The opposite effect of K+ and Na+ on the hydrolysis of linear and cyclic dipeptides

Article abstract of DOI:10.1016/j.tetlet.2018.04.073

Potassium and sodium are generally considered inert ‘spectator’ ions for organic reactions. Here, we report rate constants for the acid-promoted hydrolysis of the seven dipeptides of glycine (G) and alanine (A) and an unexpected pattern in how these rates differ in the presence of K⁺ and Na⁺. The linear dipeptides hydrolyze 12–18% percent slower in the presence of KCl versus an equal concentration of NaCl, while the cyclic dipeptides hydrolyze 5–13% faster in the presence of KCl (all P-values < 0.025). We believe this is the first report of a general organic reaction—here, amide hydrolysis—for which some substrates react faster in the presence of K⁺ and others in Na⁺. The results offer a potential reason for life's mysterious universal selection of intracellular potassium over sodium.

Full text of DOI:10.1016/j.tetlet.2018.04.073

Accepted Manuscript
The opposite effect of K+ and Na+ on the hydrolysis of linear and cyclic dipep-
tides
Thomas D. Campbell, Clara A. Hart, Rio Febrian, Mark L. Cheneler, Paul J.
Bracher
PII:
S0040-4039(18)30559-8
https://doi.org/10.1016/j.tetlet.2018.04.073
TETL 49936
DOI:
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
10 March 2018
25 April 2018
27 April 2018
Please cite this article as: Campbell, T.D., Hart, C.A., Febrian, R., Cheneler, M.L., Bracher, P.J., The opposite effect
of K+ and Na+ on the hydrolysis of linear and cyclic dipeptides, Tetrahedron Letters (2018), doi: https://doi.org/
10.1016/j.tetlet.2018.04.073
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Thomas D. Campbell, Clara A. Hart, Rio Febrian, Mark L. Cheneler, Paul J. Bracher

1
Tetrahedron Letters
The opposite effect of K+ and Na+ on the hydrolysis of linear and cyclic dipeptides
Thomas D. Campbella, Clara A. Harta, Rio Febriana, Mark L. Chenelera, and Paul J. Brachera
a Department of Chemistry, Saint Louis University, St. Louis, MO 63103 U.S.A.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Potassium and sodium are generally considered inert ‘spectator’ ions for organic reactions. Here,
we report rate constants for the acid-promoted hydrolysis of the seven dipeptides of glycine (G)
and alanine (A) and an unexpected pattern in how these rates differ in the presence of K+ and
Na+. The linear dipeptides hydrolyze 12–18% percent slower in the presence of KCl versus an
equal concentration of NaCl, while the cyclic dipeptides hydrolyze 5–13% faster in the presence
of KCl (all P-values < 0.025). We believe this is the first report of a general organic reaction—
here, amide hydrolysis—for which some substrates react faster in the presence of K+ and others
in Na+. The results offer a potential reason for life’s mysterious universal selection of
intracellular potassium over sodium.
Available online
Keywords:
Peptides
Potassium
Prebiotic Chemistry
Origin of Life
2018 Elsevier Ltd. All rights reserved.
———
 Corresponding author. e-mail: paul.bracher@slu.edu

2
Tetrahedron
1. Introduction
the hydrolysis of peptides. And if we wish to understand the
potential generation of protein biopolymers from the coupling of
amino acids in water, we will have to understand the influence of
hydrolysis as a competing deleterious side reaction.21
While all living systems on Earth enrich K+ from their
external environments—and the vast majority of cells have
higher intracellular concentrations of K+ than Na+—there exists
no definitive explanation for this preference.1-3 Features that are
ubiquitous in biology are likely to have evolved early in the
development of life, perhaps as early as the first living system.4
Examples of potential biochemical ‘fossils’ from life’s origin that
have been the focus of origin-of-life research include lipid
membranes,5, 6 nucleic acids,7, 8 proteins/polypeptides,9-11 and
common metabolic cycles.4, 12, 13
Selection of Substrates for Study. We chose to focus our initial
work on dipeptides in order to begin with the simplest system
available. While a single amino acid can dimerize into only two
dipeptides—the linear and cyclic forms—there are seven
permutations when selection is expanded to two amino acids. The
obvious candidates for study were dipeptides of glycine and
alanine. Glycine (G) is the simplest amino acid. It is the smallest
by mass, has no functional groups on its side chain, and is
Another ubiquitous feature of life is the maintenance of ion
gradients across cellular membranes.14 In modern organisms, K+
and Na+ ion gradients are critical for a number of functions.14, 15
The high concentration of potassium in cells has led some to
hypothesize that life may have developed in an environment with
high levels of potassium.3, 16 However, the observation that cells
can expend up to a third of their energy budget on Na+/K+-
ATPase suggests the intracellular enrichment of potassium is
important itself—not just a vestigial remnant of prebiotic
conditions that would have faded as life evolved.17 Still, the role
of intracellular K+ is not obvious.18 Why did life choose K+ when
it could seemingly use intracellular Na+ for the same purpose?
Few non-biological systems exist in which identical
concentrations of potassium or sodium considerably change the
properties of the system. Salts of K+ and Na+ often have different
solubilities, and the presence of equal concentrations of KCl and
NaCl can affect the solubility of organic compounds in aqueous
solutions.19 The only study we are aware of that reports a notable
difference for an organic reaction in water in the presence of K+
Scheme 1. Cyclic dipeptides—thought to be “dead ends” in the prebiotic
synthesis of polypeptides—hydrolyse to form linear dipeptides. In turn, these
acyclic compounds hydrolyse to form free amino acids. In our experiments,
we examined the hydrolysis of the seven dipeptides formed from glycine (G,
R = H) and alanine (A, R = CH3) at 70 °C in 4 m KCl or NaCl, promoted by 1
m HCl.
and Na+ is for the coupling of glutamic acid mediated by
carbonyldiimidazole.20 Natochin and coworkers observed higher
yields of oligo(glutamic acid) formation in the presence of K+
versus Na+. Motivated by this report and the premise that
universal features of biochemistry may have been important to
the origin of life, we decided to measure the influence of K+ and
Na+ on reactions of peptides.
2. Experimental Design
In selecting conditions for model prebiotic reactions, decisions
must be made to balance historical relevance with experimental
convenience. This section explains our choices regarding the
design of experiments and any simplifications or assumptions
that went into these decisions.
Selection of Reaction for Study. We elected to examine amino
acids and peptides for their (i) obvious relevance to biology, (ii)
historical interest to prebiotic chemistry, and (iii) well-studied
structure and reactivity.9 In aqueous solution, equilibrium favors
Figure 1. Example measurement of the rate constant for the acid-promoted
hydrolysis (ka) of a dipeptide. The data shown correspond to the hydrolysis
of alanylalanine (AA) under pseudo-first-order conditions, where [H+] is
roughly constant. Aliquots of the reaction (A) are sampled at timed intervals
and analyzed by NMR (B). The rate constant is determined from the slope of
plot (C). Seven replicates of this experiment were completed for each of the
seven dipeptide substrates.

3
achiral. Alanine (A) is the next smallest amino acid by mass and
has only a relatively inert methyl group for a side chain. G and A
also appear to be the amino acids most relevant to prebiotic
chemistry. A multi-factor analysis of 60 criteria by Trifonov
concluded that G and A were the first two dominant amino acids
in the chronology of evolution.22 G and A are synthesized in the
highest yields in most reported prebiotic syntheses of amino
acids, including spark-discharge experiments.9, 23 G and A are
also routinely the most abundant amino acids found on potential
extraterrestrial impactors like meteorites, asteroids, and comets,
which may have seeded early Earth from space.11
the ‘drying lagoon’ model for prebiotic chemistry in which
condensation and hydrolysis reactions transpire in trapped,
evaporating bodies of water on Prebiotic Earth.34 While the
hydrolysis of amides at room temperature and neutral pH is
notoriously slow,31 conveniently, our hydrolysis experiments
were complete in hours to days under these conditions.
While essentially no data exist regarding microenvironments
on early Earth—including the possibility of environments that
mimic the high acidity and heat of our study—there exists both
geological and biological evidence that the early Earth was
generally hotter and more acidic than today.32, 33 Several
thermoacidophilic microorganisms are known to thrive in very
acidic microenvironments on modern Earth, including those of
the genus Picrophilus, which can grow near pH 0 and up to 65
°C.35 The ability of these archaea to thrive in inhospitable
environments thought reminiscent of the hot, acidic, volcanic
settings on the early Earth suggests these organisms may be
“primordial relics from which more complex life evolved.”35
Cyclic dipeptides (2,5-diketopiperazines or DKPs, see Scheme
1) are of special relevance to origin-of-life chemistry as
potentially important species in the construction and destruction
of oligopeptides. Although conditions have been reported where
direct attack of DKPs by the free amine of an amino acid can
extend a growing peptide chain by two residues,24 the formation
of DKPs via intramolecular attack of the peptide backbone is a
principal degradation route of short oligopeptides.10, 25 The
absence of free amino and carboxyl groups in DKPs limit their
participation in peptide coupling reactions unless they first
hydrolyze, so DKPs are often invoked as “dead ends” or traps for
amino acids that remove them from a pool of molecular building
blocks.26 High yields of DKPs—to the detriment of linear
peptides—from amino acid precursors have been reported from a
variety of experiments, including those with simulated prebiotic
conditions.25, 27
There is also a theory that life evolved in an acidic, highly saline
environment because the presumed “prebiotic set” of amino acids
lacked amino acids with aromatic and basic side chains, and
peptides composed of residues from this set likely restricted the
possibility of folding to acidic and saline environments.11, 36
We previously used NMR spectroscopy to measure rate
constants for the hydrolysis of thioesters as a function of pH.37
Here, we measure pseudo-first-order rate constants for the acid-
promoted hydrolysis (ka) of each substrate at [HCl] = 1 m and
[KCl] or [NaCl] = 4 m. We define the starting material to have
hydrolyzed after any net hydrolysis of peptide bonds is observed.
A typical kinetics experiment involved removing aliquots of the
reaction mixtures at various time points, diluting them with an
equal volume of deuterium oxide, and collecting an 1H NMR
spectrum of the sample. The relative concentration of hydrolyzed
substrate was determined by comparing the integration values of
signals corresponding to the reactants and products (Figure 1).
Experimental details are provided as Supporting Information.
Selection of Reaction Conditions. It is always preferable to
match experimental conditions to those presumed to exist in the
prebiotic landscape, but some concessions must be made for
experimental convenience. We selected a temperature of 70 C
for the reactions to match the temperature of the Archean ocean
inferred from geochemical analysis of chert minerals that date to
~3.5 Ga,28 though that analysis and the question of the
temperature of the early ocean are the subject of great debate.
The concentration of the substrate was set to 50 millimolal (mm)
and the concentration of salt was set to 4 molal (m). This
concentration of salt allowed us to observe the maximum effect
of the ions on the rate of hydrolysis, as 4 m approaches the limit
of solubility of KCl and NaCl at 70 °C. We report components
with relatively high concentration in molality to maintain
consistent ionic strength and concentration (i.e., 4 M NaCl and 4
M KCl are not the same ionic strength, while 4 m NaCl and 4 m
KCl are the same ionic strength).29 The addition of 1 m HCl
allowed reasonable reaction rates, resulted in pseudo-first-order
kinetics,30, 31 and is more prebiotically relevant than alkaline
conditions.32, 33 While high concentrations of acid and salts are of
limited pertinence to extant biology, they are directly relevant to
3. Results and Discussion
The histograms in Figures 2a and 2b depict the rates of
hydrolysis for the seven dipeptides of G and A in 1 m HCl at 70
°C in the presence of 4 m KCl, 4 m NaCl, or no added alkali salt.
We compared the rates for each substrate in 4 m KCl vs. 4 m
NaCl with two-tailed t-test hypothesis testing and found all seven
differences to be statistically significant with P < 0.025. The
linear dipeptides all hydrolyzed slower in KCl (vs. NaCl), while
the reverse was observed for the cyclic dipeptides. This stark
‘flip’ in relative rates based on structure is distinctly apparent in
Figure 2c.

4
Tetrahedron
The hydrolysis of peptides and other amides has been
ions and dipeptides.41 We were not able to observe any such
studied extensively, especially in strongly acidic solutions at
elevated temperatures, as is the case in our study.30, 31, 38 The rate-
determining step of the acid-promoted mechanism is the water-
assisted attack of water on the protonated carbonyl group of the
amide undergoing hydrolysis.38 The most straightforward
interactions by changes in chemical shift (Δδ) or relaxation delay
times (ΔT1) of α-protons and methyl protons in the presence of
KCl vs. NaCl (see Supporting Information). Specific ion
interactions and effects on biological molecules have been
extensively studied and are notorious for being poorly
Figure 2. (A) Histograms of the measured rate constants (k) for the acid-promoted hydrolysis of each linear dipeptide in the presence NaCl (navy), KCl
(green), and the absence of additional salt (grey). (B) Analogous histograms for the cyclic dipeptides. (C) Histogram of the difference in k-values in KCl (kK)
vs. NaCl (kNa) divided by their average for each substrate. Upward bars correspond to substrates that hydrolyse faster in KCl than NaCl. Downward bars
correspond to substrates that hydrolyse faster in NaCl than KCl. The error bars in the first two histograms represent 90% CIs based on seven measurements.
P-values are indicated with asterisks (* – P ≤ 0.025; ** – P ≤ 0.01, *** – P ≤ 0.001).
explanation for the observed differences in rate would arise from
differences of the thermodynamic activities of one or more of the
molecules involved in the rate-determining step (water, proton,
and substrate) caused by K+ and Na+.
understood. This problem has existed since the 19th century,
when Hofmeister described the impact of salts on the solubility
of proteins.42 Current models are generally inadequate at
rationalizing the observed phenomena, and our results follow in
this longstanding, frustrating tradition.43, 44 Experiments to
uncover the mechanistic underpinnings of these initial results are
ongoing.
For equal concentrations of chloride salts in aqueous solution,
the activity of water is lower in NaCl vs. KCl (aw = 0.85 in 4 m
NaCl vs. 0.87 in 4 m KCl at 25 °C).39 While this difference in
water activity could ostensibly explain the faster hydrolysis of
the cyclic substrates in KCl vs. NaCl, such an explanation is at
odds with the observation that the linear dipeptides hydrolyzed
slower in KCl.
4. Conclusion
In summary, we report a general organic reaction—here,
peptide hydrolysis—for which the relative rates not only differ in
the presence of K+ vs. Na+, but straddle unity, as cyclic substrates
react faster in the presence of K+ but linear substrates are faster in
the presence of Na+. The observed linear/cyclic flip raises a
possible explanation for why the earliest living systems may have
selected K+ over Na+: to assist the synthesis of oligopeptide
chains by favoring increased rates of hydrolysis of DKPs and
decreased rates of hydrolysis of linear peptides. In the presence
of potassium versus sodium, dead-end DKPs are more rapidly
returned to the pool of reactive linear dipeptides, while linear
dipeptides are more slowly broken down into monomers. The
coupling of amino acids into dipeptides and longer polypeptides
represents an increase in molecular complexity. While the
differences in rate in the presence of K+ versus Na+ are modest,
they are statistically significant. And when given millions of
years, small differences in rates of hydrolysis could have had a
profound influence on the development of complexity on the
Prebiotic Earth.
In fact, this “flip” in the relative rates is a useful observation
for several reasons with regard to elucidation of the
mechanism(s) for dipeptide hydrolysis in the presence of the
salts. The flip rules out that the difference is simply due to effects
of the cations on the reactivity of water alone. While the activity
of water must contribute to the observed differences in rate, there
must be at least one other effect at play, else all the peptides—
cyclic and linear—would hydrolyze faster in the presence of the
same cation.
Proton/hydronium activities are particularly important, and
they are known to vary significantly in aqueous solutions of
different salts. The mean activity coefficient of HCl in 2 m KCl is
0.8 and in 2 m NaCl is 1.4.40 So, proton activity is higher in
concentrated NaCl vs. KCl, while water activity is higher in
concentrated KCl vs. NaCl.
If the rate-determining step for the hydrolysis in the present
system (at 1 m HCl, 4 m MCl, and 70 °C) happened to be in a
regime where water activity were dominant for one substrate and
proton activity were dominant for the other, the results could be
explained. But kinetics studies showed both the linear and cyclic
dimers to have rates first order in proton for hydrolysis in KCl
and NaCl (Figures S12 and S13).
It would be remarkable if the K+ and Na+ ions were directly
involved in the mechanism(s) through binding to the substrates,
as alkali cations are generally considered unreactive. But there
are precedents for differential binding interactions between alkali
Acknowledgments
This work was funded by Saint Louis University and the
NASA EPSCoR Missouri Research Infrastructure Development
Program funded through NASA Grant Cooperative Agreement
Number NNX15AK38A. T.D.C. is supported by NASA
Headquarters under the NASA Earth and Space Science
Fellowship Program (Award #80NSSC17K0521). T.D.C. and
R.F. have also been supported by the NSF and the NASA
Astrobiology Program, under the NSF Center for Chemical
Evolution (CHE-1504217). We are grateful to Mathew T.

5
36. L. M. Longo, J. Lee and M. Blaber, Proc. Natl. Acad. Sci. USA,
2013, 110, 2135-2139.
Graham, Dr. Wenbin Luo, and Dr. Fahu He for helpful
discussions and technical assistance.
37. P. J. Bracher, P. W. Snyder, B. R. Bohall and G. M. Whitesides,
Orig. Life Evol. Biosph., 2011, 41, 399-412.
38. R. S. Brown, A. J. Bennet and H. Ślebocka-Tilk, Accounts Chem.
Res., 1992, 25, 481-488.
References and notes
1. H. B. Steinbach, Perspect. Biol. Med., 1962, 5, 338-355.
2. E. M. Galimov, Y. V. Natochin, B. N. Ryzhenko and E. V.
Cherkasova, Geochem. Int., 2012, 50, 1048-1068.
3. A. Y. Mulkidjanian, A. Y. Bychkov, D. V. Dibrova, M. Y.
Galperin and E. V. Koonin, Proc. Natl. Acad. Sci. USA, 2012, 109,
E821-E830.
4. H. J. Morowitz, Complexity, 1999, 4, 39-53.
5. D. Deamer, J. P. Dworkin, S. A. Sandford, M. P. Bernstein and L.
J. Allamandola, Astrobiology, 2002, 2, 371-381.
6. M. M. Hanczyc and J. W. Szostak, Curr. Opin. Chem. Biol., 2004,
8, 660-664.
39. M. El Guendouzi, A. Dinane and A. Mounir, J. Chem.
Thermodyn., 2001, 33, 1059-1072.
40. H. S. Harned and B. B. Owen, The Physical Chemistry of
Electrolytic Solutions, 3rd ed., Reinhold Publishing Corporation,
New York, 1958.
41. M. T. Rodgers and P. B. Armentrout, Chem. Rev., 2016, 116,
5642-5687.
42. Y. Zhang and P. S. Cremer, Curr. Opin. Chem. Biol., 2006, 10,
658-663.
43. P. Lo Nostro and B. W. Ninham, Chem. Rev., 2012, 112, 2286-
2322.
44. P. Jungwirth and P. S. Cremer, Nat. Chem., 2014, 6, 261-263.
7. G. F. Joyce, Nature, 2002, 418, 214-221.
8. M. Neveu, H.-J. Kim and S. A. Benner, Astrobiology, 2013, 13,
391-403.
9. B. M. Rode, Peptides, 1999, 20, 773-786.
10. G. Danger, R. Plasson and R. Pascal, Chem. Soc. Rev., 2012, 41,
5416-5429.
11. L. M. Longo and M. Blaber, Arch. Biochem. Biophys., 2012, 526,
16-21.
Highlights
12. A. Brack, ed., The Molecular Origins of Life, Cambridge
University Press, Cambridge, 1998.
13. B. H. Patel, C. Percivalle, D. J. Ritson, C. D. Duffy and J. D.
Sutherland, Nat. Chem., 2015, 7, 301-307.



Linear dipeptides hydrolyze faster in the
presence of NaCl vs. KCl
Cyclic dipeptides hydrolyze faster in the
presence of KCl vs. NaCl
14. N. Lane and W. F. Martin, Cell, 2012, 151, 1406-1416.
15. F. Lang, G. L. Busch, M. Ritter, H. Volkl, S. Waldegger, E.
Gulbins and D. Haussinger, Physiol. Rev., 1998, 78, 247-306.
16. D. V. Dibrova, M. Y. Galperin, E. V. Koonin and A. Y.
Mulkidjanian, Biochem.-Moscow, 2015, 80, 495-516.
17. L. D. Faller, Arch. Biochem. Biophys., 2008, 476, 12-21.
18. M. Barbieri, The Organic Codes: An introduction to semantic
biology, Cambridge University Press, Cambridge, UK, 2003.
19. W.-H. Xie, W.-Y. Shiu and D. Mackay, Mar. Environ. Res.,
1997, 44, 429-444.
Life’s preference for K+ over Na+ may have
supported the growth of polypeptides
20. M. V. Dubina, S. Y. Vyazmin, V. M. Boitsov, E. N. Nikolaev, I.
A. Popov, A. S. Kononikhin, I. E. Eliseev and Y. V. Natochin, Orig.
Life Evol. Biosph., 2013, 43, 109-117.
21. S. A. Benner, Orig. Life Evol. Biosph., 2014, 44, 339-343.
22. E. N. Trifonov, J. Biomol. Struct. Dyn., 2004, 22, 1-11.
23. S. L. Miller, Science, 1953, 117, 528–529.
24. M. Nagayama, O. Takaoka, K. Inomata and Y. Yamagata, Orig.
Life Evol. Biosph., 1990, 20, 249-257.
25. S. M. Steinberg and J. L. Bada, J. Org. Chem., 1983, 48, 2295-
2298.
26. M. A. Grover, C. Y. He, M.-C. Hsieh and S.-S. Yu, Processes,
2015, 3, 309-338.
27. S. Steinberg and J. L. Bada, Science, 1981, 213, 544-545.
28. L. P. Knauth and D. R. Lowe, Geol. Soc. Am. Bull., 2003, 115,
566-580.
29. T. Solomon, J. Chem. Educ., 2001, 78, 1691-1692.
30. L. Lawrence and W. J. Moore, J. Am. Chem. Soc., 1951, 73,
3973-3977.
31. R. M. Smith and D. E. Hansen, J. Am. Chem. Soc., 1998, 120,
8910-8913.
32. R. Perez-Jimenez, A. Ingles-Prieto, Z. M. Zhao, I. Sanchez-
Romero, J. Alegre-Cebollada, P. Kosuri, S. Garcia-Manyes, T. J.
Kappock, M. Tanokura, A. Holmgren, J. M. Sanchez-Ruiz, E. A.
Gaucher and J. M. Fernandez, Nat. Struct. Mol. Biol., 2011, 18, 592-
596.
33. T. Ushikubo, N. T. Kita, A. J. Cavosie, S. A. Wilde, R. L.
Rudnick and J. W. Valley, Earth Planet. Sci. Lett., 2008, 272, 666-
676.
34. K. E. Nelson, M. P. Robertson, M. Levy and S. L. Miller, Orig.
Life Evol. Biosph., 2001, 31, 221-229.
35. C. Baker-Austin and M. Dopson, Trends Microbiol., 2007, 15,
165-171.