[[OOL part 2 - same author]]
Time Out
James
Tour is a synthetic organic chemist at Rice University.
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IN 1952, Stanley Miller and Harold Urey derived a
number of racemic amino acids from a handful of small molecules. These were
electrifying results because they suggested that the methods of synthetic
chemistry might finally explain the origins of life. The excitement was
justified, but premature.
1 Origins
of life (OOL) research has, to be sure, become progressively more
sophisticated, but its goal—to explain the origins of life—remains as
distant today as it was in 1952. This is not surprising. The protocols in use
have remained unchanged: buyhighly purified chemicals; mix them
together in high concentrations and in a specific order under carefully devised
laboratory conditions; derive a mixture of compounds; and publish a
paper making bold claims about OOL. These protocols are as unrealistic as they
are unimproved.
This essay comprises an argument, but it also contains an
appeal to the OOL community. The history of science suggests that on occasion
what is required for research to flourish is not further research—at least to
the extent that further research involves doing the same thing. This is one of
those times.
Needed for Life
FOUR MOLECULES are needed for life: nucleotides,
carbohydrates, proteins, and lipids. Nucleotides are composed of a trimeric
nucleobase-carbohydrate-phosphate combination, and once polymerized, constitute
DNA and RNA. Five different nucleobases comprise the entire alphabet for DNA
and RNA. The nucleotides and their subsequent DNA and RNA structures are
homochiral, yielding one of two possible enantiomers. Amino acids are most
often homochiral. When amino acids are polymerized, they form proteins and
enzymes. Proteins and enzymes also display a tertiary homochirality. Lipids are
dipolar molecules with a polar water-soluble head and a non-polar
water-insoluble tail. They, too, are most often homochiral. Cells use
carbohydrates for energy, and carbohydrates, along with proteins, are
identification-receptors. Carbohydrates are also homochiral, and their
polymeric forms take on tertiary homochiral shapes. OOL researchers have spent
a great deal of time trying to make these four classes of molecules, but with
scant success.
Constructing the molecules necessary for life from their
prebiotic precursors represents one goal of OOL research; putting them
together, another. Some of synthetic chemistry is pedestrian, and some
ingenious. Fundamental questions remain unaddressed. Claims that these
structures could be prepared under prebiotic conditions in high enantiomeric
purity using inorganic templates, or any presumed templates, have never been
realized. The carbohydrates, amino acids, lipids, and other compounds within
each of these classes require specific methods in order to control their
regiochemistry and stereochemistry. The differences in reaction rates often
require chiral systems acting upon chiral molecules. If this were possible
under prebiotic conditions, it is odd that it cannot be replicated by synthetic
chemists.
They have, after all, had 67 years to try.
Synthetic Hyperbole
CONSIDER THE class of experiments that deal with the
assembly of chemicals into what are referred to as protocells—“a
self-organized, endogenously ordered, spherical collection of lipids proposed
as a stepping-stone to the origin of life.”
2 In
2017, a team from the Origins of Life Initiative at Harvard University
performed a type of polymerization reaction in water known as the reversible
addition–fragmentation chain transfer.
3 This
reaction type is not seen in nature, and neither are the monomers that figure
in the experiment. Still, this is standard chemistry. Polymers are made by a
controlled radical polymerization reaction, where two different monomer types
are added sequentially to a chain bearing both a hydrophobic and a hydrophilic
block. Researchers observed polymeric vesicles forming during
polymerization—interesting, but not extraordinary. The vesicles grew to
bursting as researchers kept the radical chain growing through ultraviolet
light activation. There is, in this, nothing surprising: the forces between the
growing vesicle and the surrounding water dictate a critical growth volume
before the vesicle ruptures.
The claims should have ended there.
Here is how the work was portrayed in the published article:
The observed net oscillatory vesicle population grows in a
manner that reminds one of some elementary modes of sustainable (while there is
available “food”!) population growth seen among living systems. The data
supports an interpretation in terms of a micron scale self-assembled molecular
system capable of embodying and mimicking some aspects of “simple” extant life,
including self-assembly from a homogenous but active chemical medium, membrane
formation, metabolism, a primitive form of self-replication, and hints of
elementary system selection due to a spontaneous light triggered Marangoni
instability [provoked by surface tension gradients].
4
These claims were then rephrased and presented to the public
by the Harvard Gazette:
A Harvard researcher seeking a model for the earliest cells
has created a system that self-assembles from a chemical soup into cell-like
structures thatgrow, move in response to light, replicate, and
exhibit signs of rudimentary evolutionary selection [emphasis added].
5
This degree of hyperbole is excessive.
6 Nothing
in this experiment had growing cell-like structures with replication, or that
exhibited aspects of evolutionary selection.
Teams from the University of California and the University
of New South Wales recently conducted lipid bilayer assembly experiments,
publishing a summary of their work in 2017.
7 They
combined nucleotides and lipids in water to form lamellae, with the nucleotides
sandwiched between the layers. Nucleotides are trimers of
nucleobase-carbohydrate-phosphate, and, in this case, both nucleotides and
lipids were purchased in pure homochiral form. Both teams then demonstrated
that a condensation polymerization of the nucleotides can take place within the
lamella upon dehydration. Polymerization takes place by means of a reaction
between pre-loaded phosphate and the purchased stereo-defined alcohol moiety
found on a neighboring nucleotide. Similar reactions, they conjectured, may
have occurred at the edge of hydrothermal fields, volcanic landmasses providing
the necessary heat for reactions.
The chemistry that figures in these experiments is
unremarkable. Bear in mind that derivatives were all pre-loaded. To provide the
essential concentrations for the reactions, researchers removed the water, thus
driving the intermolecular reactions to form oligomers that resembled nucleic
acids. The problem with condensation polymerization is obvious: any alcohol
can compete for the reactive electrophilic site. In the case under
consideration, researchers added no other alcohols. They were scrupulous, but
the system was stacked. Condensation polymerization reactions need to be very
pure, free of competing nucleophilic and electrophilic components. Witness the
Carothers equation, which defines degrees of polymerization based upon monomer
purity.
8 If
there happened to be amino acids or carbohydrates mixed with the nucleotides,
they would terminate or interrupt the growth of the oligonucleotides. What is more,
the researchers did not confirm the integrity of the structures they claimed to
have derived. If carefully analyzed, these structures would likely have shown
attacks from unintended hydroxyl sites. Since their sequences are essentially
random, short oligonucleotides are not realistic precursors to RNA. An alphabet
soup is not a precursor to a poem. The authors go on to suggest that the
lamella sandwiching oligonucleotides eventually break off to form lipid bilayer
vesicles. These contain the oligonucleotide-within-vesicle constructs, which
they call protocells. The conversion of planar lamella into multilamellar
vesicles as they hydrate is well established, but shearing forces are generally
required to form the requisite lipid bilayer vesicle. For this reason, yields
were likely to be low.
9 It is
hard to imagine finding highly purified homochiral nucleotides trapped in a
pure lipid lamella on the prebiotic earth.
But set all that aside. These vesicles bear almost no
resemblance to cellular lipid bilayers. Lipid bilayer balls are not
cellular lipid bilayers. One would never know this from reading the authors’
account. “Then, in the gel phase,” they write, “protocells pack together in a
system called a progenote and exchange sets of polymers, selecting those that
enhance survival during many cycles.”
10 Chemicals,
of course, are indifferent to their survival. No mechanism is described to
demonstrate how protocells would bear different sets of polymers or exchange
polymers among them. Terms from biology have generally been misappropriated in
a way that makes no chemical sense. This is not an isolated or incidental
defect. It reappears when the authors write that “[t]he best-adapted protocells
spread to other pools or streams, moving by wind and water…”
11 Best-adapted?
Microbial communities apparently “evolve into a primitive metabolism required
by the earliest forms of life.” Molecules do not evolve, and nothing is being
metabolized. Condensation polymerization is a simple chemical reaction based
upon the addition of nucleophiles to electrophiles with loss of water. Such a
reaction is never referred to as a form of metabolism within synthetic
chemistry.
Terminology is one thing, non-sequiturs quite another.
“After much trial and error,” the authors write, “one protocell assembles the
complicated molecular machinery that enables it to divide into daughter cells.
This paves the way for the first living microbial community.” How is the
molecular machinery made? They do not say. The mechanisms needed for cellular
division are complex, requiring cascades of precisely functioning enzymes.
There is nothing between what the authors demonstrate and what they claim to
have established, and nothing they propose “paves the way for the first living
microbial community.”
The Emerging Cell
A FUNCTIONING CELL contains a complex non-covalent
interactive system. Nobody knows how a cell emerges from its molecular
components. An interactome is the set of molecular interactions in a given
cell.
12 Interactions
may be between proteins, genes, or molecules. Information is transferred within
the cell through these molecular interactions. Electrostatic potentials permit
information to flow through non-covalent molecular arrays, but these arrays
require specific orientation.
13 The
interactome defines these intermolecular orientations, alignments that are
unattainable through random mixing.
Peter Tompa and George Rose have calculated that if one
considers only protein combinations in a single yeast cell, the result would be
an estimated 1079,000,000,000 combinations.
14 The
authors understand that this is a very large number, one that precludes
“formation of a functional interactome by trial and error complex formation
within any meaningful span of time.” What Tompa and Rose call “a complicated
cellular sorting/trafficking and assembly system” is required. Sophisticated
scaffolding notwithstanding, “in the absence of energy even this well developed
infrastructure would be insufficient to account for the generation of the
interactome, which requires a continuous expenditure of energy to maintain
steady state.” In their concluding paragraph, Tompa and Rose remark that
[t]he inability of the interactome to self-assemble de
novo imposes limits on efforts to create artificial cells and organisms,
that is, synthetic biology. In particular, the stunning experiment of
“creating” a viable bacterial cell by transplanting a synthetic chromosome into
a host stripped of its own genetic material has been heralded as the generation
of a synthetic cell (although not by the paper’s authors). Such an
interpretation is a misnomer, rather like stuffing a foreign engine into a Ford
and declaring it to be a novel design. The success of the synthetic biology
experiment relies on having a recipient interactome … that has high
compatibility with donor genetic material. The ability to synthesize an actual
artificial cell using designed components that can self-assemble spontaneously
still remains a distant challenge.
The fact is that interactomes add a massive layer of
complexity to all cellular structures. It is one that underscores the
difference between a real cell and the protocells or extant cells made by OOL
researchers.
In 2010, a team led by Craig Venter made a copy of a known
bacterial genome and transplanted it into another cell.
15 In
2016, they did something better, removing all but 473 genes from a natural
genome and transplanting it into another cell.
16 Venter
and his team were circumspect; the press was enthusiastic. More recently,
Henrike Niederholtmeyer, Cynthia Chaggan, and Neal Devaraj have made what they
term, “mimics of eukaryotic cells.”
17 Science declared
them “the most lifelike artificial cells yet.”
18 Microcapsules
made of plastic and containing clay were prepared using microfluidics
techniques. Clay has a high affinity for binding DNA. Thus when DNA was added
to the solution, it diffused through the semi-porous plastic microcapsules and
bound to the clay. The requisite RNA polymerases, together with the ribosomes,
tRNA, amino acids, enzymatic cofactors, and energy sources were either
purchased or extracted from living systems. The expected chemical
reactions did result in protein synthesis. Newly formed proteins
diffused from their microcapsules of origin to other microcapsules. The nearer
the neighboring microcapsule, the greater the exchange of reagents between
them. Diffusion between microcapsules the authors dubbed quorum sensing. The
chemistry would work no matter the container, whether a test tube or a
large-scale industrial production tank. If the experimental design is clever,
the synthesis is unremarkable. Phys.org reported these modest results
in markedly flamboyant terms, referring to “gene expression and communication
rivaling that of living cells.”
19 There
is no rivalry here. All of the active chemical components were extracted from
living systems. If these are “the most lifelike artificial cells yet,” this
serves only to underscore the point that no one has ever come close to the real
thing.
20
Life as a Lucky Fluke
IN AN ARTICLE entitled “How Did Life Begin?” Jack
Szostak asks whether the appearance of life on earth is “a lucky fluke or an
inevitable consequence of the laws of nature.”
21 It
is a good, but premature question, a point obvious from his own appreciation of
current research. Having vetted the usual suspects of asteroids, dust clouds,
volcanoes, lightning, and time, Szostak appeals to “a concentrated stew of
reactive chemicals”:
Life as we know it requires RNA. Some scientists believe
that RNA emerged directly from these reactive chemicals, nudged along by
dynamic forces in the environment. Nucleotides, the building blocks of RNA,
eventually formed, then joined together to make strands of RNA. Some stages in
this process are still not well understood. … Once RNA was made, some strands
of it became enclosed within tiny vesicles formed by the spontaneous assembly
of fatty acids (lipids) into membranes, creating the first protocells. As the
membranes incorporated more fatty acids, they grew and divided; at the same
time, internal chemical reactions drove replication of the encapsulated RNA.
22
The thesis that “RNA emerged directly from these
reactive chemicals, nudged along by dynamic forces [emphasis added]” is painful
to a synthetic chemist. A complex pathway of reactions would have been needed
incorporating purification, assembly, polymerization, and sequencing. Nothing
emerged directly in Szostak’s scenario, let alone something as complex as RNA.
Phrases such as “nudged along by dynamic forces” have no meaning in terms of
synthetic chemistry. Nucleotides never form and join together to make strands
of RNA without complex protection and deprotecting steps. It is perfectly true
that “[s]ome stages in this process are still not well understood,” if only
because we are clueless about the chemistry needed on a prebiotic earth.
In the diagram to which Szostak appeals, the compounds
listed as simple sugars are, in fact, glycerol and ethylene glycol. There are
known routes to convert them to simple sugars, but only in gross relative and
absolute stereochemically mixed states, and as a mixture of several different polyols.
23 Carbohydrate
synthesis is a difficult prebiotic problem.
24Szostak’s
carbohydrates would be useless in their mixed states, and separations are hard.
The diagram’s cyanide derivatives are unrecognizable as cyanide derivatives. In
an act of grace, let us attribute these chemical structural errors to the
faulty renderings of a staff artist. The chemical errors are Szostak’s own.
There is simply no way that heat and light can directly make a nucleotide from
simple sugars and cyanide derivatives. Such glossy presentations have become
the standard of the OOL community when it tries to build upon the careful work
of exacting synthetic chemistry.
I have discussed these issues with OOL researchers, and I am
amazed that they fail to appreciate the magnitude of the problem in building
molecules. They see little difficulty in accepting a chemical synthesis where a
desired product is mixed with a large array of closely related yet undesired
compounds. They seem unaware that separations would be enormously complex, and
subsequent reactions unavailing. In a 2018 article for Progress in
Biophysics and Molecular Biology, Edward Steele et al. concede the following.
The transformation of an ensemble of appropriately chosen
biological monomers (e.g. amino acids, nucleotides) into a primitive living
cell capable of further evolution appears to require overcoming an information
hurdle of superastronomical proportions, an event that could not have
happened within the time frame of the Earth except, we believe, as a miracle.
All laboratory experiments attempting to simulate such an event have so far led
to dismal failure.
“At this stage of our scientific understanding,” they write,
“we need to place on hold the issue of life’s actual biochemical origins—where,
when and how may be too difficult to solve on the current evidence [emphasis
added].”
25 All
is not lost. If life on earth did not arise on earth, “[i]t would thus seem
reasonable,” Steele et al. remark, “to go to the biggest available ‘venue’ in
relation to space and time. A cosmological origin of life thus appears
plausible and overwhelmingly likely.” Why chemical reactions that are unlikely
on the earth should prove likely somewhere else, Steele et al. do not say.
Facing Facts
JOHN SUTHERLAND, one of OOL’s giants and the most skilled
synthetic chemist to engage in OOL research, has recently proposed that
“chemical determinism can no longer be relied on as a source of innovation, and
further improvements have to be chanced upon instead.”
26 Chanced
upon? It appears that Sutherland has come to appreciate the depths of the
problems facing OOL researchers. In 2017, Ramanarayanan Krishnamurthy et al.
showed that diamidophosphate can phosphorylate nucleosides, nucleotides, and
stereo-scrambled lipid precursors. These can further result in the formation of
random oligonucleotides and oligopeptides. The fundamental challenges with
respect to synthesis and assembly remain unaddressed. Krishnamurthy was rightly
measured in writing about “the pitfalls of extrapolating extant biochemical
pathways backwards all the way to prebiotic chemistry and vice versa.”
27 In
2018, Clemens Richert argued that “the ideal experiment does not involve any
human intervention.”
28 This
is a step in the right direction. So, too, is the fact that he scrupled at the
pure chemicals used by the OOL community.
It is time for a temporary time out. Why not admit what we
cannot yet explain: the mass transfer of starting materials to the molecules
needed for life; the origin of life’s code; the combinatorial complexities
present in any living system; and the precise non-regular assembly of cellular
components?
It would be helpful if leading researchers, among them very
sophisticated synthetic chemists, were to step back, pause, and join forces. If
the origins of life remain a mystery, two goals are within reach: an
agreement about the rational standards by which OOL research should be judged,
and a candid acknowledgment of the problems that remain to be overcome. A
statement of this sort would be reassuring in its candor.
29
Wikipedia, “
Miller–Urey Experiment”; Stanley Miller, “
A
Production of Amino Acids under Possible Primitive Earth Conditions,” Science 117,
no. 3,046 (1953), doi:10.1126/science.117.3046.528; Stanley Miller and Harold
Urey, “
Organic Compound Synthes on the Primitive Earth,” Science 130,
no. 3,370 (1959), doi:10.1126/science.130.3370.245.
↩
Alvin Powell, “
Mimicking Life in a Chemical Soup,” The Harvard
Gazette, March 31, 2017; Harvard University, “
Researcher Creates Chemical System that Mimics Early Cell
Behavior,” Phys.org, April 3, 2017; Ava Jones, “
Harvard Scientist Discovers Phoenix Vesicles in Quest to Mimic
Life,” University Herald, April 4, 2017; and “
Researcher Creates Chemical Complement that Mimics Early
Dungeon Behavior,” Health Medicine Network, n.d.
↩
The author thanks Paul Nelson for helpful insights. Walt
Shaw and Steve Burgess of Avanti Polar Lipids kindly provided information on
lipid bilayer assemblies.
↩
Published on July 11, 2019 in Volume 4, Issue 4.
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