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Specialty Chemistry Forums => Biochemistry and Chemical Biology Forum => Topic started by: ablake on July 30, 2021, 07:02:34 PM

Title: transamination of allo-isoleucine d10,15N to M+9
Post by: ablake on July 30, 2021, 07:02:34 PM
Does anyone have a protocol suggestion for making M+9 standard stock from allo-isoleucine d10,15N? Final product can be isoleucine d9 or allo-isoleucine d9. Someone suggested transamination reaction before, but I am not a chemist. Does this involve adding glutamate or isoleucine (or keto acid analogue of isoleucine) and transaminase on a warm water bath?
Title: Re: transamination of allo-isoleucine d10,15N to M+9
Post by: Babcock_Hall on July 31, 2021, 11:48:27 AM
The specifics of your question are unfamiliar to me.  Can you explain the meaning of M+9?  I would guess that it relates to the number of deuterium atoms. In general transamination refers to a process catalyzed by an aminotransferase enzyme.  Aminotransferases interconvert one amino acid and one ketoacid into different a different amino acid and a different ketoacid.  Almost all of these enzymes have pyridoxal phosphate as a coenzyme, which will temporarily hold onto the nitrogen atom.  There are isotope exchange reactions to consider, which may be your intent.  Offhand, I would suppose that the alpha-deuterium atom will be lost to solvent over time.  It is less clear to me what will happen to the 15N label, but it will probably depend on what else you include.  You may wish to include a buffer for best results with any enzyme-catalyzed process.
Title: Re: transamination of allo-isoleucine d10,15N to M+9
Post by: ablake on August 02, 2021, 03:14:53 PM
Can you explain the meaning of M+9?
Thank you so much for your reply! It is tremendously helpful and a great explanation of the reaction. The M+9 refers to the mass of allo-isoleucine + 9 (from deuterium presumably) that you would be looking for on mass spectrometry. A little more background on my question: when injecting allo-isoleucine d10,15N (aka allo-isoleucine M+11; cambridge isotopes) into the body, you would expect to find mainly this M+11 in plasma, but instead you find M+9. Mass spec cannot distinguish allo-isoleucine from isoleucine, so we know this M+9 is one or the other that comes from the injected compound (M+11) that has lost probably the 15N and one deuterium in a transamination reaction in the body. I want to replicate this reaction on the standard allo-isoleucine d10,15N.

So, from what you said, it sounds like the reaction would be: alpha-keto-isoleucine + allo-isoleucine d10,15N -->(aminotransferase + pyridoxal phosphate in PBS)-->alpha-keto-allo-isoleucine d1,15N + isoleucine d9???
Title: Re: transamination of allo-isoleucine d10,15N to M+9
Post by: Babcock_Hall on August 02, 2021, 07:32:04 PM
I will refer to a ketoacid using the name of its cognate amino acid, as you do.  I will assume 100% loss of the deuterium on carbon-2 to solvent, but I don't know whether or not this is strictly true.  Here is my educated guess.

alpha-keto-isoleucine + [d10,15N]allo-isoleucine  ::equil:: [15N]isoleucine + [d9]alpha-keto-allo-isoleucine.

In other words, I think that the N-15 label will transfer to the former alpha-ketoacid to make it into an alpha-amino acid.  But the deuterium will stay with the carbon skeleton.

On the other hand, suppose one were to perform an aminotransferase reaction using alpha-keto-alanine (also known as pyruvate).  Then the products would be [15N]-alanine and \ [d9]alpha-keto-allo-isoleucine.