Photoinitiated synthesis of self-assembled vesicles

Article abstract of DOI:10.1021/ja5006256

The aqueous photochemistry of 2-oxooctanoic acid (a single-tailed surfactant) results in the synthesis of a double-tailed surfactant product followed by spontaneous self-assembly into vesicles. The photochemical mechanism is detailed here, and the reaction products are identified using mass spectrometry. Then, the self-assembled vesicles are characterized using dynamic light scattering, fluorescence microscopy, and NMR. Further, their stability over time and in the presence of MgCl₂ salt is demonstrated. This work contributes to membrane evolution through the provision of a prebiotic route for the synthesis of plausible membrane components and subsequent self-assembly of a primitive enclosure.

Full text of DOI:10.1021/ja5006256

Communication
pubs.acs.org/JACS
Photoinitiated Synthesis of Self-Assembled Vesicles
Elizabeth C. Griffith,†,‡ Rebecca J. Rapf,†,‡ Richard K. Shoemaker,† Barry K. Carpenter,§
and Veronica Vaida*,†,‡
†Department of Chemistry and Biochemistry, University of Colorado at Boulder, UCB 215, Boulder, Colorado 80309, United States
‡CIRES, UCB 215, Boulder, Colorado 80309, United States
§Physical Organic Chemistry Centre, Cardiff University, Cardiff CF10 3AT, United Kingdom
S
* Supporting Information
utes to understanding the evolution of primitive membrane
structures.
Figure 1 illustrates the photochemical mechanism resulting in
the production of the dimer molecule OOA-OOA (Figure 1A)
ABSTRACT: The aqueous photochemistry of 2-oxoocta-
noic acid (a single-tailed surfactant) results in the synthesis
of a double-tailed surfactant product followed by
spontaneous self-assembly into vesicles. The photo-
chemical mechanism is detailed here, and the reaction
products are identified using mass spectrometry. Then, the
self-assembled vesicles are characterized using dynamic
light scattering, fluorescence microscopy, and NMR.
Further, their stability over time and in the presence of
MgCl2 salt is demonstrated. This work contributes to
membrane evolution through the provision of a prebiotic
route for the synthesis of plausible membrane components
and subsequent self-assembly of a primitive enclosure.
he unique properties of aqueous solutions of aggregated
Tamphiphiles have been a subject of considerable interest
in a wide range of fields.1−4 In addition, enclosures are
universally considered necessary for life.5 The search for the
emergence of such primitive cellular constructs in the origin of
life on Earth requires the existence and/or synthesis of a
prebiotically plausible membrane component (a surfactant),
followed by self-assembly into a stable enclosed structure.6
Although much work has been performed studying the
properties of vesicles composed of various surfactants,7−10 the
synthesis of such self-assembled structures and their compo-
nents under plausible prebiotic conditions has received much
less attention. Here we show the photochemical synthesis of a
double-tailed surfactant from a single-tailed one followed by
spontaneous self-assembly into stable vesicles. The single-tailed
surfactant utilized, 2-oxooctanoic acid (2-OOA, an 8-carbon
oxo-acid), is a plausible prebiotic molecule: oxo-acids and other
molecules of similar chemical functionality have been found in
meteoritic samples,11,12 and the short, 8-carbon chain of 2-
OOA is among the most prevalent length synthesized in the
common Fischer−Tropsch type synthesis of lipids (C7−C9 are
most common).13,14 With no further perturbation of the
reaction system, as the photochemistry proceeds, the products
spontaneously self-assemble into stable vesicles. These vesicles
were found to be monodisperse (200 nm in diameter), to be
temporally (over several months) and thermally (between at
least 4 - 22 °C) stable, and to persist in the presence of MgCl2
salt. This work provides a potential photochemical route for the
synthesis of membrane components and a stable primitive
enclosure under prebiotically relevant conditions and contrib-
Figure 1. Photochemical synthesis of double-tailed surfactant (OOA-
OOA) from single-tailed surfactant (2-OOA). (A) Photochemical
reaction mechanism of 2-OOA to produce OOA-OOA dimer. (B)
Mass spectrum of photolysis products with peaks due to OOA-OOA
dimer (317.2 m/z) and unreacted 2-OOA (157.1 m/z) indicated.
as well as its detection by mass spectrometry (Figure 1B). 2-
OOA first absorbs light through its carbonyl chromophore with
an absorption maximum of 320 nm (Figure S1), resulting in the
production of the dimer molecule OOA-OOA (m/z 317.2 in
Figure 1B). Analogous to the well-known photochemistry of
pyruvic acid15−17 (which has the same reactive functionality as
2-OOA), the photoexcited 2-OOA molecule can react with a
ground-state 2-OOA molecule to efficiently form the radical
intermediate 2-OOA·. Then, two 2-OOA· radicals recombine to
form the OOA-OOA dimer. The products of photolysis, when
Received: January 20, 2014
Published: February 24, 2014
© 2014 American Chemical Society
3784
dx.doi.org/10.1021/ja5006256 | J. Am. Chem. Soc. 2014, 136, 3784−3787

Journal of the American Chemical Society
Communication
extracted into chloroform, were observed to be more surface
active than 2-OOA prior to photolysis using Langmuir trough
methods (Figure S2), confirming the greater hydrophobicity of
the photolysis products. In addition to the double-tailed dimer
OOA-OOA, another dimer product was detected at 247.1 m/z
in the mass spectrum (see Supporting Information for full
characterization and reaction mechanism, Figures S3 and S4).
This product is formed through a Norrish Type II photo-
chemical reaction mechanism, but remains as a single-tailed
surfactant with similar hydrophobicity to 2-OOA, and is thus
not discussed further in detail here.
In addition, the diffusion ordered spectroscopy (DOSY)
NMR (Figure 3) spectrum detects these particulates as being
Prior to exposure to light, the 2-OOA solution is clear and
devoid of detectable particles (image on left in Figure 2A and
Figure 3. DOSY NMR illustrating the diffusion coefficients observed
in the mixture of photolysis products, including the superposition of
decay profiles for rapidly and slowly diffusion components, in the 0.5−
1.5 ppm range of the NMR spectrum. (A) Full DOSY plot. (B) Decay
of NMR signal with increase in gradient strength.
much larger than any monomers in solution and to be very
slowly diffusing (Figure 3A), yielding a diffusion coefficient of 4
× 10−8 cm2/s, consistent with the value of 3 × 10−8 cm2/s
determined by DLS. This is in contrast to the measured
diffusion coefficient of the aqueous free monomer (2-OOA),
measured by DOSY NMR to be 7 × 10−6 cm2/s. When looking
at the decay of the NMR signal with gradient strength (Figure
3B), it is also apparent that the signals due to the hydrocarbon
chain protons specifically near the ends of the tails exhibit a
two-component decay profile: one short decay with sharp
signals sitting atop broadened signals with a very long decay.
This is indicative of two types of environments present in
solution, one of monomers and one of similar molecules with
confined motion. Additional information about the confined
molecules can be obtained by noting the absence in signal from
the protons closest to the polar head groups at large gradient
strength. This is common in lipid vesicles,19 where the NMR
signals from the protons on the rigid head groups are
broadened due to dipolar relaxation, while the signals from
the more flexible tails exhibit less dipolar broadening and are
thusly easier to detect. Therefore, taken together with the size,
monodispersity, and stability, these data indicate that the
particulates detected in solution are ordered structures
consistent with self-assembled vesicles.
Figure 2. Characterization of self-assembled vesicles. (A) Photograph
of clear 2-OOA solution before photolysis (left) and opalescent
solution after photolysis (right). (B) Fluorescence microscope image
of vesicles stained with Rhodamine 6G. (C) Dynamic light scattering
(DLS) of vesicles formed illustrating mean radius of 100 nm.
fluorescence microscope image in Figure S5A). Although the
critical bilayer concentration (CBC) of 2-OOA is not known,
similar surfactants have CBCs greater than 100 mM,18 and thus
vesicles are not expected, and indeed were not observed
(Figure S5), at the low concentration of 6 mM used in this
work. Over the course of photolysis, however, the solution
becomes opalescent (image on right in Figure 2A) indicating
the presence of particulates in solution. These particles were
observed microscopically using both phase contrast microscopy
(Figure S6) and fluorescence microscopy after staining with the
membrane stain Rhodamine 6G (Figure 2B), appearing to be
spherical. Further, they were determined to be monodisperse
with a narrow size distribution centered around 200 nm in
diameter using Dynamic Light Scattering (DLS, Figure 2C).
These particles were stable in solution for several months, over
the temperature range 4−22 °C and in the presence of MgCl2
salt (Figure S7). At 200 nm in diameter, they are too large to be
micelles, and their long-term stability in solution as well as their
apparent spherical shape suggests that disordered aggregates of
monomers are unlikely.
This work illustrates the advantageous environment provided
by the water surface region. 2-OOA, as a soluble surfactant,
partitions significantly to the water surface (Figure S8),
resulting in an inhomogeneous distribution of 2-OOA
molecules in solution. The surface excess concentration of 2-
OOA allows for a local concentration that is greater than the
3785
dx.doi.org/10.1021/ja5006256 | J. Am. Chem. Soc. 2014, 136, 3784−3787

Journal of the American Chemical Society
Communication
nominal bulk concentration of 6 mM. This increases the
probability of contact between reactive species, thereby
increasing the reaction yield. In addition, it allows for
orientation of the hydrocarbon tail of 2-OOA, resulting in a
greater propensity to undergo the photochemical reaction
mechanism illustrated in Figure 1A forming the OOA-OOA
dimer, rather than the Norrish type II reaction (Figure S4)
resulting in a single-tailed dimer. The Norrish type II reaction
necessitates more motion of the hydrocarbon tail, allowing it to
fold over on itself, a situation requiring a more dilute solute
environment (less likely in the surface region). Water surfaces,
such as those found on oceans, lakes, and atmospheric aerosol
particles, have been implicated as unique prebiotic reaction
environments previously.20−27 Here, we add an additional
example of the advantageous environment provided by the
water surface for chemistry.
AUTHOR INFORMATION
■
Corresponding Author
vaida@colorado.edu
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
E.C.G., R.J.R., and V.V. would like to acknowledge funding for
this work from the National Science Foundation (CHE-
1011770). E.C.G. also acknowledges a NASA Earth and Space
Science Graduate Fellowship as well as a Marian Sharrah
Graduate Fellowship from the University of Colorado, Boulder.
The authors would also like to thank Kevin Dean for the
acquisition of microscope images, Dan Gu for the acquisition of
mass spectra, and Annette Erbse for aid in using DLS.
Thus far, simple vesicles (enclosures) composed of fatty
acids have been used as a model system for primitive cellular
constructs; they have the ability to mimic many functions of
modern life, and their monomers (fatty acids) are plausible
components on early Earth.5−8,10,13,28,29 However, such
protocells are unlikely to have been the first enclosures due
to the very specific environmental constraints put on their
synthesis and stability (requisite pH, high concentration,
intolerance of vesicles to salts).30,31 Modern cells are enclosed
in a membrane composed of primarily double-tailed surfactants
having none of the constraints put on fatty acid vesicles;
however, their prebiotic synthesis is often thought to be too
difficult due to their chemical complexity.18 Here we provide an
alternative, sunlight-driven route to primitive enclosures in
addition to what has been traditionally considered. Beginning
with a dilute solution of a short single-chained soluble
surfactant, well below its CBC, simulated sunlight prompts
synthesis of a double-tailed surfactant followed by self-assembly
without external perturbation. No complicated synthetic steps
or preparations or high concentrations of surfactant are
required. In addition, the resultant vesicles are stable over
time and persist in the presence of Mg2+ ions, a likely
component of the early ocean.32 In contrast, the more common
models of fatty acid vesicles are unstable in the presence of
divalent cations.31
Regardless as to whether or not the first enclosures were
composed of single-tailed surfactants, a transition is needed at
some point in molecular evolutionary history to membrane
constituents with a double-tailed structure, as they are prevalent
throughout modern biology. Once mixed membranes com-
posed of both single- and double-tailed surfactants are
facilitated, even with minimal double-tailed surfactant, unique
competition and subsequent evolution have been seen to
emerge.33 The present study illustrates a simple, prebiotically
plausible transition from single-tailed to double-tailed surfac-
tants using only the energy provided by the early sun.
Therefore, the work presented here provides a possible route
to the prebiotic synthesis of a simple enclosure as well as a
potential contribution to primitive membrane evolution.
REFERENCES
■
(1) Menger, F. M.; Shi, L.; Rizvi, S. A. A. J. Colloid Interface Sci. 2010,
344, 241.
(2) Maibaum, L.; Dinner, A. R.; Chandler, D. J. Phys. Chem. B 2004,
108, 6778.
(3) Whitelam, S.; Rogers, C.; Pasqua, A.; Paavola, C.; Trent, J.;
Geissler, P. L. Nano Lett. 2008, 9, 292.
(4) Lee, O.-S.; Cho, V.; Schatz, G. C. Nano Lett. 2012, 12, 4907.
(5) Szostak, J. W.; Bartel, D. P.; Luisi, P. L. Nature 2001, 409, 387.
(6) Pohorille, A.; Deamer, D. Res. Microbiol. 2009, 160, 449.
(7) Hanczyc, M. M.; Fujikawa, S. M.; Szostak, J. W. Science 2003,
302, 618.
(8) Luisi, P. L.; Walde, P.; Oberholzer, T. Curr. Opin. Colloid Interface
Sci. 1999, 4, 33.
(9) Apel, C. L.; Deamer, D. W.; Mautner, M. N. Biochim. Biophys.
Acta 2002, 1559, 1.
(10) Mansy, S. S. Cold Spring Harbor Perspectives in Biology 2010, 2,
14.
(11) Cooper, G.; Reed, C.; Nguyen, D.; Carter, M.; Wang, Y. Proc.
Natl. Acad. Sci. U.S.A. 2011, 108, 14015.
(12) Pizzarello, S. Rend. Fis. Acc. Lincei 2011, 22 (2), 153−163.
(13) McCollom, T. M.; Ritter, G.; Simoneit, B. R. T. Origins Life Evol.
Biosphere 1999, 29, 153.
(14) Rushdi, A. I.; Simoneit, B. R. T. Origins Life Evol. Biosphere 2001,
31, 103.
(15) Griffith, E. C.; Carpenter, B. K.; Shoemaker, R. K.; Vaida, V.
Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 11714.
(16) Guzman, M. I.; Colussi, A. J.; Hoffmann, M. R. J. Phys. Chem. A
2006, 110, 3619.
(17) Leermakers, P. A.; Vesley, G. F. J. Am. Chem. Soc. 1963, 85,
3776.
(18) Monnard, P. A.; Deamer, D. W. Anatomical Record 2002, 268,
196.
(19) Veatch, S. L.; Polozov, I. V.; Gawrisch, K.; Keller, S. L. Biophys. J.
2004, 86, 2910.
(20) Griffith, E. C.; Tuck, A. F.; Vaida, V. Acc. Chem. Res. 2012, 45,
2106.
(21) Griffith, E. C.; Vaida, V. Proc. Natl. Acad. Sci. U.S.A. 2012, 109,
15697.
(22) Dobson, C. M.; Ellison, G. B.; Tuck, A. F.; Vaida, V. Proc. Natl.
Acad. Sci. U.S.A. 2000, 97, 11864.
(23) Tverdislov, V. A.; Yakovenko, L. V. Moscow University Physics
Bulletin 2008, 63, 151.
(24) Lerman, L. In Water and Life: The Unique Properties of Water;
Lynden-Bell, R. M., Morris, S. C., Barrow, J. D., Finney, J. L., Harper,
C. L., Jr., Eds.; CRC Press: Boca Raton, FL, 2010; p 259.
(25) Tuck, A. Surv. Geophysics 2002, 23, 379.
(26) Goldacre, R. J. In Surface Phenomena in Chemistry and Biology;
Danielli, J. F., Parkhurst, K. G. A., Riddiford, A. C., Eds.; Pergamon
Press: New York, 1958; p 12.
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental methods as well as supporting data and reaction
mechanisms are presented in the Supporting Information. This
material is available free of charge via the Internet at http://
pubs.acs.org.
3786
dx.doi.org/10.1021/ja5006256 | J. Am. Chem. Soc. 2014, 136, 3784−3787

Journal of the American Chemical Society
Communication
(27) Fallah-Araghi, A.; Meguellati, K.; Baret, J.-C.; Harrak, A. E.;
Mangeat, T.; Karplus, M.; Ladame, S.; Marques, C. M.; Griffiths, A. D.
Phys. Rev. Lett. 2014, 112, 028301.
(28) Chen, I. A.; Roberts, R. W.; Szostak, J. W. Science 2004, 305,
1474.
(29) Stano, P.; D’Aguanno, E.; Bolz, J.; Fahr, A.; Luisi, P. L. Angew.
Chem., Int. Ed. 2013, 52, 13397.
(30) Morigaki, K.; Walde, P. Curr. Opin. Colloid Interface Sci. 2007,
12, 75.
(31) Monnard, P. A.; Apel, C. L.; Kanavarioti, A.; Deamer, D. W.
Astrobiology 2002, 2, 139.
(32) Anbar, A. D. Science 2008, 322, 1481.
(33) Budin, I.; Szostak, J. W. Proc. Natl. Acad. Sci. U.S.A. 2011, 108,
5249.
3787
dx.doi.org/10.1021/ja5006256 | J. Am. Chem. Soc. 2014, 136, 3784−3787