Biomedical Engineering Reference
In-Depth Information
O
O
O
OH
O
O
O
O
NH
2
N
H
O
N
H
NH
2
O
O
O
O
O
O
N
H
O
HN
OH
c
d
a
b
O
O
O
HN
HN
HN
HN
O
O
O
O
O
O
SC(Ph)
3
H
2
N
N
HN
H
2
N
O
O
Ph
Ph
SH
SC(Ph)
3
7
10
6
8
9
3
Reaction 5.1 Synthesis of Nε
-Hcy-Lys isopeptide (3). Reaction conditions: (a) Ar, (Boc)
2
O,
NaHCO
3
, CHCl
3
, reflux/1.5 h, 95 %. (b) H
2
, 10 % Pd/C, EtOH, rt/16 h, 97 %. (c) Et
3
N/HOBt/
DMAP/EDCI, DMF, rt/24 h, 90 %. (d) Ar, TFA:H
2
O:TIS:Phenol 88:5:2:5, rt/4 h, 95 % (Reprinted
from [311])
hydroxybenzotriazole ester of
-N-Boc-S-Trityl-Hcy-OH (9), using DMAP/EDCI
as the coupling reagents. Removal of trityl- and Boc-protecting groups is achieved
in one step using a mixture TFA:H
2
O:TIS:phenol 88:5:2:5 under argon atmosphere.
The target Nε
α
-Hcy-Lys isopeptide (3) is isolated as a white solid in 79 % overall
yield and purity
96 % as determined by HPLC.
1
H NMR,
13
C NMR, and mass
spectrometry data are consistent with the expected structure [311].
>
5.3.1.2 Physicochemical Properties
The isopeptide Nε
-Hcy-Lys is a solid, white powder, easily dissolving in water.
Acidified aqueous solution of the isopeptide (0.1 M) is stable at least 2 weeks at
+4
C. The thiol group of Nε
-Hcy-Lys oxidizes reversibly to a disulfide form, which
does not move from the origin of the thin-layer chromatography plate [73]. The
bulk of plasma Nε
-Hcy-Lys exists in the disulfide form [72]. Similar to other low
molecular thiols, Nε
-Hcy-Lys shows affinity to nucleophilic substitution reaction
with 2-haloquinolinium or 2-halolepidinium salts [72]. 2-chloro-1-methylqui-
nolinium tetrafluoroborate (CMQT) undergoes facile reaction with Nε
-Hcy-Lys to
give a stable thioether with a characteristic UV spectrum with an absorption
maximum at 355 nm. The reactivity of its sulfhydryl was exploited to develop an
Nε
-Hcy-Lys plasma assay [72, 86].
5.3.1.3 Biological Formation
Metabolic pathway leading to Nε
-Hcy-Lys is initiated by the conversion of Hcy to
Hcy-thiolactone catalyzed by MetRS. Hcy-thiolactone spontaneously reacts with
protein lysine residues generating N-Hcy-protein. That proteolytic degradation of
N-Hcy-protein affords Nε
-Hcy-Lys was shown by incubation of N-Hcy-hemoglo-
bin with mouse liver extracts. Nε
-Hcy-Lys is formed only in complete incubation
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