Formation
of an amide from a carboxylic acid and an amine often results in overall loss of free
energy however there is a high activation energy to be overcome. To make the synthesis at
all viable this energy must be lowered. This is achieved either by catalysis or by
formation of carboxylic acid derivatives (denoted RCOX), effectively starting at a higher
energy on the free energy reaction pathway (Figure). The nature of the leaving group 'X' governs the
value of the activation energy. It would thus seem desirable to form amino acid
derivatives with a strongly electron withdrawing 'X', making the carbonyl carbon more
prone to nucleophilic attack and thereby achieving high reaction rates at ambient
temperatures.
Initially
the azide and chloride groups were proposed as the 'X' substituent. While the azide proved
to be almost entirely suited to its task, the acid chloride suffers from the problem of
being "over-activated". Due to the
ease of elimination of the chloride ion, the carbonyl is prone to attack from even weak
nucleophiles. They are thus prone to hydrolysis and cannot be conveniently stored.
Another
more subtle side reaction is the loss of chiral integrity at the a-centre
of the activated amino acid. This can occur through two mechanisms:
1)
Direct
abstraction of the a-proton.
2)
Formation
of an oxazolone (also known as an azlactone), increasing the acidity of the a-proton.
Both
these situations result in the formation of a planar carbanion with reprotonation possible
from both faces (Figure).
It
becomes clear then that a balance is needed between highly reactive species, such as the
acid chloride, and almost unreactive derivatives, for example the alkyl esters. Along with
this it would be convenient to have a "user friendly" derivative which could be
prepared either in advance and stored, or an activating species that ca be prepared in situ without fear of unwanted side reactions.
Many solutions to this dilemma have been proposed based on formation of an anhydride or of
electron withdrawing esters, often phenyl groups with electron withdrawing substituents. A few representative examples of these are listed
in table 1. The use of coupling agents will be discussed below.
One
further rate determining factor is the ease with which the intermediate acyl-amine complex
can be deprotonated. If deprotonation does not occur then the ammonium ion will always be
the better leaving group and the starting materials will be reformed. For this reason
those activated species capable of accepting a proton from the intermediate will result in
a higher reaction rate. A few commonly used activating species are summarised in the figure. Coupling is achieved by combination of the
activated carboxyl group of one amino acid with the free amino group of a second.
In
addition to these, there are several reagents that activate the carboxyl group in situ, i.e. in the presence of the nucleophile.
Many of these are based on the chemistry of the carbodiimide group where the neighboring
C=N bonds are susceptible to nucleophilic attack by the carboxyl group forming an isourea
with dehydration be favored by the formation of a highly stable urea (Figure).
There
is a risk of the amine nucleophile itself reacting with the carbodiimide resulting In the
formation of an undesired guanidine but this reaction is sufficiently slow to be
unimportant in the conditions conventionally used for peptide synthesis. The classic
dicyclohexyl derivative proposed by Sheehan and Hess has been most commonly used,
producing a urea that is almost totally insoluble In conventional solvents. It does,
however, prove to have a low degree of solubility in the presence of other dissolved
material and is remarkably persistent in its contamination of the coupling product. For
this reason a number of other derivatives which produce ureas which are either water
soluble or are soluble in dimethylformamide (DMF) such as
1-ethyl-3-(3’-dimethylaminopropyl)carbodiimide (EDCI, also known as WSC for water
soluble carbodiimide) and diisopropylcarbodiimide (DIPCDI) have been developed.
It
has become increasingly popular to include catalytic auxiliary nucleophiles in acylation
mixtures (Figure). The most commonly used
additive is l-hydroxbenzotriazole (HOBt), although N-hydroxysuccinimide (HOSu) and
N-hydroxy-5-norbene-endo-2,3-dicarboxamide (HONB) are also used. These are introduced in
carbodiimide-mediated couplings to reduce possible side reactions, including racemisation,
and to increase reaction rate when using active esters. In addition to providing excellent
leaving groups, all these additives are capable of acting as proton acceptors aiding
deprotonation of the ammonium ion intermediate and thereby greatly increasing reaction
rate. Although added in equimolar quantities
to the acylating component, the additive is catalytic and therefore remains in essentially
a fixed concentration throughout the coupling step. This ensures that the highly activated
isourea derivative is short lived.
An
alternative method for generating the transient active esters, such as the benzotriazolyl
ester, is through use of a phosphonium reagent like PyBOP.
(PyBOP’s full name is benzotriazole-1-yloxy-trisphosphonium hexafluorophosphate, this name is
rarely used!). This reagent is used in tandem
with a tertiary amine base which abstracts the acidic carboxyl proton. The generated carboxylate anion attacks the
positively charged phosphorous atom, substituting for the benzotriazolyl group. The benzotriazolyl anion itself acts as a
nucleophile at the acyl centre. In the
resulting substitution reaction the OBt ester and a phosphonamide are formed (figure).
