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Methoxymethyl Isocyanate as a New Agent for Reversible Protection of SH Groups in Protein and Peptide Chemistry.

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the overall rate (6.Table 1). This latter influence, which
is a consequence of a relative stabilization of the metal-carbon b o d , is also-known from homogeneous catalysist3,'I.
teine, glutathione, papain and for the trypsin-kallicrein
inhibitor from bovine organs after its selective reduction
at Cys "Cys t31.
Received: May 14, 1973 [Z 849 IE]
German version: Angew. Chem. 85,827 (1973)
[I]
C. Henrici-Oliuiand S.Oliui, Angew. Chem. 83, 121 (1971);Angew.
Chem. internat. Edit. 10, 105 (1971),and references therein.
[2] G. Henriri-Oliuiand S. OliuP, Angew. Chem. 85, 148 (1973);Angew.
Chem. internat. Edit. 12, 153 (1973).
[3] G. Henrici-Oliub and S. Oliut?, Advan. Polym. Sci. 6, 421 (1969).
[4] H . L. K r m , Reprints 5th Int. Congr. Catalysis, Palm Beach, Florida
1972.
[5] G.Henrici-Oliuiand S. Olio;, Angew. Chem. 83,782 (1971);Angew.
Cbem. internat. Edit. 10, 776 (1971).
Methoxymethyl Isocyanate as a New Agent
for Reversible Protection of SH Groups
in Protein and Peptide Chemistry
By Hmald Tschesche and Helmut Jering[*l
The usual blocking reagents for use during alkylation,
mercuration or oxidation of the SH group of cysteinet']
lead to irreversible formation of the derivative of the thiol
groupWe have found that methoxymethyl isocyanate (I a) is
excellently suited for rapid and selective carbamoylation
of cysteine-SH at pH 4-5 in an aqueous medium and
at room temperature. The reaction yielding (2a) occurs
very fast and is complete in less than 2 rnin when an
excess of the reagent is used. The a- and amino groups
d o not react under the stated conditions. The N-methoxymethyl-S-carbamoyl derivative (2a) of cysteine is sufficiently stable at pH 6 (see Table 1). For comparison it
should be noted that the half-life-of the unsubstituted
S-carbamoyl derivative (2b) from cyanate and cysteine
is only about 10 rnin at 25°C and pH 6c21.
...-H N CIH - C O - - .YH2
S-CCPNHR
+
OHo
(2)
(a), R
= H3C-OCH2;
(6). R
= H
Methyl, ethyl, and tat-butyl isocyanate react only very
slowly at pH 4 - 7 in an aqueous medium and are thus
barely useful for our purpose.
Unblocking of the SH group with removal of the N-methoxymethyl-S-carbamoyl protecting group is easily achieved
under mild conditions in an aqueous alkaline medium
(see Table 1): at pH 9.6 its removal from cysteine is complete
in 30 rnin and from glutathione in 60 min.
We have used methoxymethyl isocyanate (I a) as protecting group for reversible blocking of the SH group in c y s
I'[
D o z Dr.H. Tschesche and Dip1.-Biol. H. Jering
Organisch-Chemisches Laboratorium der Technkchen Universitit
Miinchen,
Lehrstuhl fur Organische Chemie und Biochemie
8 Miinchen 2, Arcisstrase 21 (Germany)
756
Table 1. Removal of the protecting group from cysteine (Cys) or ptutathione (GSH) at room temperature.
PH
t
[min]
9.6
8.6
7.6
6.0
100
100
30
180
Liberation of SH c%]
CYS
GSH
100
100
67
2
80
55
20
1.5
The enzymatic activity of papain was instantaneously
blocked by reaction of methoxymethyl isocyanate with
the reactive thiol group of Cys". The proteolytic activity
oftheenzymedeactivated at pH 5.6 was restored by incubation at pH 8.8 and 20°C within 60 rnin to 50%.
When the trypsin-kallicrein inhibitor from bovine organs
has been selectively reduced by sodium tetrahydridoborate,
the two SH groups formed from the disulfide bridget3'
react quantitatively with (1 a) under the conditions described,and the two-fold S-carbamoylated inhibitor is completely inactive against trypsin. Removal of the SH-protecting groups at pH 9.5 and 20°C in the presence of glycylglycine with subsequent oxidation of the neighboring thiol
groups to the disulfide bridge by access of oxygen at
the same pH regenerates the activity of the inhibitor completely within 60 min.
General experimental directions:
1p o l of the peptide or protein is dissolved in 0.5d
of 0 . 2 ~sodium succinate buffer (pH 4.0, papain at pH
5.6), and for each mol of cysteine 3 5 0 p o l (for papain
2Opmol only) of methoxymethyl isocyanate ( I a)['', dissolved in dimethylformamide [504 of (la) in 150 4
of DMF], is added with stirring at room temperature.
Quantitative reaction occurs within 2 min with simultaneous mild evolution of C02. The mixture is stirred
for a further 3 min for complete destruction of the reagent,
and the solution is then subjected to gel filtration on Sephadex G-I5 or Bio Gel P-2 in 10%acetic acid so as to
separate the products and buffer salts. The N-methoxymethyl-S-carbamoyl derivative (2a) can be preserved in
acid solution or lyophilized from acid solution. Disulfide
bridges reduced under nitrogen by NaBH, can be treated
with ( i a ) directly in the reaction mixture after buffering at
pH 4 for removal of the acid used to destroy the excess
of reducing agent.
For removal of the SH-protecting group, 1 pnol of the
protected peptide or protein is dissolved in 0.5ml of 0.1 M
tris/HCI buffer (pH 9.5), l m g of glycylglycine is added
as cyanate trap, and the solution is set aside at room
temperature for 60 min. To avoid undesired reoxidation
of the SH groups this removal is carried out under nitrogen.
Received: June 18, 1973 [Z 863 IE]
German version: Angew. Chem. 85,765(1973)
[l] G. E. Means and R . E. Femeyr Chemical Modification of Proteins.
Holden-Day, San Francisco 1971.
[2] G. R. Stark, U! H . Stein, and S. Moore, J. Biol. Chem. 235, 3177
(1.960); G. R. Stark, ibid. 239, 1411 (1964).
[S] L. F . Kress and M. Loskowski, Sr., J. Biol. C h w . 242, 4925 (1%7).
[4] We thank Bayer AG, Leverkusen, for a sample.
Angew. Chem. internat. Edit.
1 Vol. 12 (1973) 1 No. 9
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