Homochiral oligopeptides via a lattice-controlled polymerisation in racemic crystals of valine N-carboxyanhydride suspended in aqueous solutions

Article abstract of DOI:10.1002/chem.200700994

As part of our program on the biochirogenesis of homochiral peptides, we report the formation of racemic parallel (p) β sheets composed of alternating R and S chains of up to 14-15 repeat units of the same handedness through the polymerisation of (R,S)-valine N-carboxyanhydride (NCA) crystals suspended in aqueous solutions of a primary amine as the initiator. The occurrence of such a lattice-controlled reaction accompanied by a reduction in volume implies the operation of a mechanism that differs from that of the common solid-state polymerisation in vinyl systems. The topotacticity of the reaction is explained through the operation of a multistep nonlinear process comprising lattice control coupled with an asymmetric induction in the formation of homochiral short peptides followed by their self-assembly into racemic p β sheets, which operate as efficient templates in the ensuing process of enantioselective chain elongation at the polymer/crystal interface. The composition of the diastereoisomeric libraries of oligopeptides was determined by MALDI-TOF and MALDI-TOF-TOF MS analyses of the products obtained from monomers enantioselectively labelled with deuterium. The structure of the p β sheets could be determined by initiating the polymerisation reaction with water-soluble esters of enantiopure α-amino acids or short peptides. The same reaction performed with the monomer crystals suspended in hexane yielded a complex mixture of diastereoisomeric oligopeptides, thus highlighting the indispensable role played by water in controlling the stereoselectivity of the reaction. By contrast, polymerisation of (R,S)-leucine NCA crystals, with a different packing arrangement that presumably does not endorse the formation of periodic peptide templates, yielded, both in aqueous and hexane suspensions, libraries of peptides dominated by heterochiral diastereoisomers.

Full text of DOI:10.1002/chem.200700994

DOI: 10.1002/chem.200700994
Homochiral Oligopeptides via a Lattice-Controlled Polymerisation in
Racemic Crystals of Valine N-Carboxyanhydride Suspended in Aqueous
Solutions
[a]
RanEliash, Jose G. Nery,[a] Irena Rubinstein,[a] Gilles Clodic,[b] GØrard Bolbach,[b]
Isabelle Weissbuch,[a] and Meir Lahav*[a]
Dedicated to Professor David Reinhoudt on the occasion of his 65th birthday
Abstract: As part of our program on
the biochirogenesis of homochiral pep-
tides, we report the formation of race-
mic parallel (p) b sheets composed of
alternating R and S chains of up to 14–
15 repeat units of the same handedness
through the polymerisation of (R,S)-
valine N-carboxyanhydride (NCA)
crystals suspended in aqueous solutions
of a primary amine as the initiator. The
occurrence of such a lattice-controlled
reaction accompanied by a reduction in
an asymmetric induction in the forma-
tion of homochiral short peptides fol-
lowed by their self-assembly into race-
mic p b sheets, which operate as effi-
cient templates in the ensuing process
of enantioselective chain elongation at
the polymer/crystal interface. The com-
position of the diastereoisomeric libra-
ries of oligopeptides was determined
by MALDI-TOF and MALDI-TOF-
TOF MS analyses of the products ob-
tained from monomers enantioselec-
tively labelled with deuterium. The
structure of the p b sheets could be de-
termined by initiating the polymeri-
sation reaction with water-soluble
esters of enantiopure a-amino acids or
short peptides. The same reaction per-
formed with the monomer crystals sus-
pended in hexane yielded a complex
mixture of diastereoisomeric oligopep-
tides, thus highlighting the indispensa-
ble role played by water in controlling
the stereoselectivity of the reaction. By
contrast, polymerisation of (R,S)-leu-
cine NCA crystals, with a different
packing arrangement that presumably
does not endorse the formation of peri-
odic peptide templates, yielded, both in
aqueous and hexane suspensions, libra-
ries of peptides dominated by hetero-
chiral diastereoisomers.
volume implies the operation of
a
mechanism that differs from that of the
common solid-state polymerisation in
vinyl systems. The topotacticity of the
reaction is explained through the oper-
ation of a multistep nonlinear process
comprising lattice control coupled with
Keywords: amino acids · beta-sheet
structures · biochirogenesis · lattice-
controlled reactions · oligopeptides
Introduction
of homochiral biopolymers from racemic monomers, such as
the primeval polypeptides and polynucleotides composed
from repeat units of the same handedness.[1–3] Homogeneous
polymerisation of racemic monomers in organic solvents
yields libraries of atactic polymers.[4] Under such conditions,
the formation of isotactic chains of at least eight homochiral
repeat units, commonly considered as necessary for the for-
mation of a-helix or pleated-b-sheet templates to generate
long homochiral peptides, is not possible.[4–7] Recently, we
proposed a reaction pathway for the formation of isotactic
peptides that can override this drawback. This pathway com-
prises the self-assembly of racemic monomers into crystal-
line motifs in which the two enantiomers are suitably ar-
ranged for a lattice-controlled polymerisation reaction be-
tween molecules of the same handedness.[8,9] This require-
ment, however, encounters intrinsic complications, as the
The origin of homochirality in the biological world provides
an as yet unsolved riddle of prebiotic chemistry and early
evolution. Of particular interest is the prebiotic formation
[a] Dr. R. Eliash, Dr. J. G. Nery, I. Rubinstein, Dr. I. Weissbuch,
Prof. Dr. M. Lahav
Department of Materials and Interfaces
The Weizmann Institute of Science
76100 Rehovot (Israel)
Fax : (+972)8-934-4138
E-mail: meir.lahav@weizmann.ac.il
[b] G. Clodic, Dr. G. Bolbach
Platforme SpectromØtrie de Masse et ProtØomique
UniversitØ Pierre et Marie Curie
75252 Paris, Cedex 05 (France)
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FULL PAPER
emergence of short polymers, which are denser than the
crystalline monomer, is accompanied by damage of the peri-
odic order of the latter at the sites where chain elongation
proceeds. Note that only a limited number of systems in-
cluding diethylenes,[10,11] diacetylenes[12] and nickel coordina-
tion complexes[13,14] were reported, during years of research,
to undergo lattice-controlled polymerisation beyond dimers
and trimers. As a result of this destruction, the longer poly-
mers formed in the disordered environment comprise repeat
units of both handednesses. This disadvantage might be
overcome if the short polymers of homochiral sequence (iso-
tactic) would self-assemble into ordered architectures that
could exert steric control on the ensuing process of chain
elongation occurring at the polymer/crystal interface.[15–17]
The operation of such interplay requires an understanding
of the role played by the structure of the monomer crystal
at the early stages of the reaction, its role in dictating the
self-assembly of the formed peptides into supramolecular ar-
chitectures, such as a helices or b sheets, and the ultimate
role played by the latter as stereospecific or enantioselective
templates in the ensuing stages of polymer propagation.
Here, we provide support for this hypothesis for the solid-
state polymerisation of racemic crystals of valine N-carbox-
yanhydride (ValNCA, Scheme 1) and leucine N-carboxyan-
hydride (LeuNCA) suspended in hexane or water solutions
of a primary-amine initiator. Previous reports on the poly-
merisation of these crystals suspended in hexane have not
described the diastereomeric
Scheme 1. Chemical formulae of ValNCA, LeuNCA and their oligopepti-
des of homochiral sequence (isotactic), as initiated by either n-butyl-
amine or methyl esters of various a-amino acids or short peptides.
distribution of the products
and the mechanism of the re-
action.[18–20]
Results and Discussion
Polymerisationof
(
R,S)-
ValNCA crystals suspended in
hexane and initiated by n-bu-
tylamine:
The
racemic
ValNCA crystal structure re-
ported by Kanazawa and co-
workers[21] appears in the or-
thorhombic polar space group
Pca21 (a=8.938, b=10.207, c=
7.788 Š, Z=4) with two R and
two S molecules in the unit
cell, as shown in Figure 1. The
molecules are packed in a bi-
layer motif perpendicular to
the b axis. The bilayers are
composed of alternating paral-
lel rows of R and S molecules
with a single sense of polarity
along the c axis. Within each
Figure 1. a–c) The packing arrangement of (R,S)-ValNCA viewed along the c, b and a axes. d) Two adjacent
isotactic R and S tetrapeptides viewed along the a axis, as resulting from the proposed preferred pathway of
the polymerisation reaction along the polar c axis, shown in (c).
row, the molecules are related by a twofold screw axis along
the c direction, and alternating molecular rows are related
to each other by a c glide plane perpendicular to the a axis.
On the basis of this packing arrangement, one anticipates
that a preferred geometric lattice-controlled reaction should
take place along the c axis to produce racemic mixtures of
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10141

M. Lahav et al.
oligopeptides of homochiral (isotactic) sequence. The spac-
ing that separates the N atom of one molecule from the aC-
(carbonyl) atom of a favourably oriented neighbouring mol-
In the MALDI-TOF mass spectrum, the oligopeptides of
homochiral sequence (isotactic), labelled Rn and Sn, appear
at the wings of each group of signals originating from vari-
ous diastereoisomers of a given length n, in which the sig-
nals between the wings represent the oligopeptides that con-
tain repeat units of both handednesses, labelled RhSd in
which h+d=n. Previous studies have demonstrated that the
molar fractions of the diastereoisomeric peptides of a given
length are proportional to the relative intensity of their sig-
nals.[8,9] A histogram of the experimental molar fractions of
the oligopeptides of each length and, for comparison, a his-
togram calculated with the assumption of a theoretical bino-
mial distribution are shown in Figure 3a and b. It is clear
that the distribution of the obtained diastereomeric oligo-
peptides differs substantially from the binomial one. Ac-
cording to the experimental results, racemic mixtures of ho-
mochiral oligopeptides were obtained. Moreover, for the
tetra- and pentapeptides (n=4, 5), the isotactic chains are
the dominant diastereoisomers (Figures 2a and 3a), in keep-
ing with the lattice-controlled
ecule of the same handedness along the c axis is 4.14 Š. An-
other, but less probable, pathway of polymerisation might
be along the a axis where the spacing which separates the
same N and C atoms of two adjacent unfavourably aligned
heterochiral molecules is much longer at 4.88 Š. On the
basis of these purely geometric considerations, we anticipat-
ed that the packing of the (R,S)-ValNCA crystal is appropri-
ate for the generation of isotactic polymers through a reac-
tion along the polar c axis.
Racemic crystals of (R,S)-ValNCA, in which the S enan-
tiomer was enantioselectively tagged with eight deuterium
atoms, were polymerised by suspending them in hexane con-
taining n-butylamine as an initiator. The resulting diastereo-
isomeric oligopeptides were analysed by MALDI-TOF mass
spectrometry and one such spectrum is shown in Figure 2a.
Oligopeptides with up to 14–15 repeat units were detected.
polymerisation
envisaged
above. However, the concen-
tration of the heterochiral pep-
tides, as compared to that of
the isotactic ones, increases
with the increase in length for
n=6–15, presumably due to
the lattice damage. Nonethe-
less, the departure from the bi-
nomial distribution is signifi-
cant even for the long oligo-
peptides and it suggests the
operation of an enantioselec-
tion process that results in the
formation of detectable isotat-
ic peptides of 14–15 repeat
units.
Polymerisationof
(
R,S)-
ValNCA crystals suspended in
aqueous solutions of n-butyla-
mine: A salient difference was
found in the distribution of the
diastereoisomeric oligopepti-
des obtained when the poly-
merisation reaction was per-
formed on (R,S)-ValNCA crys-
tals suspended in an aqueous
solution of the initiator, under
otherwise identical conditions.
Optical photographs of crystals
under such reaction conditions
(Figure 4a and b) also show
CO2 bubbles. The distributions
of the diastereoisomeric oligo-
peptides, as determined from
the MALDI-TOF mass spec-
Figure 2. MALDI-TOF mass spectra of the products of polymerisation, initiated with n-butylamine, of (R,S)-
ValNCA crystals suspended a) in hexane (insoluble product) and b) and c) in an aqueous solution of the initia-
tor (water-insoluble product and water-soluble product, respectively). The signals corresponding to various dia-
stereoisomers of each length n are labelled: Isotactic oligopeptides are labelled Rn and Sn. Heterochiral oligo-
peptides are labelled RhSd in which n=h+d. The labels represent the m/z range of the matrix signals that
*
were omitted for clarity.
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Controlled Polymerisation in Racemic Crystals
FULL PAPER
and heterochiral tetra- and pentapeptides were found to be
soluble in water; the mass spectrum is shown in Figure 2b.
The relative concentration of isotactic versus atactic water-
insoluble tetra- and pentapeptides (Figure 2c) is lower than
that for such peptides obtained in the hexane medium (Fig-
ure 2a). On the other hand, beyond hexa- or heptapeptides
and up to the longest detectable ones of 15 repeat units, the
isotactic peptides are dominant, as shown in the insets of
Figure 2c in which these peptides are represented at the
wings of the groups of signals corresponding to diastereoiso-
mers of length n=11–15 and labelled Rn and Sn.
On the basis of the geometrical considerations proposed
above, we may envisage that if the solid-state polymeri-
sation reaction takes place preferentially between homochi-
ral monomer molecules along the c axis, the crystal lattice
will force the short peptide chains to self-assemble into race-
mic parallel (p) b sheets composed of alternating oligo-R
and oligo-S chains. The formation of b sheets is supported
by FTIR spectra that display a characteristic stretching vi-
bration at 1632 cmꢀ1 and by X-ray diffraction patterns dis-
playing d-spacings of 4.7 and 6.9 Š, a fingerprint for b-
sheets and a 9.7 Š d-spacing corresponding to intersheet
stacking. Further independent chemical evidence for the for-
mation of racemic parallel b sheets is provided below.
Figure 3. a) Histogram of the mole fraction of the diastereoisomeric oli-
gopeptides of each length obtained in the polymerisation of (R,S)-
ValNCA crystals suspended in hexane and initiated with n-butylamine.
b) Histogram calculated by assuming a theoretical binomial distribution
of the products.
Proposed polymerisationpathway : The formation of short
isotactic oligopeptides as the dominant components, pro-
duced by suspending the monomer crystals both in hexane
and in aqueous solutions of the initiator, implies that, in
either of the suspending solvents, the early stages are con-
trolled by the crystalline lattice of the monomer. There is a
notable difference in the concentrations of the longer isotac-
tic oligopeptides that are produced in hexane and in water,
a difference that may be rationalised as follows. The smaller
concentration of short isotactic peptides produced when the
monomer crystals were suspended in aqueous solution
might be due to either partial destruction of the monomer-
crystal surfaces caused by minute NCA hydrolysis or by dis-
solution of the short atactic polymers in water. These two
processes do not occur in hexane. On the other hand, the
formation of increased concentrations of longer isotactic
peptides when monomer crystals were suspended in aqueous
solutions of the initiator implies that water induces the self-
assembly of supramolecular templates that exert asymmetric
control in the ensuing steps of the chain-elongation reaction.
The solubility of the heterochiral short peptides in water in-
creases the accumulation of isotactic oligopeptides that un-
dergo self-assembly within the monomer crystal. A small
amount of oligopeptides could arise from polymerisation of
the solubilised monomer prior to the n-butylamine initiation
reaction in the solid state. However, parallel studies of poly-
merisation in aqueous solutions to which the initiator was
added five minutes after the complete monomer dissolution
demonstrated the formation of racemic antiparallel (ap) b
sheets.[4]
Figure 4. Optical photographs of (R,S)-ValNCA crystals suspended in an
aqueous solution of n-butylamine initiator and showing CO2 bubbles
formed in the course of the reaction.
trum, are shown in Figure 2c, for the water-insoluble prod-
ucts.
In contrast to the polymerisation in hexane, when the re-
action was performed on crystals suspended in an aqueous
solution of n-butylamine, all homochiral di- and tripeptides
According to the packing arrangement of the (R,S)-
ValNCA monomer crystal, the racemic isotactic peptides
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M. Lahav et al.
should self-assemble into racemic p b sheets, although they
are anticipated, on the basis of the results of polymerisation
in aqueous solution,[4] to be energetically less stable than the
ap b sheets. This self-assembly is illustrated in Figure 5, in
Polymerisationof
(
R,S)-ValNCA crystals suspended in
aqueous solutions of enantiopure a-amino acid esters as
chiral initiators: To further confirm the formation of racemic
p b sheets, we performed the polymerisation reactions with
such initiators that should differentiate between the forma-
tion of the various possible architectures, racemic p or ap b
sheets and enantiomorphous pleated b sheets, as a result of
their spontaneous separation. If racemic p b sheets are
formed, an initiator resembling a peptide repeat unit that is
linked at the C terminus of the chain should reside far away
from the growing NH2 end and should not interfere with
chain elongation of the isotactic peptides. Therefore, we an-
ticipate that the enantiomeric excess (ee) of the formed long
oligopeptides should remain constant as a function of pep-
tide length. Such behaviour should be at variance with that
for ap b sheets, be they racemic or pleated. In these archi-
tectures, the initiator repeat unit should reside in close prox-
imity to the growing NH2 group of an adjacent chain and
should induce steric hindrance in the growth process. Conse-
quently, such an impediment on the growing chains will
result in desymmetrisation of the racemic mixtures of the
long peptides. In the case of racemic ap b sheets, isotactic
chains of the same absolute configuration as the initiator
will be formed in lower concentration.[17] If enantiomor-
phous pleated sheets are formed, those composed of isotac-
tic oligopeptides of the same absolute configuration as the
initiator will be formed in excess.[4]
The MALDI-TOF mass spectra of the water-soluble and
water-insoluble diastereoisomeric peptides generated in the
polymerisation reaction of (R,S)-ValNCA crystals suspended
in aqueous solutions containing the (S)-Val-OMe initiator
are shown in Figure 6a and b. Oligopeptides comprising up
to 14repeat units were detected and we noticed that the
water-soluble isotactic di- and tripeptides formed in excess
are of the same absolute configuration as that of the initia-
tor (Figure 6a). On the other hand, beyond the tetrapeptides
(Figure 6b), there is a reversal in sign of the ee value of the
isotactic oligopeptides and this ee value remains almost un-
changed for the longer peptides, as shown in Figure 7a. Poly-
merisation with the (R)-Val-OMe initiator yielded a mirror-
image behaviour (Figure 7a). Similar results were obtained
when the polymerisation reaction was performed with vari-
ous initiators, including methyl esters of phenylalanine (Fig-
ures 7b and 8), tryptophan or the valine di- and tripeptides,
(Val)3-OMe and (Val)3-OMe (Figure 9).
The following possible effects have been considered for
the rationalisation of the observed asymmetric induction in
the initiation steps of the reaction; these effects would result
in a reversal in the ee value of the oligomers beyond the
dimer stage: 1) Preferred recognition of the enantiopure ini-
tiator by one of the heterochiral monomer sites at the crys-
tal/solution interface. 2) A steric effect induced by the enan-
tiopure initiator at the crystal/solution interface that enan-
tioselectively hampers the interactions with the monomer
site of the same handedness. 3) Preferred solubility of the
short diastereoisomers ISSn relative to that of ISRn in water,
which would increase the concentration of the major diaste-
Figure 5. Computer model of two adjacent isotactic R and S hexapeptides
assembled into a racemic parallel b sheet formed within the crystalline
lattice of the (R,S)-ValNCA monomer. For clarity, the C atoms of the R
monomer molecules and those of the R peptide are shown in orange; in
addition, the isopropyl side chains of the two peptides are omitted.
which two parallel isotactic chains of opposite handedness
can be accommodated in this architecture without introduc-
ing high strain along the a direction of the monomer lattice.
The growing NH2 groups, present at one edge of the sheet,
are hydrated through hydrogen bonding with the water mol-
ecules. These groups react at the polymer/monomer inter-
face with the hydrated chiral monomer molecules through
the formation of diasteroisomeric transition states. In addi-
tion, at the locus of the propagation reaction, the released
CO2 molecules dissolve in water to form carbonic acid,
which can interact with the NH2 end groups to further
modify the structure of the reaction site for chain elonga-
tion. On the other hand, when the reaction is performed
with monomer crystals suspended in hexane, either the b
sheets are not formed or, if they do form, the propagation
reaction between a presumably nonsolvated peptide end
and a nonsolvated NCA monomer molecule at the partially
destroyed polymer/NCA crystal interface engenders a lower
stereospecificity.
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Controlled Polymerisation in Racemic Crystals
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reoisomer at the crystal sur-
face. 4) A change in conforma-
tion of the R and S repeat
units of the short diastereoiso-
meric peptides to favour differ-
ent propagation pathways.
Some insight into the initia-
tor recognition by the hetero-
chiral crystal sites was ob-
tained from a computational
study of the energy required
for an (S)-Val-OMe initiator
molecule to dock at the two
enantiomeric sites on various
crystal surfaces. Single crystals
of (R,S)-ValNCA are (010)
plates elongated along the c
axis and delineated by (111),
¯
(111) and (110) side faces, as
Figure 6. MALDI-TOF mass spectra of the product of polymerisation of (R,S)-ValNCA crystals suspended in
an aqueous solution of (S)-Val-OMe: a) water-soluble material, b) water-insoluble material. Note that to high-
shown in Figure 10. Although
the (010) and (010) faces are
light the signals that belong to the oligopeptides, those originating from the matrix are marked with labels.
*
¯
the most expressed, they are of
lower relevance for our pur-
pose as the initiator molecules that dock at these faces will
not be able to initiate the polymerisation reaction along the
polar ꢀc direction due to an inappropriate orientation of
the NH2 group of the initiator with regard to the aC=O of
the ValNCA molecule. On the other hand, the two less-ex-
¯
¯
¯ ¯
pressed enantiotopic side faces (111), (111), (111) and (111)
are more relevant to the polymerisation process as they
comprise sites of both handednesses, and binding of an (S)-
Val-OMe initiator, IS, to such crystal sites followed by initia-
tion of the polymerisation reaction leads to the formation of
diastereomeric intermediates. The lowest docking energy of
an IS molecule to a ValNCA site of any handedness on the
two cleaved crystal surfaces was obtained, as expected, for
an S site, with docking being somewhat more favourable on
¯
the (111) face than on the (111) face. This result is in keep-
ing with the formation of an excess of short peptides of the
same absolute configuration as the initiator, for example,
[S2IS]>[R2IS] or, by analogy, [R2IR]>[S2IR]. However, the
docking energy of an IS molecule into an R site, in one out
of four possible sites on the same crystal faces, is larger by
only 1 kcalmolꢀ1, a fact implying that formation of both the
SIS and RIS diastereoisomers is possible. The result of this
computation can provide a rationale for the observed rever-
sal in the ee value of longer isotactic peptides.
The possible steric effect of the initiator repeat unit in the
two diasteroisomeric dipeptides could be ignored as a simi-
lar reversal in the ee value was obtained by using various in-
itiators (Figures 7 and 9).
The analysis of the water-soluble short peptides by
MALDI-TOF MS indicates that there is not a substantial
difference, if any, in the degree of solubility of the diastereo-
isomeric peptides ISS2, ISR2 and ISS3, ISR3 as the relative
compositions in the water-soluble product mixture (Fig-
ure 6a) and in the water-insoluble product mixture (Fig-
Figure 7. Plots of the ee value of Sn =100([S]ꢀ[R])/([S]+[R]) for isotactic
oligopeptides of each length n obtained in the polymerisation of (R,S)-
ValNCA crystals suspended in an aqueous solution of enantiopure initia-
tor (50 mol%molꢀ1): a) Val-OMe and b) Phe-OMe of S absolute configu-
~
&
ration ( ) and R absolute configuration ( ). We represent the excess of
oligo-S chains as an ee value instead of a diastereomeric excess, de, value
by not considering the repeat unit of the initiator at the C terminus, for
convenience. Error bars indicate the average of five samples.
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M. Lahav et al.
groups of the adjacent S or R
monomer molecules in the
crystal will be different. To
obtain additional insight into
the reaction pathways at the
early propagation stages, we
analysed the sequences of the
heterochiral oligopeptides by
MALDI-TOF-TOF MS/MS.
MALDI-TOF-TOF
MS/MS
analysis of the heterochiral
peptides: The sequence of the
various diastereoisomeric het-
erochiral peptides obtained
from the polymerisation of
(R,S)-ValNCA crystals sus-
pended in an aqueous solution
of the initiator (S)-PheOMe
were studied by MS/MS (see
the Experimental Section).
The fragmentation analysis al-
lowed us to determine the
most
abundant
sequences
(MAS) of the obtained diaste-
reoisomers, as summarised in
Table 1. From these results, we
noticed that all heterochiral
peptides are composed from
Figure 8. MALDI-TOF mass spectra of the diastereoisomeric peptides obtained from the polymerisation of
(R,S)-ValNCA crystals suspended in aqueous solutions of a) (S)-Phe-OMe and b) (R)-Phe-OMe initiator.
blocks of repeat units of the same handedness. When IS is
used as the initiator, the MAS in the family (h,1) (in which
h represents the number of repeat units of R configuration
(protonated) and 1 is the deuterated S repeat unit) is either
(R)hSIS or S(R)hIS, in which the single S unit resides either
at the N terminus and operates as a terminator or is linked
directly to the IS initiator. By contrast, for the family (1,d)
(in which d represents the number of repeat units of S con-
Figure 9. Plot of the Sn ee value for isotactic oligopeptides of each length
n obtained in the polymerisation of (R,S)-ValNCA crystals suspended in
an aqueous solution (with 85 mol%) of (S)-(Val)3-OMe initiator.
ure 6b) are the same. Furthermore, the solubility of ISS4 is
lower than that of ISR4; the concentration of the former is
higher in the solid phase. If differences in solubility were re-
sponsible for the reversal in sign of the ee value, the longer
peptides would be enriched with ISSn, which is at variance
with the experimental result (Figure 7).
We are thus left with the fourth possibility mentioned
above, which considers the removal of the mirror symmetry
of the R and S monomer molecules located at the crystal/so-
lution interface after formation of the diastereoisomers ISS
and ISR. As a result, the contact of the growing NH2 moiet-
ies of these two diastereoisomers with the reactant carbonyl
Figure 10. Photograph of crystals grown from THF/hexane and the com-
puter-calculated growth morphology of (R,S)-ValNCA crystals.
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Controlled Polymerisation in Racemic Crystals
FULL PAPER
Table 1. The most abundant sequences (MAS) for the heterochiral oligo-
peptides obtained from the polymerisation of (R,S)-ValNCA suspended
in an aqueous solution of (S)-Phe-OMe (IS) initiator as determined by
MALDI-TOF-TOF MS/MS. The most abundant sequences appear in
bold.
of either handedness. On the other hand, RIS reacts with
monomers of the same handedness along the translation c
axis.
On the basis of these results, we infer that the isotactic
oligo-R chains should be formed in excess as the SIS dipep-
tide proceeds along two propagation pathways and reacts
with either S or R monomers, thus decreasing the concentra-
tion of the oligo-S chains (Figure 11). This deduction sup-
ports the observed reversal in the ee value at the early
stages of the polymerisation.
A
ACHTREUNG
[a]
A
ACHTREUNG
A
ACHTREUNG
RSRRRRRIS
SRRRRRSSIS
G
G
A
ACHTREUNG
A
ACHTREUNG
To summarise the reaction mechanism with the enantio-
pure initiator in these polymerisation experiments, we may
trace the following sequential steps. First is an asymmetric
induction of the enantiopure initiator with either an R or an
S monomer molecule at the crystal/liquid interface to form
two diastereoisomeric dipeptides that react differently with
their adjacent neighbours in the crystal to yield a nonrace-
mic composition of the short dipeptides. Once short isotactic
oligopeptides are formed, they assemble into racemic p b
sheets, as dictated by the crystalline lattice of the monomer.
The operation of asymmetric induction in the initiation step
induces a desymmetrisation of the racemic mixtures of the
formed isotactic oligopeptides, in spite of their parallel ori-
entation in which the enantiopure initiator is located far
away from the NH2 growing groups. The ensuing chain elon-
gation proceeds with almost equal rates for the two enantio-
meric chains, thereby leaving the ee value unchanged as
function of length. This process occurs at the growing
SSSSSRRRIS
A
ACHTREUNG
G
ACHTREUNG
E
ACHTREUNG
SRRRRRIS
SSRRRRIS
A
ACHTREUNG
A
ACHTREUNG
SSSRRRIS
SSSSRRIS
SSRSRRRRRIS, SSSRRRRRRIS
(5,4) RRRRRSSSSIS, RSSSSRRRRIS,
(2,4) RRSSSSIS,
G
SSSSRRRRRIS
R
ACHTREUNG
N
ACHTREUNG
SRRRRRRIS
SSSRRRRIS
SSSSSSRRRIS
(5,2) RRRRRSSIS
ACHTREUNG
A
ACHTREUNG
A
SRRRSSSIS,
RRSRSSSIS
A
SRRSSSSIS,
SSSSSRRIS
sheets/crystal interface presumably through
a diffusion
mechanism of the monomers towards the growing rims of
A
[a] For example, the sequence SRRRIS stands for NH2-(S)-Val-(R)-Val-
(R)-Val-(R)-Val-IS.
figuration (deuterated) and 1
is the protonated
R repeat
unit), the MAS with d>5 are
R(S)dIS in which the single R
repeat unit is found only at the
N terminus. The diastereoiso-
mer with the R repeat unit at-
tached to the initiator was not
detected. This result suggests
that, whereas the RIS dipeptide
reacts exclusively with
R
repeat units, the SIS dipeptide
reacts with both S and R mo-
nomer molecules; this implies
that, once the latter dipeptide
is formed, it assumes a confor-
mation in which the S repeat
unit is dislocated from its origi-
nal site in the crystal, such that
its NH2 group can react with
adjacent monomer molecules
Figure 11. Computer models of the propagation steps during polymerisation of (R,S)-ValNCA crystals initiated
with (S)-Val-OMe, IS: a) Diastereomeric RIS and SIS dipeptides, the former with only one possible propagation
pathway along the R monomer columns and the latter with two possibilities, yielding b) diastereomeric RRIS,
SSIS and RSIS tripeptides. For clarity, the C atoms of the R monomers are coloured in orange.
Chem. Eur. J. 2007, 13, 10140 – 10151
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10147

M. Lahav et al.
the peptides. From this stage on, the stereospecificity of the
reaction is controlled primarily by the structure of the grow-
ing rims of the oligopeptides. As the composition of the
long peptides is very different when the reaction is per-
formed by suspending the crystals in hexane, the hydration
of both the monomer and the growing rims is imperative for
the formation of the isotactic peptides.
To cast additional insight on the role played by the crys-
talline lattice in the distribution of the diastereoisomeric oli-
gopeptides, we investigated the solid-state polymerisation of
racemic crystals of LeuNCA.
perpendicular to the b axis. Should the reaction proceed
along the b axis, the S molecules will react in the +b direc-
tion and the R molecules in the opposite ꢀb direction. The
spacing between a nitrogen atom of one molecule and the
aC(carbonyl) atom of a neighbouring molecule of the same
A
handedness along the b axis is 4.47 Š. Reactivity along this
direction should produce racemic ap b sheets. However, ac-
cording to the packing arrangement of this crystal, there is
another polymerisation pathway along the a axis to produce
a syndiotactic polymer composed from alternating R and S
repeat units. Furthermore, during the chain elongation pro-
cess, the reaction may proceed along either pathway to yield
atactic polymers.
Polymerisationof ( R,S)-LeuNCA crystals suspended in
water and hexane: The packing arrangement of (R,S)-
LeuNCA crystals reported by Kanazawa[22] (monoclinic, a=
9.878, b=5.640, c=15.448 Š, b=105.8448, space group P21/
c, Z=4) is shown in Figure 12 as viewed along the b and a
axes.
The molecules form bilayer structures that are parallel to
the (001) plane of the crystal. Each bilayer is composed of
antiparallel ordered alternating rows of R and S molecules
along the unique b axis. Rows of R or S molecules, related
by a twofold axis parallel to the b axis, form the bilayers.
The S molecules are related to the R molecules by a c glide
Polymerisation experiments were performed on racemic
crystals, in which the S enantiomer was enantioselectively
tagged with ten deuterium atoms, suspended both in hexane
and in water containing the n-butylamine initiator. The dia-
stereoisomeric distributions of the oligopeptides for both
suspending liquids are shown in Figure 13. (R,S)-LeuNCA
crystals, which are insoluble in hexane, yield a complex li-
brary dominated by the long heterochiral peptides (Fig-
ure 13b). When the same reaction was performed in water
containing the n-butylamine initiator, the isotactic isomers
were more abundant in the formed oligopeptides (Fig-
ure 13a) in comparison to those obtained in hexane but
were less abundant than those obtained from the (R,S)-
ValNCA crystals in water. Such a distribution is in keeping
with the double reaction pathway that is possible in the
(R,S)-LeuNCA crystal, which makes it difficult to engender
the b-sheet templates required for the formation of the long
homochiral peptides. However, water enhances, in compari-
son to hexane, the formation of the isotactic peptides, as a
result of the hydration of the monomer and of the growing
ends of the chains, as well as due to the higher solubility of
the atactic short peptides than that of the isotactic ones. In
this system, we cannot ignore the possibility that a small
number of the isotactic peptides were formed in the aqueous
solution due to some dissolution of the monomer.[4]
Conclusion
In this study, we demonstrate the formation of isotactic oli-
gopeptides of valine containing up to 14–15 repeat units of
the same handedness by starting with (R,S)-ValNCA crystals
suspended in aqueous solutions containing an amine initia-
tor. This reaction is stereospecific, in contrast with previous-
ly reported nonstereospecific solid-state polymerisation re-
actions,[23,24] although the polymeric phase is denser than the
monomer crystal. This different behaviour can be rational-
ised by assuming a multistep mechanism comprising 1) lat-
tice control in the early stages of the reaction to yield short
racemic oligopeptides enriched with the isotactic ones and
2) self-assembly of the latter into supramolecular p b sheets
followed by 3) a chain-elongation step in which the mono-
mer molecules diffuse towards the structured rims of the
newly formed polymeric phase. The stereospecificity of the
Figure 12. Packing arrangement of (R,S)-LeuNCA viewed along a) the b
axis and b) the a axis. Black arrows indicate feasible reaction pathways in
the early stages. The short contacts between the reactive atoms are
marked. All of the C atoms of molecules of R absolute configuration are
coloured in orange.
10148
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Chem. Eur. J. 2007, 13, 10140 – 10151

Controlled Polymerisation in Racemic Crystals
FULL PAPER
Experimental Section
Materials: ValNCA and LeuNCA
monomers were prepared according
to the procedure of Daly and
PochØ,[29] with minor adjustments.
(R,S[D8])-ValNCA: Finely ground
(R)-Val (117.2 mg, 1 mmol, 0.5 equiv)
and (S[D8])-Val (98%, Cambridge
Isotope
Laboratories;
125.2 mg,
1 mmol, 0.5 equiv) were suspended in
dry THF and heated to reflux under
argon. Solid bis(trichloromethyl)car-
bonate
(triphosgene;
237.4mg,
0.8 mmol, 1.2 equiv) was added and
reaction was continued for 0.5 h. Tri-
phosgene
portions
(0.4mmol,
0.6 equiv) were consecutively added
every 0.5 h until a clear solution was
obtained (usually two or three por-
tions were needed). The reaction was
then continued for one additional
hour. The clear solution was allowed
to cool to room temperature and the
THF was evaporated under reduced
pressure. CH2Cl2 (ꢁ5 mL) was
added,
followed
by
hexane
(ꢁ50 mL), with stirring. A white pre-
cipitate appeared almost immediately
and the suspension was cooled to 48C
for 2–3 h, after which the crystals
were filtered off. The small needle-
Figure 13. MALDI-TOF mass spectra of diastereoisomeric oligopeptides obtained from the polymerisation of
(R,S)-LeuNCA suspended in a) aqueous and b) hexane solutions of n-butylamine.
like
(R,S[D8])-ValNCA
crystals
(272.9 mg, 93% yield) were analysed
by IR spectroscopy. All other NCAs
were prepared in a similar manner.
chain-elongation process is defined by the structures of the
rims of the supramolecular p b sheets that operate as tem-
plates. The monomer crystal structure also defines the mode
of organisation of the peptide chains as racemic b sheets.
The templates may be either parallel, as described here for
ValNCA, or antiparallel as reported in a previous study of
the polymerisation of (R,S)-PheNCA.[16,17] The packing ar-
rangement of (R,S)-LeuNCA presumably does not permit
the formation of either architecture as the isotactic peptides
are not formed in substantial quantities from this starting
material.
The present study also demonstrates that the suspending
solvent plays an important role in determining the diastereo-
isomeric distribution of the oligopeptides. At least two ef-
fects played by water have to be taken into consideration:
firstly, the ability of the hydrophobic peptide chains to self-
assemble preferentially in an aqueous millieux as compared
to hexane and, secondly, the formation of chiral hydrated re-
gions at the growing NH2 groups that increase the enantio-
meric recognition for the hydrated monomers.
Methyl esters of amino acids and peptides: (R)- and (S)-Valine methyl
ester (Val-OMe) hydrochloride, (R)- and (S)-leucine methyl ester (Leu-
OMe) hydrochloride, (R)- and (S)-phenylalanine methyl ester (Phe-
OMe) hydrochloride and (R)- and (S)-tryptophan methyl ester (Trp-
OMe) hydrochloride were purchased from Sigma and the corresponding
free amines were prepared by suspending the material in dry CH2Cl2 and
bubbling ammonia gas through the suspension for a few minutes. The re-
sulting ammonium chloride was filtered off and the CH2Cl2 was removed
by evaporation.
The methyl ester hydrochlorides of di- and tripeptides ((Val)2, (Val)3, and
(Leu)2; Sigma or Bachem) were prepared as follows: Dry methanol
(20 mL) was placed in a 50 mL round-bottomed flask equipped with a
drying tube and a magnetic stirring bar and acetyl chloride (3.2 mL) was
added dropwise. The solution was allowed to cool to room temperature
and, for example, the (Val)3-OMe, (Val)3-OH tripeptide (500 mg,
1.58 mmol, 1 equiv) was added. The reaction was monitored by TLC
(30% aqueous NH3:isopropyl alcohol 3:7) and was continued for two to
three days. After completion of the reaction, the MeOH was evaporated
under vacuum, then CH2Cl2 was added and evaporated; this procedure
was repeated until the remaining material was transformed from an oil
into a white solid. Hexane was added and evaporated several times until
a loose white precipitate appeared. The material was filtered off to give
(Val)3-OMe·HCl (538 mg, 92% yield). The hydrochloride salts were re-
crystallised from CH2Cl2/hexane at 48C overnight.
The operation of the multistep mechanism proposed here
was instrumental in the generation of homochiral peptides
from amphiphilic activated a-amino acids polymerised in
aqueous solutions.[4] These studies might be of relevance to
the formation of homochiral primeval peptides as NCA acti-
vation of a-amino acids is currently considered as a relevant
precursor step.[25–28]
Polymerisationprocedure : The appropriate NCA material was weighed
into a glass vial or polypropylene centrifuge tube (1–2 mg, or 10 mg when
more material was needed for analyses). Stock solution (0.75 mL for
each milligram of NCA) of the appropriate free amine in water or
hexane was added and the reaction was stirred for 24–72 h. The stock sol-
utions were prepared so that the amount of free amine added will equal
0.85 mol equivalents of the amount of NCA. Some of the peptide methyl
esters did not dissolve completely in the hexane and a well-stirred sus-
Chem. Eur. J. 2007, 13, 10140 – 10151
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10149

M. Lahav et al.
pension was used. After 24–72 h, the vessels were subjected to centrifuge
and the solvent was carefully decanted and filtered to remove any residu-
al precipitate. The remaining precipitate and the solution were both
dried, hexane was removed by a stream of dry nitrogen and water was re-
moved by lyophilisation.
MALDI-TOF mass spectroscopy measurements and analyses:
Sample preparation: A small amount of the polymerisation product (ap-
proximately 0.05–0.1 mg) was placed in a 500 mL polypropylene micro-
centrifuge tube and trifluoroacetic acid (TFA, 20 mL) and THF (80 mL)
were added in succession to obtain clear solution I. A matrix solution
(0.5 mL, dithranol solution in chloroform/saturated NaI solution in THF
1:1) was placed on the target plate and left to dry; on top of this, solutio-
n I (0.5 mL) was deposited (double deposition).
The MALDI-TOF positive-ion mass spectra were obtained in reflector
mode from a Bruker Reflex III instrument equipped with an N2 laser.
Only singly charged ions, [M+Na]+, with the expected isotopic pattern
were observed. The final mass spectra resulted from the signal average of
at least a few hundred laser shots in different spots of the target, to
ensure reliable statistics about the ion peak. Good statistics were ob-
tained when the isotopic distribution of an ion species corresponded to
that expected from calculation. Mass assignments were made by using
both the m/z value and the isotopic distribution.
^
Figure 14. MALDI-TOF-TOF mass spectra data for the (6,0) ( ) and
&
(0,6) ( ) oligopeptides of (R,S)-ValNCA initiated with (S)-Phe-OMe.
The observed abundance (the peak intensity divided by the sum of all
the peak intensities) of the y-type fragments is plotted against the mass.
The following notation code is used for the peptide molecules: (h,d) des-
ignates all molecules composed of h repeat units of R configuration (pro-
tonated) and d repeat units of S configuration (deuterated), with n=h+
d being the total number of repeat units. The relative abundance (ra) of
each type of oligopeptide (h,d) was obtained by dividing the intensity of
Table 2. Table of the experimental y-type and a-type fragmentations of
the (3,1) oligopeptides obtained from the polymerisation of (R,S)-
ValNCA suspended in an aqueous solution of (S)-Phe-OMe (IS) initiator
measured in the MALDI-TOF-TOF MS/MS, as compared with a random
polymerisation and the corresponding sequences. The resulting most
abundant sequences (MAS) are shown in bold.
the signals from a particular molecule I(h,d) by the total intensity of the
A
ions from all of the molecules of the same length, n. For example, the rel-
ative abundance of the tetrapeptide (4,0), composed of four (R)-Val
repeat units, was calculated from Equation (1).
y-Type fragmentations
[e]
[f]
no.
F
(m/z)[a]
m/z[b]
I[c]
O[d]
In
On
In/On
1
2
3
4
5
6
7
507.339
499.289
408.27
400.221
309.202
301.152
202.0840
507.357
499.292
408.268
400.233
309.177
301.139
0
1279
4276
2284
12572
848
3
1
2
2
1
3
0.23
0.77
0.15
0.85
0.14
0.86
0.75
0.25
0.5
0.5
0.25
0.75
0.31
3.08
0.31
1.69
0.57
1.14
rað4,0Þ ¼ Ið4,0Þ=fIð4,0ÞþIð3,1ÞþIð2,2ÞþIð1,3ÞþIð0,4Þg
ð1Þ
The relative abundance of the tetrapeptide (1,3), composed of one R
(protonated) and three S (deuterated) Val repeat units, is calculated from
Equation (2).
5129
4 0
1
0
y-Type fragmentations
2nd 3rd
rað1,3Þ ¼ Ið1,3Þ=fIð4,0ÞþIð3,1ÞþIð2,2ÞþIð1,3ÞþIð0,4Þg
ð2Þ
Sequence 1st
4th
RRRSIS
RRSRIS
RSRRIS
SRRRIS
507.339 408.27
507.339 408.27
507.339 400.221 301.152 202.084
499.289 400.221 301.152 202.084
309.202 202.084
301.152 202.084
The ionisation yield is expected to be very similar for (h,d) oligopeptides
of the same length n=h+d due to their similar chemical properties and
detection efficiency. Thus, the ion intensities of the different (h,d) diaste-
reoisomeric oligopeptides of the same length n are directly and reliably
comparable.
a-Type fragmentations
no.
F
(m/z)
m/z
I
O
In
On
In/On
1
2
3
4
5
6
7
399.318
300.249
292.2
201.181
193.131
102.112
94.063
399.308
300.233
292.193
201.175
193.123
0
14373
7195
2123
2408
1138
0
4
3
1
2
2
1
3
1
1
1
MALDI-TOF-TOF mass spectra: The spectra were obtained with an Ap-
plied Biosystems 4700 Proteomics MALDI-TOF-TOF mass spectrometer
fitted with an NdYag laser operating at 355 nm and at a frequency of
200 Hz. The window for selecting ions in MS/MS was set to 6 U (mass
unit) The spectra were collected automatically for each original (h,d)
peak. Only y-type and a-type fragmentations with masses consistent with
sodium ionisation [M+Na]+ were observed. The y-type fragmentation is
when the peptide bond is cleaved and the positively charged Na+ ion re-
mains attached to the C terminus and an a-type fragmentation is when
ꢀ
0.77
0.23
0.68
0.32
0
0.75
0.25
0.5
0.5
0.25
0.75
1.03
0.91
1.36
0.64
0
0
0
0
0
a-Type fragmentations
2nd 3rd
Sequence 1st
4th
the Ca C(carbonyl) bond is cleaved and the charge remains on the N
G
RRRSIS
RRSRIS
RSRRIS
SRRRIS
94.063
94.063
94.063
193.131 292.2
193.131 300.249 399.318
201.181 300.249 399.318
399.318
terminus. From the fragmentation of the homochiral peptides ((n,0) and
0,n)) in which only one sequence is present, it is clear that the experi-
mental abundance of each fragment is not equal to the expected occur-
rence, which should be identical for all fragments. However, it is clear
that the fragmentation is not affected by an isotopic effect or by the
nature (R or S) of the residues since the fragment abundances of (n,0)
and (0,n) isotactic peptides are very similar with respect to their relative
masses (Figure 14).
102.112 201.181 300.249 399.318
[a] F(m/z) is the calculated fragment mass. [b] m/z is the measured peak
A
mass. [c] I is the integrated intensity of the peak. [d] O is the occurrence
of the fragment for random distribution; it is the number of times this
fragment appears in the adjacent table. [e] In is the normalised intensity.
[f] On is the normalised occurrence. Note that fragments with high In/On
values and the most abundant sequences are shown in bold font.
A program was written to process the MS/MS spectral data (mass, inten-
sity) and to compare these data to possible sequences for each (h,d)
10150
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Chem. Eur. J. 2007, 13, 10140 – 10151

Controlled Polymerisation in Racemic Crystals
FULL PAPER
oligopeptide expected from a random polymerisation. The comparison of
the experimental fragment abundance to the normalised occurrence for a
given sequence allows the extraction of information on the different pos-
sible sequences of the (h,d) oligopeptides, as shown in Table 2. For the
(3,1) oligopeptide, the y-type fragment at m/z 499 is three times more
abundant than the normalised occurrence of the sequences giving the
same fragment (m/z 499) and expected for a random process (Table 2).
This fragment indicates that the three R repeat units are mainly bound to
the initiator IS whereas the y-type fragment at m/z 507, which has a very
low abundance, indicates that the single S repeat unit is less likely to be
bound to the initiator. From the data of both fragmentation types, the
most abundant sequences (MAS) can be defined (see example for the
(3,1) oligopeptide in Table 2) for oligopeptides of length n=4–9 (see
Table 1).
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Received: June 29, 2007
Published online: October 15, 2007
Chem. Eur. J. 2007, 13, 10140 – 10151
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
10151