Diastereoselectivity in prebiotically relevant 5(4H)-oxazolone-mediated peptide couplings

Article abstract of DOI:10.1039/c3cc49580a

A stereochemical study of a potentially prebiotic peptide-forming reaction was carried out as the first part of a systems chemistry investigation of potential paths for symmetry breaking. Substantial diastereomeric excesses result from a fast epimerization of the 5(4H)-oxazolone intermediate in aqueous solution. The Royal Society of Chemistry.

Full text of DOI:10.1039/c3cc49580a

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Diastereoselectivity in prebiotically relevant
5(4H)-oxazolone-mediated peptide couplings†
Cite this: Chem. Commun., 2014,
50, 3100
Damien Beaufils,a Gregoire Danger,b Laurent Boiteau,a Jean-Christophe Rossia and
´
Robert Pascal*a
Received 18th December 2013,
Accepted 28th January 2014
DOI: 10.1039/c3cc49580a
www.rsc.org/chemcomm
A stereochemical study of a potentially prebiotic peptide-forming the identification of cyclic networks capable of amplifying ee.
reaction was carried out as the first part of a systems chemistry This possibility requires configuration to be considered as a mole-
investigation of potential paths for symmetry breaking. Substantial cular property that can be reproduced from one chiral centre to another
diastereomeric excesses result from a fast epimerization of the one and then, that complies with the specific kind of stability
5(4H)-oxazolone intermediate in aqueous solution.
(dynamic kinetic stability, DKS) associated with entities that grow
exponentially.9 It requires energy to be provided to the self-
The formation of prebiotic peptides as well as that of other organizing system and to be coupled with an amplification loop.10,11
biopolymers remains an essential issue in the chemistry of the In addition, any reproduction of chiral configuration implies that certain
origins of life since the polymerization of building blocks is asymmetric centres undergo an inversion of configuration or that new
usually an uphill process in water.1–4 The formation of peptides at ones are generated from achiral centres. In this communication, we
equilibrium is indeed limited to that of short oligomers. Therefore, focus on the possibility of reproduction of chiral configuration
chains having a length sufficient to allow the formation of secondary during the formation of peptide linkages occurring through
structures have a very low probability to be formed. Far-from- 5(4H)-oxazolone intermediates that are well known to undergo
equilibrium conditions are thus required for peptide polymerization, epimerization easily.12 Activation of peptides by carbodiimides
indicating that the reactions of free a-amino acids must be and cyanamide was previously demonstrated to take place via
promoted by activating agents or, alternatively, prebiotically the intermediacy of a 5(4H)-oxazolone in diluted aqueous
available activated a-amino acid derivatives must be used as solution in the pH range 5–7.13 Starting from a racemic mixture
starting materials.5 With regard to the issue of chirality, any of reagents, the chiral instability of 5(4H)-oxazolone offers a
possibility of stereoselection in this process could have led to possibility of reproduction of configuration under the influence
homochiral segments in peptides at least. Moreover, cyclic of neighbouring asymmetric centres if stereoselective reactions
networks of reactions can be formed if polymerization is can be associated with epimerization. Scheme 1 displays the two
associated with hydrolytic processes on geological time scales, possibilities of diastereoselection involving the unstable asym-
which may lead to symmetry breaking in the amino acid pool.3 metric centre of 5(4H)-oxazolones under the influence of the
Model dynamic systems allowing symmetry breaking have been configuration of either the N-terminal part of the peptide chain
proposed,6,7 and the recycling of materials proved to be efficient.7 In or from the nucleophilic segment. Here, we report that a notable
this view, an enantiomeric excess (ee) may be amplified through
autocatalytic cycles, when stereoselective steps are present.3
A
significant stereoselectivity has for instance been observed for the
cleavage of the N-terminus in peptides leading to diketopiperazines.8
We embarked on a systems chemistry experimental approach into
a
´
´
Institut des Biomolecules Max Mousseron, UMR 5247, CNRS—Universite
Montpellier 1 & Montpellier 2, CC17006, Place E. Bataillon, 34095 Montpellier,
France. E-mail: rpascal@univ-montp2.fr
b
´
´
Spectrometries et Dynamique Moleculaire, Physique des Interactions Ioniques et
´
´
Moleculaires, UMR 7345, CNRS—Aix-Marseille Universite,
´ ˆ
Scheme 1 Stereoselectivity in peptide couplings via 5(4H)-oxazolones
can be influenced by the chiral environment from both the N-terminal
segment and the C-terminal segment.
Centre Saint-Jerome—case 252, 13397 Marseille, Cedex 20, France
† Electronic supplementary information (ESI) available: Synthesis, analytical
procedures and coupling experiments. See DOI: 10.1039/c3cc49580a
3100 | Chem. Commun., 2014, 50, 3100--3102
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the reactant or product states. The results displayed in Table 1 thus
express the preferred stability of the homochiral transition states
obtained upon addition of water (or hydroxide ion) to the 5(4H)-
oxazolone 2. The diastereomeric ratio of the product turned out to
become rapidly independent of the configuration of the starting
material, which indicates that the epimerization rates at least
approach the rates of subsequent hydrolysis, if, consistent with the
above mentioned hypothesis, they do not exceed it. The steady state
d.r. was not significantly changed by substituting water for H-Gly-
NH2 as an alternative non-chiral nucleophile (ESI,† Table S2) leading
to the tripeptide Ac-L-Tyr(Me)-(D,L)-Ala-Gly-NH2. As a whole, these
results show that the penultimate residue in dipeptide 1 notably
influences the stereoselectivity of the reaction of the intermediates
with nucleophiles. Though d.r. values remain unexceptional, it is
worth noting that the homochiral configuration was preferred in
every case.
The configuration of the nucleophile component during the
EDC-promoted coupling (Scheme 3) of Ac-L-Tyr(Me)-OH 3 has a
stronger influence (Table 2, method A and ESI†). The results are
pH-dependent. At low pH, initial rates are fast but the yields level off
rapidly (ESI,† Fig. S2) probably because EDC is consumed through
acid-catalyzed hydrolysis.14 In contrast, above pH 6.5, reactions
became tedious and the determination of d.r. values of products
obtained in low yields turned out to be imprecise (ESI,† Fig. S2). No
difference in d.r. is observed from reactions of both enantiomers at
pH 6.5 (and at higher values of pH within the limits of the accuracy
of measurements at low conversion yields, ESI,† Table S3) and values
exceeding a ratio of 65/35 in favour of the homochiral species were
obtained in every case. The observation that the stereoisomeric ratio
is, in most cases, independent of the configuration of the starting
material indicates that epimerization of the oxazolone intermediate
takes place faster than the subsequent reaction with nucleophiles.
The change in configuration at the a-carbon of Tyr(Me) indicates
that epimerization of the 5(4H)-oxazolone 4 intermediate competes
with further reactions with nucleophiles. Since there is obviously no
free energy difference between the two enantiomers of intermediate
4 in the ground state, the diastereomeric ratios of the products are
under kinetic control and determined by the stability of the transi-
tion state of the reaction with nucleophiles.
Scheme 2
preference for the homochiral configuration is observed in both
cases as a result of kinetically selective processes.
The formation of diastereomers was monitored by HPLC using
starting materials 1a–c (Scheme 2) involving a tyrosine residue with
a chromophoric group and in which the phenol group was
methylated to avoid any possibility of reaction with electrophiles.
Before studying the coupling reaction, the epimerization of
dipeptides as a result of activation was investigated. The dipeptides
Ac-L-Tyr(Me)-Xaa-OH 1a–c bearing different C-terminal residues were
allowed to epimerize in 0.1 M 2-(N-morpholino)-ethane sulphonic
acid (MES) buffers upon the addition of 1-ethyl-3-(3-dimethylamino)-
propyl-carbodiimide (EDC, 1 eq.). In every case, a new HPLC peak
corresponding to that of the product of epimerization was formed.
The diastereomeric ratio (d.r.) was monitored by HPLC and the
addition of EDC was repeated (3 to 4 times) until a steady state was
reached (i.e. until no change in d.r. was observed after EDC addition),
which was confirmed by the comparable values obtained starting
either from L,L-1a or from L,D-1a (Table 1). A preference in favour of
homochiral peptides as a result of EDC activation was observed in
every case and the greater value determined for leucine derivative 1c
probably results from a bulky side-chain. It is noteworthy that d.r.
values at a steady state cannot correspond to equilibrium values
corresponding to the thermodynamic stability of diastereomers
L,L-1 and L,D-1. Making the hypothesis that the interconversion of
5(4H)-oxazolone epimers L,D-2 and L,L-2 is fast, the value of d.r. would
actually correspond to the ratio of the rates of hydrolysis of this
mixture into the products and does not result from the difference in
the free energy between the diastereomers of the 5(4H)-oxazolone
intermediates L,D-2 and L,L-2 in the ground state. Under the effect of
activation, it is actually the relative free energies of the transition
states of the hydrolysis reaction that should determine the product
ratio. The measured d.r. values are therefore kinetically rather than
thermodynamically controlled and correspond to a steady state and
are likely to be different from equilibrium values whether related to
At lower values of pH, coupling rates were observed to
increase, but at pH 5.5 the final diastereomeric ratio was lower
Table
1
Epimerization of the C-terminal residue of dipeptides
Ac-L-Tyr(Me)-Xaa-OH 1a–c (1 mM in 0.1 M MES buffer pH 5.5 at r.t.) upon
stepwise addition of EDC (1 equiv. every 24 h, 3 days)
Final diastereomeric ratioa
(homochiral/heterochiral)
Entry
Xaa
1
2
3
4
L-Ala
D-Ala
L-Val
L-Leu
59/41 (L,L-1a/L,D-1a)
56/44 (L,L-1a/L,D-1a)
58/42 (L,L-1b/L,D-1b)
70/30 (L,L-1c/L,D-1c)
a
Scheme 3
Determination by integration of HPLC peaks (detection at 273 nm).
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Table 2 Diastereomeric ratios (ratio of HPLC peak areas of homochiral vs.
heterochiral isomers) of peptide products obtained by the coupling of
1 mM Ac-Tyr(Me)-OH 3 or the corresponding 5(4H)-oxazolone 4 with
different nucleophiles (10 mM)
The results of the present study show that 5(4H)-oxazolone-
mediated peptide couplings can lead to significant diastereo-
meric excesses in favour of homochiral structures. Noticeable
d.r. values are obtained in every case. However, a stronger
influence of the configuration of the nucleophilic component
was observed and the results seemed to be improved for
chains of increased lengths suggesting that the occurrence of
secondary structures in peptides may improve the stereo-
selectivity as already demonstrated during the polymerization
of N-carboxyanhydrides.16 The 5(4H)-oxazolone-mediated
coupling remained efficient in spite of the low concentration
of reagents used, demonstrating that the process may have
played a role in abiotic environments and have led at least to
the formation of homochiral domains in random peptides
starting from racemic monomers. The possibility of associating
stereoselectivity in oligomerization with stereoselective peptide
cleavage is a requirement for obtaining an amplification of
chirality leading to a global symmetry breaking.7 The results
provided here are presently not sufficient to establish this
possibility that also depends on the occurrence and stereo-
selectivity of processes in which the peptide products can be
reprocessed. However, they constitute a preliminary step in a
systems chemistry study of a potential process for symmetry
Conditionsb
Entry Nucleophilea (pH, t)
Conversion
yielde (%)
Methodc d.r.d
1
2
3
4
5
6
7
8
H-L-Ala-NH2
H-D-Ala-NH2
H-D/L-Ala-NH2 5.5, r.t.
H-L-Ala-NH2
H-D-Ala-NH2
H-D/L-Ala-NH2 6.5, r.t.
H-L-Ala-NH2
H-D-Ala-NH2
H-D/L-Ala-OH
H-D-Ala-OH
H-D-Ala-NH2
H-L-Ala-NH2
H-D-Ala-NH2
H-D-Ala-OH
H-(L-Ala)2-OH 7.5, r.t.
H-(L-Ala)3-OH 7.5, r.t.
H-(L-Ala)4-OH 7.5, r.t.
H-D-Ala-NH2
H-(L-Ala)3-OH 7.5, 0 1C
H-(L-Ala)4-OH 7.5, 0 1C
5.5, r.t.
5.5, r.t.
A
A
A
A
A
A
B
B
A
B
B
B
B
B
B
B
B
B
B
B
54/46
43/57
8
6
54/46 14
72/28 15
73/27 12
72/28 18
70/30 28
70/30 29
71/29 15
69/31 23
75/25 32
75/25 33
75/25 46
71/29 47
75/25 20
77/23 41
77/23 42
80/20 60
82/18 53
81/19 53
6.5, r.t.
6.5, r.t.
6.5, r.t.
6.5, r.t.
6.5, r.t.
6.5, r.t.
6.5, 0 1C
7.5, r.t.
7.5, r.t.
7.5, r.t.
9
10
11
12
13
14
15
16
17
18
19
20
7.5, 0 1C
a
b
10 mM concentration. 100 mM MES buffers (pH 5.5 and pH 7.5),
c
100 mM MOPS buffer (pH 6.5). Method A: 1 mM Ac-Tyr(Me)-OH
d
3+1 mM EDC; Method B: preformed oxazolone 4. Ratio of homochiral breaking through a peptide protometabolism associating a
e
vs. heterochiral isomers. Determined from the final peak areas of
favourable kinetic stereochemical preference with the possibility
peptides and Ac-Tyr(Me)-OH 3.
of reversion of the configuration of an unreacted precursor.
`
We thank Dr Monique Calmes for assistance in performing
at the same time as it was partly dependent on the configuration
of the alaninamide nucleophile (Table 2, entries 1–3). A similar
observation was made by coupling 3 with the enantiomers of free
amino acids H-Ala-OH, H-Leu-OH, H-Val-OH (ESI†). Considering
that neither the reactants nor the products undergo a change in
the ionization state between pH 5.5 and 6.5, it is difficult to
account for this result without considering the possibility of a
change in the diastereoselectivity determining step.
chiral HPLC analyses and COST Actions CM0703 ‘‘Systems
chemistry’’ and CM1304 ‘‘Emergence and Evolution of Complex
Chemical Systems’’.
Notes and references
1 R. Pascal, L. Boiteau and A. Commeyras, Top. Curr. Chem., 2005, 259,
69–122.
We also performed experiments (method B) in which the
5(4H)-oxazolone 4 was used instead of Ac-L-Tyr(Me)-OH 3 and
EDC. In this case the starting material was racemic, as shown
by the chiral HPLC analysis of its hydrolysis product in pure
water (ESI†). For intermediate 4 (Table 2, method B), coupling
yields increase at high pH indicating that EDC-mediated activation
becomes less efficient as the pH increases, which is consistent with
the fact that carbodiimide-mediated activation is acid-catalysed.15
The d.r. values are influenced by temperature and the best results
were obtained at 0 1C and pH values of 6.5 and 7.5 with d.r.
reaching or exceeding 80/20. The nature of the nucleophile also has
an influence on the diastereoselectivity. Alaninamide produced
2 P. L. Luisi, The Emergence of Life: From Chemical Origins to
Synthetic Biology, Cambridge University Press, Cambridge, UK, 2006.
3 G. Danger, R. Plasson and R. Pascal, Chem. Soc. Rev., 2012, 41,
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4 K. Ruiz-Mirazo, C. Briones and A. de la Escosura, Chem. Rev., 2014,
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U. S. A., 2004, 101, 16733–16738.
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9 A. Pross, What is life? How chemistry becomes biology, Oxford University
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10 A. Pross and R. Pascal, Open Biol., 2013, 3, 120190.
11 R. Pascal, A. Pross and J. D. Sutherland, Open Biol., 2013, 3, 130156.
12 N. L. Benoiton, Biopolymers, 1996, 40, 245–254.
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15 (a) D. F. DeTar, R. Silverstein and F. F. Rogers, J. Am. Chem. Soc.,
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homochiral oligo-alanines were used. No significant influence of
the MES buffer concentration was observed (ESI†). At pH values of
6.5 and 7.5, d.r. values (defined as the ratio of homochiral products
vs. heterochiral products) were independent of the configuration of
the carboxylic acid component 3 or 4.
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3102 | Chem. Commun., 2014, 50, 3100--3102
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