Racemic β-sheets as templates for the generation of homochiral (isotactic) peptides from aqueous solutions of (RS)-valine or -leucine N-carboxy-anhydrides: relevance to biochirogenesis

Article abstract of DOI:10.1002/chem.200801477

As part of our program on biochirogenesis of homochiral peptides from racemic precursors, we report the feasibility of obtaining peptides with homochiral sequences composed of up to 25 residues of the same handedness in the polymerization of racemic valine or leucine N-carboxyanhydrides in aqueous solutions, as initiated by amines. The composition of the oligopeptides was determined by MALDITOF mass spectrometry, and the sequences of some of the heterochiral diastereoisomers were studied by MALDI-TOF MS/MS performed on samples in which the S enantiomers of the monomer were tagged with deuterium atoms. The process comprises several steps: 1) a Markov mechanism of asymmetric induction in the early stages of the polymerization yields libraries of racemic oligopeptides enriched with isotactic diastereoisomers, together with oligopeptide sequences containing enantiomeric blocks of homochiral residues; 2) the short peptides self-assemble into racemic colloidal architectures that serve as regio-enantioselective templates in the ensuing process of chain elongation; 3) homochiral residues of the amino acids located at the periphery of these colloidal aggregates exert efficient enantioselection, which results in the formation of long isotactic oligopeptides. The final diastereoisomeric distribution of the peptides depends upon the composition of the templates, which is determined by the concentration of the initiator. The racemic mixtures of isotactic peptides can be desymmetrized by using enantiopure methyl esters of α-amino acids as initiators.

Full text of DOI:10.1002/chem.200801477

FULL PAPER
DOI: 10.1002/chem.200801477
Racemic b-Sheets as Templates for the Generation of Homochiral (Isotactic)
Peptides from Aqueous Solutions of (RS)-Valine or -Leucine N-Carboxy-
anhydrides: Relevance to Biochirogenesis
Irina Rubinstein,[a] Gilles Clodic,[b] Gerard Bolbach,[b] Isabelle Weissbuch,[a] and
Meir Lahav*[a]
Abstract: As part of our program on
biochirogenesis of homochiral peptides
from racemic precursors, we report the
feasibility of obtaining peptides with
homochiral sequences composed of up
to 25 residues of the same handedness
in the polymerization of racemic valine
or leucine N-carboxyanhydrides in
aqueous solutions, as initiated by
amines. The composition of the oligo-
peptides was determined by MALDI-
TOF mass spectrometry, and the se-
quences of some of the heterochiral
diastereoisomers were studied by
MALDI-TOF MS/MS performed on
samples in which the S enantiomers of
the monomer were tagged with deute-
rium atoms. The process comprises sev-
eral steps: 1) a Markov mechanism of
asymmetric induction in the early
stages of the polymerization yields li-
braries of racemic oligopeptides en-
riched with isotactic diastereoisomers,
together with oligopeptide sequences
containing enantiomeric blocks of ho-
mochiral residues; 2) the short peptides
self-assemble into racemic colloidal ar-
chitectures that serve as regio-enantio-
selective templates in the ensuing pro-
cess of chain elongation; 3) homochiral
residues of the amino acids located at
the periphery of these colloidal aggre-
gates exert efficient enantioselection,
which results in the formation of long
isotactic oligopeptides. The final diaste-
reoisomeric distribution of the peptides
depends upon the composition of the
templates, which is determined by the
concentration of the initiator. The race-
mic mixtures of isotactic peptides can
be desymmetrized by using enantio-
pure methyl esters of a-amino acids as
initiators.
Keywords: chirality · oligopeptides ·
self-assembly · sheet structures ·
template synthesis
Introduction
cally pure compounds are a prerequisite for the evolution of
living species and that “mirror-symmetry breaking” must
have taken place before the emergence of life.[2–6] In the ab-
sence of reliable fossils, it is difficult to track down this his-
torical event; however, by performing laboratory experi-
ments, one may cast light on possible reaction pathways that
demonstrate the feasibility of converting racemic activated
monomers of amino and nucleic acids into homochiral bio-
polymers. Such a scenario involves two central problems:
first, the production of long polymeric chains composed
from residues of the same handedness (isotactic chains) by
starting from racemic reagents and overriding the difficulties
of binomial kinetics in the polymerization of multicompo-
nent monomers in an isotropic medium; second, the ach-
ievement of mirror-symmetry breaking in the racemic mix-
tures of isotactic peptides.
One of the open questions about the origin of life deals with
the timing and possible scenarios that might have been re-
sponsible for the emergence of homochiral biopolymers in
the achiral prebiotic environment. Two central surmises
have been proposed: one suggests that homochirality
emerged after the development of the primeval biological
system,[1] whereas the second maintains that enantiomeri-
[a] Dr. I. Rubinstein, Dr. I. Weissbuch, Prof. Dr. M. Lahav
Department of Materials and Interfaces
The Weizmann Institute of Science
76100 Rehovot (Israel)
Fax : (+972)8934-4138
E-mail: meir.lahav@weizmann.ac.il
Previously, a number of models have been proposed to
highlight the problem of biochirogenesis of peptides on
Earth, including those by Bonner and co-workers,[7] by
Brack,[8] and by Luisi and co-workers.[9,10]
[b] G. Clodic, Dr. G. Bolbach
Plate-Forme Spectromꢀtrie de Masse et Protꢀomique
Universitꢀ Pierre et Marie Curie
75252 Paris Cedex 05 (France)
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In our approach, we have followed a working hypothesis
that comprises several steps: the formation of libraries of
short oligomers composed from blocks of homochiral se-
quence, followed by the self-assembly of the oligomers
through hydrogen bonding into aggregates that operate as
regio-enantiospecific templates and control the ensuing pro-
cess of chain elongation.[11–15] Such a scenario has common
features with the process of crystallization, which comprises
the steps of crystal nucleation and stereospecific growth.
Herein, we report a regio-enantioselective process in the
polymerization of N-carboxyanhydrides of racemic leucine
(Leu-NCA) or valine (Val-NCA) in aqueous solution to
yield isotactic oligopeptides.
Results
Polymerization of racemic Val-NCA or Leu-NCA, enantio-
selectively tagged with 8 and 10 deuterium atoms, respec-
tively, was performed in aqueous solution by dissolving the
monomer in water and, after 5 min for the first system and
1 min for the second, adding 5, 10, or 25% (mol/mol) of ini-
tiator, namely n-butylamine or enantiopure methyl ester of
Val (Val-OMe), Leu (Leu-OMe), or phenylalanine (Phe-
OMe), under a continuous vortex. The reaction started at
room temperature as a clear solution; however, foam ap-
peared after 30s to a few minutes, due to CO2 evolution,
and the solution became turbid as a result of the formation
of colloidal peptide aggregates. The X-ray diffraction pat-
terns of these colloids were measured, either in situ with the
aqueous solution in a capillary, as shown for racemic Val-
NCA initiated by n-butylamine in Figure 1a, or at the end of
the reaction after centrifugation, water decantation, and
drying (Figure 1b, c). These patterns are very similar, are in-
dependent of the concentration of the initiator, and display
Bragg peaks at d spacings of 4.7 ꢂ for both the Val and Leu
oligopeptides, indicative of interchain hydrogen bonding be-
tween the peptide bonds, and of 9.7 and 12 ꢂ for oligo-Val
and oligo-Leu, respectively, indicative of the peptide inter-
layer spacing. The dry oligopeptides also displayed a d spac-
ing of 6.9 ꢂ, which corresponds to an extended peptide
backbone.
Figure 1. X-ray powder diffraction patterns measured from a) a heteroge-
neous reaction mixture of (RS)-Val-NCA initiated with 25% (mol/mol)
n-butylamine inserted in a capillary; b and c) the water-insoluble oligo-
peptides obtained in the polymerization of (RS)-Val-NCA and (RS)-Leu-
NCA, respectively, with 25% (mol/mol) n-butylamine.
The FTIR spectra of both systems showed the C=O
amide I stretching-vibration band at n˜ =1633 cmꢀ1 and the
ꢀ1
ꢀ
N H amide II band at n˜ =1540 cm . The XRD and FTIR
Figure 2. AFM image of the colloidal architectures formed after 10 min
of polymerization of (RS)-Val-NCA in an aqueous solution in the pres-
ence of n-butylamine and subsequent transfer to a mica support.
measurements are in keeping with the formation of b-sheet-
like structures that are stacked in architectures through the
hydrophobic side groups. Additional evidence is provided
by AFM images (for example, Figure 2) of the colloidal ag-
gregates formed after ꢁ10 min of polymerization and de-
posited on a mica support. The morphology appears fila-
ment-like, of 40–60 nm in width, and with thicknesses in the
range of 1.0–2.8 nm.
The MALDI-TOF mass spectra of the oligopeptides ob-
tained from the polymerization of racemic Val-NCA with
25, 10, and 5% (mol/mol) of n-butylamine initiator are
shown in Figure 3a–c. In the spectra, the isotactic oligopepti-
des, (of homochiral sequence), labeled Rn and Sn, are locat-
ed at the wings of each group of signals originating from
various diastereoisomers of a given length n, whereas those
peptides containing heterochiral residues, labeled RhSd in
which h+d=n, are located between them. In our previous
studies, we have demonstrated that the molar fractions of
the diastereoisomeric oligopeptides of a given length are
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Formation of Isotactic Peptides
FULL PAPER
Figure 4. Histograms showing the distribution of the diastereoisomers of
each length n obtained in the polymerization of racemic Val-NCA in
water as initiated by n-butylamine at a) 25 and b) 5% (mol/mol). c) His-
togram showing the calculated values with assumption of a binomial dis-
tribution of the diastereoisomers.
Figure 3. MALDI-TOF MS analysis of the Na+-cationized oligopeptides
obtained from the polymerization of racemic Val-NCA initiated with n-
butylamine at a) 25, b) 10, and c) 5% (mol/mol). Oligopeptides initiated
by traces of water are labeled with
.
*
and 5% (mol/mol) of n-butylamine initiator, along with the
corresponding values calculated for a theoretical binomial
distribution. At 25% initiator, the isotactic peptides are the
most dominant diastereoisomers for longer oligopeptides
comprising 7–14 residues (Figure 4a). At 5% initiator, the
mole fractions of the isotactic peptides appear to be some-
what smaller than those of the corresponding heterochiral
ones, (Figure 4b); however, they are significantly higher
than those calculated by assuming a theoretical binomial
distribution (Figure 4c). The observed peptide distribution
implies the operation of enantioselection in the chain-elon-
gation process.
The MALDI-TOF mass spectra of oligopeptides obtained
in the polymerization of racemic Leu-NCA in THF and
water with n-butylamine initiator are shown in Figure 5. In
the organic solvent, the reaction takes place under homoge-
neous conditions.[16] The isotactic di- and tripeptides are
formed more commonly than in a random process (Fig-
ure 5a) as a result of the operation of a Markov mechanism,
by which a homochiral residue at the N terminus of the pep-
tide chain adds preferentially, upon reaction, another amino
acid residue of the same handedness.[9,10] Such partial asym-
proportional to the relative intensities of their signals.
Therefore, one can clearly deduce from the spectra in
Figure 3 that the diastereoisomeric distribution of the oligo-
peptides strongly depends on the concentration of initiator
used in the reaction. The signal intensities corresponding to
the isotactic oligopeptides increase significantly, in compari-
son with those of their heterochiral counterparts, with an in-
crease in the amount of initiator. The common feature of
these spectra is that the short peptides comprise all possible
diastereoisomers in similar molar fractions, independent of
the concentration of initiator. On the other hand, the molar
fractions of the isotactic diastereoisomers beyond the hep-
tamers (7–14-mers) and of those containing one heterochiral
residue, labeled (nꢀ1) and (1ꢀn), increase with length. This
result significantly departs from the binomial distribution
for either the Val or Leu reaction systems.
Histograms of the molar fractions (Figure 4), calculated
from the signal intensities in the mass spectra, show the dis-
tribution of the diastereoisomers of each length n obtained
in the polymerization of racemic Val-NCA in water with 25
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M. Lahav et al.
Figure 6. Plot of the normalized homochiral enhancement of the isotactic
^
oligo-Val chains of various lengths n obtained with 25 ( ) and 5% (mol/
&
mol) ( ) of n-butylamine. The insert shows a magnified plot for n=2–7.
Some of the short peptides are water-soluble, as shown by
the MALDI-TOF MS of the supernatant solution after re-
moval of the water-insoluble fraction (Figure 7a–c). The
water-soluble fraction contains primarily the di- and tripep-
tide diastereoisomers and only heterochiral tetra- to hexa-
peptides, whereas the isotactic penta- and hexapeptides are
water insoluble. The importance of this result is considered
in the discussion of the mechanism of the reaction (see
below).
Figure 5. MALDI-TOF MS analysis of the oligopeptides obtained from
polymerization of racemic Leu-NCA a) in THF with 25% (mol/mol) of
n-butylamine; b and c) in water with 25 and 10% (mol/mol) of n-butyla-
mine, respectively. Oligopeptides initiated by traces of water are labeled
with
.
*
metric induction introduced by isolated chains should drive
the reaction towards random diastereoisomeric distribution
of the long oligopeptide chains. By contrast, the distribution
of the longer oligopeptides obtained by polymerization in
water (Figure 5b and c) shows a significant departure from
the random distribution. This difference in the diastereoiso-
meric distribution of the oligopeptides in the two solvents
suggests the occurrence of self-assembly of the short hydro-
phobic isotactic and atactic oligopeptides into clusters in
water, but not in the organic solvent; these clusters then op-
erate as templates and induce regio-enantioselection in the
later stages of the reaction.
The homochiral enhancement obtained in the polymeri-
zation of racemic Val-NCA initiated with 25 and 5% (mol/
mol) of n-butylamine was evaluated by dividing the experi-
mental molar fractions of isotactic oligopeptides of each
length with those calculated for a theoretical binomial distri-
bution (Figure 6). Thus, the homochiral enhancement for
dodecapeptides has a value of 550 at 25% initiator and only
200 at 5% initiator, but it increases to ꢁ7000 for the 18-
mer peptides.
Figure 7. MALDI-TOF spectra of the water-soluble oligopeptides ob-
tained from the polymerization of racemic Val-NCA in water with a) 25,
b) 10, and c) 5% (mol/mol) n-butylamine initiator. Isotactic oligopeptides
are marked arrows and labeled Rn or Sn. Oligopeptides initiated by traces
of water are labeled with
.
*
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Formation of Isotactic Peptides
FULL PAPER
To obtain additional insight into the sequences of the het-
erochiral peptides, they were analyzed by MALDI-TOF-
TOF MS. In this analysis, fragmentation of the ions by a
metastable decomposition and/or collision-induced dissocia-
tion showed two main fragmentation series (see the Experi-
mental Section). Data analysis shows that the n-butylamine
initiator (I), located at the C terminus, is linked to R or S
residues. For the heterochiral oligopeptides, at 25% initia-
tor, the fragmentation studies clearly highlight the presence
of a block sequence. Typically, for n=6, the overexpressed
ꢀ
sequences (N to
C terminus) were SRRRRR I and
ꢀ
ꢀ
ꢀ
RRRRRS I for (5,1), SSRRRR I and RRRRSS I for (4,2),
ꢀ
ꢀ
ꢀ
RRRSSS I and SSSRRR I for (3,3), RRSSSS I and
ꢀ
ꢀ
ꢀ
SSSSRR I for (2,4), and RSSSSS I and SSSSSR I for (1,5).
The single residue of opposite handedness for the oligopep-
tides labeled (nꢀ1,1) and (1,nꢀ1), in which n=4–9, is locat-
ed at either the N or the C terminus of the chains. Similarly,
for the oligopeptides comprising two residues of opposite
handedness, labeled (nꢀ2,2) and (2,nꢀ2), the sequence has
two blocks grouping residues of same handedness. For
longer oligopeptides, n>7, with a similar number of R and
S residues, blocks of 2–4 residues of the same handedness
were observed.
A more complex picture was observed for MALDI-TOF-
TOF analysis of heterochiral peptides obtained in the poly-
merization of (RS)-Val-NCA with 10 or 5% n-butylamine.
The oligopeptides bearing one heterochiral residue, (nꢀ1,1)
and (1,nꢀ1) in which n=5–9, showed this residue in a
rather random location. For the diastereoisomers that con-
tain more than one residue of opposite handedness, smaller
blocks of 2 or 3 residues of the same handedness were
found, in contrast to the results obtained with 25% initiator.
Figure 8. MALDI-TOF MS analysis of the Na+-cationized oligopeptides
obtained from the polymerization of racemic Val-NCA initiated with S-
Val-OMe at a) 25, b) 10, and c) 5% (mol/mol).
Desymmetrization of the racemic mixtures: In our attempts
to desymmetrize the racemic mixtures of the isotactic pep-
tides, we performed the polymerization reactions with the
methyl esters of Val, Leu, or Phe of either absolute configu-
ration. The distributions of the diastereoisomeric oligopepti-
des obtained in the polymerization of racemic Val-NCA
with 25, 10, and 5% S-Val-OMe are shown in Figure 8 and
the corresponding results for racemic Leu-NCA with 25, 10,
and 5% S-Leu-OMe are in Figure 9. The enantiomeric
excess (ee) values of the isotactic peptides as a function of
length for the Val and Leu systems with 25% initiator are
shown in Figure 10 (left). From these plots, we notice that
the enantiopure initiator engenders asymmetric induction in
the initiation of the polymerization and the ee value de-
creases around the tetramer–hexamer region. Beyond this
length, a reversal in the ee value occurs, such that the oligo-
peptide chains composed from residues of the opposite
handedness to that of the initiator are formed in excess.
Note that, by symmetry, S- and R-Val-OMe yielded mirror-
symmetry-related results.
When the polymerization of either racemic Leu-NCA or
racemic Val-NCA was initiated with 10 or 5% methyl ester,
the di- to hexapeptides were formed with an excess of
chains composed from residues with the same absolute con-
Figure 9. MALDI-TOF MS analysis of the Na+-cationized oligopeptides
obtained from the polymerization of racemic Leu-NCA initiated with S-
Leu-OMe at a) 25, b) 10, and c) 5% (mol/mol).
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M. Lahav et al.
Figure 10. Plots of ee% Sn =100([Sn]ꢀ[Rn])/([Sn]+[Rn]) of the isotactic oligopeptides for each length n obtained from the polymerization of racemic Val-
~
&
NCA (top) and racemic Leu-NCA (bottom) in water with 25, 10, or 5% (mol/mol) of R initiator ( ) or S initiator ( . Error bars are the average of five
samples.
figuration as the initiator. The ee value increases for the
tetra- to hexapeptides and then it decreases slowly to almost
racemic composition (Figure 10, middle and right).
conditions. Once the tetra-, penta-, and hexapeptides have
been formed, they self-assemble into water-insoluble colloi-
dal aggregates, such that the ensuing process of chain elon-
gation continues heterogeneously at the colloidal-particle/
solution interface. Nevertheless, in spite of this complica-
tion, one may outline some of the mechanistic details of the
reaction by analyzing, by mass spectrometry, the final com-
position of the diastereoisomeric oligopeptides and the se-
quences of some of the chains containing heterochiral resi-
dues.
The polymerization of racemic Leu-NCA with 25% R- or
S-Leu-OMe initiator (Figure 9a) demonstrates the formation
of detectable isotactic peptides of 25 residues in length. The
distribution of the short peptides is comparable to that ob-
tained from racemic Val-NCA; however, the peptides
formed beyond the hexa- and heptamers are primarily iso-
tactic oligopeptides or those containing one residue of oppo-
site handedness (Figure 9a), which, according to the
MALDI-TOF MS/MS analysis, is located either at the N ter-
minus or adjacent to the initiator at the C terminus of the
peptide chains.
Stereospecific templates from oligopeptides comprising en-
antiomeric blocks: The MALDI-TOF MS analysis of whole
reaction mixtures shows that the di-, tri-, and tetramers are
enriched with isotactic diastereoisomers composed of resi-
dues of the same handedness (Figure 4), in comparison with
a random distribution. This result implies that the NH2 end
group of the ultimate homochiral amino acid exerts asym-
metric induction in the early stages of the chain-propagation
process, which occurs in the isotropic solution, by preferen-
tially selecting residues of the same handedness. The short
peptides are water soluble, as indicated by the MALDI-
TOF MS analysis of the aqueous solution after removal of
the precipitate (Figure 7).
Discussion
The feasibility of forming racemic mixtures of oligopeptides
of homochiral sequence, of up to 25 residues, during poly-
merization in aqueous solutions of racemic Leu-NCA and
Val-NCA has been demonstrated. These isotactic peptides
are formed in concentration orders of magnitudes higher
than those anticipated from a random polymerization reac-
tion. Moreover, the racemic mixtures of isotactic peptides
could be desymmetrized if the polymerization was initiated
by enantiopure methyl esters of a-amino acids.
The relative composition of the short peptides and their
solubility in water determines the structure and chemical
properties of the colloidal self-aggregates that operate as
templates in the formation of the longer peptides. By chang-
ing the concentration of the initiator, one varies the diaste-
The mechanism of this reaction is very complex because it
starts and proceeds in its early stages under homogeneous
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Formation of Isotactic Peptides
FULL PAPER
reoisomeric composition of the
short peptides, and consequent-
ly, the composition and chemi-
cal properties of the colloidal
aggregates. When 5 or 10% ini-
tiator is used, peptides longer
than 25 residues were observed.
The diastereoisomeric libraries
are composed from complex
mixtures of isotactic and atactic
peptides. Nevertheless, the rela-
tive composition of the longer
peptides as a function of length
indicates that they depart sig-
nificantly from
a theoretical
random distribution (Figure 4b
and c). Thus, with 5% initiator,
the normalized homochiral en-
hancement, defined as the
molar fraction of isotactic pep-
tides normalized to those for
random
polymerization
(Figure 6) increases with chain
length and reaches a value of
ꢁ7000 for the 18-mer isotactic
peptides. In addition, the rela-
tive composition of the isotactic
versus heterochiral diastereo-
isomeric peptides decreases
only slightly as a function of
chain length. The histograms of
Figure 11. Schematic representation of the evolution of the most representative sequences of pentapeptide dia-
stereoisomers as suggested by MALDI-TOF-TOF MS data analysis of the products obtained from the poly-
merization of racemic Val-NCA initiated with 10% (mol/mol) S-Val-OMe.
the diastereomeric distribution of the polymerization prod-
ucts of racemic Val-NCA in water initiated by 5% n-butyla-
mine (Figure 4b) suggest that the reaction assumes a regio-
enantioselective pathway beyond a certain length and that
most of the heterochiral residues are inserted in the chains
in the early stages of the polymerization and are thus pri-
marily located close to the C terminus of the peptide chains.
Further support for this model is obtained from the
MALDI-TOF-TOF MS/MS results for the oligopeptides ob-
tained in the polymerization of racemic Val-NCA initiated
by 10% S-Val-OMe, as represented by an evolving tree of
penta- to undecapeptides (amenable to the analysis) in
Figure 11. The oligopeptides composed from homochiral
amino acid of opposite handedness, they either undergo
chain termination or, presumably, form an additional block.
On the basis of these results, we propose that, once a li-
brary of short racemic oligopeptides composed of short
blocks of enantiomeric residues is formed, the oligopeptides
self-assemble into colloidal templates. The FTIR spectra
and X-ray diffraction patterns measured from these tem-
plates suggest the formation of b-sheet-like architectures,
due to hydrogen-bond formation between chains composed
of short homochiral blocks of opposite handedness and
sheet stacking through hydrophobic contacts. Although the
composition of these colloidal particles is racemic, all of the
chains expose NH2 end groups of homochiral amino acid
residues of either handedness to the periphery. As a result
of the asymmetric induction exerted by these groups, the
process of chain elongation continues to be regio-enantiose-
lective. The newly formed isotactic blocks on the surfaces of
the colloidal templates can continue to self-assemble be-
tween themselves or with short peptides from the solution
to yield more robust sheets.
ꢀ
residues, S5 IS and R5-IS, evolved into longer isotactic ones,
ꢀ
ꢀ
S11 IS and R11 IS, but also into oligopeptides composed
mainly from two blocks of homochiral sequences. All of the
shortest heterochiral sequences present in the insoluble frac-
tion have the two-block sequence with S residues linked to
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ ꢀ
IS (R SSSS IS, RR SSS IS, RRR SS IS, and RRRR S IS)
and they evolved into heterochiral sequences containing two
ꢀ
ꢀ
blocks of opposite handedness, such as RRRRRR SSSS IS,
ꢀ
ꢀ
ꢀ ꢀ
RRRRRRRR SS IS,
RRRRRRRR SSS IS,
and
Rippled b-sheets as templates for the formation of isotactic
peptides: To increase the concentration of longer isotactic
oligopeptides, it was imperative to find conditions under
which one may generate, at an early stage of the reaction,
colloidal templates that are composed primarily from short
ꢀ ꢀ
RRRRRRRRRR S IS. One notices that, as a result of the
asymmetric induction of the S initiator, the formation of
short chains containing an excess of S residues close to the
initiator is favored. When these long blocks react with an
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M. Lahav et al.
isotactic oligopeptides. The analysis of the water-soluble
fractions of the polymerization of racemic Leu-NCA and
racemic Val-NCA demonstrates that the isotactic tetra- to
hexapeptides (Figure 7) were less soluble than the hetero-
chiral oligopeptides. Consequently, if enhanced concentra-
tions of these isotactic peptides are obtained during the
early stages of polymerization, one can design polymeri-
zation conditions whereby these peptides can separate from
the others as ordered b-sheet templates that can promote
the formation of long isotactic peptides. Indeed, when the
reaction was performed with 25% enantiopure Val-OMe or
Leu-OMe, we obtained isotactic peptides and those that
contained one residue of opposite handedness attached to
the initiator as the dominant diastereoisomers.
The powder X-ray diffraction, AFM, and FTIR measure-
ments cannot differentiate, within these colloidal particles,
between parallel (p) and antiparallel (ap) b-sheets or be-
tween racemic and pleated sheets. The racemic (rippled) b-
sheets might be more favored for kinetic and thermodynam-
ic reasons. In kinetic terms, provided that the rippled and
pleated sheets are of the same stability, the rate of self-as-
sembly of racemic b-sheet aggregates should depend upon
the concentrations of the S and R chains, whereas the for-
mation of the enantiomorphous pleated sheets, because they
are thermodynamically separated phases, should depend
upon the concentration of only one of the enantiomers
(Figure 12).
spectively, suspended in an organic solvent or water contain-
ing the initiator. On the other hand, NMR spectroscopy
studies of short racemic oligopeptides forming b-sheet-like
architectures in chloroform have shown that the interactions
between residues residing in neighboring chains of the same
handedness are more stable than those of opposite handed-
ness by 0.6–0.8 kcal per residue.[25] Consequently, it is of in-
terest to prove experimentally the structure of the b-sheet
templates under the present reaction conditions. For this
purpose, we took advantage of the structural differences be-
tween the various motifs. Homochiral rims delineate the rip-
pled b-sheet templates and, therefore, they can induce
regio-enantioselection in the formation of the longer pep-
tides. Such enantioselection can be shown schematically for
the arrangement of the NCA monomer molecules at the
growing enantiomeric sites of the template. An NCA mole-
cule of R configuration can react enantioselectively with the
NH2 group of an R chain, with assistance in the transition
state from two hydrogen bonds, one between the to-be-re-
ꢀ
acted C=O carbonyl group and an N H group of a neigh-
ꢀ
boring S chain and the second between the N H group of
the NCA molecule and the C=O bond of the amide C-ter-
minal end group of
a different neighboring S chain
(Scheme 1). Such interactions will organize the NCA mono-
mer molecule with the alkyl group away from the surface of
the template and will bring the to-be-reacted C=O group
into a proper orientation to form a new residue of the same
absolute configuration as that of the growing peptide chain.
Such a transition state should reduce the energy of activa-
tion in comparison with that of the reaction of an NCA mo-
nomer molecule with an isolated peptide chain. At the same
time, if an R-NCA molecule approaches the NH2 reactive
group of an S chain, as required for chain elongation, it will
sense steric hindrance between its iPr group and the iPr
group of an adjacent R chain (Scheme 1).
Figure 12. Schematic representation of the self-assembly of a racemic
mixture of short isotactic peptides into either rippled b-sheets or racemic
mixtures of enantiomorphous pleated b-sheets.
The rippled b-sheets are composed of alternating isotactic
peptide chains of opposite handedness. Indeed, Pauling and
Corey demonstrated the possibility of the formation of such
b-sheets.[17] Support for the proposed structures of our tem-
plates is also provided by the racemic rippled ap b-sheets
observed in the crystal structures of the glycine tripeptide[18]
and the high-molecular-weight polyglycine I polymorph.[19–21]
These achiral peptides of glycine have the option of crystal-
lizing in the form of either pleated or racemic b-sheets and
they prefer to form the racemic rippled ap b-sheet motif.
Another reported example is a solubility study showing that
the mixing of equal amounts of water-soluble polylysine of
opposite handedness results in the precipitation of the race-
mate.[22,23]
Scheme 1. Schematic representation of an R-Val-NCA molecule interact-
ing with the NH2 end group of an R chain (in black) and an S chain (in
red) confined in a rippled ap b-sheet template. These interactions ac-
count for the enantioselectivity of the chain-elongation process.
The formation of rippled ap b-sheets and rippled p b-
sheets has been reported in the polymerization of racemic
crystals of (RS)-Phe-NCA[11,12,24] and (RS)-Val-NCA,[13] re-
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Formation of Isotactic Peptides
FULL PAPER
Experimental support for the preferred formation of rip-
pled ap b-sheets, rather than a racemic mixture of pleated
sheets, could be obtained from the reversal in the ee value
of the oligopeptide chains as a function of length when
enantiopure initiators are used. Such polymerization initia-
tors should yield different diastereoisomeric oligopeptides
according to different possible motifs, as illustrated schemat-
ically in Figure 13.
rippled ap b-sheets. A comparison between the symmetrical
chain elongation in the presence of an achiral initiator and
the inhibition in chain elongation of an S strand introduced
by an S initiator linked at the C terminus of an adjacent R
strand (R5-S-OMe) is modeled in Figure 14.
Figure 13. Different types of ap b-sheets comprising four strands of ho-
mochiral sequences with an S* residue of the initiator attached at the C
terminus. a) Racemic b-sheet of alternating oligo-R and oligo-S strands,
in which the S* residue of the initiator is attached to the oligo-R chains
and impedes the growth of adjacent S chains. b and c) Enantiomorphous
pleated b-sheets of either oligo-S or oligo-R strands: b) The S* initiator
attached to the oligo-S chains in a pleated motif will not hinder the
growth of chains of the same absolute configuration as that of the initia-
tor; c) the S* residue is attached to the C terminus of the oligo-R chains
and should hinder the growth of these chains. The horizontal arrows rep-
resent the direction of chain elongation and the inclined lines represent
sites where the S* initiator residue impedes the growth of adjacent
strands
Figure 14. Top and side views of rippled ap b-sheets composed of alter-
nating R and S strands with an achiral initiator (left) and with an S-Val-
OMe initiator (right) that causes growth impediment due to steric hin-
drance.
Additional evidence was provided by a comparison be-
tween the distributions of the homochiral oligopeptides ob-
tained from the polymerization of enantiopure S-Val-NCA
in water initiated with either the R- or S-Val-OMe initiators,
but otherwise under same conditions. The polymerization of
enantiopure monomers can yield only the pleated b-sheets.
MALDI-TOF MS analysis of the oligopeptides clearly dem-
onstrates (Figure 15) that, beyond octamers, after the self-
assembly into pleated b-sheets, the S oligopeptides obtained
with S-Val-OMe are in excess over those obtained with R-
Val-OMe; this result is in keeping with the formation of
pleated ap b-sheets and is in contrast with the results ob-
tained in the polymerization of racemic monomers. No
The analysis of the architectures in Figure 13 suggests
that, if the reaction is initiated by an enantiopure methyl
ester of, say, S absolute configuration, this residue will
reside at the C terminus of isotactic peptide chains com-
posed from repeat homochiral residues of either handed-
ness. Such a residue will be integrated coherently within the
chains composed of S residues and will not interfere with
the growth of the nearer peptide chains comprising R resi-
dues (Figure 13a). On the other hand, the presence of the
same initiator at the C terminus of an R chain creates a
defect at the rim of the growing template. In the case of a
racemic ap b-sheet (Figure 13a), such an initiator residue
will introduce steric hindrance to the growing NH2 group of
the neighboring peptide chains composed from S residues,
which will result in an excess of the isotactic oligopeptide
chains composed of R residues, that is, of a configuration
opposite to that of the initiator. An opposite effect is antici-
pated in the case of enantiomorphous pleated-sheet forma-
tion because an S initiator will integrate straightforwardly
within the b-sheets composed from S strands (Figure 13b),
whereas it will engender asymmetric hindrance and rate im-
pediment in the growth of R strands (Figure 13c). The oper-
ation of the hindrance mechanism in the formation of rip-
pled ap b-sheets was reported in the polymerization of
(RS)-Phe-NCA crystals.[12]
The above-described experimental results from the poly-
merization of either racemic Val-NCA or racemic Leu-
NCA, first dissolved in water and then treated with 25%
enantiopure initiator, showed a reversal in the ee value of
the long oligopeptides with respect to that of the short ones
(Figure 10, left), a result in keeping with the formation of
Figure 15. MALDI-TOF mass spectra of the oligopeptides obtained from
the polymerization in water of S-Val-NCA with a) R-Val-OMe and b) S-
Val-OMe initiators. The signal corresponding to the octapeptides is indi-
cated by an arrow. In (b), there are relatively large amounts of short (n=
5-–8) oligopeptides initiated by hydrolyzed Val-NCA, labeled with ; this
*
is in keeping with slower polymerization.
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M. Lahav et al.
steric hindrance by the initiator linked at the C terminus is
anticipated in parallel b-sheets, be they racemic or pleated,
since this group will be located far away from the NH2
growing chain ends.
ease of formation of the racemic b-sheets and their role as
efficient templates in the generation of homochiral peptides
suggests that they might have emerged prior to the a-helices
or the pleated b-sheets[33] in times of early evolution.
Studies on the design of other synthetic routes for the for-
mation of homochiral peptides from racemic a-amino acids
are under current investigation.
Another factor to be taken into consideration is a possible
kinetic effect that might influence the ee value of isotactic
peptides as a function of length. On one hand, the enantio-
pure initiators exert an asymmetric induction in the early
stages of the reaction to enrich the solution with short oligo-
peptides composed from residues of the same handedness,
but on the other hand, the solution is depleted of monomers
of opposite handedness. As a result of the complexity of the
polymerization process, it is difficult to evaluate the role of
this kinetic effect. However, if the kinetic effect were re-
sponsible for the dramatic reversal in the ee value as a func-
tion of length for the polymerization with 25% initiator, we
should have observed a related effect in the reactions with 5
and 10% initiator. Under the latter conditions, the ee value
decreased gradually for the longer oligopeptides, but no re-
versal in ee value was observed (Figure 10). These results
are clearly dependent on the nature of the templates formed
at different concentrations of initiator. With 5 and 10% ini-
tiator, the templates also contain heterochiral oligopeptide
chains, but comprise block sequences of homochiral residues
that compel the chains to assemble in motifs that are very
different from those of the rippled b-sheets formed with
25% initiator.
Experimental Section
Materials
N-Carboxyanhydrides of valine and leucine: Val-NCA and Leu-NCA
were prepared according to the method of Daly and Pochꢀ, with minor
adjustments.[34] Finely ground R-Val or R-Leu (1 mmol, 0.5 equiv) and S-
Val or S-Leu (d8 or d10, 98%, Cambridge Isotope Laboratories; 1 mmol,
0.5 equiv) were suspended in dry THF and heated to reflux under argon.
Solid triphosgene (0.8 mmol, 1.2 equiv) was added and after 0.5 h triphos-
gene portions (0.4 mmol, 0.6 equiv) were consecutively added every 0.5 h
until a clear solution was obtained (2 or 3 portions were usually needed).
The reaction was then continued for an additional hour. The clear solu-
tion was allowed to warm to room temperature and the THF was evapo-
rated under reduced pressure. CH2Cl2 (approximately 5 mL) was added
and then hexane (about 50 mL) was added with mixing. A white precipi-
tate appeared almost immediately; the suspension was cooled to 48C for
2–3 h and then the crystals were filtered off. The small needle-like crys-
tals of NCA were characterized by FTIR spectroscopy.
Methyl esters of amino acids: The free amine was prepared from commer-
cially available monohydrochlorides of the amino acid methyl ester
(Sigma) by suspending the material in dry CH2Cl2 and bubbling with am-
monia gas for a few minutes. The resulting solid NH4Cl was filtered off
and the CH2Cl2 was removed by evaporation.
Polymerization procedure: Val-NCA or Leu-NCA (10 mg) was weighed
into a microcentrifuge tube and dissolved in water (1 mL). The appropri-
ate amount of n-butylamine or amino acid methyl ester was added after
5 min for Val-NCA and 1 min for Leu-NCA. The reaction was stirred vig-
orously for 24 h. The tubes were then subjected to centrifugation and the
supernatant was carefully decanted and filtered to remove any residual
precipitate. The precipitate and the supernatant were dried by lyophiliza-
tion.
Conclusion
The formation of long hydrophobic oligopeptides of homo-
chiral sequence in the polymerization of racemic NCA mon-
omers, in the presence of initiator, in water, and in the ab-
sence of enzymes, is demonstrated. Although pure NCAs
are unstable derivates of a-amino acids, they have been pro-
posed as possible intermediates for the formation of the
early peptides.[26–30]
MALDI-TOF mass spectra and analysis: For sample preparation, a small
amount of the sample (<1 mg) was placed into a polypropylene micro-
centrifuge tube and dissolved in trifluoroacetic acid (TFA; 20 mL). THF
(80 mL) was then added. A volume (0.5 mL) of a 1:1 (v/v) mixture of the
matrix (dithranol) solution in chloroform and a saturated solution of NaI
in THF was placed on the target plate and dried. The solution to be ana-
lyzed (0.5 mL) was then deposited. MALDI-TOF positive-ion mass spec-
tra were obtained in reflector mode with a Bruker Reflex III instrument
equipped with an N2 laser. Mass spectra resulted from a signal average of
at least several hundred laser shots on different spots of the target, to get
reliable statistics about the ion peaks. Mass assignments of Na-cationized
oligopeptides were made by using both the m/z values and the isotopic
distributions. The following notation code is used for the peptide mole-
cules: oligopeptide (h,d) assigns all molecules composed of h residues of
R configuration (protonated) and d residues of S configuration (deuter-
ated), with n=h+d being the total number of repeat units. The ionization
yield is expected to be very similar for oligopeptides (h,d) of the same
length n, due to their similar chemical properties and identical Na+-ion
affinity. One proof is given by a random polymerization for which the rel-
ative abundance of the different Na-cationized oligopeptides (h,d) of the
same length perfectly fitted the binomial law. In addition, similar detec-
tion efficiencies are expected, due to very close masses and velocities in
the TOF analysis. Thus, the ion intensities of the different diastereoiso-
meric oligopeptides (h,d) of the same length are directly and reliably
comparable. The relative mole fraction of each type of oligopeptide (h,d)
was obtained by dividing the intensity of the signals from a particular
The generation of isotactic peptides comprises several
steps that must operate in tandem. The present mechanism
suggests that the simple formation of short racemic b-sheets
and their operation as efficient stereoselective templates for
the formation of isotactic peptides might suggest a plausible
scenario by which the homochiral peptides emerged prior to
the early enzymes. One of the important results in this study
is that, although the templates can be composed of different
racemic mixtures of short isotactic and atactic peptides com-
prising four to six residues and depending upon the initial
concentration of the initiator, they can still exert regio-enan-
tioselection in the chain-elongation processes. Such a mech-
anism has great advantages over the theoretical model pro-
posed by Wald[31] of using a-helices as templates for the
spontaneous formation of long isotactic peptides,[32] because
spontaneous self-assembly into helices would require the
formation of peptides that have at least eight residues of the
same handedness before they can exert asymmetric induc-
tion in the elongation of the peptide chains. The relative
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Formation of Isotactic Peptides
FULL PAPER
molecule by the total intensity of the signals from all molecules of the
same length n. For example, the relative abundance of tetrapeptide (1,3),
composed of one R (protonated) and three S (deuterated) residues, was
calculated according to Equation (1):
[5] V. Avetisov, V. Goldanski, Proc. Natl. Acad. Sci. USA 1996, 93,
11435–11442.
[6] L. E. Orgel, Nature 1992, 358, 203–209.
[7] P. Franck, W. A. Bonner, R. N. Zare in Chemistry for the 21st Centu-
ry (Eds.: E. Keinan, I. Schecter), Wiley-VCH, Weinheim, 2000,
pp. 175–208, and references therein.
[8] A. Brack, Chem. Biodiversity 2007, 4, 665–679, and references
therein.
intensity of ð1,3Þ
ð1Þ
Mole fraction of ð1,3Þ ¼
intensity of ð4,0Þþð3,1Þþð2,2Þþð1,3Þþð0,4Þ
[9] M. Blocher, T. Hitz, P. L. Luisi, Helv. Chim. Acta 2001, 84, 842–847.
[10] T. Hitz, M. Blocher, P. Walde, P. L. Luisi, Macromolecules 2001, 34,
2443–2449.
[11] J. G. Nery, G. Bolbach, I. Weissbuch, M. Lahav, Chem. Eur. J. 2005,
11, 3039–3048.
MALDI-TOF-TOF spectra were obtained from an Applied Biosystems
4700 Proteomics instrument by using a time–ion selector over a mass
window (ꢂ3 u) centered on the first isotope. Fragmentation of these ions
by metastable decomposition and/or collision-induced dissociation (N2,
2ꢃ10ꢀ7 Torr, ion energy=1 keV) showed that two main fragmentation
series are produced for all of the initiators. These two series comprise
either the N or the C terminus of the oligopeptide and are Na+ cation-
ized (Scheme 2).
[12] G. J. Nery, R. Eliash, G. Bolbach, I. Weissbuch, M. Lahav, Chirality
2007, 19, 612–624.
[13] R. Eliash, J. G. Nery, I. Rubinstein, G. Clodic, G. Bolbach, I. Weiss-
buch, M. Lahav, Chem. Eur. J. 2007, 13, 10140–10151.
[14] I. Rubinstein, R. Eliash, G. Bolbach, I. Weissbuch, M. Lahav,
Angew. Chem. 2007, 119, 3784–3787; Angew. Chem. Int. Ed. 2007,
46, 3710–3713.
[15] R. A. Illos, F. R. Bisogno, G. Clodic, G. Bolbach, I. Weissbuch, M.
Lahav, J. Am. Chem. Soc. 2008, 130, 8651–8659.
[16] H. R. Kricheldorf, Angew. Chem. 2006, 118, 5884–5917; Angew.
Chem. Int. Ed. 2006, 45, 5752–5784.
[17] L. Pauling, R. B. Corey, Proc. Natl. Acad. Sci. USA 1953, 39, 253–
256.
[18] T. Srikrishnan, N. Winiewicz, R. Parthasarathy, Int. J. Pept. Protein
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[19] B. Lotz, J. Mol. Biol. 1974, 87, 169–180.
[20] S. Munoz-Guerra, J. Puiggali, A. Rodriguez, J. A. Subirana, J. Mol.
Biol. 1983, 167, 223–225.
Scheme 2. The two main fragmentation series observed for the Na+-cat-
ionized oligopeptide ions. These are referred to as the y-type and a-type
series.
[21] A. V. Kajava, Acta Crystallogr. Sect.D 1999, 55, 436–442.
[22] J. H. Fuhrhop, M. Krull, G. Bꢄldt, Angew. Chem. 1987, 99, 707–708;
Angew. Chem. Int. Ed. Engl. 1987, 26, 699–700.
[23] Similar kinetic effects can also account for the abundant formation
of racemic crystals, particularly among the hydrophobic amino acids
in nature, in comparison with conglomerates of enantiomorphous
crystals.
[24] J. G. Nery, G. Bolbach, I. Weissbuch, M. Lahav, Angew. Chem. 2003,
115, 2207–2211; Angew. Chem. Int. Ed. 2003, 42, 2157–2161.
[25] D. M. Chung, J. S. Nowick, J. Am. Chem. Soc. 2004, 126, 3062–3063.
[26] A. Brack, Biosystems 1982, 15, 201–207.
The y-type fragmentation series [yk+NaꢀH]+ allows, typically, the explo-
ration of the sequence of residues at the N terminus up to the half-way
point. The a-type fragmentation series [akꢀH+Na]+ allows the explora-
tion of the sequence at the C terminus, also over the length of half of the
oligopepetides. Thus, both series have to be used to get sequence infor-
mation. In all MALDI-TOF-TOF experiments, the number of laser shots
was typically 10000 on 50 different spots on the target. This measurement
allows us to obtain reliable statistics on the ion intensities.
[27] C. Huber, G. Wꢅchtershꢅuser, Tetrahedron Lett. 2003, 44, 1695–
1697.
[28] D. Hafenbradl, M. Keller, G. Wꢅchtershꢅuser, K. O. Stetter, Tetrahe-
dron Lett. 1995, 36, 5179–5182.
Acknowledgements
This work was supported by the Israel Science Foundation, the Clore
Center for Biological Physics, and The Helen & Milton A. Kimmelman
Center for Biomolecular Structure & Assembly. G.B. thanks Le Conseil
Regional Ile-de-France and UPMC. We thank Dr. Alla Shainskaya and
her team from the MS laboratory of the Weizmann Institute of Science.
[29] A. Commeyras, H. Collet, L. Boiteau, J. Taillades, O. Trambouze, H.
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Selsis, M. Dobrijevic, Polym. Int. 2002, 51, 661–665.
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[31] G. Wald, Ann. N. Y. Acad. Sci. 1957, 69, 352–368.
[32] Such a model has not yet been proved experimentally in the poly-
merization of racemic amino acid activated derivatives.
[33] A. Brack, G. Spach, J. Mol. Evol. 1979, 13, 35–46.
[34] W. H. Daly, D. Pochꢀ, Tetrahedron Lett. 1988, 29, 5859–5862.
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Received: July 21, 2008
Published online: October 27, 2008
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11009