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Investigation of Binding of Inhibitors to Ribonuclease by NMR Spectroscopy.

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plished with the system propionic acid/water/carbon tetrachloride in a flow apparatus specially developed for this
purpose [21. The resulting kinetic data are plausible if the
boundary layer reaction occurring on transfer of a caroxylic
acid is formulated as a series of three individual reaction
stepsW For the passage through the boundary layer from
water to carbon tetrachloride, these steps are:
1) Adsorption of the carboxylic acid molecule, which exists
as the monomer in water;
2) dimerization of the adsorbed molecules; and
3) desorption of the dimer into the cc14 phase.
The same processes occur in the reverse order on passage in
the opposite direction. The dependence of the rate constants
on the length of the fatty acid residues lends support to the
above interpretation. This dependence corresponds quantitatively to established adsorption phenomena.
Lecture at Stuttgart (Germany) on January 9. 1969 IVB 194 IEI
German version: Angew. Chem. 81,401 (1969)
[*] Priv.-Doz. Dr. W. Nitsch
Institut fur Technische Chemie der Technischen Hochschule
8 Miinchen, Arcisstrasse 21 (Germany)
[I] H . Furst and W. Nirsch, unpublished.
[2] W. Nitsch and K. Matschke, Chemie-1ng.-Techn. 40, 625
(1968).
[3] W. Nitsch, Chemie-1ng.-Techn. 38, 525 (1966).
Cyclization of Unsaturated Fatty Acids
By V. Wc(f[*1
The cyclization of 9,12,15-c,c.c- (linolenic acid), of 9,12,15t,t,t- (linolenelaidic acid), of 9,11,13-c,i,t- (a-eleostearic acid),
and of 9,11,13-t,t,t-octadecatrienecarboxylicacid (p-eleostearic acid) has been studied in alkaline diethylene glycol
solutions at 180-220 ‘C. An alkali-induced ionic reaction
occurs in which the non-conjugated acids undergo allylic rearrangement, whereas cis-trans rearrangement and doublebond shifts are observed in systems already containing two
or three conjugated double bonds or in cases in which such
conjugated systems are formed.
The a,y,c-t,c,t-triene formed in this manner is cyclized in a
thermal multi-center process to give 5,6-disubstituted 1,3cyclohexadiene. Compounds of this kind occur in equilibrium
with all conceivable cyclohexadienes and cyclohexenes having
conjugated semi-cyclic double bonds. About 10% of the cyclic dienes form o-substituted aromatic carboxylic acids. The
aromatization proceeds by loss of either two hydrogen
atoms, or one hydrogen atom and one side chain, or two side
chains. Interpretation of the mechanism is greatly facilitated
by the diverse but highly characteristic distribution of the
lengths of the alkyl and ester side chains for each of the
acids investigated.
Lecture at Freiburg (Germany) on January 24, 1969 IVB 195 1El
German version: Angew. Chem. 8, 402 (1969)
[*I Prof. Dr. V. Wolf
Unilever Forschungslaboratorium
2 Hamburg 50, Behringstrasse 154 (Germany)
New Investigations of the Biosynthesis of
Flavonoids
By H. Grisebach [*I
trans-Cinnamic acid which is formed from L-phenylalanine
by the enzyme phenylalanine-ammoniumlyase is an important precursor for the formation of flavonoids. Its hydroxylation to 4-hydroxycinnamic acid Cp-coumaric acid) is catalyzed by a microsome fraction from peas“]. This enzyme belongs to the mixed-function hydroxylyases; it could be shown
392
that the H atom originally in the para-position migrates into
the ortho-position to the hydroxyl group (“NIH shift”). A
similar reaction occurs in the biosynthesis of several flavonoids.
Further hydroxylation ofp-coumaric acid, giving caffeic acid,
can be catalyzed by an enzyme (or several enzymes) of phenolase type[21. This enzyme, purified from spinach beet, can
also hydroxylate several flavonoids at the 3’-position of the
B-ring.
According to our results, chalcones are the central intermediates in the biosynthesis of flavonoids. The enzyme chalcone-flavanone-isomerase, first enriched by E. Wong, has been
found to be present in all plants so far studied. The substrate
specificity of several isoenzymes, which are found in plants,
were investigated. It remains unsolved whether the further
changes start from the chalcone or the flavanone precursors.
Kinetic experiments with the pair [“C]chalcone-[3H]flavanone have been carried out in this connection.
Coumestanes and related natural products are biogenetically
to be counted among the isoflavones since, like isoflavanones, the latter can be converted into coumestanes. Coumestrol and a new coumestane have been isolated from Mung
bean (PhaseoIus aureus).
Dihydrodaidzein can be converted into daidzein in seedlings
of the Mung bean. Dihydroisoflavones are, however, probably not intermediates in the biosynthesis of isoflavones. The
fate of the hydrogen was studied in the conversion of 7,4‘dihydroxyt2- 14C-3 -3Hzjflavanone and 5,7,4‘- trihydroxy[2-14C-2-3H]flavanone; the H atom on C-2 is retained but a
H atom is lost from C-3.
Work by W. Burr in our Institute indicates that some isoflavones and coumestrols in Cicer arietinum and Phaseolus
aureus undergo a turnover whose biological half-life is of the
order of 50 hours. Thus synthetic and degradative reactions
will have to be considered in future studies of the metabolism
of these compounds.
Lecture at Berlin on February 24, 1969 IVB 197 1El
German version: Angew. Chem. 81,403 (1969)
[*I Prof. Dr. H. Grisebach
Biologisches Institut I1 der Universitat
78 Freiburg, Schanzlestrasse 9/11 (Germany)
[l] D. W. Russel and E. E. Conn, Arch. Biochem. Biophys.,
No. 1 2 2 ; ~ 256
.
(1967).
[2] P. F. T . Vuughan and V . S. Butt, Biochem. J., in press.
Investigation of Binding of Inhibitors to
Ribonuclease by NMR Spectroscopy
By H . Ruterjans [*I
Hitherto, high-resolution nuclear resonance spectra have
been little used for the structural analysis of such macromolecules. The signals are broad and overlap because of the small
mobility of the individual molecular groups. Only a limited
number of the signals in the N M R spectra can be unequivocally assigned to particular protons of the protein. As example
can be cited the signals of the C-2 protons of the histidineirnidazole rings, which are shifted beyond the spectral region
of the remaining protons to lower field. Furthermore, the
signals of these protons shift about 1.0 ppm to higher field
during the deprotonation to the uncharged imidazole ring; if
the chemical shift of the imidazole C-2 protons is plotted
against the p H value, “titration curves” and p K values of the
histidines are obtained.
In the case of bovine pancreatic ribonuclease A, different pK
values are obtained for the four histidines present in the enzyme. From N M R investigations on carboxyalkyl derivatives
of RNase A and on the subtilisin-cleaved enzyme (RNase S),
these pK values can be assigned to the histidines 12, 48, 105,
and 119 of the polypeptide chainrll. The pK values of the
histidines 12 and 119 of the active site are unusually low; they
increase by about 1.0 and 0.7 respectively if the NaCl concenAngew. Chem. internat. Edit. 1 Vol. 8 (1969) 1 No. 5
tration is increased from 0.1 M to 0.4 M, whereas the pK value
of His 105 increases by only 0.2 o n changing the ionic strength
by this amount. This finding indicates that a positively
charged group, probably the &-aminogroup of the lysine 41,
is immediately adjacent to the two imidazole rings of His 12
and His 119. Furthermore, from an accurate analysis of the
titration curves of His 12 and His 119 it can be shown that the
imidazole rings of these two histidines are coupled by a
hydrogen bond in a specific conformation of the enzyme.
There are many indications that this specific conformation
of the RNase A is the catalytic form of the enzyme.
If competitive inhibitors, such as cytosine 3’-monophosphate
or cytosine 2’-monophosphate, are added to a solution of
RNase A, the C-2 NMR-signals of His 12 and His 119
shift to lower field, indicating protonation of the imidazole
rings. On addition of inhibitors both histidines remain
protonated until decomposition of the RNase-inhibitor
complex at p H values of about 7-9. The chemical shift
and line width of the two signals are different. The fact
that this shift of the C-2 NMR-signals to lower field is also
observed on addition of sodium phosphate and sodium pyrophosphate, lends further support to the conclusion that the
&-aminogroups of Lys 41 and of the positively charged imidazole rings of His 119 probably fix the doubly negatively
charged phosphate group of the inhibitor 121.
Lecture at Munster on January 27, 1969 [VB 196 1EI
German version: Angew. Chem. 81, 402 (1969)
..
[“I
~-
Dr. H. Riiterjans
Institut fur physikalische Chemie der Universitat
44 Miinster, Schlossplatz 4 (Germany)
[ l ] D . H. Meadows, 0. Jardetzky, R . M . Epand, H . H. Ruterjans,
and H . A . Scheraga, Proc. nat. Acad. Sci. USA 60, 166 (1968).
[ 2 ] H . H . Riiterjans and H . Witzd, European J. Biochem.
9, 118 (1969).
Molecular Structure from Rotational Spectra
and angles determine the three principal moments of inertia,
upon which the rotational energy terms of the freely rotating
molecule (gas phase) alone depend. Once the spectrum has
been assigned then the principal moments of inertia are
known.
Molecules containing isotopes have different moments of
inertia because of the different masses; consequently, they
have different spectra despite the fact that they have the same
geometry. If the spectra of a largeenough number of isotopically substituted molecules have been analyzed a sufficiently
large set of principal moments of inertia can be accumulated
to derive from it the structural parameters.
Rotation-vibration interactions give rise to difficulties.
Whenever possible one attempts the determination of an “rSstructure” (s = substitution): For the location of an atom only
the difeerences of the principal moments of inertia before and
after the isotopic substitution of that particular atom are used.
Thus the greater part of the vibration-r 3tation interaction
which is independent of isotopic substitution is eliminated.
Only one atom at a time may be substituted. At least partial
r,-structures have been given for some 500 smaller or moderately sized molecules. The accuracy of the bond lengths lies
between 0.001 and 0.01 A. For the determination of thephysically defined “equilibrium structure” (minimum of the potential surface), explicit consideration of vibration-rotation interaction is necessary, i.e. the analysis of the rotational spectra
also in the excited states of each vibration of all molecular
isotopes is required. The effort for such an investigation to
be carried out is justified only in the case of the smallest
molecules. It is believed, however, that the above r,-structure
comes very near the equilibrium structure.
I n addition to providing data about geometry, rotation spectroscopy also furnishes a great deal of further data concerning
structure: definite dipole moment components, internal mobility, etc. The instrumentation is characterized by high resolution but low intensity of the spectra recorded, and by the use
of waveguide techniques, as well as monochromatic, tunable
radiation sources.
Lecture at Kiel on November 14, 1968 [VB 192 IE]
German version: Angew. Chem. 81, 401 (1969)
By H. D. Rudolph [*I
Rotation spectroscopy, in addition to electron-, X-ray-, and
neutron diffraction, furnishes data concerning the geometry
of a molecule. For known atomic masses, the bond lengths
[*] Doz. Dr. H. D. Rudolph
Physikalisches Institut der Universitat
78 Freiburg, Hermann-Heder-Strasse 3 (Germany)
SELECTED ABSTRACTS
Isoxazol-Qine derivatives such as ( I ) have been prepared‘for
the first time by M. Seidl, R. Huisgen, and R. Knorr by 1,3dipolar cycloaddition of nitrones to alkynecarboxylic esters,
e.g. methyl propiolate, ethyl phenylpropiolate, and dimethyl
acetylenedicarboxylate. The five-membered heterocycle produced can be stabilized in different ways. Thus the thermolabile compound ( I ) , m.p. 53-55OC. is converted into a
betaine by cleavage of the five-membered ring in boiling
glacial acetic acid. On the assumption that the isoxazol-4-ine
ring could be stabilized by incorporation of the double bond
The-synthesis-of substituted isothiazoles-from‘isoxazoles has
been reported by D. N . McGregor, U . Corbin, J . E . Swigor,
and L. C . Cheney. The isoxazole ring ( I ) is first opened up by
reduction with Raney Ni-Hz (in CH,OH, CzH50H; at ca.
r, 1
R
R’
NH2 0
3 atm) and the enamine ketone (2) thus formed is converted
into the isothiazole (3) by treatment with P2S5 followed by
oxidation with e.g. chloranil. The yields are between 38 and
90 %. / Tetrahedron 25,389 (1969) / -Ma.
[ ~ 1000
d p]
of a benzene ring, the previously unknown benz[d]isoxazolines were synthesized from nitrones and arynes e.g. dehydrobenzene. Compound (2),yield loo%, melts at 59-59.5 “C. /
Chem. Ber. 102,904 (1969) / -Ma.
[Rd 999 IE]
Angew. Chem. internat. Edit. J Vol. 8 (1969)
No. 5
The synthesis and pyrolysis of 6,7-diazatricyclo[3.2.2.02~4]non-gene (2) has been investigated by M . Martin and W.
R. Roth. The synthesis is accomplished by Diels-Alder addition of 1,3,5-cycloheptatriene and 4-phenyl-l,2,4-triazoline-
393
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investigation, spectroscopy, nmr, ribonuclease, inhibitors, binding
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