Biomimetic peptide bond formation in water with aminoacyl phosphate esters

Article abstract of DOI:10.1039/c1ob05660c

Aminoacyl phosphates, biomimetic analogues of aminoacyl adenylates, react efficiently with amino acid esters to form dipeptides with retention of stereochemical integrity. The reactions are selective and occur readily in the presence of nucleophiles other than amino groups on their side chains. Aminoacyl phosphate esters that lack an amino-protecting group are also suitable for peptide bond formation, leading to a simplified overall process.

Full text of DOI:10.1039/c1ob05660c

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Biomimetic peptide bond formation in water with aminoacyl phosphate esters†
Raj S. Dhiman, Liliana Guevara Opinska and Ronald Kluger*
Received 27th April 2011, Accepted 21st June 2011
DOI: 10.1039/c1ob05660c
Aminoacyl phosphates, biomimetic analogues of aminoacyl
adenylates, react efficiently with amino acid esters to form
dipeptides with retention of stereochemical integrity. The
reactions are selective and occur readily in the presence of
nucleophiles other than amino groups on their side chains.
Aminoacyl phosphate esters that lack an amino-protecting
group are also suitable for peptide bond formation, leading
to a simplified overall process.
Scheme 1 Peptide bond formation via BocPheEP and an amino acid
ester.
product was recovered using solid phase extraction cartridges with
activated carbon as the stationary phase. ESI-MS data confirmed
that the dipeptide is formed exclusively without further reactions
that would result in oligomerization. In extending the study, we
find that aminoacyl phosphate esters are also efficiently coupled
to other amino acid esters (Table 1).
In all cases the coupling reaction occurs rapidly and the
conversion of the amino acid ester to the dipeptide by acylation
is nearly quantitative. The precipitation of the product greatly
simplifies the process as the material is isolated by filtration. This
result is consistent with the development of ideal reactions in water
where two reactants that are completely soluble combine to form
a product which is insoluble; this approach serves to reduce waste
generated during work-up procedures.6
We find that the reactions are selective for peptide bond forma-
tion in the presence of other reactive nucleophiles. The reaction
of the aminoacyl phosphate with serine ethyl ester produces the
dipeptide with no acylation of the hydroxyl of the side chain
(based on results of the hydroxamic acid test7). However, where
cysteine ethyl ester is the substrate, there is greater potential for
side chain acylation due to the presence of a significant proportion
of the thiolate relative to the thiol at pH 8.0. Nonetheless, the
coupling reaction results exclusively in formation of the peptide.
This conclusion is based on qualitative analysis (TLC analysis and
Ellman’s test) as well as comparative analysis of the products of
the reaction in which N-acetyl cysteine serves as the substrate. This
selectivity may result from a direct reaction of the more reactive
amine or an initial aminoacylation of the thiolate followed by S to
N acyl migration, a common pattern in cysteine-centred ligation
strategies.8
We report the use of aminoacyl phosphates as the key to
a biomimetic protocol for producing peptides in water. Our
method is based on an analogue of the common intermediate
in biochemical pathways that efficiently produces peptides from
amino acids. In these pathways the initial activation of an amino
acid by reaction with ATP generates an enzyme-bound aminoacyl
adenylate.1 In this intermediate, the carboxylic acid is activated
as an acyl phosphate monoester and this acylates a second
substrate that is bound to the enzyme.2 The transient nature
of the acyl phosphate monoester in enzymes is not due to any
inherent instability: acyl phosphate monoesters are long-lived in
water.3 Due to this stability, several reports in the literature have
described the application of synthetic acyl phosphate monoesters
in aqueous acylation reactions.4,5 The known characteristics make
them logical candidates for a central role in a biomimetic approach
to peptide synthesis; they provide an opportunity for selective re-
actions and a reduced dependence on protecting groups. Thus, we
investigated the efficiency of the reactions of aminoacyl phosphate
monoesters with amino acid esters. We find that dipeptides are
formed readily in buffered aqueous solutions, establishing a basis
for a general protocol that can be extended for multiple steps in
aqueous peptide coupling.
Scheme 1 presents the overall reaction scheme. As an initial test
of the general approach, reactions were performed with N-t-Boc-
protected phenylalanyl ethyl phosphate (BocPheEP) and glycine
ethyl ester (GlyOEt).
Analysis of the residual aminoacyl phosphate by 31P-NMR
reveals that it is fully converted within one hour (pH 8 HEPES
buffer). The reaction produces BocPheGlyOEt as a precipitate
that is readily isolated by filtration (55% isolated yield). Additional
A recent report from our laboratory noted that aminoacyl phos-
phates without a protecting group on nitrogen provide selective
lanthanide-promoted esterification of nucleosides and nucleotides
without oligomerization of the reagent.9 This prompted us to
attempt peptide coupling with deprotected aminoacyl phosphate
monoesters (Scheme 2).
Thus, in neutral buffer, PheEP forms oligopeptides and dike-
topiperazines that result from self-condensation. These reactions
must be suppressed in order for this class of reagent to be
Davenport Chemical Laboratories, Department of Chemistry, University
of Toronto, Toronto, Ontario, M5S 3H6, Canada. E-mail: rkluger@
chem.utoronto.ca
† Electronic supplementary information (ESI) available: Detailed experi-
mental procedures and NMR data for coupling reactions of BocPheEP.
See DOI: 10.1039/c1ob05660c
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Table 1 Summary of peptide coupling reactions of BocPheMP and amino acid esters
Amino acid ester
GlyOEt
Product
Isolated yield
55%
ESI-MS data
351.1 (M + H+)
AlaOMe
ValOMe
LeuOMe
27%
43%
80%
351.1 (M + H+)
379.1 (M + H+)
393.2 (M + H+)
SerOEt
CysOEt
33%
50%
381.1 (M + H+)
397.1 (M + H+)
Table 2 ESI-MS analysis of peptide coupling reaction between PheEP
and GlyOEt (1 : 1 ratio)
Compound
ESI-MS data
Scheme 2 Peptide coupling between phenylalanine ethyl phosphate
(PheEP) and GlyOEt.
251.1 (M + H+)
398.2 (M + H+),
545.1 (M + 147),
692.1 (M + 2 ¥ 147),
839.1 (M + 3 ¥ 147)
utilized for the desired peptide coupling process. We find that
in the reaction of PheEP with GlyOEt in the presence of 50%
molar excess of the amino acid ester leads to efficient coupling
with suppression of side reactions (ESI-MS peak identification in
Tables 2 and 3). A control experiment with PheEP in 50% molar
excess gave products due to the side reactions. The results indicate
that an excess of the non-activated substrate is needed to suppress
side reactions. Our results are consistent with the general reactivity
patterns of acyl phosphates monoesters with amines (bnuc = 0.9).10
The amino groups of the aminoacyl phosphate and the amino acid
ester have similar pKa values and without any other structural
distinction they would be expected to react at comparable rates.
However, the anionic character of the phosphate adds a barrier
to self-condensation. In combination with higher concentrations
of the attacking amine, this distinction results in selective reaction
of the amino acid ester in an otherwise competitive situation.
We then extended this approach to reactions with alanine and
again found that an excess of the amino acid is required before
peptide bond formation is observed.11 Reactions with the use of
additional amino acid substrates are expected to follow the same
295.1 (M + H+)
pattern and the utility of deprotected materials is currently the
subject of further studies.
Activated amino acids are potentially subject to racemization
due to the increased acidity of their a-protons compared to
those of the free acid. This can be prevented by mediating the
reactivity of the activated amino acid by changing the activating
group or by the use of additives.12 It has already been established
that activation of an amino acid as an acyl phosphate ester
does not lead to racemization of its aminoacyl component.13
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Table 3 ESI-MS analysis of peptide coupling reaction between PheEP
and GlyOEt (1 : 1.5 ratio)
Acknowledgements
We thank NSERC Canada for support through a Discovery Grant.
Compound
ESI-MS data
251.1 (M + H+)
References
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This was demonstrated by base hydrolysis of the aminoacyl
phosphate, followed by derivatization with Mosher’s acid chloride.
The resultant materials, when tested against authentic samples,
did not undergo any racemization. By extension, as the peptide
coupling reaction is performed under mildly basic conditions,
there should be no racemization in the dipeptide products. Further,
we examined the1H-NMR spectrum of an aminoacyl phosphate
ester in D2O and found that there is no exchange of the a-proton,
which is a necessary consequence of racemization and is thus
a reliable test. The anionic charge of the phosphate may also
contribute to configurational stability by hydrogen bonding to the
a-proton. Similar effects account for the configurational stability
of acyl azides.14 The anionic charge also repels hydroxide, which is
the necessary catalyst for proton removal.
In conclusion, we have demonstrated that aminoacyl phos-
phate monoesters function as effective acyl donors in water in
their reactions with amino acid esters. The pattern is clearly
suitable as a basis for peptide coupling in water in general.
Furthermore, the process permits formation of amides from
amines in the presence of other reactive nucleophiles. Based
on these results, we are broadening the scope of our peptide
coupling protocol as well as expanding the method to related
applications.
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 5645–5647 | 5647
©