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| | From:  MikeKL5 (Original Message) | Sent: 11/24/2004 1:56 PM |
Hey Steve, We attempted a synthesis in my Advanced Synthesis class which, quite frankly, was an utter disaster. :-) If you don't mind, I was wondering if you could take a look at the synthesis procedure and let me know your thoughts. The basic premise of the synthesis was the reduction of 1,3-dinitrobenzene to 3-nitroanaline. The enzyme at work was not given in the synthesis, and researching the literature has not turned up anything useful which might indicate what enzyme is supposed to be accomplishing this reduction. Bakers Yeast was used as the source of the mystery enzyme. In the synthesis we took Bakers Yeast, added it to water, and heated it to 70 C for 5 minutes. We then added a solution of 250 mg 1,3-dinitrobenzene dissolved in 20 mL of methanol to the yeast, as well as a solution of 2 g NaOH in 5 mL of water. We then incubated the solution at 70 C for two hours. We attempted to extract the product from the resulting goop with several extractions of dichloromethane. The goop formed an emulsion with the dichloromethane (naturally), and we tried to ameloriate this situiation by adding sat aq NaCl. Eventually we were able to get several dichloromethane extractions, and we attempted to purify the product via chromatography with silica gel. I ended up with two solutions, each representing a large band on the silica gel. Each of these products was essentially impossible to crystallize, and each seemed to be more of a waxy substance than a crystal. Since I got only a few milligrams of this stuff it was impossible to run an NMR, IR, or anything else, and I'm reporting the synthesis as a total failure. I didn't think that this synthesis would work from the beginning. I showed the synthesis to my enzymology professor he also did not think that the synthesis should have worked. I think that the "products" were various cellular compounds (hence the waxiness), and that if Bakers Yeast does have an enzyme in it that can catalyze the reduction of 1,3-dinitrobenzene to 3-nitroanaline (very possible) that it was rapidly denatured under the conditions used in the reaction. My advanced synthesis professor is a very experienced synthesist of the old school... Let me put it this way; when I approached him with the idea that maybe the reaction conditoins were too harsh for the enzyme his response was that "well, it's an enzyme, it's just a catalyst, so it will work". I have the utmost respect for the man, and I would like to have as many informed opinions as possible before I write a paper which basically will say between the lines that the synthesis procedure was not well planned. I think that the synthesis would have worked if we had used lower temperatures, gentler reagents to lyse the cells, and that we should have purified by some type of column chromatography as is used in biochem labs. What are your thoughts? Thanks for reading such a long post! MikeKL5 |
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Reply
| | | From: ·Steve· | Sent: 11/24/2004 7:14 PM |
Hi Mike, tough lab! I agree that concentrated NaOH could denature the active reductase in the yeast, not to mention prolonged high temperature. Perhaps this is a tried and true procedure nonetheless. If you have a lot of yeast and subsequent breakdown products of the yeast present with only a small yield of product, pure material will be difficult to obtain, but the CH2Cl2 extraction and chromatography procedure sounded like a good method to isolate the compound.
Were either of the bands you collected yellow in color? That is the color of 3-nitroaniline. Also, do you have access to a solution cell for the IR instrument? If you do, you could take an IR of a CH2Cl2 solution of your substances, against a CH2Cl2 blank, and compare to the spectrum of an authentic sample of 3-nitroaniline, if available. Otherwise, with such a small amount of compound to work with, your options are limited. A proton or C-13 NMR spectrum would be useful also, again if you could compare the spectrum to that of an authentic sample. You might be able to distinguish signals of 3-nitroaniline against a presumed background of impurity signals. That would at least let you know if your material contains at least some correct product.
The enzyme must be some kind of reductase. Some keywork searching did not turn up anything relevant to the experiment, except that a number of reductases, such as oxidoreductases, apparently can effect the reduction of the nitro group to the amino group. But unfortunately I could not tell what specific enzyme in Bakers Yeast that is.
Another topic for further study!
Steve
Nitroreductase An enzymes produced by fecal bacteria which is implicated in the formation of aromatic amines from procarcinogens. These amines can be converted into cancer causing compounds such as N-hydroxy and nitroso compounds in the body tissues. . . . . .
Nitroreduction is an initial step in the metabolism of a variety of structurally diverse nitroaromatic compounds, including nitrofurans, nitropyrenes, and nitrobenzenes (1-5). Enzymes that catalyze this process are termed nitroreductases and are classified into two groups: oxygen-sensitive and oxygen-insensitive (6). The former enzymes, such as NADPH-cytochrome P-450 oxidoreductase (EC 1.6.2.4) and NADPH-b5 oxidoreductase (EC 1.6.2.2), catalyze the one-electron reduction of nitro moiety in which case the anion free radicals are formed (7-9). These enzymes are termed oxygen-sensitive, because the resulting radicals are easily reoxidized to the parent compounds by O2 in a futile redox cycle, which generates superoxide. Thus, these enzymes can mediate the reduction of nitroaromatics only under anaerobic conditions (3). The latter enzymes, such as NAD(P)H-quinone oxidoreductase (formerly called DT-diaphorase, EC 1.6.99.2) and nitroreductases of enteric bacteria, catalyze the two-electron reduction of the nitro moiety through nitroso and hydroxylamine intermediates to the fully reduced amino compounds (10, 11). Although this process does not produce superoxide, some of the hydroxylamine intermediates are mutagenic and carcinogenic (12, 13).
. . . . .
Glutaredoxins are small, heat-stable oxidoreductases, first discovered in Escherichia coli as GSH-dependent hydrogen donors for ribonucleotide reductase (1). They have proposed roles in many cellular processes, including protein folding and regulation, reduction of dehydroascorbate, protection against reactive oxygen species and sulfur metabolism (2�?). Glutaredoxins form part of the glutaredoxin system, comprising NADPH, GSH, and glutathione reductase, which transfers electrons from NADPH to glutaredoxins via GSH (5).
Yeast contains two classic glutaredoxin genes, designated GRX1 and GRX2, that share 40�?2% identity and 61�?6% similarity with those from bacterial and mammalian species (6). Strains deleted for both GRX1 and GRX2 are viable but lack heat-stable oxidoreductase activity using -hydroxyethyl disulfide (HED)1 as a model disulfide substrate. Previous studies have shown that the yeast glutaredoxins are active as antioxidants and are required for protection against reactive oxygen species. |
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Reply
| | | From: MikeKL5 | Sent: 11/25/2004 1:10 AM |
Steve,
Wow, maybe you should have been doing this synthesis instead of I. lol!
Yes, one band was yellow in color. However, it did not correspond to the
"product" as tested on silica gel. By that I mean we tested the reactant,
reaction mixture, and purified "product" on a silica gel plate before doing
the Chromatatron silica gel separation. A Chromatatron (sp?) is a device
that spins a plate covered in silica gel, and the solution is filtered
through the silica gel on the plate. Under UV light there was a second,
"purple" band which was following the yellow band down the silica disk, and
this band corresponded to the "product". (By product I mean what resulted
from the rxn, not necessairily what I was trying to get).
I had thought of trying to do as you suggested, and get some kind of
analysis confirming that I did indeed obtain the intended product.
Unfortunately I did not consider testing the product while dissolved in
something, as far as IR goes, and so I don't have that data. I did try a
proton NMR, and although the solution was somewhat colored from the product
I still got nothing on the NMR. A very slight peak in the aromatic range
(between 7 and 8 ppm), and I'm talking like a peak 2mm tall, on one of the
two separations, but that was it. On the other separation I got nothing.
I know that this synthesis was published this year, but the synth prof
didn't give us a reference, and searches using something called SciFinder
turned up limited information; it did not turn up an exact reference. I have
reason to believe that this synthesis was published in conjunction with ACS,
but I cannot find anything specific. It's a new synthesis (definately 2004),
and all of my professors are continually encouraging me to "validate new
information for myself", but I think that at my level of understanding I
should probably try to find my mistake rather than just dismiss something
that got pubslished in conjunction with ACS as wrong. :-) I'm definately
missing something, but I can't figure out what.
Thank you for all of your time and help. I really appreciate it, and
you have no idea how comforting to know that I can e-mail SOMEONE and at
least ask the questions.
Thanks again,
MikeKL5
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| | | From: ·Steve· | Sent: 11/25/2004 2:55 AM |
>> A very slight peak in the aromatic range (between 7 and 8 ppm), and I'm talking like a peak 2mm tall << Oh well... After thinking about it, the NMR spectrum may not be helpful anyway, since there aren't any characteristic "marker" signals in the product. An expanded C-13 NMR in the aromatic region, compared to the spectrum of an authentic sample of 3-nitroaniline, might or might not have enabled you to verify whether any of this product was formed.
We synthesize ortho- and para-nitroaniline routinely in organic lab, and do a TLC on silica gel with CH2Cl2 on the product. Both bands are distinctly yellow in color, and still look that way under UV light (not purple). That's why I was interested in any yellow substances in your product mixture after chromatography. But I gather that the yellow band corresponded to that of m-dinitrobenzene starting compound in your chromatotron control? 8-(
>> The basic premise of the synthesis was the reduction of 1,3-dinitrobenzene to 3-nitroanaline <<
I suppose it is definite that 3-nitroaniline really forms in this reaction, at least, as reported by the original authors? And that further reduction of the other nitro group does not occur? Otherwise, this sounds like a "throw it together and cook it for awhile and see what happens" type of experiement. I also wonder if it is necessary to denature the yeast, if that was the purpose of the rather high concentration of NaOH in the reaction mixture. When extracting amines with an organic solvent like CH2Cl2 from an aqueous solution, the solution is normally made basic by adding NaOH or NH3(aq) after the reaction to ensure that the amine is in the neutral "free base" form. You have the right idea - in original research, you're the boss, and can vary things like amounts and concentrations, pH and temperature, etc., in order to find a procedure that works the best. Very often, following someone else's procedure for the first time "blindly" gets the same result you got, I can say from (repeated) experience!
Steve
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Reply
| | | From: MikeKL5 | Sent: 11/27/2004 5:06 PM |
Steve,
Thanks a million for all of your help. I really appreciate it. I hadn't
thought about making the solution basic to ensure that the amine is in the
free-base form, although it still seems like we added way more NaOH than was
necessary for that purpose. I'll be sure to let you know what I get on the
paper when I get it back in a week or two
Thanks again for all your help,
MikeKL5.
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| | | From: ·Steve· | Sent: 11/27/2004 7:01 PM |
OK Mike, I'll keep my fingers crossed! Hopefully, the instructor will appreciate your valid reasons given for not getting the desired product and not base the grade entirely on yield. After all, that's part of research! Steve |
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