Biomedical Engineering Reference
In-Depth Information
the final methyl labelling pattern is a mixture of isotopomers (CH
3
,CH
2
D,
CHD
2
and CD
3
) which results in asymmetric peaks in the (
1
H,
13
C) correlation.
More recently 2-aceto-2-hydroxybutyrate (IUPAC name, 2-ethyl-2-hydroxy-
3-oxobutanoic acid; CAS number, 3142-65-2), a derivative of acetolactate
(Figure 1.4; see Section 1.2.2.4.2 for a more comprehensive description of
acetolactate), has been used to selectively label c
2
-methyl groups of isoleu-
cine.
42,43
Greater than 95% labelling of isoleucine c
2
-methyl groups can be
achieved by adding 100 mg L
21
of 2-aceto-2-hydroxybutyrate to an E. coli
expression culture growing in perdeuterated minimal medium. 2-aceto-2-
hydroxybutyrate is produced as a racemic mixture but only the 2-(S)-aceto-2-
hydroxybutyrate enantiomer is metabolised by E. coli.
While the level of protonation of the c
2
-methyl groups is high, unexpected
cross-labelling to the pro-(R) methyl groups of leucine and valine occurs. This
scrambling results in weak pro-(R) methyl group correlations in (
1
H,
13
C)
correlation spectra.
42,43
Isotopic scrambling can be eliminated by adding
saturating amounts (200 mg L
21
) of deuterated a-ketoisovalerate, a precursor
of leucine and valine (see Section 1.2.2.4.1), at the same time as 2-aceto-2-
hydroxybutyrate.
43
An adaptable synthesis scheme has been published that allows the
production of 2-aceto-2-hydroxybutyrate with different isotopic composi-
tion.
43
Using [1,2,3,4-
13
C]-labelled 2-aceto-2-hydroxybutyrate, for example,
'linearises'
the
isoleucine
side-chain
and
enables
efficient
transfer
of
magnetisation between nuclei in the backbone and c
2
-methyl group.
1.2.2.4 Leucine and Valine
Leucine and valine are produced from two molecules of pyruvate (Figure 1.4).
Their biosynthesis shares several early steps and precursors. The isopropyl
group present in both leucine and valine is generated before the two pathways
diverge and consequently labelling techniques directed towards these residues
result in equal labelling of them both.
As discussed in Section 1.2.2.3.2, [3-
13
C]pyruvate
41
can be used to
isotopically label the prochiral methyl groups of leucine and valine (and the
c
2
-methyl group of isoleucine) but, while cheap, this approach leads to the
production of methyl group isotopomers and complicated NMR spectra.
Consequently, alternative strategies have been proposed. To date these focus
on the use of two metabolic precursors common to both leucine and valine
biosynthesis: a-ketoisovalerate (IUPAC name, 3-methyl-2-oxobutanoic acid;
CAS
number,
759-05-7)
and
2-acetolactate
(IUPAC
name,
2-hydroxy-2-
methyl-3-oxobutanoic acid; CAS number, 71698-08-3).
1.2.2.4.1
a-Ketoisovalerate
a-Ketoisovalerate is the last precursor common to both valine and leucine
biosynthesis (Figure 1.4). a-Ketoisovalerate is converted directly into valine by
