A NEW BROMINE-CONTAINING REAGENT
551
were prepared in laboratory glassware filled with argon Identified tryptic peptides of lysozyme
using water saturated with argon.
MH+
calculated determined
Trypsin TPCK was from Sigma (United States). IR
Amino acid sequence
spectra were recorded on an Infralum FT-801 IR Fou-
1
rier spectrometer. H NMR spectra were registered on a
VXR-500S spectrometer (Varian). Chemical shifts (δ,
ppm) were recorded relative to 2,2-dimethyl-2-silapen-
tane-5-sulfonic acid. Chromatograms were obtained on
a Milikhrom A-02 liquid chromatograph (ZAO
HGLDNYR
874.42
874.42
1428.64
1753.91
1045.55
739.15
FESNFNTQATNR
1428.65
EkoNova, Novosibirsk) equipped with a Silasorb SPH NTDGSTDYGILQINSR
1753.84
C-18 column (75 × 2 mm). TOF mass spectra were
GTDVQAWIR
1045.54
recorded on a MX-5303 mass spectrometer with elec-
trospray ionization (Institute of Analytical Instrument
GCRL*
739.23
Making, Russian Academy of Sciences, St. Peters-
burg).
CELAAAMK*
WWCNDGR*
1127.4
1127.54
1227.53
1560.12
1284.35
5
-Bromo-2[(2-iodoacetyl)amino]benzensulfonic
1227.38
acid. a. Sodium salt of 2-amino-5-bromobenzene-
sulfonic acid was synthesized according to the modi- GYSLGNWVCAAK*
1559.61
1284.23
fied procedure [18]. 4-Bromoaniline (5 g, 29 mmol)
CELAAAMKR*
(
Aldrich, United States) was mixed with dry quartz
sand (10 ml) with a particle size less than 1 mm in a
porcelain mortar. Concentrate sulfuric acid (2.85 g,
Note: * Peptides containing the modified cysteine.
2
9 mmol) was added, and the mixture was carefully
ground. The reaction mixture was placed in a round-
bottom flask and heated to 170−190°ë at 10 mm Hg
pressure for 5 h. Then, the flask was cooled, and the
reaction mixture with the sand was transferred into a
KOH solution (2 g per 250 ml of water), stirred for
2
932, 2855 (methylene group), 528 (C–Br), 680 (C–I);
1
H NMR (δ, ppm): 3.00 (2 H, s, CH ); 7.00–8.30 (3 H,
2
m, Ar), and 10.50 (1 H, s, NH). Found, %: C 22.0,
H 1.2, N 3.1, S 7.4. Calcd. for C H BrINNaO S, %:
C 21.7, H 1.4, N 3.2, S 7.3.
Alkylation of the NCMLDY peptide (Asn-Cys-
8
6
4
3
0 min, and filtered. Hydrochloric acid (10%) was
added dropwise to the filtrate to pH 3. The precipitate
was filtered and dried in a vacuum. 2-Amino-5-bro- Met-Leu-Asp-Tyr). A solution of the NCMLDY pep-
mobenzenesulfonic acid was obtained in yield 4.54 g tide (1 mM, 10 µl) in 0.1 M hydrochloric acid was
(
62%); mp (with decomposition) 275–280°ë. This placed in a glass tube of a 250-µl volume; 0.1 M
value corresponded to the literature data [19]. The NaHCO (50 µl), H O (120 µl), and 5 mM solution of
3
2
resulting compound (5 g, 19.8 mmol) was mixed with AIBSA (20 µl) in 50% acetonitrile were added to the
water (5 ml) and NaHCO (1.67 g, 19.8 mmol). Water reaction mixture under vigorous stirring. The reaction
3
was removed by azeotropic distillation with toluene was monitored by HPLC (Fig. 1) at the interval of
(
50 ml), and the residue was separated and dried in a 12 min. The chromatographic conditions were: 0.05 M
vacuum. Sodium salt of 2-amino-5-bromobenzene- KH PO (pH 6.0) in 5% aqueous acetonitrile served as
2
4
sulfonic acid (5.43 g) was prepared in a quantitative eluent A and methanol served as eluent B. The column
yield; IR (KBr, ν, cm ): 3072, 3051, 3020, 1482, 1467, was eluted with a gradient of B in A (from 15 to 75%
–1
–
for 9 min) at a flow rate of 200 µl/min; the column tem-
perature was 45°ë; detection was at 200, 210, 220, 250,
8
17, 720 (benzene ring), 1230, 1185, 1147, 1033 (SO )
3
group), 3423, 3300, 1624, 1289, 1119, 627 (amino
group), 528 (C–Br).
2
60, 270, 290, and 330 nm. Areas of peaks of the
NCMLDY peptide (í 4.7 min) and the reaction prod-
r
b. AIBSA. A solution of sodium 2-amino-5-bro- uct (í 7.6 min) were determined at a wavelength of
r
mobenzenesulfonate (5.43 g) in DMF (75 ml) was 220 nm. The fraction of the reaction product was pre-
cooled to 0°ë, and iodoacetic acid (4.81 g, 25.9 mmol) paratively separated, and its structure was confirmed by
(ACROS) was added to the solution. The mixture was ESI TOF MS.
stirred to solution, and N,N'-dicyclohexylcarbodiimide
7.69 g, 37.3 mmol) (Aldrich) was added. The reaction
The rate constant of the reaction was determined
(
from the slope of the time dependence of log(C /C ),
0
mixture was kept for 24 h at 0°ë. The precipitate was
removed. The filtrate was evaporated at a residual pres-
sure of 1 mm of Hg, and the residue was recrystallized
from ethanol. The purity of the product was determined
by HPLC. AIBSA was prepared as its sodium salt in
where t is time from the reaction start, min; C the start-
0
ing area of the NCMLDY peptide peak; and ë is the
area of NCMLDY peptide peak at the time t.
Alkylation of lysozyme. Lysozyme was reduced by
−1
yield of 2.5 g (29%); mp 238–241°ë; IR (KBr, ν, cm ): mercaptoethanol by a standard procedure [17]. The
–
2
3
290, 1674, 1524 (amide), 1239, 1185, 1150, 1025 (sul- thiol groups of reduced lysozyme (5.6 × 10 mM) were
fonic group), 3038, 3025, 3060, 821 (benzene), 2971, modified by AIBSA (30 mM of AIBSA in 0.1 M Tris-
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 33 No. 6 2007