[ablake]

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?

[Babcock_Hall]

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.

[ablake]

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???

[Babcock_Hall]

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  [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.