Download 2. ACTIVATION OF CARBOXYL GROUPS IN

ACTIVATION OF CARBOXYL GROUPS IN PEPTIDE SYNTHESIS
2.
-
2.1
A REVIEW
Introduction
Over the last two decades there has been a rapid growth
in interest in the chemistry of peptides and proteins. 1-2
The study of the mechanisms of hormone action and the
enzyme-substrate,
antigen-antibody
and
protein-DNA
inter-
actions have now moved to the forefront of contemporary
chemistry.
One of the m a i n d i f f i c u l t i e s f a c i n g the
researchers in this field is the isolation of sufficient
amount of the peptide or protein in a pure form.
The
isolation of protein from natural sources is laborious and
often provides only tiny quantity of material.
Among the
contemporary methods for peptide and protein synthesis
biotechnological techniques are currently used, out of which
the most useful method is the cloning of genes.3 - 4
They
are powerful techniques but their drawbacks still create
problems.
Although, such methods can be used to provide
modified protein structure,5 they are really inappropriate
for developing analogous systems which are routinely
necessary for investigating the structure-activity relationships.
The advances in peptide synthesis over the last five
decades have made the synthesis of larger peptides and
proteins a realistic possibility.
Chemical synthesis is
probably the most practical way o f providing sufficient
amount of material, and in addition, allows the systematic
variation of structure necessary for the development of
peptides for various biochemical studies.
Analogous to
peptides,
specifically
modified
structures
containing
labelled amino acids and also peptide mimetics are more
efficiently made by chemical synthesis.
However, for larger
peptide molecules 6 , the synthesis of chemically homogeneous
m a t e r i a l i s a c h a l l e n g i n g field but
success in this
endeavour cannot necessarily be guaranteed.
Organic chemists since Emil
p is her' have noticed the
problems that chemical syntheses of proteins entail.
syntheses of insulin8 , ribonuclease '
A
analogues1'
and also of lysozyme
are milestones in this field.
larger p e p t i d e s
in
The
The synthesis of
s o l u t i o n , i n which
the
various
intermediate compounds are purified and characterised is, in
p r i n c i p l e , t h e c o r r e c t w a y of obtaining homogeneous
material. ''-I4
However, this synthetic approach of larger
peptides is not without problems such as insolubility o f
peptides in various solvents, epimerisation at the activated
C-terminal end, lack of powerful methods of purification
etc.
In spite of these, synthesis in solution is one of the
important methods of peptide synthesis and continues to find
various applications.
The large scale
peptides with unusual amino acids
of shorter
,
peptides16 and the semisynthesis of
all often carried out in solution
Synthesis of peptide with a
amino acid residues is a fairly com
sequence of
ess.
The crux
of peptide synthesis lies in the 'activation of carboxyl
component' followed by aminolysis for the formation of
peptide bond.
To convert carboxylic acids into activated
form. their hydroxyl group must be replaced by an electron
withdrawing substituent ( X ) to enhance the polarisation ot
the carboxyl group and thereby the electrophilicity at the
carbon centre.
This facilitates the nucleophi lic attack by
the amino and alcoholic groups.
The following are the important conventional methods
used for carboxyl activation.
2.2
Acid chlorides
The first attempt on the activation process was made by
Emil
ish her^
peptide
at the cradle of this century itself and
bonds w e r e
method "ll'.
formed using
this acyl chloride
Here the chloride ion is used as the electron
withdrawing moiety.
Acyl chlorides 1 were formed by using
phosphorous pentachloride or thionyl chloride.
2.3
Acid azides
Parallel to the acyl chloride method, acid azide method
was developed by curtius'',
which remains even today as a
p o w e r f u l and practical approach for the synthesis o f
peptides.
T h e ' o r i g i n a l steps o f activation involves
h y d r a z i n o l y s i s o f a l k y l esters and conversion of the
hydrazides to acid azides 2 with the help of nitrous acid.
In recent years direct conversion of carboxylic acids
to acid azides with the help of diphenyl phosphoryl azide
(3) or hydrazide21,22
serves as an attractive alternative.
The reagent obtained from diphenyl phosphoryl chloride and
sodium azide, proved to be very effective for the synthesis
of peptides.
Even in solid phase peptide synthesis the
azide method was implemented by Felix and Merrifield. 2 3
H e r e , stepwise synthesis o f peptides by the initial
attachment of an amino acid, t-butyloxycarbonyl hydrazide
through its
oC
-amino group to a polystyrene resin was
investigated.
N,N3-Bis(2-oxo-3-oxazolidinyl) phosphorodiamide
(BOPA, 4) is another useful coupling agent. 2 4
azide
The peptides
prepared by this method are of high purity and the extent of
recemization is same as that of DCC/HOBt method.
2.4
Anhydrides
A very simple and most efficient method of activation'
is the treatment of amines with anhydrides of carboxylic
acids 5.
Symmetrical anhydrides are nowadays prepared b y
treating carboxylic acid with phosgene.
The most successful
generation of mixed anhydrides is by treating carboxylic
acids with alkyl chlorocarbonates.
The resulting mixed
25-27
anhydrides are readily used in the synthesis of peptides.
A
particular
advantage
of alkyl carbonic acid mixed
anhydrides is that the by-products of the acylation reaction
can easily be removed from the reaction mixture.
A very interesting development of the method of mixed
anhydrides is the application
of
1,2-dihydroquinoline
derivatives in the s y n t h e s i s of peptides. 28-30
N-
Ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ, 6 ) which
can be readily o b t a i n e d f r o m ethyl chloroformate and
quinoline, give
carboxylic acids.
rise to mixed anhydrides on reaction with
Various peptides have been synthesised in
good yields by this procedure.
A polymer modification of
EEDQ has been developed and various peptides have been
prepared using this31 reagent.
A closely related reagent N -
isobutyloxycarbonyl-2-isobutyloxy-1,2-dihydroquinoline
(IIOQ, 713'
is used nowadays for peptide synthesis which is
powerful than EEDQ.
2.5
Esters
Another well established mt.rhod to prepare amides of
blocked amino acids or peptides is by treating their alkyl
esters with ammonia.
Electron withdrawing inductive effect in the alcoholic
component of the ester renders the carbon atom of the
carboxyl group more electrophilic and more ready to attack
nucleophiles.
T h u s , methyl e s t e r s could be further
activated by ~ ~ S s t i t u t i owith
n
strongly electron withdrawing
groups.
Aminolysis of cyanomethyl esters 3 3
was
found
suitable for the preparation of peptides.
Aryl esters were proved to be more promising and it was
observed that reactivity would be increased using proper
electron withdrawing substitutions.
nitrophenyl esters (8
&
Thus,
eand para-
9) were well studied as the tools
for peptide synthesis.3 4 - 3 7
Along with the nitrophenyl esters, chloro and fluor0
phenyl esters (10. 11
L
peptide synthesis.3 8 - L C
12) were found to be very useful for
N-Hydroxysuccinimide
and
N-hydroxyphthalimide
esters
( 1 3 , 1 4 ) used more
f r e q u e n t l y than p - n i t r o p h e n y l and
chlorophenyl esters for p e p t i d e s y n t h e s i s . 4 1 - 4 3 T h e
synthesis of N-hydroxysuccinimide e s t e r s p r e s e n t no
difficulties and they provide completely racemization free
peptides
.
Esters derived from aldoximes ( 1 5 ) an& ketoximes ( 1 6 )
w e r e a l s o s t u d i e d in peptide s y n t h e s i s . 4 4
Esters o f
k e t o x i m e s m a d e it p o s s i h l e to s y n t h e s i z e s e r i n e and
threonine peptides without protecting
the hydroxyl
group
Interesting results have been obtained from esters derived
from pyridine-4-aldoximes.
Much attention has been devoted to activated esters
whose reactivity is explained by i n t r a m o l e c u l a r base
catalysis.
Prominent among these was the ester derived from
8-hydroxyquinoline (17).4 5
The effect o f the intramole-
cular base catalysis is manifested to a greater extent in
esters based on 2-hydroxypyridine (la), 2-mercaptopyridine
(19) and lLhydroxypiperidine(20). 46-40
Esters of l-hydroxybenzotriazole ( H O B t ) ( 2 1 ) and
indole-3-acetic acid
agents.4 9 7 5 0
(22) arc
extremely
potent
acylating
Enhanced reactivity of the former group is
attributed to the anchimeric assistance.
2.6
Coupling Reagents
Introduction of carbodiimides, especially dicyclohexyl
carbodiimide (23) as reagent for the formation of peptide
bond w a s a turning point
synthesis.5 1
in the history
of peptide
These compounds can be directly added to a
mixture o f carboxylic acid and amine s o that
1~
sit2
activation and coupling occurs.5 2
Eventhough, activation and coupling in a single step
make this process so-popular, they providr pcptide scgments
with loss of optical purity.
Also, the reaction leads to
thc formation of unreactive by-products such as N-acyl ureas
(24).
Both these difficulties to a possible extent can be
overcome by the addition of auxiliary nucleophiles such as
1-hydroxybenzotriazole and N-hydroxysuccinimide (25) to the
reaction system.53.51,
4 e0
O!-!-~"G
24
25
It should be noted that since HOBt is regenerated
during acylation, its concentration remains almost constant
during coupling.
A more efficient coupling agent, 1-hydroxy
7-azobenzotriazole (HOAt, 2 6 ) is used nowadays for the
effective coupling of peptides. 55
The success achieved in using DCC stimulated the search
for better
coupling
agents.
Among
them,
carbonyl
diimidazole (27) was found to be an effective one which
mediates
coupling
intermediate. 5 6 9 5 7
through
a
reactive
N-acyl
Since rigorous anhydrous conditions are
needed both in the preparation of the reagent and in bond
formation, it appears to be too demanding for general use
Castro
BOP
reagent
a.introduced
a little more ideal reagent,
[benzotriazol-1-yl-oxy-tris-(dimethylamino)-
phosphonium hexafluorophosphate] (28) for peptide synthesis
in solution phase. 5 8 , 5 9
The rate of coupling using BOP was
greater than DCC and other methods.
The BOP coupling using
the solid phase procedures 6 0 - 6 2 proceeded more rapidly and
to a greater d e g r e e of completion f o r p e p t i d e bond
formations.
hexafluoro phosphate (HBTU, 29) was found to be an efficient
c o u p l i n g a g e n t w h i c h p r o v i d e s peptides w i t h m i n i m a l
racemization and side reactions.6 3
Just like BOP reagent,
this is highly useful for rapid activation in solid phase
peptide synthesis.
Since 1940, N-carboxyanhydrides (NCA) have been used in
the formation of peptide bonds and were extensively applied
to the synthesis of poly ( A - a m i n o acids). 64-66
Eventhough,
several small peptides were prepared using this unprotected
NCAs, they are not suitable for larger ones.6 7
Urethane
protected NCAs (UNCA) should result in readily controlled
acylation reactions.
Among
them
(9-fluorenylmethoxy)
carbonyl protected NCAs (F-moc NCAs) have been used in the
successful synthesis of a decapeptide in a flow reactor.68
Similarly g-butyloxycarbonyl (Boc) protected NCAs gave
excellent results and were comparable with BOP and HBTU. 69
2.7.
Photochemical Activation
In the conventional a c t i v a t i o n m e t h o d o l o g y , the
carboxyl functional group is usually converted into the
corresponding acyl h a l i d e , mixed a n h y d r i d e or active
ester. 1 8 * 2 5 9 3 3 In these c a s e s , t h e c a r b o x y l function
possesses enhanced reactivity towards nucleophilic attack.
All
these approaches require
somewhat drastic
conditions
which involve the use of a base. Sometimes the substrate may
be sensitive to these conditions and c a n create serious
difficulties.
Therefore, the use of activating groups which
could be accomplished u n d e r n o n - d e s t r u c t i v e neutral
conditions, a v o i d i n g the need for r i g o r o u s chemical
treatment is a suitable alternative.
Photochemical
reactions could provide an ideal way of activation of the
carboxyl groups under mild, neutral conditions.
photochemical
activation procedures
find
These
promising
applications i n the field of p e p t i d e a n d macrolide
synthesis.7 0 - 7 3
A photochemical activating group contains a chromphore
which is sensitive to light, but relatively stable to most
of
the w i d e
encountered.
variety
of
chemical
reagents
commonly
The wavelen~th of the radiation is absorbed
only by the activating group and i t should not affect other
parts of the molecule.
Morcover, the photochemical reaction
of the activating chromophore should no way affect the
substrate molccule and the photoproduct should be readily
separable.
In
the
photochemical
activation
approach,
the
functional group is derivatized with a light sensitive
chrornophore, which can serve as a latent activator of the
functional group.7 4
On irradiation with light of suitable
wavelength, the functional group is converted to an active
form and the light sensitive chromophore is removed.
The
active species in which the functional group possesses
enhanced
reactivity
towards the desired
effective synthesis of peptides,7 5 , 7 6
reaction permits
macrolides 7 7 , 7 8
and
carbohydrates 7 9 3 8 0 under m i l d , neutral photochemical
conditions.
Photolabile groups have been used frequently in peptide
synthesis as protecting and or activating groups and as
handles of polymeric support.
The photolysis reaction
is very selective and highly efficient, especially when it
is carried out under homogeneous conditions and even when
the group is attached to a polymer.
It has been shown that
photolysis at 350 nm causes no damage towards the most light
sensitive amino acids such as Trp and Tyr.8 4
The following
review gives an insight into the photolabile
carboxyl
activating groups, touching upon their applications in this
field.
Amit et al.8 5 , 8 6
found that N-acyl derivatives of 5 -
bromo-7-nitroindoline (Bni, 3 0 ) and aromatic or simple
aliphatic acids undergo efficient photosolvolysis to yield
acids, esters or amides depending on the nucleophile present
during irradiation.
Photosolvolysis of the 5-bromo-7-nitro-
1-indolino group was obseryed even using light above 400 nm.
When
were
N-acyl derivatives of 5-bromo-7-nitroindoline
irradiated in the presence of nuclrophiles such as
amines, alcohols etc. the respective products were isolated.
This reaction has been developed for the synthesis of
peptides. 87
Goissis et a1.88 could synthcsitc protected
peptide acids and esters by photosolvolysis of l-peptidyl-5bromo-7-nitroindolines.
ii)
2-Thionothiazolidines
-
Burton and whiteBg has reported the photochemical
activation
of
carboxyl
thionothiazolidines (31).
group
via
N-acyl
2-
Photolysis of 31 in the presence
of alcohols resulted in the formation of the corresponding
csters in very good yield.
The N-acyl derivatives are
prepared by the treatment
of the sodium salt of 2 -
thionothiazolidine with the corresponding acyl chlorides in
benzene.
It was explained that the kinetic product formed
in this reaction is the S-acyl derivative,
a facile S - - >
which undergoes
N thermal rearrangement to yield the
N-
acylated product (Scheme2.1), 90,91
0
II
___*
0J-R
R-C-OR'
Scheme 2.1
A mechanism involving
\(-hydrogen abstraction by the
sulphur atom is postulated for this activation process
(Scheme 2.2).
This mechanism which involves a ketene
intermediate is analogous to the mechanistic pathway for the
photolysis of g-phenylethyl
Barton and coworkers.9 2
thiobenzoates
investigated by
This activation approach using
2-
thionothiazolidines has been extended to the synthesis of
amides and peptides.93-95
Scheme 2 . 2
iii)
5-Azido-1.3.4-oxadiazo-
A conceptually novel approach to the synthesis of
peptides based
on
the
photochemistry
of
2-substituted
involves incorporation of the
5-azido-1,3,4-oxadiazole
carboxyl group of a suitably protected amino acid in the
heterocyclic system.96797
oxadiazoles
afford
On irradiation, these 1 , 3 , 4 -
the
activated
acyl
cyanide
corresponding to the carboxylic acid, with the extrusion of
two molecules of gaseous nitrogen (Scheme 2.3).
irradiation of
2-substituted
When the
5-azido-1,3,4-oxadiazoles
was carried out in the presence of nucleophiles such as
amines and alcohols, the corresponding products result with
an overall yield of 80-100%.
N-N
%
-N
Scheme 2.3
iv)
O x ~ ~ z o ltri.amide
erearrangement
In order to illustrate the generation of activated
carboxyllc species, Wasserman
a &.
sensitized
of oxazoles to triamides.
photooxygenation
developed the dye-
Under mild conditions the oxazoles convert to triamides,
eventually each of the carbon atoms in the oxazole ring
transforms to a carboxylate derivative (Scheme 2.4).98,99
Scheme 2.4
By proper choice of the substituents R1, R 2 and R 3 it
was possible to limit the reaction of the triamide with
nucleophiles to one of the three carboxyl groups. l o o This
selective reaction has been utilised by Wasserman
the synthesis of macrolide lactones
of peptides.
a.for
and in the synthesis
In peptide condensation, the carboxyl group of
an N-acylated amino acid was protected by conversion to an
oxazole derivative, which on photooxygenation regenerated
the carboxyl group in the activated triamide form.7 8
ACTIVATION OF CARBOXYL GROUPS
USING 2-MERCAPTOBENZOTHIAZOLE