Hello folks. A lot has been speculated about the procedure in US patent 2,236,574 (biochemical manufacture of ketosteroids), I've seen the procedure posted here and on other forums:
https://thinksteroids.com/community/threads/process-of-making-testosterone.134361616/#post-1190550
and as usual I have seen a lot of speculation but little understanding of the process. Some think this will be child's play, but I don't think these persons have experience with attempting to filter or extract anything from a yeast suspension. Usually a centrifuge is used for these kind of processes. I will give it a try as soon as I can get to the welder's supply and buy a tank of oxygen. But before we do anything we must have a clear understanding of what is going on, that s why I researched the literature first. Especially since it is a patent, and patents are notorious for deliberately "forgetting" important details or adding "mistakes", so competitors won't be able to replicate their findings. A procedure from an academic journal is trustworthy and tries to be as exact as possible, as it is peer-reviewed, while patents are filed with the motive of owning not only a certain process but as many similar ones as possible, so the descriptions are often as vague as possible.
What makes this biosynthesis unusual is that two steps are performed by the same organism. Normally this organism, plain baker's or beer brewing yeast (Saccharomyces cerevisae) is under standard contitions only capable of performing reductive reactions, this is well known as yeast in used both industrially as in the lab as a selective reducing agent. Such a process is called a biochemical hydrogenation. In this particular case we are using "impoverished" yeast.
There's an excellent early review (Wieland 1932) on the preparation and properties of "impoverished" yeast ("verarmter" Hefe). I've included it here, but it is in German. I will translate here what is important for our goal. Impoverished yeast can be used as a biochemical dehydrogenation agent, thus promoting an oxidative process. The gem of this patent is, that it used both these qualities of yeast in situ, meaning that the intermediates do not have to be isolated. In this case of the impoverished yeast, after it has done its dehydrogenation of DHEA (forming 4-androstene-3,17-dione), the yeast is "fed" (adding the sugars) and does its usual selective hydrogenation, in our case only the ketone at the 17-position is reduced, forming testosterone. All this in the same vessel, the process is similar to brewing beer.
The procedure posted here has been adapted by an overly enthousiast arm-chair chemist, so let me post the real one from the patent:
8 grams of yeast (Milan flocculent ferment) are suspended in 30 cc of water, treated with 10 cc of N/5 Na2HPO4 solution and 10cc of N/5 KH2PO4 soluton and shaken for 20 hours in an oxygen atmosphere at 32°C. Then 200 mg of dehydroandrosterone [DHEA], suspended in 30 cc of water are introduced and the mixture was shaken for a further 48 hours in an oxygen atmosphere.
Thereupon a solution of 25 grams of invert sugar in 150 cc of water is introduced and the reaction mixture is allowed to stand in a fermentation vessel for 3 days at room temperature.
The reaction mixture is then extracted with ether and the ethereal solution washed with water, caustic soda lye, N/1 hydrochloric acid and water. After drying over sodium sulphate the ethereal solution is evaporated. The remaining residue is recrystallised from acetone and petrol ether. There are obtained 120 mg of a substance of M.P. 151°C, which is shown to be identical with testosterone.
Let us first look at the type of yeast used in the patent, there they call for "Milan flocculated yeast". According to wikipedia yeast flocculation typically refers to the clumping together (flocculation) of brewing yeast once the sugar in a wort has been fermented into beer. In the case of "top-fermenting" ale yeast (Saccharomyces cerevisiae), the yeast creates a "head" on the top of the liquid, unlike with "bottom-fermenting" lager yeast (Saccharomyces pastorianus) where the yeast falls to the bottom of the brewing vessel. [...] For flocculation to occur the yeast must be flocculent and certain environmental conditions (such as agitation, absence of sugars, a microamount of Ca2+, ethanol, etc.; Jin and Speers 1999) must be present.
https://byo.com/mead/item/62-7-fascinating-facts-about-yeast
This site contains most information we need about the type of yeast to use. It appears that the cheapest option, regular baker's yeast, will probably not do for the first step of the process (the dehydrogenation). Most species of yeast are not flocculent. It is thought the reason brewer’s yeast is flocculent is the natural selection process that has taken place in brewing, dating back hundreds of years. The German review (Wieland 1932) also mentions that they used for all their research a flocculent type of bottom-fermenting, lager yeast (Saccharomyces carlsbergiensis) donated to them by the Münchener Löwenbrauerei, and that the uniformity of that material allowed them to use it instead of the more preferable flocculent types of top-fermenting yeast. They also mention that the yeast can be collected and re-used after the experiment, but that this should only be repeated "a couple of times". I was thinking that, in the case separation of the yeast proved too problematic, perhaps a bottom-fermenting type would be preferable with an eye on the subsequent extraction with ether (or toluene etc... any suitable organic solvent that floats on water). Or if it is heavily flocculent and top-fermenting, the yeast could be harvested "by hand" before extraction? Just thinking out loud, experiment will show.
Also, not every type of beer yeast is flocculent! From above-mentioned website: For example London is known to be home of a very flocculent yeast. This yeast will form very large clumps even before fermentation is finished. This intensive flocculation sometimes necessitates that a brewer rouse the yeast to get it back into solution to finish the fermentation. On the other hand this simplifies filtration and yeast recovery. Other ale strains such as American/California strains are powdery and do not flocculate out until the beer is chilled. These strains tend to be more attenuative since they are in suspension for a longer period of time. On the other side of the scale, German ale yeast strains from Bavaria that are used to produce hefe-weizens are usually non-flocculent, and this is a desired characteristic of this beer. One aspect worth noting is that hefe-weizen flavors closely resemble wild yeast flavors, and these yeast flocculate like wild yeast. So it is clear that the choice of yeast is an important step.
Next is the addition of a buffered (phosphates) aqueous solution. The person who adapted the process said " if possible", but they really should not be omitted, they are vital to this particular process! Both salts are cheap and easily available, and so widely used as buffer agents they will certainly not raise any eyebrow. It is best to prepare stock-solutions of both beforehand, N/5 = 0.2M.
Two very important details are omitted in the patent, when it comes to the first step. First of all the water, without phosphate buffers added, should first be saturated with toluene. This is as simple as shaking the aqueous solution with toluene in a separatory funnel. The patent mentions on the first page: As particularly advantageous has proved the application of the so-called "impoverished” yeast as is obtainable for example according to Wieland, “Annalen 'der Chemie,” vol. 492, page 183 et seq. by shaking yeast suspensions in toluene-water with oxygen. But nowhere in the preparation this is mentioned. Toluene-saturated water is used as an antiseptic, as under conditions to generate "impoverished" yeast, bacterial infection often occurs without it. Also, toluene-saturated water does not affect yeast performance and oxygen uptake, in contrast to other antiseptic agents such as chloroform or thymol.
This is the procedure to produce "impoverished" yeast according to Wieland, translated from German:
We were able to fully confirm the observation already made by Firth and Lieben that the aerobic respiration in the yeast cell can be greatly accelerated by mechanical agitation. We have shaken suspensions of 2 grams of fresh, thoroughly washed yeast in 35 cc of buffered, semi-saturated toluene-water in a big, flat 150 cc volume Barcroft-Warburg apparatus for 15 hours at 30°C (thermostat) in an oxygen atmosphere. The inner flask [of the Barcroft-Warburg apparatus] contained a 50% KOH solution to prevent any dilution of the atmosphere with CO2.
We can forget about the Barcroft-Warburg apparatus (unless you have exceptional skills as a glass blower) but we see here the other "detail" omitted in the patent: the carbon dioxide gas generated by the living yeast has to be absorbed from the atmosphere. The Germans used a saturated KOH solution, but a strong NaOH solution (sold as drain cleaner) will do just as fine. The idea is that this base solution can never contact the shaking/stirring yeast suspension but is placed above it, or the oxygen atmosphere is bubbled through it. The bubbling isn't necessary, a solution placed above it in a closed vessel will readily absorb any CO2 generated (which chemically reacts and forms carbonate/bicarbonate).
Basically we need a flask-type reactor, placed on a magnetic sirrer, with a balloon on top to provide a slight overpressure of oxygen gas. Attached inside the top of the flask is a small vessel containing some concentrated NaOH-solution. The whole is best placed in a water-bath heated and regulated at 32°C with the type of submergable heating element/thermostat using for tropical fish aquaria.
The second part of the reaction is much easier, a regular bucket-type fermentor with a water-trap for homebrewing beer is used. The suspension is added to this together with the solution of invert sugar. As invert sugar is just a 50/50 (by weight) mixture of glucose and fructose, that can be used too. But better not start messing with brown sugar and honey like advised by our adaptor, these are impure substances that contain things like wax and molasses that can end up in your final extract. Not to mention the horrible emulsions things like honey can cause. Extracting a yeast emulsion will be enough a headbreaker without a centrifuge. Better choose as flocculent a yeast as possible.
I think that is about it when it comes to the outlines. I have almost everything and will be able to follow up this post with some practical experiments. It was a long-winded post, but I assure you everything in it is essential as preparation for the actual work to come.
https://thinksteroids.com/community/threads/process-of-making-testosterone.134361616/#post-1190550
and as usual I have seen a lot of speculation but little understanding of the process. Some think this will be child's play, but I don't think these persons have experience with attempting to filter or extract anything from a yeast suspension. Usually a centrifuge is used for these kind of processes. I will give it a try as soon as I can get to the welder's supply and buy a tank of oxygen. But before we do anything we must have a clear understanding of what is going on, that s why I researched the literature first. Especially since it is a patent, and patents are notorious for deliberately "forgetting" important details or adding "mistakes", so competitors won't be able to replicate their findings. A procedure from an academic journal is trustworthy and tries to be as exact as possible, as it is peer-reviewed, while patents are filed with the motive of owning not only a certain process but as many similar ones as possible, so the descriptions are often as vague as possible.
What makes this biosynthesis unusual is that two steps are performed by the same organism. Normally this organism, plain baker's or beer brewing yeast (Saccharomyces cerevisae) is under standard contitions only capable of performing reductive reactions, this is well known as yeast in used both industrially as in the lab as a selective reducing agent. Such a process is called a biochemical hydrogenation. In this particular case we are using "impoverished" yeast.
There's an excellent early review (Wieland 1932) on the preparation and properties of "impoverished" yeast ("verarmter" Hefe). I've included it here, but it is in German. I will translate here what is important for our goal. Impoverished yeast can be used as a biochemical dehydrogenation agent, thus promoting an oxidative process. The gem of this patent is, that it used both these qualities of yeast in situ, meaning that the intermediates do not have to be isolated. In this case of the impoverished yeast, after it has done its dehydrogenation of DHEA (forming 4-androstene-3,17-dione), the yeast is "fed" (adding the sugars) and does its usual selective hydrogenation, in our case only the ketone at the 17-position is reduced, forming testosterone. All this in the same vessel, the process is similar to brewing beer.
The procedure posted here has been adapted by an overly enthousiast arm-chair chemist, so let me post the real one from the patent:
8 grams of yeast (Milan flocculent ferment) are suspended in 30 cc of water, treated with 10 cc of N/5 Na2HPO4 solution and 10cc of N/5 KH2PO4 soluton and shaken for 20 hours in an oxygen atmosphere at 32°C. Then 200 mg of dehydroandrosterone [DHEA], suspended in 30 cc of water are introduced and the mixture was shaken for a further 48 hours in an oxygen atmosphere.
Thereupon a solution of 25 grams of invert sugar in 150 cc of water is introduced and the reaction mixture is allowed to stand in a fermentation vessel for 3 days at room temperature.
The reaction mixture is then extracted with ether and the ethereal solution washed with water, caustic soda lye, N/1 hydrochloric acid and water. After drying over sodium sulphate the ethereal solution is evaporated. The remaining residue is recrystallised from acetone and petrol ether. There are obtained 120 mg of a substance of M.P. 151°C, which is shown to be identical with testosterone.
Let us first look at the type of yeast used in the patent, there they call for "Milan flocculated yeast". According to wikipedia yeast flocculation typically refers to the clumping together (flocculation) of brewing yeast once the sugar in a wort has been fermented into beer. In the case of "top-fermenting" ale yeast (Saccharomyces cerevisiae), the yeast creates a "head" on the top of the liquid, unlike with "bottom-fermenting" lager yeast (Saccharomyces pastorianus) where the yeast falls to the bottom of the brewing vessel. [...] For flocculation to occur the yeast must be flocculent and certain environmental conditions (such as agitation, absence of sugars, a microamount of Ca2+, ethanol, etc.; Jin and Speers 1999) must be present.
https://byo.com/mead/item/62-7-fascinating-facts-about-yeast
This site contains most information we need about the type of yeast to use. It appears that the cheapest option, regular baker's yeast, will probably not do for the first step of the process (the dehydrogenation). Most species of yeast are not flocculent. It is thought the reason brewer’s yeast is flocculent is the natural selection process that has taken place in brewing, dating back hundreds of years. The German review (Wieland 1932) also mentions that they used for all their research a flocculent type of bottom-fermenting, lager yeast (Saccharomyces carlsbergiensis) donated to them by the Münchener Löwenbrauerei, and that the uniformity of that material allowed them to use it instead of the more preferable flocculent types of top-fermenting yeast. They also mention that the yeast can be collected and re-used after the experiment, but that this should only be repeated "a couple of times". I was thinking that, in the case separation of the yeast proved too problematic, perhaps a bottom-fermenting type would be preferable with an eye on the subsequent extraction with ether (or toluene etc... any suitable organic solvent that floats on water). Or if it is heavily flocculent and top-fermenting, the yeast could be harvested "by hand" before extraction? Just thinking out loud, experiment will show.
Also, not every type of beer yeast is flocculent! From above-mentioned website: For example London is known to be home of a very flocculent yeast. This yeast will form very large clumps even before fermentation is finished. This intensive flocculation sometimes necessitates that a brewer rouse the yeast to get it back into solution to finish the fermentation. On the other hand this simplifies filtration and yeast recovery. Other ale strains such as American/California strains are powdery and do not flocculate out until the beer is chilled. These strains tend to be more attenuative since they are in suspension for a longer period of time. On the other side of the scale, German ale yeast strains from Bavaria that are used to produce hefe-weizens are usually non-flocculent, and this is a desired characteristic of this beer. One aspect worth noting is that hefe-weizen flavors closely resemble wild yeast flavors, and these yeast flocculate like wild yeast. So it is clear that the choice of yeast is an important step.
Next is the addition of a buffered (phosphates) aqueous solution. The person who adapted the process said " if possible", but they really should not be omitted, they are vital to this particular process! Both salts are cheap and easily available, and so widely used as buffer agents they will certainly not raise any eyebrow. It is best to prepare stock-solutions of both beforehand, N/5 = 0.2M.
Two very important details are omitted in the patent, when it comes to the first step. First of all the water, without phosphate buffers added, should first be saturated with toluene. This is as simple as shaking the aqueous solution with toluene in a separatory funnel. The patent mentions on the first page: As particularly advantageous has proved the application of the so-called "impoverished” yeast as is obtainable for example according to Wieland, “Annalen 'der Chemie,” vol. 492, page 183 et seq. by shaking yeast suspensions in toluene-water with oxygen. But nowhere in the preparation this is mentioned. Toluene-saturated water is used as an antiseptic, as under conditions to generate "impoverished" yeast, bacterial infection often occurs without it. Also, toluene-saturated water does not affect yeast performance and oxygen uptake, in contrast to other antiseptic agents such as chloroform or thymol.
This is the procedure to produce "impoverished" yeast according to Wieland, translated from German:
We were able to fully confirm the observation already made by Firth and Lieben that the aerobic respiration in the yeast cell can be greatly accelerated by mechanical agitation. We have shaken suspensions of 2 grams of fresh, thoroughly washed yeast in 35 cc of buffered, semi-saturated toluene-water in a big, flat 150 cc volume Barcroft-Warburg apparatus for 15 hours at 30°C (thermostat) in an oxygen atmosphere. The inner flask [of the Barcroft-Warburg apparatus] contained a 50% KOH solution to prevent any dilution of the atmosphere with CO2.
We can forget about the Barcroft-Warburg apparatus (unless you have exceptional skills as a glass blower) but we see here the other "detail" omitted in the patent: the carbon dioxide gas generated by the living yeast has to be absorbed from the atmosphere. The Germans used a saturated KOH solution, but a strong NaOH solution (sold as drain cleaner) will do just as fine. The idea is that this base solution can never contact the shaking/stirring yeast suspension but is placed above it, or the oxygen atmosphere is bubbled through it. The bubbling isn't necessary, a solution placed above it in a closed vessel will readily absorb any CO2 generated (which chemically reacts and forms carbonate/bicarbonate).
Basically we need a flask-type reactor, placed on a magnetic sirrer, with a balloon on top to provide a slight overpressure of oxygen gas. Attached inside the top of the flask is a small vessel containing some concentrated NaOH-solution. The whole is best placed in a water-bath heated and regulated at 32°C with the type of submergable heating element/thermostat using for tropical fish aquaria.
The second part of the reaction is much easier, a regular bucket-type fermentor with a water-trap for homebrewing beer is used. The suspension is added to this together with the solution of invert sugar. As invert sugar is just a 50/50 (by weight) mixture of glucose and fructose, that can be used too. But better not start messing with brown sugar and honey like advised by our adaptor, these are impure substances that contain things like wax and molasses that can end up in your final extract. Not to mention the horrible emulsions things like honey can cause. Extracting a yeast emulsion will be enough a headbreaker without a centrifuge. Better choose as flocculent a yeast as possible.
I think that is about it when it comes to the outlines. I have almost everything and will be able to follow up this post with some practical experiments. It was a long-winded post, but I assure you everything in it is essential as preparation for the actual work to come.
