Cannabis Science And Adventures

By UvaE

Created Aug 13 2012 – 2:00am

“A chemist”, Primo Levi wrote in an essay on carbon, “only has to leaf through a treatise, and memories rise up in bunches.” When my wife became curious about the whereabouts of former fellow chemistry graduate students, I suddenly remembered one of my peers nicknamed “Padma”. In the lab where I was working on an undergraduate project, he was completing his doctorate on the synthesis of spiroaxane sesquiterpenoids. Spiroaxanes are natural products that are found mostly in various marine organisms and mushrooms. The general class of compounds known as sesquiterpenoids contain 15 carbon atoms; they’re made up of 3 basic units of isoprene and can serve as protective agents or chemical messengers.Padma had the talent of being able to smile through frustration, and it served him well. His thesis adviser was no longer active in research, and when he had been, his specialty was photochemistry, not organic synthesis, which meant that Padma was doing the fruitful part of problem-solving on his own. While writing up his thesis, he had to put up with one of those time-murdering, primitive word processors of the e

arly 1980’s, and when he needed additional data for an intermediate product, he had to resign himself to synthesizing some of them all over again. Finding every original intermediate or hoping that none of them had decomposed was hopeless: the refrigerator and storage cabinets for chemicals was a disaster area. No wonder he was 32 by the time he got his doctorate, but his persistence paid off. In three subsequent decades, he published several worthwhile papers in both academia and industry, and he’s currently an executive for an American pharmaceutical company.One day, one of his friends, Thomas, who had done some graduate work in our lab but had gone elsewhere for his Ph.D, needed an infrared spectrum of something he had synthesized. Interestingly, this “something” had, unlike some of the other products in our lab, neither decomposed nor been misplaced. The “something” was one of the cannabinoids found in hemp. When I accompanied Padma to the instrument room to run the spectrum, I took some of the thick, brownish liquid, spread it on a piece of paper and placed in my shirt pocket. The next day at home, when I wanted to showoff the chemical to friends, I found the shirt with empty pockets on the laundry line.

Cannabinoids is the term given to the group of compounds found in the resin of cannabis plants. Unlike sesquiterpenoids, which are built from three isoprene units, cannabinoids are biosynthesized from only two activated building blocks, and the ensuing 10-carbon intermediate is then fused with a phenolic compound known as olivetolic acid. In Padma’s time, as reflected in the content of his thesis, it was believed that mevalonic acid was the activated isoprene unit for biosynthesis. It’s now known to be isopentenyl pyrophosphate. Marijuana’s active molecules surprised early investigators because, unlike psychoactive drugs in other plants, those of cannabis are not alkaloids.

Later in the fall, one of my friends, Mike, suddenly took an interest in our lab and asked for a tour. I let him in, showed him around, but he was not exactly captivated by a demonstration of the rotary evaporator. He wanted to know how to turn on an electronic balance, and when I did it for him, he unwrapped some aluminum foil around a big brown lump to verify if his supplier had given him his money’s worth. A characteristic, spicy odor came through the wrapping.

The lump was hashish, a mixture of cannabis trichomes and other plant material. Also found in many other plants, trichomes are hairs that often produce characteristic compounds. For instance, tomato leaf trichomes produce sesquiterpenes; mint and basil ones secrete their characteristicsweet-smelling oils and the antimalarial drug artemisinin, another sesquiterpene, comes from the trichome glands of the Chinese herb sweet wormwood.

In Cannabis, trichomes are found mostly on flower buds but also on various other mature plant parts. As revealed in the images some trichomes are non-productive; the bulbous ones contain annabinoids-secreting glands. There’s a positive correlation between the total number of glands per leaf as compared and the total cannabinoid content of the leaf. At their densest, leaf trichomes, however, produce, at best, only one fourth of the cannabinoids made by those of female flowers.

The presence of an illicit drug in the lab made me pretty nervous, especially in that quantity. I made sure nobody was peering through the glass in the door to our lab, and I told Mike he should leave before he got me kicked out of college. Not yet stoned—his eyes did not have that common, glazed look—he had a smile suggesting that contrary to my concerns, nothing could possibly go wrong.

Our university was probably where Mike, a non-college student, purchased his stash. One day, in that same year, I was in the hallway, reading on a black couch, when someone walked up to me. Without a book or back pack, he had one of those four o’clock shadows at 11 in the morning, a thick accent, and a couple of look-alikes behind him. He asked me to move over. I obliged. Unknowingly, I had been sitting on a slit of the couch’s fake leather. He slid his hand into it, and from underneath the yellow-brown foam, he pulled out a lump surrounded by foil, much like the one my friend had weighed.

The sneaky behavior and waste of energy is in part due to irrational marijuana laws. In addition, they create tedious paperwork and funding obstacles for motivated researchers who hope to learn more about the medicinal potential of cannabis. In 1838, when it was legal, William B. O’Shaughnessy found clinical evidence for some of the Far Eastern medicinal folk use of marijuana. More recently, in 2009, one of the non-psychoactive ingredients, cannabidiol(CBD), proved to be affective against colitis in mice. For events in between the following outline from a review paper by Vincenzo Di Marzo highlights the most important developments.

The author points out that the British have made the most contributions (highlighted in blue) to the field. The 1980s medicines Marinol and Cesamet , based on cannabinoids, are still used to treat nausea and weight-loss in patients with cancer and AIDS. But some marijuana smokers feel that the plant itself is more effective than the use of isolated compounds. In the next decade, two receptors for cannabinoids were found in humans and other mammals along with two animal compounds(annadamide and 2-4G) that interact with those receptors. The compounds’ molecules have “tails” that are identical to those of tetrahydrocannabinol (THC), cannabis’ psychoactive ingredient. This so-called endocannabinoid research has led to the development of the drugs Sativex and Rimonabant. Sativex is a mouth spray used for symptomatic relief of neuropathic pain in people with multiple sclerosis. The active ingredients are THC and the mouse-colitis-reliever CBD. The appetite suppressant, Rimonabant, did not enjoy the same success, and in 2009 was withdrawn from the market.

New drugs were not the only fruits of endocannabinoid investigations. They led to the elucidation of the previously unknown mechanism of retrograde signalling. If a neuron simultaneously receives different neurotransmitters(GABA and glutamate, specifically) from different presynaptic neurons, it will not fire. But if the postsynaptic neuron releases the cannabinoid 2-4G, it will interact with a cannabinoid receptor on the presynaptic neuron and prevent the release of GABA. The postsynaptic neuron will then be able to signal the next cell.

It is unfortunate that in the public imagination, such intriguing findings are clouded by an atmosphere of information that either exclusively focuses on the unscientific rationalization of existing laws or on the exaggerated claims about what it can cure.

SOURCES:

• Francesca Borrelli and al. Cannabidiol, a safe and non-psychotropic ingredient of the marijuana plant Cannabis sativa, is protective in a murine model of colitis .J Mol Med (2009) 87:1111–1121 (entire article; free of charge)
• Monika Fellermeier , Wolfgang Eisenreich , Adelbert Bacher and Meinhart H. Zenk. Biosynthesis of Cannabinoids Incorporation Experiments with 13C-labeled glucoses. Eur. J. Biochem. 268, 1596±1604 (2001) (entire article; free of charge)
• Anthony L. Schilmiller and al. Studies of a Biochemical Factory: Tomato Trichome Deep Expressed Sequence Tag Sequencing and Proteomics. Plant Physiology July 2010 vol. 153 no. 3 1212-1223 (entire article; free of charge)
• Di Marzo V. A brief history of cannabinoid and endocannabinoid pharmacology as inspired by the work of British scientists. Trends Pharmacol Sci. 2006 Mar;27(3):134-40. Epub 2006 Feb 13.(entire article; free of charge)
• Nicoll and Bradley. The Brain’s Own Marijuana. Scientific American. December 2004 (entire article; free of charge)
• Turner JC, Hemphill JK, Mahlberg PG. Interrelationships of glandular trichomes and cannabinoid content. II. Developing vegetative leaves of Cannabis sativa L. (Cannabaceae).Bull Narc. 1981;33(3):63-71.
• Newsweek How procedural and legal restrictions make studying the effects of medical marijuana difficult. Nov 3, 2010

Changing Ideas About The Origin Of Life

For life to begin—to borrow from Addy Pross’ definition—a kinetically stable, self-sustained reaction network is needed, one that is tied into a replication mechanism. When 20th century scientists accepted and elaborated on J.B.S Haldane’s primordial soup hypothesis, their guesses and suggestive experiments centered mostly around the mature field of organic chemistry. But biochemistry as a science was still in its infancy. Their hunches were like those of aliens trying to account for our transition from hunter gatherer-groups to civilization without understanding the roles of agriculture, division of labor and writing. So what major biochemical insights — related to the origin of life — have we gained since Nobel Laureate Melvin Calvin’s 1969 book Chemical Evolution summarized the fashionable ideas of that decade?

(1) Primordial Catalysts Were Probably Not Proteins In the 1960’s, RNA’s role as a catalyst and replicator was greatly underestimated. Unaware of retroviruses, or at least of their reproductive mechanism, biochemists believed that information only flowed from DNA to RNA, and they perceived proteins to be the only biological catalysts. After the discovery of ribozymes revealed that tertiary RNA molecules could speed up their own production, it was hypothesized that these were life’s first catalysts because they could have evolved from single-stranded RNA molecules, which are structurally simpler than both DNA and proteins. But the current popular notion that RNA was essentially the sole primeval replicator and catalyst has also come under attack. As an alternative, the first catalysts may have also included inorganic ions. Hydrothermal vents are rich in  iron (II) sulfide (FeS) and nickel (II) sulfide, both of which can speed up biochemical reactions. In existing hydrothermal bacteria and archaebacteria, these compounds are part of protein complexes, but the ions are the reactive catalytic centers for a remarkable exergonic reaction. Hydrogen gas from the vents combines with dissolved carbon dioxide in the sea (and an acetyl-less CoA ) to produce water and acetylCoA (a key molecule involved in releasing energy from sugars; in fatty acid synthesis etc). The overall reaction releases 59 kJ of free energy for every 2 moles of fixed CO₂,  enough to drive the synthesis of ATP.

Another argument against RNA exclusivity from Lane, Allen and Martin is that each time RNA makes a copy of itself in a “primordial soup” its concentration drops so the rate of reaction can only be maintained if nucleotides are continuously replenished. This brings us to the issue of energy.

(2) First Energy Source Likely Involved Proton Gradients In the same way that a room only remains tidy and dustless with continuous effort, life forms are capable of maintaining order only in the presence of a continuous energy supply. Even before life arises, the required conversions of small molecules to larger ones are endothermic. Whereas the original hypotheses were careful enough to exclude oxygen from the original mix because an oxidizing atmosphere would break up newly made molecules, the irony is that the proposed energy sources, ultraviolet and lightning, would also destroy newly synthesized molecules. The excessive heat and low pH’s from deep, volcanic hydrothermal vents do not lead to a viable energy alternative.  But Lane, Miller and Allen point out that there is another hydrothermal vent which gets its heat from the mid Atlantic’s tectonic boundaries, known as the Lost City, where olivine mineral (a combination of magnesium and iron silicates) turns to serpentine (hydroxylated iron and magnesium silicates).

This is the source of hydrogen gas used by the previously mentioned bacteria to “fix” carbon dioxide into acetyl, a part of a vital metabolite. The hydroxides formed are not inconsequential because, with the help of simple membranes, they provide a natural pH-gradient, essentially a voltage, one that was more pronounced in ancient seas due to CO₂ concentrations that were 1000 times higher than their modern counterpart. Remarkably that gradient is comparable to the one created by the biochemical processes in today’s cells. Forty years ago, this idea that chemiosmosis was the energy-provider for earth life’s first cells could not be put forth because no one understood how the universal reaction-facilitator, ATP(adenosine triphosphate), was made from ADP(adenosine diphosphate). But given that proton gradients power ATP production in all kingdoms of life: in respiration, photosynthesis and in rotating motors of bacterial flagella, the hypothesis is now plausible. The enzyme ATP synthase is a molecular machine whose “blades” are rotated by H⁺ that are put in motion by coulombic repulsion. The enzyme-portion attracts and combines ADP with a phosphate group and the spinning nanomachine releases the ATP.

DiMauro has spent 10 years working on the chemistry of  1-carbon amide formamide (H₂NCOH), subjecting it to a variety of conditions and mineral catalysts. He has produced all four nucleic acids and a variety of carboxylic acids. What works best is when he uses a pH of 9 to 10 and temperatures in the 80–160 ◦C range, conditions that are found in non-volcanic hydrothermal vents.

(3) Knowledge of New Bacterial Kingdoms Downplays Role of Fermentation In First Cells The authors of How did LUCA make a living? Chemiosmosis in the origin of life. BioEssays. January 2010 refute the popular notion that fermentation was used by the first cells to release chemical energy from food molecules. Aside from the idea that fermentation seems to be a derived chemical process,  when comparing bacteria to archaea, there are also major differences in the gene sequences of fermentation enzymes. On the surface they seem like similar processes, but in reality the release of energy in oxygen’s absence evolved separately and independently.  It’s essentially convergent evolution, the way Old World Euphorbia and New World cacti have similar adaptations but are not related. Clostridia-type fermentations (Clostridia are sulfite reducing bacteria that include tetanus-producing bacteria), which represent ancient lineages, actually involve chemiosmosis, which of course exploits ion gradients across the cytoplasmic membrane and rotor–stator type ATPases (enzymes that cleave ATP to place a good leaving group on otherwise nonreactive molecules). The same is true of fermentation in most free-living anaerobic bacteria.

Conclusion Writing in 1969, Calvin said:

As long as we are limited to biology as it is on the earth, it is going to be difficult for us to be sure that such a system occurred in the way described in this book. We shall have to find other places in the universe, preferably nearby, in which this process is going on and has not gone all the way, so that we can observe it at some other stage of its development. this is why I am interested in lunar and planetary exploration.

In four decades no such places have been found yet, but at least something esoteric has been discovered at the bottom of our own oceans. It’s far from direct evidence, which of course eludes everyone because the molecular precursors to primordial life left no traces. But along with more detailed knowledge of biochemistry, the Lost City has inspired hypotheses that bring us closer to a non-fictitious narrative of our chemical history.

SOURCES:

• William F. Martin   Edited by Miguel Teixeira and Ricardo O. Louro   Hydrogen, metals, bifurcating electrons, and proton gradients: The early evolution of biological energy conservation  Febs Letters Volume 586, Issue 5, 9 March 2012, Pages 485–493
• Nick Lane, John F. Allen, William Martin. How did LUCA make a living? Chemiosmosis in the origin of life. BioEssays. January 2010 (whole article can be read free of charge)
• Michael J. Russell, William Martin. The Rocky Roots of the Acetyl-coA Pathway  TRENDS in Biochemical Sciences Vol.29 No.7 July 2004 (whole article can again be read free of charge)
• Calvin, M. (1969). Chemical evolution: Molecular evolution towards the origin of living systems on the earth and elsewhere. Oxford: Clarendon Press. QH325 .C26

Neutralization In Sexual Intercourse

Judging from the nature of  google searches that bring most people to this article, I realize that readers are looking for a method of contraception. Hate to disappoint you, but this article is about how nature protects sperm against acid. In other words it’s about how nature increases the likelihood of conception—ironically, the opposite of what you’re interested in! 

Not all chemical reactions involve a gain or loss of electrons. Substances that release hydrogen ions react with those which accept them. This is the basis of a neutralization reaction, and reactions of this type occur everywhere, in and out of the laboratory, from soils to sex organs.

Imagine the acidic species, the H⁺ releasers, as food for pac-men, who are the hydroxide ions(OH^–), common H⁺ acceptors.  For a neutralization reaction to go to completion, all of the pac-men have to be fed. The stuffed pacman is a molecule of water.However, the reality is a little more complex than the analogy. The unfed pacmen and their food are not originally separate or isolated species. They have counterparts which react to produce a salt. If the salt is incapable of generating either a basic or acidic ion in water, then we will have a perfectly neutral solution. Otherwise, the neutralization reaction will leave behind either an acidic or basic(alkaline) solution, albeit one that’s closer to pH 7 than the unneutralized one.

The first neutralization reaction associated with sex occurs within the penis.  Urine usually leaves behind an acidic residue in the urethra, a passageway shared by the male’s reproductive and excretory systems. There are a few unbuffered organic acids in urine that usually make it slightly acidic. They include benzoic acid, oxalic acid (which comes from oxalates in plant material) and acetic acid, which resurfaces a little later in our story. But the acids are neutralized by the pea-sized Cowper’s glands’ pre-ejaculatory fluid, which contains an alkaline secretion. This preserves semen’s alkaline pH due to spermine, spermidine and the foul-smelling putrescine and cadaverine. The important quartet of amines is secreted by the seminal vesicles next to the prostate gland. Sperm cells are vulnerable to acid because their cells are little more than naked genetic nuclei, and a low pH will denature DNA.Whereas  semen contains 2 to 4 times as much hydroxide as what’s found at pH = 7 (water is in equilibrium with very small and equal amounts of H⁺ and OH^–), vaginal fluid’s pH normally ranges from 3.8 to 4.5. This protects the warm, dark canal from most bacteria, who prefer a pH range of 6.5 to 7.5. So the vagina should never be washed with soap, which would neutralize protective acids.

Soap is alkaline because its main ingredient is a salt formed from the neutralization of a fatty acid. The positive portion of this salt is sodium ion, which is relatively inert in water. But the negative ion neutralizes H⁺ from water, causing the latter to churn out more hydroxide ions.

When a woman is sexually stimulated, the blood vessels around the vagina become engorged. The ensuing pressure on the surrounding tissues cause the vaginal walls to exude tiny droplets. Their main acidic ingredient is acetic acid, also found in vinegar. The droplets eventually coalesce and their other components serve to lubricate the sexual canal during intercourse. After male ejaculation, the semen’s amine bases protect the sperm cells by neutralizing nearby H⁺ from the acetic acid.

There you have it: another instance of how it is impossible to escape chemistry. I must admit, though, that I have not reached the stage where neutralization enters my consciousness while I’m loving my wife. I only think about the H⁺-OH^–-sex connection in bed when she’s asleep or reading.

According to one double-blind study, involving you-guessed it–50 college students, aside from the always prevalent acetic acid, a minority of women also produce elevated levels of minor organic acids such as butanoic and propanoic acids, especially during the first half of their menstrual cycles. Butanoic acid, also known as but

yric acid, is a component of body odor.  The production of all acids in the vagina decreased for women who were on oral contraceptives.

One might be tempted to get other college women to participate in a seemingly more fruitful study, pun intended. Maybe, if you coat a tampon with the necessary enzymes and ethyl alcohol, it will react with both acetic and butyric acids from their secretions to produce the fruity, non toxic ethyl acetate and ethyl butyrate, which smells like pineapple. Problem? That’s right. Eliminating acid will lead to vaginal infections. Well, you can always try it on armpits, assuming you can get the enzymes to work.