UNEXPECTED DIFFERENCES BETWEEN D- AND L- TYROSINE LEAD
TO CHIRAL ENHANCEMENT IN RACEMIC MIXTURES
Dedicated to the memory of Prof. Shneior Lifson – A great liberal thinker.
MEIR SHINITZKY1∗, FABIO NUDELMAN1 , YANIV BARDA1 ,
RACHEL HAIMOVITZ1, EFFIE CHEN2 and DAVID W. DEAMER2
1 Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel;
2 Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA, U.S.A.
(∗ author for correspondence, e-mail: [email protected])
(Received 16 April 2002; accepted in revised form 6 June 2002)
Abstract. We report here an unexpected difference in the solubilities of D- and L-tyrosine in water,
which could be discerned by their rate of crystallization and the resulting concentrations of their
saturated solutions. A supersaturated solution of 10 mM L-tyrosine at 20 ◦ C crystallized much more
slowly than that of D-tyrosine under the same conditions, and the saturated solution of L-tyrosine
was more concentrated than that of D-tyrosine. Supersaturated solutions of 10 mM DL-tyrosine in
water formed precipitates of predominantly D-tyrosine and DL-tyrosine, resulting in an excess of
L-tyrosine in the saturated solution. The experimental setups were monitored independently by UVabsorption, radioactivity tracing, optical rotation and X-ray diffraction. The process of nucleation
and crystallization of D- and L-tyrosine is characterized by an exceptionally high cooperativity. It
is possible that minute energy differences between D- and L-tyrosine, originating from parity violation or other non-conservative chiral discriminatory rules, could account for the observations. The
physical process that initiated chiral selection in biological systems remains a challenging problem
in understanding the origin of life, and it is possible that chiral compounds were concentrated from
supersaturated racemic mixtures by preferential crystallization.
Keywords: chiral selection, crystallization, enantiomeric discrimination, origin of life
1. Introduction
The absolute identity of bulk chemical and physical properties of chiral isomers
is an axiom of stereochemistry. However, properties reported in the literature for
pairs of enantiomers under identical conditions occasionally reveal cases where
this identity is not preserved. Such deviations from chiral identity tend to emerge
in condensed assemblies such as concentrated solutions, aggregates, neat fluids
or crystals. For instance, Thiemann and Wegener (1970) reported a ‘remarkable
phenomenon’ in which a selective enantiomeric precipitation was observed when a
saturated aqueous solution of racemic sodium ammonium DL-tartarate was cooled.
The authors concluded that the enantiomorphic crystals must differ in their lattice energy. Thiemann and Darge (1974) reviewed the older literature and found
a number of reports in which selective precipitation of one enantiomer from a
saturated racemic mixture solution was observed and left unexplained. Phase transOrigins of Life and Evolution of the Biosphere 32: 285–297, 2002.
© 2002 Kluwer Academic Publishers. Printed in the Netherlands.
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itions in single crystals of D- and L-alanine were found to be distinctively different despite their identical unit cells, which was attributed to differences in the
electroweak force induced by parity violation (Wang et al., 2000). In a study of
liquid mixtures of stereoisomers (Atik et al., 1981) a clear difference in the partial
specific volumes of α-methylbenzylamine was measured. Micellar aggregates of
N-stearoyl D- or L-serine in water acquire a strong circular dichroism (CD) band
which was attributed to formation of a chiral surface in the micelles (Shinitzky and
Haimovitz, 1993). However, the absolute magnitude of this CD signal markedly
differed between the D and L enantiomeric micelles, which was interpreted as a
difference of packing tightness. Upon disruption of the micelles by the addition of
ethanol, the CD band of the monomer was regained with an identical absolute
magnitude for these enantiomers. Similar differences were observed in the CD
spectra of R(-)-2-butanol and S(+)-2-butanol which were found to be considerably
different in absolute magnitude in the neat phase but diminished upon dilution with
water (Shinitzky, 1989).
The deviation from bulk identity of chiral isomers in condensed states imposes
an important challenge, which we tested in the following study with D- and Ltyrosine. This amino acid was selected on the basis of commercial availability in
high purity, limited solubility in water, robust optical activity and convenient UV
absorption spectrum.
2. Materials and Methods
2.1. C HEMICALS
D-tyrosine, L-tyrosine and DL-tyrosine, all of >99% purity, were obtained from
Fluka (Fluka Chemie AG, Germany). D- and L-tyrosine were crystallized 3 times
from sterile double distilled water (DDW) under argon in the dark. The crystals
were dried under vacuum and kept in dark containers. DL-tyrosine was washed
several times with acetone and dried thoroughly. It was not recrystallized in order to avoid preferential enantiomeric crystallization. [3 H]-L-tyrosine (40–60 Ci
mmol−1 ) was obtained from Amersham (Amersham Pharmacia Biotech., U.K.).
It was possible that traces of chiral biological material remained in commercial
samples of D- and L-tyrosine and could affect the results reported here. For this
reason DL-tyrosine was chemically synthesized by a modification of a procedure
described by Greenstein and Winitz (1961). O,N-diacetyl-L-tyrosine was prepared
by acetylation of L-tyrosine with acetic anhydride. This was dissolved in a mixture of acetic acid and acetic anhydride and boiled for 1 hr to ensure complete
racemization (Greenstein and Winitz, 1961). Hydrolysis of the acetyl groups was
carried out in 2.5 N HCl under reflux for 2 hr. After neutralization with concentrated ammonium hydroxide, DL-tyrosine precipitated and collected. Several bulk
purifications were then carried out by dissolving the precipitate in boiling water
DIFFERENCES BETWEEN D- AND L- TYROSINE LEAD TO CHIRAL ENHANCEMENT
287
and immediately cooling to 4 ◦ C. Proton-NMR analysis and paper chromatography
indicated +99% purity.
2.2. I NSTRUMENTS
UV absorption at 274.5 nm was measured with a single 1 mm quartz cuvette.
Radioactivity was recorded with a Packard β-counter model TRI-CARB 2100TR.
Optical rotation with sodium band light was recorded in a 10 cm path with a
Perkin Elmer polarimeter model 341. Single crystal X-ray diffraction and analysis
of D- and L-tyrosine were performed by Marilyn Olmstead at the University of
California, Davis, and results are recorded in the Cambridge Crystal Structure
registry.
3. Results
In a preliminary screening of water solubility of amino acids, we observed that
D- or L-tyrosine are considerably less soluble than D- or L-phenylalanine. This
surprising finding could be attributed to a crystallization process in supersaturated solutions of tyrosine which is driven by an exceptionally high cooperativity
induced by the hydroxyl residue. This property could potentially detect minute
differences in enantiomorphic contact between the two enantiomers of tyrosine, as
has been claimed for alanine (Wang et al., 2000). A systematic testing indicated a
marked and reproducible difference in the crystallization rates of D- and L-tyrosine
from 10 mM supersaturated aqueous solutions. These solutions were prepared by
heating to 100 ◦ C and were allowed to cool to 25 ◦ C in a water bath within 15 min.
Thereafter, the clear solutions were first adjusted to identical 10 mM concentrations
using UV absorbance measurements (see below) and then distributed as sets of four
1.0 mL aliquots in Eppendorf tubes which were sealed and covered with aluminium
foil. One set was kept at 20±0.5 ◦ C and the other at 4 ◦ C. At varying time intervals
each set of 4 was centrifuged, the supernatants were collected and their absorption
at 274.5 nm was determined in a 1 mm cuvette (ε274.5 = 145 M−1 mm−1 ).
The 10 mM solutions of D- and L-tyrosine at 20 and 4 ◦ C formed crystals over
periods of hours to days. As shown in Figure 1, D-tyrosine crystallized much faster
than L-tyrosine. As the crystals formed, the supernatant solution approached saturation of 2.8 mM after 48 hr, while L-tyrosine reached saturation of 4.5 mM only
after approximately 10 days. At 4 ◦ C the rates of crystallization were considerably
faster for both enantiomers while the difference in the saturated concentrations of
D- and L-tyrosine (1.9 and 2.5 mM, respectively) was maintained (Figure 1).
The marked differences in the rates of crystallization displayed in Figure 1 was
clearly visualized in the test vessels, as demonstrated photographically in Figure 2.
Analogous crystallization tests carried out in a variety of glass or plastic vessels
yielded in all cases crystallization profiles similar to those displayed in Figure 1 (an
288
M. SHINITZKY ET AL.
Figure 1. Rates of crystallization of supersaturated aqueous solutions (10 mM) of L-tyrosine () and
D-tyrosine () at 20 ◦ C (upper panel) and 4 ◦ C (lower panel). Each measured point represents
average ±S.D. of quadruplicate of the fraction remaining in the supernatant. In all of them the
difference between L- and D-tyrosine was highly significant (p < 0.0001).
DIFFERENCES BETWEEN D- AND L- TYROSINE LEAD TO CHIRAL ENHANCEMENT
289
Figure 2. Photographic presentation of 10 mM aqueous solutions of D- and L-tyrosine at 20 ◦ C in
glass vessels, taken at 0, 6, and 24 hr. Analogous solutions in Eppendorf plastic tubes (presented in
Figure 1) yielded similar precipitation views (not shown).
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M. SHINITZKY ET AL.
example is shown in Figure 2). It seems therefore that the unexpected differences
in the rates of crystallization of D- and L-tyrosine presented in Figure 1 is not
associated with a specific chiral discriminatory element present on the surface of
the test vessel.
The apparent differences in solubility profiles during crystallization provided
the rationale for the chiral enhancement experiments described below. Racemic
mixtures of supersaturated 10 mM DL-tyrosine in DDW were prepared either by
mixing equal volumes of 10 mM D- and L-tyrosine just after dissolving at 100 ◦ C,
or by using the commercial racemate. Each set was doped with 1 µM [3 H]-Ltyrosine to yield ∼106 cpm mL−1 . The mixtures were allowed to crystallize at
20±0.5 ◦ C as described above. The absorbance, A, of the supernatants was assayed
at 274.5 nm, which represented the total amount of D+L tyrosine while the content
of L-tyrosine was monitored by the radioactivity of [3 H]-L-tyrosine. Denoting Y as
the fraction determined by A274.5 and X as the fraction of radioactivity remaining
in the supernatant, then for the general case
D
Y
D
=
+1 −1,
(1)
L t
X
L o
and D
are the ratios of D-tyrosine to L-tyrosine in the supernatant
where D
L t
L o
at
time
t
and
time
o,
respectively.
For the specific case of the above experiments,
D
=
1,
yielding
Equation
(2)
from
which we calculated the change in proporL o
tion of the D and L enantiomers during the course of precipitation:
2Y
D
−1.
(2)
=
L t
X
The results obtained with 10 mM racemic mixtures of 50% D- + 50% L-tyrosine
or DL-tyrosine at 20 ◦ C are shown in Figure 3. The precipitation process in these
mixtures was considerably faster than those of the pure isomers (Figure 1) as
was noticed in earlier studies (Winnek and Schmidt, 1935, 1936) and presumably
consisted predominantly of precipitation of DL aducts and D-tyrosine. Selective
precipitation of L-tyrosine (5 mM initial concentration at 20 ◦ C) could be neglected
as implied from the results presented in Figure 1. A progressive enrichment of the
L enantiomer in solution resulted in a supernatant having approximately 60% Ltyrosine and 40% D-tyrosine in both experiments, using either the racemic mixture
prepared by mixing D- and L-tyrosine, or the commercial racemate (Figure 3).
Enantiomeric impurity, i.e. the presence of a trace of the opposite chiral isomer,
could in principle have affected the results presented in Figures 1–3. We therefore
tested the preferred precipitation of D-tyrosine in mixtures with excess D- or Ltyrosine under similar conditions to those described in Figure 3. The (D/L)o in
these mixtures extended from 0.43 to 2.33. On day 6, the supernatants were separated, their A274.5 and cpm were measured and (D/L)t was evaluated according to
Equation (1). As a control test we also evaluated Y /X of 10 mM L-tyrosine which
DIFFERENCES BETWEEN D- AND L- TYROSINE LEAD TO CHIRAL ENHANCEMENT
291
Figure 3. Rates of precipitation of commercial racemic DL-tyrosine (upper panel) and a racemate
made of 50% D-tyrosine plus 50% L-tyrosine (lower panel) obtained with supersaturated (10 mM)
aqueous solutions at 20 ◦ C. The D-tyrosine plus L-tyrosine remaining in solution was monitored
by light absorption () while the remaining L-tyrosine was monitored independently with a trace
of [3 H]-L-tyrosine (). The proportions of L- and D-tyrosine in the supernatant were evaluated
according to Equation (2) and are indicated in the figure.
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M. SHINITZKY ET AL.
Figure 4. Solubility discrimination in solutions of 10 mM D + L tyrosine of different proportions.
The figure presents the ratio of fraction of light absorption (Y) and radioactivity of 3 [H]-L-tyrosine
(X) remaining in solution after 6 days at 20 ◦ C. The change in ratio of the enantiomers at time 0,
(D/L)o and after 6 day, (D/L)t , was evaluated by Equation (1). The experiment was carried out in
groups of 10 samples and the results are presented as average ±S.D.
corresponded, as expected, to 1.0±0.04. The results of this set are presented in Figure 4 and clearly indicate that in all tested mixtures Y /X was lower than 1, further
demonstrating the preferred solubility of L-tyrosine over that of D-tyrosine while
excluding the effect of enantiomeric impurity. The ratio (D/L)o /(D/L)t given in
Figure 4 for each test can be taken as the ‘discrimination factor’ for maintaining Ltyrosine in the supernatant of supersaturated solution of D+L tyrosine. In all 5 cases
presented in Figure 4 this factor was in the range of 1.40–1.51, which indicates a
40–51% excess build up of L-tyrosine in the supernatant of supersaturated racemic
mixtures of tyrosine. It further indicates that L-tyrosine retains a saturation profile
higher than that of D-tyrosine (as shown in Figure 1) irrespective of the ratio D/L.
The results presented in Figures 3 and 4 predict that optical activity of opposite
signs will be acquired in both the precipitate and the supernatant of a supersaturated solution of DL-tyrosine as it undergoes crystallization. This was tested
independently in the following experiment in which the concentration of the measured mixture was increased 10-fold to permit an accurate determination of optical
rotation. A supersaturated solution of 5 mM D-tyrosine + 5 mM L-tyrosine was
prepared as described above. A 12 mL sample of the racemate was removed, and
DIFFERENCES BETWEEN D- AND L- TYROSINE LEAD TO CHIRAL ENHANCEMENT
293
Figure 5. Optical rotation α (Na band), measured at 23 ◦ C, obtained with a supersaturated solution
of 5 mM D-tyrosine plus 5 mM L-tyrosine immediately after preparation and after 72 hr when precipitation took place (Figure 3). Ten samples of 1 mL each were collected, combined and dissolved
in 1 mL HCl 1 N as described in the text. Each point was recorded 8–12 times. The proportions of
L- and D-tyrosine of each set were evaluated according to Equation (5).
the rest was distributed in 12 Eppendorf tubes in portions of 1 mL. The samples
were kept at 20 ◦ C for 72 hr and then centrifuged. The supernatants and the precipitates were collected and pooled. The initial solution sample, and the pools of
precipitates and superntants were lyophilized to complete dryness and then dissolved in 1.2 mL of 1 N HCl. The optical rotation, α, of each concentrated sample
was recorded 10 separate times, each consisting of 20 sec averaging, and absorbance at 274.5 nm was determined in parallel. The results are presented in Figure 5.
In an analogous experiment with synthetic DL-tyrosine (see the section Materials
and Methods), the α values obtained were +4.0±1.41 mdeg for the precipitate and –
3.1±1.45 mdeg for the supernatant. This finding indicates again that the precipitate
had an excess of D-tyrosine while the supernatant had an excess of L-tyrosine.
The simultaneous values of α and A274.5 were translated to the ratio D/L as
follows:
α = [α]
O.D
g
= [α] ·mw·10−3
.
mL
ε
(3)
294
M. SHINITZKY ET AL.
In a mixture of D + L,
[α]·mw·10−3 O.D
+α
D
ε
,
=
O.D
−3
L
[α]·mw·10
−α
ε
(4)
where [α] is the specific rotation presented as an absolute value. For the case of Dand L-tyrosine mw = 181, [α] = 10.6◦ and ε = 145 M−1 mm−1 yielding:
D
1.32·10−2 O.Dlmm + α
=
.
L
1.32·10−2 O.Dlmm − α
(5)
The recorded α values, their mean ± S.D. and their corresponding percentage of Dand L-tyrosine (evaluated from Equation (5)) are presented in Figure 5. The results
clearly confirm the unequal distribution of D- and L-tyrosine in the supernatant
and the precipitate formed during crystallization. Furthermore, the acquired optical
rotation in the supernatant corresponds to an excess of L-tyrosine very close in
magnitude to that recorded in the independent experiment presented in Figure 3.
X-ray diffraction of single crystals indicated that L-tyrosine crystallized from
water has an orthorombic structure of P21 21 21 space group with a density of 1.41 g
cm−3 (Mostad et al., 1972). However, crystal data reported for D-tyrosine corresponded to a slightly different orthorombic space group of P21 22, with a density
of 1.45 g cm−3 (Khawas, 1986). We therefore determined the crystal structure of
D- and L-tyrosine by single crystal X-ray diffraction and found that both the space
group and the unit cell dimensions of D-tyrosine were identical to those reported
for L-tyrosine (Olmstead et al., 2001, unpublished results). We could therefore
conclude that the effects reported above correspond to a difference in kinetics of
crystal formation in the isotropic supersaturated solutions, rather than unexpected
differences in L and D crystal structures.
A selective biochemical or chemical degradation of D- or L-tyrosine could
provide a trivial explanation to our results. As stated above, the tyrosine solutions
were prepared in sterile DDW, deoxygenated with argon and kept in the dark. Possible decompositions were nevertheless checked by simultaneous measurements
of A274.5, optical rotation (sodium band) and thin layer chromatogrpahy (with 70%
methanol on Whatman #3 paper, detection by iodine vapor or autoradiography). Of
the various tyrosine solutions at equilibrium no changes in these parameters were
detected up to 14 days after preparation. We concluded that the tyrosine solutions
remained chemically intact during the course of the experiments.
4. Discussion
‘Chiral discrimination’ in saturated racemic solutions, where parallel crystallization of each of the enantiomers takes place, was first observed in the pioneering
work of Pasteur (1848). In some cases where highly cooperative intermolecular
DIFFERENCES BETWEEN D- AND L- TYROSINE LEAD TO CHIRAL ENHANCEMENT
295
interactions dictate the rate of crystallization, a sporadic microscopic nucleation
can initiate the separation of one type of enantiomeric crystals out of a ‘racemic
conglomerate’. In principle, such spontaneous chiral resolution cannot favor any
of the enantiomers and their selection is at random (for reviews, see Bonner, 1991;
Bonner et al., 1999). However, a catalytic quantity of an unrelated chiral element
can favor to some extent a specific resolution of one enantiomer out of the racemic
milieu (Buhse et al., 2000; Gillard and da-Luz-de-Jesus, 1979; Hazen et al., 2001;
Klabunovskii and Thiemann, 2000; Kondepudi et al., 1990; Soai et al., 1999).
Trivial factors like an environmental or inherent impurity which bears a chiral
element could act as such catalysts.
In cases where the solubility of the racemic mixture is lower than that of the
enantiomers a slight excess of one enantiomer can increase in proportion upon
reaching saturation followed by continous evaporation of the solvent (Morowitz, 1969). Earlier conjectures (Hegstrom et al., 1980; Kondepudi and Nelson,
1985; Mason and Tranter, 1984; Szabo-Nagy and Keszhelyi, 1999; Wang et al.,
2000) proposed that the violation of parity in β emission may be reflected in a
minute energy difference between chiral enantiomers. However, only after a vigorous autocatalytic process can such a difference be expanded to experimentally
detectable levels. It was also suggested that a process of secondary crystallization could provide a platform for such a cooperative amplification (Hazen et al.,
2001; Kondepudi et al., 1990) but it remains doubtful whether the cooperativity
associated with crystallization could, in general, be powerful enough to account
for chiral selection. Exceptionally high cooperativity of crystallization, as for Dand L-tyrosine could nevertheless help in discerning inherent energy differences
between enantiomers. However, if autocatalysis associated with the condensation
mechanism is the determining step in the selective chiral enhancement, a small
excess of the opposite enantiomer could in principle diminish it or even reverse it,
which does not comply with our experimental findings (Figure 4).
The results presented in this study seem therefore to violate the axiom that chiral
isomers are identical in their bulk energetic quantities. They are supported by a
few earlier reports in which an unexpected chiral selection was observed (Atik et
al., 1981; Thiemann and Darge, 1974; Thiemann and Wagener, 1970; Shinitzky
and Haimovitz, 1993). In these reports, as well as in our study, a condensed system with two phases is involved where one phase undergoes a selective dynamic
process. Whatever the underlying cause of this remarkable and unexpected phenomenon, our results indicate that a specific chiral component can be separated
from a racemic mixture by a spontaneous crystallization.
At the current state of our conceptual understanding of chirality it is difficult to
propose an explanation different from those proposed above that can account for
such a selective chiral separation. In a search for a plausible mechanism for chiral
enhancement in a condensed racemic mixture we previously suggested a nonconservative expansion of quantum singularity to chiral centers. This singularity
was proposed to be expanded in condensed assemblies and could induce detectable
296
M. SHINITZKY ET AL.
difference in enantiomorphic contact energy (Shinitzky, 1989). This hypothesis,
however, has neither experimental nor theoretical proof.
It will be interesting to extend our observations with tyrosine enantiomers to
amino acids that were likely to have been present in the prebiotic inventory of organic compounds, such as alanine. With respect to the latter, it is of interest to note
the observation where polymerization of DL-alanine resulted in the unexpected
emergence of optical rotatory dispersion spectrum typical of excessive domains of
poly L-alanine (Thiemann and Darge, 1974). The possibility of spontaneous chiral
resolution offers a symmetry-breaking mechanism by which one of the enantiomers
can selectively be removed from a racemic millieu leaving behind a sufficiently
concentrated enantiomer to establish the initial chirality of molecular systems involved in the origin of life. We note that Cronin and Pizzarello (1997) discovered
an excess of L-isomers in several of the amino acids present in a carbonaceous
meteorite, which may be related to the results described here.
Acknowledgements
The authors wish to thank Marilyn Olmstead for her expert analysis of D- and
L-tyrosine single crystals, and Mark Anderson and Alex Helman for their help in
obtaining powder X-ray diffraction data. This research was supported in part by
NASA Grant SC-00-35.
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