., 1991,56,2635.
185. Perlow D. S.; E;rb J . M.; Gould P.; Tung R. D.; Freidinger R. M.;
Willams P. D.; Veber D. F. J. Org. Chem., 1992, 57, 4394.
186. Carpino L. A.; Beyermann M.; Wenschuh H.; Bienert M. Ace.
Chem. Res., 1996, 29, 268.
187. Wenschuh H.; Beyermann M.; Haber H.; Seydel J.-K.;
Krause E.; Bienert M.; Carpino L. A.; El-Faham A,; Albericio
F. J. Org. Chem., 1995, 60, 405.
188. Wenschuh H.; Beyermann M.; Krause E.; Brudel M.; Winter R.;
Schumann M.; Carpino L. A.; Bienert M. J. Org. Chem., 1994,
59, 3275
189. Wenschuh H.; Beyermann M.; Krause E.; Carpino L. A,;
Bienert M. Tetrahedron Lett., 1993, 34, 3733.
190. Mital M . ; Srir~ivastava N.; Kumar A. Indzan J. Chem., 1991,
30B, 1 129
191. Srivastava A.; Misra S.; Haq W.; Katti S. 8 . ; Mathur K. B.
Indian J. Chem., 1991, 30B, 1131.
192. Frerot E.: Cos1.e J . ; Pantaloni A.; Dufour M.-N.; Jouin P.
Tetrahedron Lett., 1991, 47, 259.
193. Coste J . ; Ferot E.; Jouin P. J. Org. Chem., 1994, 59, 2437.
194. Kutoh T.; Ueki M. Int. J. Peptide motein Res., 1993, 42,
264.
195. Carpino L. A.; Mansour E. S.; El -Faham A. J. Org. Chem.,
1993, 58, 41162:.
196. Belleau B.; Malik G. J. Am. Chem. Soc., 1968, 90, 1651.
197. Slaab H. A. Leibigs., Ann. Chem., 1957, 609, 75.
198. Bodanszky M . ; Funk K . W.; Fink M. L. J. Org. Chem., 1973,
38, 3565.
199. BodanszIq A.;. Hodanszky M.; Chandramouli N.; Kwei J. 2.;
Martinez J. ; Tolle J. C. J. Org. Chem., 1980, 45,72.
200. Merrifield R. B.; Vizioli L. D.; Boman H. G. Biochemistry,
1982, 2 1 , 5020.
20 1. Chang C. D.; Felix A. M.; Jimenez M. H.; Meienhofer J. Int.
J. Peptide Protein Res., 1980, 15, 485.
202. Li P.; Xu J . C. ,J. Peptide Res., 2000, 55, 1 10.
203. Henklein P.; Beyermann M.; Bienert M.; Knorr R. 'Peptides
199G' (Giralt E., Andreu D., Eds.), ESCOM, Leiden, The
Netherlands, 1991, 67.
204. Carpino L. A.; El -Faham A. J. Org. Chem., 1994, 59, 695,
205. Kiso Y . ; Fujiwara Y.; Kimura T.; Nishitani A,; Akaji K. Int. J.
Peptideprotein Res., 1992, 40, 308.
206. Colin H.; Guiochon G. J. Chromatography, 1977, 141, 289.
Mant C. T.; Hedges R. S. 'HPLC of Peptides and Proteins:
Separation, Andysis and Conformation', CRC Press, Boca
Raton, FL, 1991.
Buttner K.; Blondelle S. E.; Ostresh J. M.; Houghten R. A.
Biopolymers, 1992,32, 575.
Mant C. T.; Kor~dejewski L. H.; Cachia P. L.; Monera 0. D.;
Hodges R. S. Methods in Enzymology, 1997, 289, 426.
Ozols J . Methods in Enzymology, 1990, 182, 587.
Barber M.; Borcioli R. S.; Elliot G. J . ; Sedgwick R. D.; Tyler
A. N. Anal. Che~n., 1982, 54, 645.
Seifert J r . W. E.; Caprioli R. M. Methods in Enqmology,
1996, 270, 453.
Moore W. T.; Caprioli R. M. .'Techniques in Protein chemisw
I f (Villafranca J. J . , Ed.), Academic Press, San Diego, C A,
1991, 511.
Beranova-Gior($anni S.; Desiderio D. M. Methods in ELqmoIop,
1997,289,47(3.
Packman L. C.; Quibell M.; Johnson T. Peptide Res., 1994,
7, 125.
Burdick I). J . ; Stults J. T. Methods in Enqmology, 1997,
289. 499.
Banks J r . J . I?.; Dresch T. Anal, Chem., 1996, 68, 1480.
Posthumus NI. A,; Kistemaker P. G.; Meazelaar H.L.C., Ten
Neuver De Rr,auw M. C. Anal. Chem., 1978, 50, 985.
Hillenkamp F.; Karas M.; Bearis R. C.; Chait 8. T. And
Chem., 1991, 63, 1193.
Noble D. Anal. Chem., 1995 ,67 ,497
Woody R. W. 'The Peptides' Academic Press, Orlando,
Florida, 1985, 7, 15.
Simons E. R. Spectroscopy in Biochemistry (Bell J . E., Ed.),
CRS Press, Boca Raton, Florida, 1981, 1, 63.
Y u V. S.; Prendergast F. G. Anal. Biochem., 1997, 248, 234.
Tanfani F.; Kochan 2.; Swerczynski J . ; Zydowo M. M.;
Bertoli E. Biqgolymers, 1995, 36, 569.
Wuthrich K. NMR of Proteins and NucLic Acids, Wiley-
Interscience, New York, 1986.
Evans J . N . S. Biomolecular NMR Spectroscopy, Oxford
University Press Inc., New York, 1995.
Ducruix A.; Giege R. Crystallization NucIeic Acids and
Proteins: A Practical Approach, Oxford University Press,
Oxford, 1992.
Mutter,M.; Altmann, K.H.; Bellof, D.; Florsheimer, A.;
Herbert,J.; Huber, M.; Kleim, B.; Strauch, L. Proceedings
of the 9th Aniencan Peptide Symposium; Deber, C, M.;
Hruby,V , J ; IKopple, K. D, Eds.; Pierce, Chemical Company:
Rockford, IL,1985; 397.
Live, D. H.; Kent, S.U.H. In Peptides: Structure and Function;
Hruby, V. B., Rich, D. H., Eds.; Pierce Chemical Company:
Rockford, lL, 1983; 65-68.
230. Westfall F.C.; Robenson, A. B. J. O r - Chem. 1970,35, 2842.
231. Chou, F.C.H.; Kobenson, A. B. J. Am. Chem. Soc. 1971, 93,
267.
232. Narita, M.; Isokawa, S.; Matsuzawa, T.; Miyauchi, T.
Macromolecules 1985, 18(7), 1363- 1366.
233. Isokawa, S.; Kobayashi, N.; Nagano, R.; Narita, M.
Makromol. C15em., 1984, 185, 2065.
234. Morawetz, H.:P. Pure Appl. Chem. 1979, 51, 2307
235. Morawetz, H. In Chemical Reactions on Polymers; Benharn,
J.J.K., Kensette, J.F. Eds.; ACS Symposium Series 363;
American Chemical Society: Washington,DC, 1988; 317.
236. Ford, W. T.; Ellum, F. D. J. Polym. Sci., Part A. Polym. Chem.
1990, 28, 931-934.
237. Hancock, W. S.; Prescott, D. J.; Vagelos R.; Marshall, G.R. J.
Org. Chem. 1973, 38(14), 774-781.
238. Sann, V.K., Kent, S.B.H.; Merrifield, R.B. J. Am. Chem. Soc.
1980, 102, 6,5463-5470.
239. Flory, P.J. Macromolecules. 1979, 12, 1 19.
240. Bayer, E. Nac:hr. Chem. Tech. 1972, 20, 492
241. Bayer, E., Mutter, M. Chem. Ber. 1974, 107.
242. Kent, S.B.H. Arm. Rev. Biochem. 1988, 57, 959-989.
243. Atherton, E.; Wooley, V.; Sheppard, R.C. J. Chem. Soc.,
Chem. Commu.n. 1988, 970-97 1.
244. Meister, S . N [ . ; Kent, S.B.H. In Peptides: Structure and
Function, Proceedings of @h American Peptide Symposium;
Hruby, V.J.; Rich, D.H., Eds.; Pierce Chemical Company:
Rockrod, IL, :1984; 103-106.
245. Narita, M.; Isokawa, S. PolymerJ. 1983, 15, 25.
246. Narita M.; Doi, M.; Sugasawa, H.; Ishikawa, K. Bull. Chem.
SOC. Jpn. 1985, 58, 1473-1479.
247. Narita, M.; Chen, J-Y.; Sato, H.; Kim, Y. Bull. Chem. Soc.
Jpn., 1985, 58, 2494-2501.
248. Narita, M.; Islnikawa, K.; Sugasawa, H.; Doi, M. Bull. Chem.
SOC. Jpn. 1985, 58, 1731-1737.
249. Paterson, Y.; Rumsey, S.M.; Benedetti, E.; Ne'methy, G.;
Scheragam, H.A. J. Am. Chem. Soc. 1981, 103, 2947-2955
250. Przbycein, T.; Bailey. J. Biochem. Biophys. Acta, 1991, 103,
1076.
251. Ismail, A, , M.antsch, H.; Wong, P.T.T. Biochem. Biophys.
Acta 1992, 183, 1121.
252. Zettlmeissl, Ci.; Rudolph, R.; Jaenicke, R. Biochemistry
1979, 18, 5567.
Top Related