Site-selective deuteration of amino acids through dual protein catalysis
A method for site-selective deuteration of amino acids using a protein system having an aminotransferase (e.g., DsaD) and/or a small partner protein (e.g., DsaE). A non-deuterated amino acid is contacted with deuterium and an aminotransferase or a combination of an aminotransferase and a partner protein, to yield a Cα-deuterated or a Cα- and Cβ-deuterated amino acid. Cβ-deuterated amino acids can be accessed by contacting a Cα- and Cβ-deuterated amino acid with non-deuterium hydrogen and an aminotransferase to wash out the deuterium at the Cα carbon atom by the non-deuterium hydrogen.
1 . A method of making a deuterated amino acid, the method comprising contacting a non-deuterated amino acid having an α-position carbon atom and a β-position carbon atom with deuterium and a protein selected from the group consisting of a branched-chain aminotransferase and a combination of a branched-chain aminotransferase and a partner protein, for a time and at a temperature to selectively deuterate the non-deuterated amino acid at the α-position carbon atom to yield a Cα-deuterated amino acid or to selectively deuterate the non-deuterated amino acid at both the α-position and the β-position carbon atom to yield a Cα- and Cβ-deuterated amino acid, wherein the branched-chain aminotransferase is a protein comprising an amino acid sequence at least 85% identical to SEQ ID NO: 1, and wherein the partner protein is a protein comprising an amino acid sequence at least 85% identical to SEQ ID NO: 3 or SEQ ID NO: 4.
2 . The method of claim 1 , wherein:
the branched-chain aminotransferase is a protein comprising an amino acid sequence of at least 95% identical to SEQ ID NO: 1; and
the partner protein is a protein comprising an amino acid sequence of at least 95% identical to SEQ ID NO: 3 or SEQ ID NO: 4.
3 . The method of claim 1 , wherein the non-deuterated amino acid is contacted with the deuterium and the branched-chain aminotransferase to yield a Cα-deuterated amino acid.
4 . The method of claim 1 , wherein the non-deuterated amino acid is contacted with the deuterium and the combination of the branched-chain aminotransferase and the partner protein to yield a Cα- and Cβ-deuterated amino acid.
5 . The method of claim 1 , further comprising contacting the Cα- and Cβ-deuterated amino acid with non-deuterium hydrogen and the branched-chain aminotransferase, for a time and at a temperature to selectively replace the deuterium at the α-position carbon atom with the non-deuterium hydrogen to yield a Cβ-deuterated amino acid.
6 . The method of claim 5 , wherein the non-deuterium hydrogen is provided as non-deuterated water.
7 . The method of claim 1 , wherein the deuterium is provided as deuterated water.
8 . The method of claim 1 , wherein the protein is cell-free protein.
9 . The method of claim 1 , wherein the temperature ranges from about 20° C. to about 40° C.
10 . The method of claim 1 , where in the time ranges from about 1 hour to about 24 hours.
11 . A method of making a deuterated amino acid, the method comprising contacting a non-deuterated amino acid having an α-position carbon atom and a β-position carbon atom with deuterium and a combination of a branched-chain aminotransferase and a partner protein, for a time and at a temperature to selectively deuterate the non-deuterated amino acid at both the α-position and the β-position carbon atom to yield a Cα- and Cβ-deuterated amino acid, wherein the branched-chain aminotransferase is a protein comprising an amino acid sequence at least 85% identical to SEQ ID NO: 2, and wherein the partner protein is a protein comprising an amino acid sequence at least 85% identical to SEQ ID NO: 3 or SEQ ID NO: 4.
12 . A method of making a deuterated amino acid, the method comprising contacting a non-deuterated amino acid having an α-position carbon atom and a β-position carbon atom with deuterium and a branched-chain aminotransferase, for a time and at a temperature to selectively deuterate the non-deuterated amino acid at the α-position carbon atom to yield a Cα-deuterated amino acid, wherein the branched-chain aminotransferase is a protein comprising an amino acid sequence at least 85% identical to SEQ ID NO: 1.
13 . A method of making a deuterated amino acid, the method comprising:
contacting a non-deuterated amino acid having an α-position carbon atom and a β-position carbon atom with deuterium and a combination of a branched-chain aminotransferase and a partner protein, for a time and at a temperature to selectively deuterate the non-deuterated amino acid at both the α-position and the β-position carbon atom to yield a Cα- and Cβ-deuterated amino acid; and
contacting the Cα- and Cβ-deuterated amino acid with non-deuterium hydrogen and the branched-chain aminotransferase, for a time and at a temperature to selectively replace the deuterium at the α-position carbon atom with the non-deuterium hydrogen to yield a Cβ-deuterated amino acid;
wherein the branched-chain aminotransferase is a protein comprising an amino acid sequence at least 85% identical to SEQ ID NO: 1; and
wherein the partner protein is a protein comprising an amino acid sequence at least 85% identical to SEQ ID NO: 3 or SEQ ID NO: 4.