Mechanistic Insight into the Origin of Stereoselectivity in the Ribose-Mediated Strecker Synthesis of Alanine

Article abstract of DOI:10.1021/jacs.1c03552

Enantioenriched amino acids are produced in a hydrolytic kinetic resolution of racemic aminonitriles mediated by chiral pentose sugars. Experimental kinetic and spectroscopic results combined with DFT computational studies and microkinetic modeling help to identify the nature of the intermediate species and provide insight into the stereoselectivity of their hydrolysis in the prebiotically relevant ribose-alanine system. These studies support a synergistic role for sugars and amino acids in the emergence of homochirality in biological molecules.

Full text of DOI:10.1021/jacs.1c03552

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Mechanistic Insight into the Origin of Stereoselectivity in the
Ribose-Mediated Strecker Synthesis of Alanine
̀
Luca Legnani, Andrea Daru, Alexander X. Jones, and Donna G. Blackmond*
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ABSTRACT: Enantioenriched amino acids are produced in a
hydrolytic kinetic resolution of racemic aminonitriles mediated by
chiral pentose sugars. Experimental kinetic and spectroscopic results
combined with DFT computational studies and microkinetic
modeling help to identify the nature of the intermediate species
and provide insight into the stereoselectivity of their hydrolysis in the
prebiotically relevant ribose−alanine system. These studies support a
synergistic role for sugars and amino acids in the emergence of
homochirality in biological molecules.
Scheme 1. Hydrolysis of rac-Alanine Aminonitrile to
Enantioenriched Alanine Mediated by D-Ribose
INTRODUCTION
■
The building blocks that construct the informational polymers
of life are sugars and amino acids of single chirality. How this
homochirality evolved from a presumably racemic prebiotic
world has been the subject of intense theoretical and
experimental study, focusing on both chemical1 and physical2
processes. The classic Strecker3 and formose4 reactions for the
synthesis of amino acids and sugars, respectively, along with
more recent developments such as the Powner−Sutherland
synthesis of activated pyrimidine nucleotides5 and ensuing
work by these groups6,7 have increased our understanding of
plausible prebiotic chemistry of these molecules, but the
question of chirality has often been left to the side.8 A number
of recent studies have provided insight into protometabo-
lism,9−12 particularly in reaction networks where stereo-
chemistry is not a key feature, such as in the citric acid
cycle. It seems clear, however, that the question of chirality
must be addressed in the formation of RNA, DNA, and
proteins, the molecules that form the genetic basis of life. The
provenance of the homochirality of the sugar and amino acid
building blocks for these molecules appears to be intrinsically
intertwined.13
We recently demonstrated that chiral pentose sugars can
direct the hydrolysis of racemic aminonitriles in a stereo-
selective kinetic resolution.14 Other approaches to amino acids
via aminonitriles involving mixed phases15,16 or mixed chemo/
enzymatic systems17 have been reported. We report here
detailed kinetic, spectroscopic, and computational studies of
this process for the prebiotically important alanine system
(Scheme 1) that provide insight into the mechanism of the
reaction, the nature of the intermediate species, and the origin
of stereoselectivity in the prebiotic synthesis of amino acids.
RESULTS AND DISCUSSION
■
The temporal progress of the reaction of Scheme 1 was
monitored by 13C NMR spectroscopy, where Ala-I, Ala-II, and
Ala-III give distinct signals for integration.18 Figure 1 shows
the temporal concentrations (Figure 1a and 1b) and
enantiomeric excesses (Figure 1c). Consumption of Ala-I is
rapid and occurs concomitant with production of Ala-II
(Figure 1a). No Ala-III is observed in the first 10 h. The
concentration of Ala-II rises to a value of ca. 0.1 M in the first
3 h and remains nearly constant at this level for several hours.
This represents ca. 80% of the starting aminonitrile, suggesting
that some material is sequestered as intermediate species.
These results are in accordance with our previous observations
of reaction progress for the D-ribose-phenylalanine aminonitrile
system.14
This picture changes substantially at longer reaction times,
as is shown in Figure 1b (note the change in scale of the time
Received: April 3, 2021
Published: May 12, 2021
https://doi.org/10.1021/jacs.1c03552
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© 2021 American Chemical Society
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Figure 1. Reaction monitoring of the reaction in Scheme 1 carried out with 0.125 M rac-Ala-I, 0.125 M D-ribose, and 0.01 M NaOH in D2O at
ambient temperature. pH at the outset of the reaction was 10.98. Concentration profiles of Ala-I (blue diamonds), Ala-II (orange squares), and Ala-
III (green circles) at reaction times of (a) 0−600 min and (b) 600−11000 min. Dashed line in (b) indicates the concentration of a quantitative
mass balance for (Ala-I+Ala-II+Ala-III). Note the change in time scale from part (a); (c) temporal evolution of enantiomeric excess of Ala-II
toward the D-enantiomer (orange squares) and Ala-III toward the L-enantiomer (green circles). Positive numbers for ee indicate D, negative
numbers indicate L.
axis). The concentration of Ala-II begins to decrease as the
amino acid Ala-III begins to form. However, as the dashed line
for a mass balance in Figure 1b shows, the production of Ala-
III does not match the decrease in Ala-II, suggesting that an
even more significant buildup of a reservoir of intermediate
species occurs beyond what was observed in the first 600 min
shown in Figure 1a. The concentration of Ala-III increased
steadily but remained below 10% at the extended reaction
times.
Scheme 2. Identification of Intermediates Formed in the
Reaction With 13C-Labeled Sugar and Aminonitrile18
Figure 1c follows the evolution of enantiomeric excess for
Ala-II and Ala-III over the course of the reaction. Ala-II is
formed as nearly racemic at the outset and evolves a small
excess (13% ee) to the D-enantiomer over time, while the ee of
Ala-III, initially ca. 50% ee, rises to 68% ee to the L-enantiomer
of the amino acid. A similar trend was observed for the amino
amide in our previous studies of the phenylalanine system,14
but further hydrolysis to the amino acid was not observed and
was found to require more forcing conditions in that case.
Reactions carried out at higher concentrations of D-ribose
gave similar trends, forming higher ee of D-Ala-II, and higher
production and lower ee of L-Ala-III, along with a significantly
lower mass balance.18 Using either L-ribose or D-lyxose
produced the opposite sterochemical outcome in ee of Ala-II
and Ala-III compared to D-ribose, in accordance with our
previous studies. The use of other bases, including Ca(OH)2,
gave trends similar to NaOH.14,18
We confirmed both the concentration and the ee trends of
Figure 1 by carrying out reactions starting from the
aminoamide rac-Ala-II prepared from its HCl salt, adjusted
to provide similar pH upon release of HCl.18 These results
suggest that steps between Ala-II and Ala-III are key to the
stereoselectivity in this ribose-mediated kinetic resolution. In
order to probe for intermediate species on this pathway,
reactions were carried out using 13C1-labeled D-ribose and
13C1-labeled Ala-I prepared from K13CN. Several intermediate
species were observed, and a suite of NMR techniques18
confirmed the structure of major species Ala-II-A and minor
species Ala-III-B in the pyranose form shown in Scheme 2.
The L and D enantiomers of amide/carboxylic acid in their
respective D-ribo-complexes each exist as the α and β anomers
of the sugar, for a total of four isomers each for species Ala-II-
A and Ala-III-B.
that has been found to be an umami-type taste compound.19
We were able to synthesize authentic samples of intermediate
Ala-II-A from these literature procedures, yielding separately
the four diastereomers formed from the two enantiomers of
the amino amide with D-ribose.18 Species Ala-III-B was
identified by mixing 13C1-labeled D-ribose with each pure
enantiomer of Ala-III. No Ala-II-A was observed in this
reaction, indicating that the step from Ala-II-A to Ala-III-B is
likely to be irreversible and rate-determining.
Figure 2 compares the 13C NMR spectra from the reaction
of Scheme 2 using 13C-labeled sugar and aminonitrile (bottom
spectrum) with the spectra of the synthesized compounds Ala-
II-A and Ala-III-B. Interestingly, as shown in Figure 3, under
reaction conditions the D-amide species dominate for Ala-II-A,
while for species Ala-III-B it is the two L-amino acid−sugar
compoundswhich are the species leading to the major amino
acid product L-Ala-IIIthat dominate.
In order to quantify the relative reactivity of these different
diastereomers, we carried out kinetic studies of the hydrolysis
of the authentic intermediate species Ala-II-A prepared from
rac-Ala-II and D-ribose. The absolute concentration employed
for Ala-II-A in this reaction is ca. 10-fold higher than what we
observed during reaction starting from Ala-I, and the reaction
begins with equal D-Ala-II-A and L-Ala-II-A concentrations,
differing from those developed during reaction starting from
Precedent for such species may be found in food chemistry,
where a Maillard-mimic reaction forms a glycoconjugate from
L-glutamic acid and D-glucose analogous to species Ala-II-A
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Figure 2. 13C NMR spectra of (a) reaction of Scheme 2 at 24 h using
rac-13C-Ala-I (0.250 M), 13C1-D-ribose (0.250 M) and NaOH (0.250
M); (b) species B observed from mixing D-Ala-III and 13C1-D-ribose;
(c) species B observed from mixing L-Ala-III and 13C1-D-ribose; (d)
species A synthesized using D-Ala-II and D-ribose; (e) species A
synthesized using L-Ala-II and D-ribose. L- and D- labels refer to the
alanine amide and alanine stereochemistry.
Figure 4. Hydrolysis of Ala-II-A (0.250 M) in D2O at ambient
temperature in the presence of D-ribose (0.250 M) and NaOH (0.250
M). (a) Overall temporal reaction progress of D-Ala-II-A (blue
triangles) and L-Ala-II-A (blue diamonds); (b) temporal enantiomeric
excess of Ala-II (orange squares, left axis) and Ala-III (green circles,
right axis). L- and D- labels refer to the alanine amide and alanine
stereochemistry. Positive numbers for ee indicate D, and negative
numbers indicate L.
racemic aminonitrile Ala-I. Similar results were observed when
the hydrolysis of Ala-II-A was carried out in the absence of
added D-ribose.18
The temporal concentration profiles together with the
evolution of enantiomeric excess of both Ala-II and Ala-III
in Figure 4 allow us to quantify the net rates of production of
each enantiomer of the aminoamide Ala-II and the amino acid
Ala-III starting from the intermediate species Ala-II-A as
shown in Scheme 3.18 The dominant reactions of this
Figure 3. Relative concentrations of the four diastereomers each of
Ala-II-A (blue bars) and Ala-III-B (red bars) from the 13C NMR
spectrum of the reaction of Figure 2 at 24 h starting from rac-Ala-
I-13C1 (0.250 M), D-ribose-13C1 (0.250 M), and NaOH (0.250 M) at
ambient temperature in D2O. L- and D- labels refer to the alanine
amide and alanine stereochemistry.
Scheme 3. Dominant Reaction Pathways for Reaction of
Species Ala-II-A Showing Net Relative Rates18
the aminonitrile Ala-I as shown by the blue bars of Figure 3.
Monitoring temporal consumption in the presence of D-ribose
and NaOH demonstrated that the L-aminoamide ribosyl
intermediate reacts faster than the D-aminoamide ribosyl
intermediate (Figure 4a), consistent with our results of the
reaction initiated from the aminonitrile.18 Enantiomeric excess
to Ala-III (Figure 4b) developed much more rapidly starting
from Ala-II-A compared to reactions starting from either Ala-I
(Figure 1) or Ala-II·HCl.18 It is notable that, in addition to
amino acid formation in these experiments, we observed
concomitant formation of the aminoamide Ala-II, indicating
reversibility in the formation of species Ala-II-A from ribose
and Ala-II.
intermediate are the forward reaction to form L-Ala-III
(shown by large green arrow) and the reversible back reactions
(shown by orange arrows) to form both enantiomers of Ala-II.
No racemization of the stereocenter of the amino acid was
observed from mixing enantiopure alanine Ala-III with D-
ribose, and no formation of intermediate Ala-II-A or
aminoamide Ala-II was observed. Together with the observed
preference for L-Ala-III-B and D-Ala-II-A intermediates in the
reaction of Scheme 1 starting from the rac-aminonitrile, these
results suggest that the step from Ala-II-A to Ala-III-B is
Figure 4b shows that Ala-II is formed with a D-excess and
Ala-III is formed with a larger L-excess of ca. 70% ee, also
consistent with our studies of the reaction initiated from the
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Figure 5. Reaction coordinate for the hydrolysis of Ala-II mediated by D-ribose and NaOH. ΔG values are in kcal/mol calculated with the B3LYP-
D3BJ functional (T = 298 K). L- and D- labels refer to the alanine amide and alanine stereochemistry.
irreversible and is rate-determining, as depicted in Scheme 2.
In addition, these results demonstrate the increased complexity
of the reaction mechanism beyond a conventional competitive
kinetic resolution network, which helps to rationalize the
observed enantioenrichment of the amino acid L-Ala-III.
Enantioselectivity to the L-amino acid is achieved not only
because L-Ala-II-A hydrolyzes faster than D-Ala-II-A but also
because the reverse reaction of both intermediates to reform
the aminoamide Ala-II occurs equally as readily as the
dominant irreversible hydrolysis to L-Ala-III through Ala-III-
B, which is enantio-determining (Scheme 3).
The implication of these results is that a large portion of the
pool for potential D-amino acid remains sequestered for an
extended period of time in an equilibrium between the sugar-
amide intermediates and the aminoamide D-Ala-II. The ability
to exploit such differential reactivity in a prebiotic chemistry
scenario will depend on whether a means of engaging the L-
enantioenriched amino acid in further productive reactions, for
example peptide formation, and removing it from the
environment occurs prior to the ultimate production of
significant amounts of the opposite enantiomer from the D-
intermediate reservoir.
Although these relative rates of Ala-II and Ala-III formation
explain the observed results, it is not immediately clear from
study of the structures of the intermediate species Ala-II-A and
Ala-III-B how stereoselectivity is achieved in this reaction,
given that the sole stereochemical element differentiating the L
and D amino acid products is a single methyl group on the
linear side chain of the cyclic D-aminosugar. We turned to
DFT20 calculations to gain insight into this process, in
particular the steps between Ala-II and Ala-III.18 Figure 5
shows the reaction coordinate. The first step of the mechanism
from Ala-II is the formation of the Schiff base 1 (after coupling
of Ala-II with the linear form of the sugar D-ribose, with the L-
diastereomer found to be slightly more stable than the D by 0.9
kcal/mol. Intermediate 2 is stabilized by an interaction
between 1 and Na+ by ca. 2 kcal/mol compared to the
absence of the cation. The sugar moiety then cyclizes into the
furanose or the more stable pyranose form to give the
intermediate Ala-II-A (denoted in the model as 3). Further
discussion here addresses the pyranose form.21 These cyclic
species then form the diastereomers of Ala-III-B (denoted in
the model as 5), which are significantly more stable than Ala-
II-A by ca. 16−19 kcal/mol. The step from Ala-II-A to Ala-III-
B, which eliminates NH3, is clearly irreversible, while the step
from Ala-III-B to Ala-III is reversible but exhibits a significant
free energy barrier.
The highest barriers are those for the transitions from Ala-II-
A to Ala-III-B and from Ala-III-B to Ala-III. The α-forms of
each species exhibit higher barriers than the β-forms for both
reactions. The transition from the β-forms of Ala-III-B to Ala-
III exhibit significantly lower barriers, with L-(β) being 1.4
kcal/mol lower than D-(β). Figure 6 shows transition state
structures for two of the diastereomers of Ala-III-B, where
different solvation shells exist around Na+ in the different
structures to accommodate the different shape of the α-like
and β-like structures. This explicit solvation has been
performed for every structure included into the reaction
mechanism (see SI for more details).
The barriers for each anomer in these two steps are
compared in Figure 7. The small energy difference between the
transition states for L-(β) and D-(β) forms of Ala-III-B
contributes to enantioselectivity for the L-Ala-III product. The
experimentally measured difference in concentration of these
two species shown in Figure 3, with the L-(β) form nearly 4-
fold higher in concentration than the D-(β) form, also serves to
enhance enantioselectivity.
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microkinetic steps include all those shown in the calculated
reaction coordinate of Figure 5, moving in both the forward
and reverse directions from Ala-II-A. Because we observe
decay of ribose over time, we include an irreversible step slowly
removing ribose from the network over time after its release
from steps forming Ala-II and Ala-III. Comparing the temporal
concentration profiles of the four diastereomers of Ala-II-A
observed experimentally (Figure 8a) with those obtained from
Figure 6. Transition state structures TS4αD (top) and TS4βL
(bottom), including solvation water molecules, optimized using
B3LYP-D3BJ functional in the reaction network shown in Figure 5.18
Color scheme: C = green; O = red; N = blue; H = white; Na+ = violet.
Figure 8. Comparison of temporal reaction progress of the reaction of
the four diastereomers of the species Ala-II-A (a) experimental data
from the reaction shown in Figure 4a; (b) microkinetic model carried
out at 0.187 M species Ala-II-A, 0.187 M NaOH, 55 M H2O, with
elementary steps as in Figure 5 and rate constants estimated from the
free energy calculations of Figure 5.18 L- and D- labels refer to the
alanine amide and alanine stereochemistry.
Figure 7. Free energy barriers for the transition states from Ala-II-A
to Ala-III-B (blue bars) and from Ala-III-B to Ala-III (red bars) from
the energy diagram in Figure 5.18 L- and D- labels refer to the alanine
amide and alanine stereochemistry.
Because these differences in transition state free energies are
not large, and because the errors on DFT calculations of free
energies can be significant, we turned to microkinetic modeling
to gain further insight in the chiral discrimination in this
process.22 Microkinetic modeling can be informative because it
introduces the role of concentrations as driving forces in the
reaction coordinate. The free energy calculations in Figure 5
allow us to estimate rate constants for the elementary steps in
the mechanism. While the results of such calculations may not
predict absolute values (for example, an error of 1 kcal/mol
translates to ca. one order-of-magnitude error in a calculated
rate constant), such models can be used to substantiate and
predict temporal trends as well as to account for the role of
disparate substrate and intermediate concentrations.23 We
chose to model the reaction starting from the racemic
aminosugar intermediate Ala-II-A under conditions similar to
those of the experimental reactions shown in Figure 4. The
the microkinetic model (Figure 8b), we see that the model
captures the general trends. Species L-Ala-II-(β)-A and D-Ala-
II-(α)-A show the highest concentrations and L-Ala-II-(β)-A
shows the fastest rate of consumption, leading to the major L-
Ala-III product.
The rate and equilibrium constants derived from the
microkinetic model also offer more detailed insight into the
distribution and reactivity of the four diastereomers of D- and
L-Ala-II-A, as shown in Figure 9. The equilibrium concen-
trations of the L-diasteromers of both Ala-II-A and Ala-III-B
are shifted toward the β-form, while the D-diastereomers are
shifted toward the α-form. The diastereomers of Ala-II-A are
in rapid equilibrium from the outset of the reaction; by
contrast, exchange between the diastereomers of Ala-III-B
reaches equilibrium after ca. 5% conversion to Ala-III. This
finding of rapid equilibration between the anomers together
with the high barriers for the α-anomers of both D and L Ala-
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reactions that ultimately yield amino acids is highlighted. This
work reveals how the interplay between chiral sugars and
amino acid precursors in such a reaction network might
contribute to the emergence of biological homochirality under
plausible prebiotic conditions.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.1c03552.
■
sı
Experimental details, NMR spectra of compounds, NMR
temporal profiles, details of the computational DFT and
microkinetic models (PDF)
Figure 9. Equilibrium and rate constants for the interconversion of
diastereomeric intermediates Ala-II-A and Ala-III-B from the
microkinetic model shown in Figure 8. Species 2 and 6 refer to
Figure 5. Reactions of species 6 to form Ala-III are shown as the
dominant forward step.18 L- and D- labels refer to the alanine amide
and alanine stereochemistry.
AUTHOR INFORMATION
Corresponding Author
■
Donna G. Blackmond − Department of Chemistry, Scripps
Research, La Jolla, California 92037, United States;
orcid.org/0000-0001-9829-8375; Email: blackmond@
scripps.edu
III-B to form Ala-III shown in Figure 7 indicate that the
pathway to Ala-III is channeled through the β-anomers, which
are the major species on the L-side of the network but are the
minor species on the D-side. Ala-II-(β)-A reacts fastest in the
rate-determining step forming Ala-III-B.
Authors
Luca Legnani − Department of Chemistry, Scripps Research,
La Jolla, California 92037, United States
Andrea Daru − Department of Chemistry, Scripps Research,
La Jolla, California 92037, United States
̀
The equilibration of intermediate species in reservoirs as
Ala-II-A and Ala-III-B allows the relative concentrations of L-6
and D-6 to vary only slightly over the reaction time monitored
in Figure 8. This helps explain the relatively constant
enantiomeric excess of Ala-III observed in Figure 4b, which
is unlike a conventional kinetic resolution involving simple
parallel reactions. The full microkinetic model rate and
equilibrium constants are given in the Supporting Information.
The question of the provenance of the enantioenrichment in
ribose is not addressed in this work. However, our previous
work has demonstrated ways in which glyceraldehyde, the first
chiral sugar, may be enantioenriched by both chemical and
physical methods.13 Other work has shown how enantioen-
riched glyceraldehyde can lead to enantioenriched pentose
sugars under prebiotically relevant conditions.24
These studies complement our previous work that focused
on phenylalanine, where we observed enantioenrichment
toward the D-aminoamide but did not observe formation of
the amino acid. In that case, we postulated the formation of
five-membered cyclic intermediates in analogy to the
Bucherer−Bergs hydantoin synthesis. Our more detailed
NMR studies for the alanine system in our present studies
allow us to rule out those intermediates in favor of the six-
membered aminosugar species Ala-II-A and Ala−III-B shown
in Scheme 2 and Figure 2.
Alexander X. Jones − Department of Chemistry, Scripps
Research, La Jolla, California 92037, United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.1c03552
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
D.G.B. acknowledges funding from the Simons Foundation
under the Simons Collaboration on the Origins of Life (SCOL
287625). L.L. acknowledges the DFG for a research fellowship
(Project No. 422044447). Helpful discussions with Laura
Pasternack of the Scripps NMR Facility are acknowledged.
Discussions with Jinhan Yu and help in editing the manuscript
are acknowledged. Preliminary studies by A. J. Wagner are
acknowledged.
REFERENCES
■
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CONCLUSIONS
■
Detailed kinetic, spectroscopic, and computational investiga-
tions provide a deeper mechanistic understanding of the
ribose-catalyzed enantioselective Strecker synthesis of alanine.
Identification of two sets of aminosugar intermediates lead to
the proposal of a complex parallel/consecutive reaction
pathway. DFT calculations combined with microkinetic
analysis depict a reaction network with a significant reservoir
of intermediate species and reversibility in key steps, enabling
rationalization of the observed temporal enantioselectivity. The
role of the chiral sugar in mediating these diastereoselective
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