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Studies of Intramolecular Proton Transfer to Cysteine and Related Compounds by Relaxation Kinetics.

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Studies of Intramolecular Proton Transfer to
Cysteine and Related Compounds by Relaxation
Kinetics I**]
(FB=yBco,Cc=ycco: equilibrium concentrations of B and
C, respectively) gives a straight line when plotted against
the initial concentration co (Fig. 1).
By Giinter Maass and Frens Peters“
I
The dissociative behavior of amino acids such as cysteine
and related compounds containing more than two proton
donor-acceptor functions has been repeatedly investigat.ed“ -3! Whereas the carboxyl-proton is removed in acid
solution, the amino- and thiol-protons of cysteine are
liberated in about the same weakly basic media. It is,
however, not possible, without further assumptions, to
assign the potentiometrically determined pK values pK,,
and pK,,, to individual “microscopic”pK values of the two
groups“’ that are linked by the relations K,,=K, + K, and
l/K1~~=l/K3+l/K~.
For the dissociative behavior of cysteine and analogous diand poly-basic acids a simple reaction scheme is usually
given14’according to which the protonated speciesA passes
into the deprotonated form D with loss of two protons by
way of B or C. We have found that this scheme must be
supplemented by a direct proton transfer between B and C,
whose equilibrium constant is related to the other microB
I
D
05
10
co[mol/L1 -z
Fig. 1. Dependence of the relaxation frequencies on concentration.
0 = Glutathione (pH = 9.05); 0 = cysteamine (pH = 9.7)
Thence, by extrapolation to low concentrations the time
constants for the intramolecular proton transfer can be
calculated. For cysteamine one thus obtains 1 / f = k’, + k;,
=3.0 x lo7 s-‘ and for glutathione l/r’=6.2 x lo7 s-‘. If
the intramolecular proton exchange is fast compared with
the intermolecular process the first terms can be negiected
and the reciprocal relaxation time becomes independent
of concentration ; this condition is fulfilled for cysteine.
\SH
C
scopic constants by K, = K JK2 = K,/K,. This proton
transfer can be demonstrated directly by investigation of
the chemical relaxation by means of ultrasound absorption
measurements[s.‘ 1 . Similar proton transfer reactions are
known for cysteamine HS(CH,),NH,.
The proton exchange between the two functional groups
may occur intra- or inter-mdecularly :
1o6
The pH values were so chosen that the concentrations of
forms B and C were maximal and those of forms A and D
were low. Under these conditions the reciprocal of the
relaxation time for the two parallel reactions :
[*] Priv.-Doz. Dr. G . Maass and Dr. F. Peters
Gesellschaft fur Molekularbiologische Forschung
3301 Stockheim/Braunschweig, Mascheroder Weg 1 (Germany)
and
Max-Planck-Institut fur Biophysikalische Chemie
34 Gottingen-Nikolausherg (Germany)
This work was supported by the Deutsche Forschungsgerneinschaft (SFB 75).
[**I
428
lo7
lo8
Fig. 2. Sound absorption spectra of aqueous cysteine solutions at
pH = 9.5.
The sound absorption spectrum (Fig. 2) shows a rapid
contribution, independent of concentration (effect 11), and
a second one (effect I) that is slower by an order of magnitude
and can be ascribed to the intermolecular reactions. In this
case, because of the markedly differing time constants, the
two effects can be treated as uncoupled. For cysteine the
sum of the velocity constants for intramolecular proton
exchange between the thiol and the amino group has the
value 1/r’=3.6 x 10’ s-’.
Direct calculation of the individual velocity constants k’,,
and k h l , and thus the microscopic pK values, from the
Angew. Chem. internat. Edit.
Vol. I 1 (1972)
1 No. 5
relaxation measurements .requires a knowledge of the
equilibrium constants K , . However, the microscopic constants can be determined by combination of potentiometric
and spectrophotometric titrations"] only under the limiting
assumption that the absorption by the deprotonated thiol
group is independent of the prevailing degree of protonation
of the amino group. This assumption can be tested by
kinetic methods if the reaction volume AV(=V,-V,) is
known and provided that it can be decided upon which
side the equilibrium lies. It is then possible to determine
K = FB/Fc =y/( 1 -y) unambiguously by measuring the
maximal sound absorption amplitude
( A ~ / V ) , , ,=
~ ~const.y(l -y)AV2.
Such a compound (R=C6HlI; R'=CH,, M=Au, n = l )
is formed (as trimer) when methanolic potassium hydroxide
solution (157 mg/l8.9 ml) and then, with stirring, cyclohexyl isocyanide (305 mg) are added to a suspension of
chloro(triphenylphosphane)gold(rfin methanol. After 0.5 h
the suspension is concentrated to half volume in a waterpump vacuum. The white precipitate is recrystallized from
chloroform/methanol (m. p.
190°C with gradual decomposition and violet coloration above 160°C).
However, it can be shown that for the systems studied, the
intramolecular proton exchange between neighboring
groups is faster by two or three orders of:magnitude than
the protolysis (or hydrolysis) reactionsr7](see Table) :
group ( ~ = 5 . 9 9ppm). No N-H, 0-H, or -NECstretching vibration was observed in the W spectrum, but
,
B + H+=A+C+H+
or B $ D + H + e C [ 7 ]
The 'H-NMR spectrum, in CDCl,, measured from T = - 10
to 30 ppm, contains only signals that can be assigned to
the cyclohexyl ( ~ ~ 5 and
. 7 8.3 ppm) and the methoxyl
+
@
I
I
only -C=Nand =C-OCH,
stretching vibrations at
1535 (vs, br.) and 1130 (s) cm-', respectively. The compound is non-conducting in methylene chloride. The molecular weight in chloroform amounted to 1031 and is independent of concentration (0.81 - 3.1 wt.-%).
I
Table. Relaxation times for the intramolecular proton transfers.
Compound
= rs1
Cysteine
Homocysteine
Penicillamine
Glutathione
Cysteamine
2.8
4.4 x
4.9 x
1.6 x
3.3 x
Q
These findings and the position of the -C=Nvibration indicate a formula of type (I).
stretching
10-9
10-8
10-8
lo-'
10-8
Au
zN===C
This means that the direct proton exchange between the
two functional groups with interchange'of hydrogen bonds
can occur appreciably more easily than the corresponding
reactions of the free protons or hydroxide ions.
These results are of particular interest for enzyme reactions
in which mutually reacting groups are brought into close
contact by rapid structural interchanges
s). They
show that the individual steps of intramolecular proton
transfer are as fast as, or faster than, the structural changes.
Received: January 14,1972 [Z 604 IE]
German version: Angew. Chem. 84,430 (1972)
[I] R. E. Benesch and R. Benesch, J. Amer. Chem. Soc. 77,5877 (1955).
[2] K . Waiienfels and Ch. Sfrefer, Biochem. 2. 346, 119 (1966).
131 E. Coates, C. Marsden, and B. Rigg, Trans. Faraday Soc. 65, 863
(1969).
[4] J . I: Edsalland J . Wyman: Biophysical Chemistry. Vol. 1. Academic
Press, New York 1966.
[5] M . Eigen and L. de Maeyer in A . Weissberger: Technique of Organic
Chemistry. Vol. 8/2, Interscience, New York 1963.
[6] F. Peters, Acustica, in press.
[7] M . Eigen, Angew. Chem. 75, 489 (1963); Angew. Chem. internat.
Edit. 3, 1 (1964).
Trimeric 1-(Cyclohexylimino)rnethoxymethylgold( I),
A New Type of Organometallic Compound
CsHL
,Au
'OCH,
(11
If aromatic isocyanides are used instead of cyclohexyl
isocyanide, monomeric (not trimeric) ligand-stabilized
compounds such as
[(C6H,),P]AuC(=N-C6H,-CH,)OCH,
are formed"].
Except for the unstable cyclopentadienylgold(1) and
[C6H,-c~C-Au],,
the compound ( I ) described here
is the only organogold(1)compound['] that does not require
stabilization by ligands such as phosphanes or isocyanides.
Received: December 20,1971 [Z 616 IE]
German version: Angew. Chem. 84, 482 (1972)
[1] G . Minghetti and F. Bonati, Rend. Accad. Naz. Lincei, Classe Sci.
fis. mat. nat. [VIII], 49, 287 (1970).
[2] B. Armer and H . Schmidbauer, Angew. Chem. 82, 120 (1970);
Angew. Chem. internat. Edit. 9, 101 (1970).
Ethyl y -Cyano-a-isocyanoalkanoates from
a-Metalated Ethyl Isocyano-acetates or
-propionates and AcrylonitrilesI']
By Ulrich Schollkopf and Paul-Hermann Porsch"]
No organometallic compound corresponding to the general
formula [R-N=C( OR')-],M
has hitherto been known.
y-Cyano-a-isocyanoalkanoic esters (6) deserve attention
because they have several reactive centers and can react in
many ways, e.g. by hydrolysis of the isocyano group to
yield a-amino-y-cyanoalkanoates. They are obtained by
[*] Dr. G. Minghetti and Prof. Dr. F. Bonati
[*I
By Giovanni Minghetti and Flavio Bonati[*]
Istituto di Chimica Generale dell'Universita
1-20133 Milano, via Venezian 21 (Italy)
Angew. Chem. internat. Edit. / Vol. 11 (1972) 1 No. 5
Prof. Dr. U. Schollkopf and Dip1.-Chem. P.-H. Porsch
Organisch-Chemisches Institut der Universitat
34 Gottingen, Windausweg 2 (Germany)
429
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intramolecular, compounds, transfer, kinetics, cysteine, related, relaxation, proto, studies
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