The peptide formation mediated by cyanate revisited. N-carboxyanhydrides as accessible intermediates in the decomposition of N-carbamoylamino acids

Article abstract of DOI:10.1021/ja061339+

Similar to many ureas, N-carbamoylamino acids were shown to be hydrolyzed in aqueous solution through elimination mechanisms at close to neutral pH, the nucleophilic attack of water being a minor process. Two competing elimination mechanisms can take place involving either cyanate or isocyanate transient intermediates. Peptide formation was observed and attributed to the latter pathway through the intermediacy of amino acid N-carboxyanhydride (NCA). Eventually, cyanate and its precursors (including urea) unexpectedly behave as amino acid activating agents because of their ability in amino acid carbamoylation. Owing to its ability to generate a background prebiotic production of NCAs on the primitive Earth, this reaction is suggested to have contributed to the origin of life process. Copyright

Full text of DOI:10.1021/ja061339+

Published on Web 05/17/2006
The Peptide Formation Mediated by Cyanate Revisited. N-Carboxyanhydrides
as Accessible Intermediates in the Decomposition of N-Carbamoylamino
Acids
Gre´goire Danger, Laurent Boiteau, Herve´ Cottet, and Robert Pascal*
Dynamique des Syste`mes Biomole´culaires Complexes, UMR 5073 (UniVersite´ Montpellier 2, CNRS), CC 017,
UniVersite´ Montpellier 2, Place E. Bataillon, 34095 Montpellier Cedex 5, France
Received February 24, 2006; E-mail: rpascal@univ-montp2.fr
Cyanate-promoted peptide bond formation has been observed
by heating R-amino acids (AAs) in phosphate-buffered aqueous
solutions,1a but it gave low peptide yields and its mechanism has
remained unclear; peptide formation in molten urea (140 °C) has
also been reported.1b On the other hand, AAs are easily converted
by cyanate into N-carbamoylamino acids (CAAs),2 which are
usually considered to be poorly reactive species that can only be
hydrolyzed under alkaline conditions2 or by heating at moderate
pH.3 However, CAAs 1 can be cleanly activated into amino acid
N-carboxyanhydrides (NCAs, 3) by nitrosating the urea group
(Scheme 1, pathway a), then releasing N2 and water as only
byproducts.4 Reconsidering this process, we inferred that, even
without activation, CAAs could partly, but spontaneously, be
transformed into NCAs provided that the decomposition proceeds
through elimination (Scheme 1, pathways b and c), potentially
explaining the observation of cyanate-mediated peptide formation.
This hypothesis is supported by elimination mechanisms that
account for urea decomposition at moderate pH.5 The addition-
elimination mechanism, involving HO- attack, only prevails at high
pH. Then, CAAs, as nonsymmetrical ureines, must undergo
elimination either through the release of AA or that of ammonia,
giving NCO- or isocyanate 2-H+, respectively. Finally, the
isocyanate 2-H+ is likely to be converted into NCA by cyclization
involving the carboxylate group. We show here that the decomposi-
tion of CAA in water at 80 °C actually produces peptides under
favorable conditions and gives rise to a complex behavior that could
not have been expected from a simple hydrolytic process.
Figure 1. Decomposition of 20 mM Ala CAA 1a in buffered aqueous
solution (50 mM NaHCO3/saturated CO2) at 80 °C, without added AA (9)
and in the presence of 100 mM Ala (O).
Scheme 2. Activation of R-Amino Acids by Cyanate or Related
Reagents
Scheme 1. Pathways for the Conversion of N-Carbamoylamino
Acids into Amino Acid N-Carboxyanhydrides
with an addition-elimination process. Indeed, a process reverting
cyanate into CAA 1a can account for the decrease in rate observed
in the presence of added Ala, whereas an addition/elimination
mechanism is unable to explain this observation. From these
experiments, we can conclude that the elimination pathway is
largely predominating for CAAs, at least at the temperature of the
experiment (80 °C), as already observed from other ureas.
The decomposition of Ala CAA 1a predominantly takes place
through the elimination of Ala (pKa of Ala zwitterion ) 9.87), a
The decomposition of Ala CAA 1a was studied by NMR analysis
of samples of the reaction medium. In CO2/HCO3 buffers, the
-
addition of alanine (Ala) to the reaction medium strongly depressed
the initial decomposition rate (Figure 1). A similar kinetic effect
was not induced by the addition of glycine (Gly) (Supporting
Information). Nevertheless, in the latter experiment, Gly was partly
converted into carbamoylated derivative 1b. All together, these
results support the elimination pathway in which cyanic acid (as
the reactive form of cyanate, Scheme 1) is trapped by the
nucleophilic attacks of water, Gly, or Ala, but they are incompatible
+
better leaving group than ammonia (pKa of NH4 ) 9.3). This is
confirmed by the ca. 5-fold slower rates observed for the decom-
position of symmetrical urea 4a (Scheme 2), the breakdown of
which also turned out to be accompanied by additional processes
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J. AM. CHEM. SOC. 2006, 128, 7412-7413
10.1021/ja061339+ CCC: $33.50 © 2006 American Chemical Society

C O M M U N I C A T I O N S
the cyclization of anion 2a-H+ is likely to be easier than that of
neutral acid 2a from which NCA has been isolated under acidic
conditions.4 This new pathway illustrates the theoretical possibility
of obtaining NCAs from moderately activated AA derivatives.6 It
may be helpful in understanding the processes that contributed to
the origin of life. NCAs are likely intermediates in the primordial
formation of peptides,6 so investigations aimed at finding prebi-
otically plausible synthetic pathways have been carried out recently.7
The ability of NCAs to promote phosphate and nucleotide activa-
tion8 may have contributed to the emergence of the translation
apparatus. This contribution and a putative role of energy carriers
at early stages of the evolution of living organisms6 would require
the availability of straightforward abiotic pathways of synthesis of
NCAs sufficient to feed the first living organisms with energy and
allow them to survive in varied environments. Cyanate is produced
by electric discharges in reducing mixtures of N2, CO2, and H2,9
which is consistent with a recent reassessment of the composition
of the primitive Earth atmosphere.10 Furthermore, the Bu¨cherer-
Bergs synthesis11 may have directly yielded CAAs as well.2a Then
the pathway reported here starting from CAAs extends the
availability of NCA to reducing conditions, whereas it has been
mainly demonstrated in mild oxidizing environments4 or in the
presence of oxidizing agents.7 Then, the emergence of life and early
stages of biochemical evolution may have taken advantage of the
unique features of theses simple activated forms of AAs over the
varied environments of the primitive Earth.
Table 1. Nature and Yields of Obtained Peptides Determined by
CE
entry
reactants
timea/days
residual 1a
peptide (yield)b
1
2
3
Alac
53d
28g
53g
Ala2 (<0.2%)e
Ala2 (1.7%)h
Ala2 (12.7%)h
Ala3 (1.9%)
1af
21%
42%
1af + Alac
4
5
1af + Glyc
4af+ Alac
53d
35g
7%
Ala-Gly (2.1%)
Gly2 (6.8%)
Ala2 (20.0%)h
Ala3 (1.2%)
6
7
4af + Glyc
35d
56g
Ala-Gly (18.5%)
ureaf + Alac
Ala2 (7.2%)h
a Incubation at 80 °C. b Yields relative to activated agent (by EC) unless
otherwise mentioned. c At 100 mM. d At 50 mM NaH2PO4/50 mM Na2HPO4
buffer, pH 6.8 at 25 °C. e Yield relative to Ala (by NMR). f At 20 mM.
g At 50 mM NaHCO3/CO2 buffer, pH 7.1 at 25 °C. h Mixture of isomers.
Acknowledgment. We thank the European COST action D27
“Prebiotic Chemistry and Early Evolution” for support to this work.
Supporting Information Available: Experimental procedures,
NMR spectra, plots of reaction time course, and reaction conditions.
This material is available free of charge via the Internet at http://
pubs.acs.org.
Figure 2. Capillary electrophoresis (CE) of crude reaction medium obtained
after incubating 20 mM CAA 1a with 100 mM Ala for 53 days; buffer 50
mM NaHCO3 under CO2 atmosphere. Electrophoretic conditions: fused
silica capillary, 50 µm i.d. × 27 cm (19.6 cm up to the detector).
Electrolyte: 100 mM phosphate buffer, pH 2.56. Applied voltage: + 15
kV. Sample injection: 1s, 0.5 psi.
References
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(Supporting Information). Then, peptide production via NCA is a
minor process hardly detected from the reaction of CAA 1a alone
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