Amino acid N-carboxyanhydrides: Activated peptide monomers behaving as phosphate-activating agents in aqueous solution

Article abstract of DOI:10.1021/ja048189s

The hydrolysis of valine N-carboxyanhydride (NCA) in aqueous phosphate buffers was shown to proceed through nucleophilic catalysis via an aminoacyl phosphate intermediate that displays phosphorylating capabilities through a potentially prebiotic process that simulates modern biochemical metabolic pathways. Copyright

Full text of DOI:10.1021/ja048189s

Published on Web 07/13/2004
Amino Acid N-Carboxyanhydrides: Activated Peptide Monomers Behaving as
Phosphate-Activating Agents in Aqueous Solution
Jean-Philippe Biron and Robert Pascal*
Organisation Mole´culaire EVolution et Mate´riaux Fluore´s UMR 5073 - CC 017, De´partement de Chimie,
UniVersite´ Montpellier 2, Place Euge`ne Bataillon, F-34095 Montpellier, France
Received March 30, 2004; E-mail: rpascal@univ-montp2.fr
R-Amino acid N-carboxyanhydrides (NCAs), 1, are activated
building blocks that can yield polypeptides with high degrees of
polymerization.1 This unique ability prompted investigations2 of
the prebiotic significance of NCA polycondensation even though
no convincing prebiotic pathway was available for their synthesis.
Their stereoselective polycondensation has also been considered
as a possible pathway for the abiotic origin of single-handedness
in nature.3 The disclosure of a possible mechanism for their
formation under the conditions of the primitive Earth through the
4,5
activation of carbamoyl amino acids by NO/O2 made their
prebiotic status more likely. In the course of a kinetic study of NCA
polymerization in aqueous solution,6,7 the competitive hydrolysis
was observed to be subject to buffer catalysis by various buffers
including phosphate,7 generalizing the previous observation of
acetate buffer catalysis.8 Usually, buffer catalysis corresponds to
general acid-base catalysis, but the alternative possibility of
nucleophilic catalysis has to be considered in every case.9 Nucleo-
Figure 1. Progress of the reaction of 10 mM Val-NCA in a 20 mM Na2-
HPO4/200 mM NaH2PO4 solution in D2O at 21 °C (pD 6.2) monitored by
philic mechanisms involving carboxylic-phosphoric mixed anhy-
drides have thus been reported for several reactions of acyl donors
including activated esters,10 thioesters,11 or anhydrides.12 A nu-
cleophilic mechanism would then be expected for phosphate buffer
catalysis of NCA hydrolysis (Scheme 1) because the carbamate
group must be a better leaving group than phosphate dianion from
the tetrahedral intermediate 2 as expected from the values of
ca. 513 and 7.2 for the respective pKA’s of the conjugated acids.
Because of their close relationship with aminoacyl adenylates,14
the intermediates of amino acid activation involved in protein
biosynthesis, and their potential phosphorylating ability,15 we
devised that the transient formation of aminoacyl phosphates 3
would have important consequences in the field of prebiotic
chemistry. Here we show that an intermediate displaying properties
consistent with an aminoacyl phosphate is formed from the
corresponding NCA in diluted phosphate buffer and that its
breakdown proceeds through phosphoryl transfer.
1H NMR spectrometry (300 MHz). Relative areas of CHR signals of Val-
NCA (9, δ ) 4.32 ppm, J ) 4.3 Hz), 3a (b, δ ) 3.81 ppm, J ) 4.5 Hz)
and Val (2, δ ) 3.48 ppm, J ) 4.4 Hz). Experimental points and curves
fitted assuming exponential decays for Val-NCA (2.1 × 10-3 s-1) and for
3a (5.7 × 10-4 s-1).
Scheme 1. Nucleophilic mechanism of catalysis of NCA
hydrolysis by phosphate dianion
The reaction of 10 mM Val-NCA in phosphate buffers was
monitored by NMR spectroscopy. An intermediate was indeed
observed in the course of the reaction (Figure 1), and both its
formation and its decay progressed according to first-order kinetics.
Since rates were affected by a change in the concentration of
HPO42- but not that of H2PO4- 16 then phosphate dianion must be
,
behavior was observed by 31P NMR spectroscopy. These NMR data
are also consistent with the values reported for valyl ethyl phosphate
4a.14a
The structure of the mixed anhydride 3a was confirmed by an
independent preparation from the Boc/tBu-protected derivative 4c
adapted from the synthesis of the ethyl phosphate derivative 4b.14a
Boc and tBu protecting groups were removed from 4c with
trifluoroacetic acid (TFA), and the acidic product was dissolved in
0.02 M Na2HPO4 buffered D2O displaying NMR spectra and a
the reactive species, which is in agreement with previous kinetic
measurements.7 However, when the concentration of the acid
component of the buffer was reduced, the final mixture was
complicated by the formation and precipitation of polypeptides due
to the increased pH value promoting the direct formation of peptides
from Val-NCA. In agreement with the structure of the mixed
phosphoric-carboxylic anhydride 3a (R ) CHMe2) for the
intermediate, a signal at -1.5 ppm (apart from the large peak of
inorganic phosphate, δ ) 0.2 ppm) displaying a similar kinetic
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10.1021/ja048189s CCC: $27.50 © 2004 American Chemical Society

C O M M U N I C A T I O N S
an energy-rich bond, according to Lipmann’s terminology.21 This
process may have constituted an original entry into the core of a
protometabolism in which “high-energy” species such as acyl
phosphates, thioesters, and ATP have been connected to each other,
which has been previously conceived as the consequence of a
thioester world.22 Further developments such as the research of
conditions or catalysts allowing phosphorylation of selected pre-
biotic molecules of interest are currently under investigation in our
group.
Scheme 2
Acknowledgment. This work was supported by the COST
action “Prebiotic Chemistry and Early Evolution”; working group
project D27/0001/02 “Molecular Origins of Life: Dynamic Co-
evolution of Peptides and Chemical Energetics, a Gate towards the
emergence of catalytic activity and nucleotides”. We are indebted
to Prof. Auguste Commeyras for advice and encouragement during
the course of this research. Thanks are also due to Jean-Pascal
Gimeno for experimental assistance.
kinetic behavior in agreement with that observed for the intermedi-
ate of Val-NCA hydrolysis.
Supporting Information Available: Reaction of 3a with methyl
alcohol and procedures for the synthesis of compounds 4c and 3a. This
material is available free of charge via the Internet at http://pubs.acs.org.
Because aminoacyl phosphates are carboxylic-phosphoric mixed
anhydrides with potential acyl and phosphoryl transfer abilities, two
different mechanisms of hydrolysis are conceivable (Scheme 2, R2
) H). To investigate this point, the intermediate 3a was formed in
aqueous solution, then excess methanol (R2 ) CH3), was added
and the product was analyzed by NMR spectroscopy after removal
of the solvent under reduced pressure. Methyl phosphate [1H NMR
(D2O) δ ) 3.39, J (P,H) ) 10.4 Hz; 31P NMR (D2O) δ ) 4.55, in
agreement with reported data17] was identified as a product, whereas
Val-OMe was not detected provided that NCA was no longer
present at the time of methanol addition. This result is consistent
with a cleavage of the P-O bond of the mixed anhydride. It is
also in agreement with the well-known reactivity of acetyl phosphate
near neutrality,18 which is similar to that of monosubstituted
phosphates esters that predominantly undergo a dissociative mech-
anism of phosphoryl transfer through a metaphosphate-like transi-
tion state.19
NCAs are therefore capable of activating inorganic phosphate
in diluted aqueous solution, which may have important conse-
quences for the processes that led to the emergence of life. It is
remarkable that the mixed anhydride pathway is prevalent even at
low (10-20 mM) concentrations of phosphate dianion and that high
temperatures are not required for phosphoryl transfer. Amino acid
chemistry was not expected to be able to provide a mechanism for
prebiotic phosphorylation and then to have possibly participated
in nucleotide and RNA synthesis. The connection revealed by the
mixed anhydride pathway could be an indication of an early
coevolution involving both amino acid/peptide and nucleotide/RNA
chemistries. Because of its highly evolved character, it seems
unlikely that an early connection of that kind could have been lost
at a later stage, which challenges the occurrence of an RNA-only
world where living organisms used RNA for both catalysis and
information storage.20 Furthermore, the process by which the
chemical energy present in NCA is transferred to a phosphorylated
intermediate simulates modern biological metabolic pathways. In
this view, the carbon-oxygen bond in NCAs can be considered as
References
(1) Kricheldorf, H. R. R-Amino Acid-N-Carboxy-Anhydrides and Related
Heterocycles: Syntheses, Properties, Peptide Synthesis, Polymerization;
Springer: Berlin; 1987.
(2) (a) Wen, K.; Orgel, L. E. Origins Life EVol. Biosphere 2001, 31, 241-
248 and references therein. (b) Brack, A. Biosystems 1982, 15, 201-207.
(3) Hitz, T.; Luisi, P. L. HelV. Chim. Acta 2003, 86, 1423-1434.
(4) Collet, H.; Bied, C.; Mion, L.; Taillades, J.; Commeyras, A. Tetrahedron
Lett. 1996, 37, 9043-9046.
(5) Commeyras, A.; Collet, H.; Boiteau, L.; Taillades, J.; Vandenabeele-
Trambouze, O.; Cottet, H.; Biron, J.-P.; Plasson, R.; Mion, L.; Lagrille,
O.; Martin, H.; Selsis, F.; Dobrijevic, M. Polym. Int. 2002, 51, 661-665.
(6) Plasson, R.; Biron, J.-P.; Cottet, H.; Commeyras, A.; Taillades, J. J.
Chromatogr., A 2002, 952, 239-248.
(7) Plasson, R. Ph.D. Thesis, Universite´ Montpellier 2, France, 2003.
(8) Bartlett, P. D.; Jones, R. H. J. Am. Chem. Soc. 1957, 79, 2153-2159.
(9) Jencks, W. P.; Carriuolo, J. J. Am. Chem. Soc. 1961, 83, 1743-1750.
(10) (a) O’Connor, C. J.; Wallace, R. G. Aust. J. Chem. 1984, 37, 2559-
2569. (b) El Seoud, O. A.; Ruasse, M.-F.; Rodrigues, W. A. J. Chem.
Soc., Perkin Trans. 2 2002, 1053-1058.
(11) Gill, M. S.; Neverov, A. A.; Brown, R. S. J. Org. Chem. 1997, 62, 7351-
7357.
(12) (a) Andre´s, G. O.; Granados, A. M.; de Rossi, R. H. J. Org. Chem. 2001,
66, 7653-7657. (b) Higuchi, T.; Flynn, G. L.; Shah, A. C. J. Am. Chem.
Soc. 1967, 89, 616-622.
(13) Johnson, S. L.; Morrison, D. L. J. Am. Chem. Soc. 1972, 94, 1323-1334.
(14) (a) Kluger, R.; Li, X.; Loo, R. W. Can. J. Chem. 1996, 74, 2395-2400.
(b) Kluger, R.; Loo, R. W.; Mazza, V. J. Am. Chem. Soc. 1997, 119,
12089-12094.
(15) Paecht, M.; Katchalsky, A. J. Am. Chem. Soc. 1954, 76, 6197-6198.
(16) Only traces of 3a were formed in 0.2 M NaH2PO4 in the absence of Na2-
HPO4, whereas a 10-fold reduction of the concentration of NaH2PO4
had a limited effect at a 0.01 M Na2HPO4 concentration.
(17) Wheelhouse, R. T.; Stevens, M. F. G. J. Chem. Soc., Chem. Commun.
1993, 1177-1178.
(18) (a) Bentley, R. J. Am. Chem. Soc. 1949, 71, 2765-2767. (b) Koshland,
D. E. J. Am. Chem. Soc. 1952, 74, 2286-2292. (c) Di Sabato, G.; Jencks,
W. P. J. Am. Chem. Soc. 1961, 83, 4400-4405.
(19) (a) Westheimer, F. H. Chem. ReV. 1981, 81, 313-326. (b) Hengge, A.
Acc. Chem. Res. 2002, 35, 105-112. (c) Bianciotto, M.; Barthelat, J.-C.;
Vigroux, A. J. Am. Chem. Soc. 2002, 124, 7573-7587.
(20) (a) Gilbert, W. Nature 1986, 319, 618. (b) Yarus, M. Curr. Opin. Chem.
Biol. 1999, 3, 260-267.
(21) Lipmann, F. AdV. Enzymol. 1941, 1, 99-162.
(22) (a) De Duve, C. In The Molecular Origins of Life. Assembling Pieces of
the Puzzle; Brack, A., Ed.; Cambridge University Press: Cambridge, 1998;
pp 219-236. (b) De Duve, C. Origins Life EVol. Biosphere 2003, 33,
559-574.
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