1. 1
Illuminating Biopolymer Origins through Ester-Amide
Exchange by Modeling Lactic Acid Polymerization
Catherine Psarakis, Rollins College Sheng-Sheng Yu, Dr. Martha Grover,
Georgia Institute of Technology
Introduction
The role of α-hydroxy acids in facilitating the
formation of the first peptides on Earth has been
of great interest in the study of chemical
evolution. Beginning with the electrical
discharge experiments conducted by Miller and
Urey, which demonstrated the formation of α-
hydroxy acids and amino acids from organic
molecules, numerous studies have focused on the
thermodynamic and environmental parameters
allowing for polymerization of these
monomers.1,2,3
In order to account for the ability of peptides to
polymerize under non-enzymatic conditions,
current research proposes an ester-amide
exchange mechanism, in which the
thermodynamic barrier of peptide bond
formation is overcome by the more favorable
creation of precursor ester bonds.2,4
Specifically,
Mamajanov et al. demonstrated a significantly
greater yield of oligoesters (L-malic acid) as
compared to oligopeptides under wet-dry
cycling, indicating the plausibility of esters
forming first.5
Ester-amide exchange involves an oligoester
undergoing a nucleophilic attack by the amine
group of a free amino acid to form a
depsipeptide, which is a compound containing a
mixture of ester and amide bonds. Wet-dry
cycling is crucial to ester-amide exchange,
simulating the environmental conditions
believed to have been present at interfacial
regions of land, sea, and air on primordial earth.
In theory, during the hot and dry daytime,
polymerization is facilitated by solvent
evaporation, while cold and wet conditions
during the night allow for water to be added back
into the reaction mixture so as to lower its
viscosity and push the reaction forward. The
ultimate purpose of cycling is to selectively
hydrolyze the less stable ester bonds to
preferentially build a peptide chain.4
The next phase in investigating the efficacy of
ester-mediated amide bond formation involves
determining the selectivity of certain oligoester
and oligopeptide structures from a variety of
starting hydroxy acids and amino acids available.
However, structural discrimination may only be
examined following detailed kinetic and
thermodynamic analyses of the polymerization
of specific hydroxy and amino acids separately
and then in combination.2
This study focuses on the polymerization of L-
lactic acid, an α-hydroxy acid with amino acid
analogue alanine. Besides its relevance in
evolutionary chemistry, polylactic acid (PLA)
has great industrial importance as a potential
replacement for non-biodegradable petroleum-
based synthetic polymers.6, 7
Additionally, PLA
may be a useful therapeutic delivery device due
to its ability to degrade non-toxically.8
It is important to understand the factors affecting
PLA hydrolysis so as to better model its
polymerization. Since hydrolysis may be
catalyzed by both acid and base, pH plays a
major role in the reaction, in addition to
molecular weight, temperature, and
stereochemistry.9,10
The synthesis of PLA may be accomplished
either by direct polycondensation of lactic acid
or by ring-opening polymerization of lactide.11
The Harshe et al. group has reported a kinetic
model for the former mechanism, detailing the
polycondensation of lactic acid in both closed
and open systems, and comparing the model with
experimental results obtained from gel
permeation chromatography (GPC).11
Following this study, Codari et al. have shown
the utility of high performance liquid
chromatography (HPLC) as an analytical method
to experimentally measure the polymerization
and hydrolysis of PLA as a function of its ability
to clearly separate oligomers based on their

2. 2
hydrophobicity and molecular weight.12
Furthermore, the Codari group has devised a
quantitative method to measure the concentration
of each oligomer within a sample of low
molecular weight (LW) PLA. Specifically, by
conducting hydrolysis studies of individual PLA
oligomers up to 10-mer eluted by fractional
HPLC, they were able to derive a constant
termed the “response factor” 𝑘𝑖 based on the
relationship between units (mmol) of oligomer
𝑛𝑖 and integral area 𝐴𝑖. They were then able to
establish linearity of this response factor, thus
extrapolating it to determine the degree of
polymerization (DP) for higher order oligomers.
It is with the Codari et al. calibration procedure
that the current investigation has analyzed the
polymerization of LW PLA.10,12
The purpose of this research was to contribute
towards the determination of the kinetic rate
constants for the polymerization of L-lactic acid.
The objectives included conducting hydrolysis
experiments with L-lactide at 85o
C (Scheme 1),
and from this data determining the response
factor allowing for the extrapolation of the DP
for LW PLA. HPLC was the primary analytical
technique, and was cross examined by 1
H-
NMR.13
Scheme 1. Lactide hydrolysis.
Polymerization reactions were conducted in a
closed system (Figure 1) to conserve the total
lactic acid units by preventing evaporation
during the drying process, and to promote the
formation of polymer from simple recycling of
lactic acid without external contribution.
Polymerization was carried out at several time
points (6, 18, 24, 42 hrs) in 85 o
C, and with
numerous experimental replicates.
Following extrapolation of the linear trend
characterizing the response factor for lactide
(cyclic dimer), linear dimer, and monomer, the
DP was found to increase as incubation time
increased. Conversely, the total lactic acid units
within the dry phase of the closed system
decreased as incubation time increased. It would
be beneficial in future studies to analyze the
amount of lactic acid present in the wet phase of
the reaction, as well as the polymerization at
other temperatures to ultimately characterize the
Arrhenius behavior for polymerization.
Experimental Methods
Hydrolysis Studies
(3S)-cis-3,6-Dimethyl-1,4-dioxane-2,5-dione
(L-lactide) was dissolved in a 20 vol % ACN, 80
vol % H2O solution and hydrolyzed at 85o
C.
HPLC was used to characterize the hydrolysis
reactions at several sequential time points.
Calculations were performed in Matlab to
determine the response factor 𝑘𝑖 of lactide, lactic
acid linear dimer, and monomer according to
direct proportionality with the units (mmol) of
oligomer 𝑛𝑖 and oligomer area 𝐴𝑖 as defined by
HPLC.
𝑛𝑖 = 𝑘𝑖 × 𝐴𝑖
Closed System Polymerization
100mM L-lactic acid was incubated at 85 o
C in a
closed system at multiple time points (6, 18, 24,
42 hrs). Following dry-down in the closed
system, samples were dissolved in 20 vol %
ACN-d3, 80 vol % D2O in preparation for HPLC
and 1
H-NMR.
Figure 1. Closed System Reactor.

3. 3
Polymer Characterization by HPLC
Polymers were separated based on
hydrophobicity and molecular weight by reverse-
phase chromatography on an Agilent 1290 series
apparatus (Agilent, USA) equipped with UV
detector set at 210 nm and column oven
temperature kept at 40 o
C. The mobile phase
was comprised of a mixture of water acidified
with phosphoric acid (0.1%) and LC-MS grade
Acetonitrile (OmniSolv) flowing at 0.5 ml ∙
min−1
. The gradient profile is displayed in
Table 1.
Table 1. HPLC Gradient for Polymer
Characterization.
Polymer Characterization by 1
H-NMR
LW PLA samples were characterized on a Varian
(400MHz) spectrometer (Varian, NMR
Instruments, Palo Alto, CA). Chemical shifts
were given in ppm relative to benzoic acid at 7.7
ppm.
Results and Discussion
HPLC of lactide before hydrolysis (0 min)
confirmed a pure sample of cyclic dimer, while
the linear dimer and monomeric forms became
increasingly abundant as lactide was hydrolyzed
for 40 and 100 minutes (Figure 2).
a
b
c
Figure 2. HPLC of lactide (18.8mg) hydrolysis
at (a) 0, (b) 40, and (c) 100 minutes.
For each oligomer species, the response factor 𝑘𝑖
of the HPLC in relation to the moles n and
integral area 𝐴𝑖 was calculated in Matlab using a
system of linear equations in the form below:
𝑘1 𝐴1 + 2𝑘2 𝐴2 + 2𝑘 𝑐2 𝐴 𝑐2 = 𝑛

4. 4
The reciprocals of the individual response factors
𝑘1, 𝑘2 and 𝑘 𝑐2 were then plotted as a function of
their chain lengths i to yield a linear trend with
the slope representing the general response factor
𝑘𝑖 (Figure 3).
Figure 3. Response factor attained from lactide
hydrolysis.
Following hydrolysis experiments, HPLC was
used to characterize polymerization conducted in
the closed system (Figure 4). Using the oligomer
integral areas provided by HPLC and the
extrapolated response factor for each oligomer,
the DP as well as the total lactic acid units n in
the dry phase of the system were calculated.
1
𝑘 𝑖
= 6 × 107
𝑖  𝑘𝑖 =
1.67 × 10−8
𝑖
∙
𝑚𝑚𝑜𝑙
𝑚𝐴𝑈∙𝑠𝑒𝑐
𝑛𝑖 = 𝑘𝑖 × 𝐴𝑖
𝑫𝑷 𝑯𝑷𝑳𝑪 =
𝛴(𝑛𝑖 × 𝑖)
𝛴(𝑛𝑖)
𝒏 𝑯𝑷𝑳𝑪 = 𝛴(𝑛 × 𝑖) ×
600𝜇𝑙 𝑡𝑜𝑡𝑎𝑙 𝑠𝑎𝑚𝑝𝑙𝑒 𝑣𝑜𝑙𝑢𝑚𝑒
1.5𝜇𝑙 𝑖𝑛𝑗𝑒𝑐𝑡𝑖𝑜𝑛 𝑣𝑜𝑙𝑢𝑚𝑒
1
H-NMR data (Figure 5) was also utilized to
determine the DP based on functional group
integral areas A (Figure 6).13
𝑫𝑷1
H-NMR =
𝐴 𝑏𝑎𝑐𝑘𝑏𝑜𝑛𝑒
𝐴 𝑒𝑛𝑑
+ 1
The total lactic acid units n were calculated from
1
H-NMR by first determining the concentration
𝑀 of lactic acid methyl groups as a ratio to the
concentration of benzoic acid with respect to the
relationship between concentration, peak
intensity I, and number of protons N.
𝐼𝐿𝐴−𝐶𝐻3
𝐼 𝐵𝑒𝑛𝑧𝑜𝑖𝑐 𝐴𝑐𝑖𝑑
=
𝑀𝐿𝐴−𝐶𝐻3
𝑁𝐿𝐴−𝐶𝐻3
𝑀 𝐵𝑒𝑛𝑧𝑜𝑖𝑐 𝐴𝑐𝑖𝑑 𝑁 𝐵𝑒𝑛𝑧𝑜𝑖𝑐 𝐴𝑐𝑖𝑑
The concentration was then converted to moles
of total lactic acid units.
𝒏1
H-NMR = 𝑀𝐿𝐴𝐶𝐻3
× 600𝜇𝑙 𝑡𝑜𝑡𝑎𝑙 𝑠𝑎𝑚𝑝𝑙𝑒 𝑣𝑜𝑙𝑢𝑚𝑒
Figure 4. HPLC of 24hr closed system lactic acid polymerization.

5. 5
Figure 5. 1
H-NMR of 24hr closed system lactic acid polymerization corresponding with polylactic acid
functional groups.13
Figure 6. 1
H-NMR peak assignments
corresponding with polylactic acid molecular
structure.13
The results for DP and total lactic acid units were
compared between HPLC and 1
H-NMR. The two
methods demonstrated consistency, with 9.87%
maximum error for DP and 15.00% maximum
error for total lactic acid units (Figure 7, Figure 8).
Figure 7. Degree of polymerization of lactic acid
in closed system experiments.
Figure 8. Total units of lactic acid (mmol) in dry
phase of closed system experiments.
Conclusions
The purpose of this research was to contribute to
the determination of the kinetic rate constants for
lactic acid polymerization. Within the timeframe
of the project, hydrolysis and polymerization
experiments were conducted and used to confirm
the efficacy of closed system lactic acid polymer
regeneration and to determine the DP. Further
study will involve comparing experimental results
with a theoretical computer simulation to attain
the kinetic rate constants for polymerization and
hydrolysis at 85 o
C and at other temperatures. This
work will ultimately be utilized in modeling
polymer formation via the ester-amide exchange
mechanism from a starting array of α-hydroxy
acids and amino acids.

6. 6
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Acknowledgments
This work was jointly supported by NSF and the
NASA Astrobiology program under the NSF
Center for Chemical Evolution, CHE-1004570.
Catherine Psarakis
I am a rising senior at Rollins College in Winter
Park, FL double-majoring in chemistry and music,
and plan to pursue graduate studies.