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IR Spectroscopic Determination of the Enzyme Content of Carrier-fixed Enzymes.

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tically active valienamine.I5' Other syntheses described in
the literature afford either the racemate['l or (with 3a as
starting corn pound) a diastereoisomeric
For the
decisive diastereospecific allylic amination of an alkoxysubstituted cyctohexene we chose the [2,3]-sigmatropic
shift of a sulfimide groupLH1
as reaction step['' (see Scheme
I). The starting compound is the cycIitol derivative 3a, b,
which can be obtained via Ferrier rearrangement as a 4 : 1
diastereorneric mixture in an overall yield of 40% in six
simple steps from the commercially available methyl a - D pl~copyranoside.'~.
'I
For introduction of the C , sidechain, and preparatory to the sigmatropic shift, the 4 : 1
diastereorneric mixture was converted (without separation)
in an HC1-catalyzed reaction with ethanethiol into the
thioketal derivative, which on reaction with trimethylsilyl
cyanide and tin(iv) chloride as catalyst according to a
method described by Reetz and Muller-Sfarkel' ' I afforded
4a, b (4 : I diastereorneric mixture). Reduction of the nitrile
group with diisobutylalurninurn hydride (DIBAH) led directly to the corresponding aldehyde, which OR further reduction with LiAlH4 and selective benzoytation with benzoyl cyanide (BzCN)"I furnished the cyclitol derivative
5 a , b ( I :4 diastereomeric mixture) in good yield. This
product mixture was regiospecifkally dehydrated by treatment with triphenylphosphane/diethyl azodicarboxylate
(DEAD) to compound 6 . Imination of the thioether group
with chloramine T in a two-phase process with benzyltriethylammonium chloride (BTAC) in dichlorornethane afforded directly and diastereospecifically the valienamine
derivative 7 in good yields. The protecting groups on 7
were removed with sodium in liquid
to give
valienamine, 1, which was characterized by peracetylation
to the known compound 2.1b1
IR Spectroscopic Determination of the Enzyme
Content of Carrier-fixed Enzymes
By Willi Herzog, Reinhoid Keller, * Erharr Neukurn.
and Dieter Wullbrandt
Dedicaled to Professor Heint Harnisch on the occasion
of his 60th birthday
Enzymes are attracting increasing attention as catalysts
for organic syntheses."] They are employed both in hydrolyses on an industrial scale as well as for the preparation of
reagents for analyses and syntheses on a laboratory scale.
Improvements in the preparation of the enzymes and in
their stabilization and manipulation by immobilization
continually make these catalysts all the more attractive.
In order to find optimal conditions for the immobilization of the enzymes it i s necessary to determine not only
the activity but also the enzyme content and then to calculate the specific activity. In this way one obtains activities
based on the amounts of enzyme used, thus enabling a
comparison of the catalysts. We now show with the enzyme a-chymotrypsin as example that the enzyme content
can be determined by IR spectroscopy. a-Chymotrypsin is
an endoprotease and catalyzes, inter alia, the enantiospecific ester cleavage of L-phenylalanine methyl ester.['I aChymotrypsin is preferentially immobilized on inorganic
substances such as silica gels. The support is first aminated
on the surface and then coupled via glutaric dialdehyde
with the enzyme (Fig. 1).
i
Received: January 21, 1987 [Z 2061 IE]
German version: Angew. Chem. 99 (1987) 490
CAS Registry numbers:
1. 3831-86-6; 2. 38231-89-9; 313,85716-45-6; 3b. 87984-31-4; 4a, 107960-16-7:
4 b . 108032-87-7: Sa. 108032-89-9; 5n. aldehyde precursor, 107960-17-8: Sa, alcohol precursor. 107960-18-9; Sb, 107960- 19-0; Sb, aldehyde precursor,
108032-88-8:5b, alcohol precursor, 104976-78-5: 6. 107960-20-3: 7. 1079602 1-4.
E. Truscheit, W. Fromrner. B. Junge, D. D. Schmidt. W. Wingender. Anqvw Chem. 93 ( I98 I ) 738: Angew. Chem. int. Ed. Engl. 20 (I98 1) 744;
B.
Junge, H. Boshagen, J. Stokefuss, L. Miiller in U. Brodbeck (Ed.): En: m r Inhihittws. Verlag Chemie. Weinheirn 1980. p. 123, and references
cited therein.
Y. Kameda. N . Asano, M . Yoshikawa, K. Matsui, J. Anfibin,. 33 (1980)
1 5 7 5 . T. Takehara, E. Newbrun. C. 1. Hoover, Curies Res. 19 (1985)
766.
T. lsawa, H . Yamamoto, M. Shibata. J . Anlibjot. 23 (1970) 595.
A. Kohn. R. R. Schmidt. Liebigs Ann. Chem. 1985. 775, and references
cited therein.
H. Paulsen. F. R. Heiker, Angew. Chem. 92 (1980)930; Angew. Chern.
Int. Ed. Engl. 19 (1980) 904; Liebigs Ann. Clrem. 1981. 2180.
S. Ogawr. T. Toyokuni, T. Suami. Chem. Let!. 1980, 713; 1981. 947: S.
Ogawa. T. Toyokuni. M . Omata. N. Chida. T. Suarni, Bull. Chem. Sac.
Jpn. 53 (19x0) 455; S. Ogawa, N. Chida, T. Suami, J . Org. Chem. 48
(19x3) 1203 1 T. Toyokuni,S. Ogawa, T. Suami, Bull. Chem. SOC.Jpn. 56
(19x3) I l h l .
N . Sakairi, H. Kuzuhara. Tetrahedron Lett. 23 (1982) 5327.
W. Ando. Acc. Chem. Res. 10 (1977) 179: R. W. Hoffmann, Angew.
Chetn. V I ( 1979)625; Angew. Chern. Int. Ed. Erlgl. 18 (1979) 563.
A. K o h n , Di.v.serrotion. Universitat Konstanz 1987.
D. Serneira. M . Phillippe. J.-M. Dclaurneny, A . - M . Sepulchre, D. Gero.
Qnr/rPsi.%IY8.3. 7 10. This method has been improved: A. KBhn, unpublished results (1984) (see 191); S. Mirza. L.-P.Molleyres. A. Vasella, Helu.
Chitit. Acfa 68 ( 1985) 989.
M. T. Reetz. H. Miiller-Starke. Tetraliedrun Le/r. 25 (1984) 3301.
E. J . Keisf. V. J Bartuska. L.Goodman. J . O r g . Chem. 29 (j964) 3 7 2 5 ; V.
C . Nayah. R. C. Whistler. ihid. 34 (1969) 97.
A I I ~ P H .C'lrrm.
.
Int. Ed. Engl. 26 l?987JNo. S
-
2 H,O
Fig. I . Coupting o f a n aminated silicate support via glutaric dialdehyde with
an enzyme (schematic).
The IR measurements were carried out with a PerkinElmer grating spectrometer 580B coupled to a computer
(Perkin-Elmer PC-7300 with CDS-IR software). First of
all, it was demonstrated by recording the IR spectra of
self-supporting targets of ca. 10 rng weight (diameter:
13 mm, thickness < 0.1 mm), prepared from the carrier material, the carrier material treated with glutaric dialdehyde
and carrier-fixed a-chymotrypsin (Fig. 2) with a pressure
of 100 kN,that there is no band overlap.
The spectrum of the catalyst shows a broad band at
V=3350 cm-', which is mainly due to the NH,- and NHgroups of the enzyme. Besides the absorption bands of the
CH3- and CHI-valence vibration in the region of 29702880 cm-I and those of the lattice overtone vibrations of
the carrier material at 2000-1850 cm-', the amide-I and
amide-I1 bands of the enzyme are clearly observable at
1650 and 1540 c m - ' , respectively. The integral of these
two amide bands is a valid measure of the enzyme content.
After recording the IR spectrum the target was weighed
and the spectrum normalized to 10 rng initiaf weight. Be[*] Dr. R. Keller, Dr. W.Herzog. E. Neukum, Dr. D. Wullbrandt
Hoechs! Aktiengesellschaft, Zentralforschung
D-6230 Frankfurt am Main 80 (FRG)
0 YCH VerIoqsqeseIhhqji mhH. 0-6940 Weinheim. 1987
0570-0833/87,~0505-03
S 02.50/0
483
curve was then plotted after subtraction of the background
(absorption of the propylamine-silica gel in the same region (Fig. 4).
t
60'
3
Q
a3000
LOO0
-
2000
1;oo
Skrn-']
Fig. 2. IR spectrum of the fresh catalyst 01' propylamine-silica gel, glutaric
dialdehyde and a-chymotrypsin (see text).
20-
I
cause of the relatively strong absorption of water at 1650
c m - ' the sample had to be carefully dried before being
pressed. This was carried out using a pump equipment
with (forepump) backing pump and diffusion pump, first
to a pressure of 1 mbar and then, after pulverization of the
catalyst, to a pressure of less than 5 x lo-' mbar.
For constructing the calibration curve a stock mixture of
880 mg propylamine-silica gel and 120 mg a-chymotrypsin
was prepared; this was moistened to improve mixing, and
finally dried. From this stock mixture, containing 12 wt-%
a-chymotrypsin, further aliquoted mixtures ranging down
to a content of 2 wt-% a-chymotrypsin were prepared by
addition of propylamine-silica gel (Fig. 3). In order to determine the scatter, the spectra of four pressed disks from
each mixture were recorded.
Finally an integration was carried out in the region of
the amide bands from 1736 to 1426 cm-'. The calibration
-I
0
1800
1600
1LOO
331cm"l
Fig. 3. 1R spectra of fresh catalysts ( c l . fig. 2) as a function o f t h e a-chymotrypsin content (data in wt-%).
484
0 VCH Verlugsgerellrchaff mhH. 0 - 6 9 4 0 Weinherm. 1987
4
6
8
1
0
a-Chymotrypsm lwt -%I
1
2
-
Fig. 4. Calibration curve for the I R spectroscopic dcterminutioii 01 the a chymotrypsin content of support catalysts (cf. Fig. 2). A 6 denotes arbitrary
units.
On completion of the calibration curve, the a-chymotrypsin contents of five fresh contact catalysts containing
varying amounts of enzyme were determined 1R spectroscopically (lower limit of measurement ca. 1 wt-Yo, relative
error 3%). As shown in Table I , n-chymotrypsin is completely bound to the silica gel during the catalyst preparation.
Table I. Comparison of enzyme loss to be expected after preparation of the
catalyst with the I R spectroscopically determined enzyme content.
Enzyme content [wt-%]
after catalyst prepn.
2wo
i
Enzyme content [wt-%]
I R spectroscopic
i
5
1.3
48
8
10
15
7.7
9.9
14.5
Since the catalytic activity of freshly prepared catalysts
increases during the reaction it was necessary to check
whether this is due to loss of enzyme or deactivation of the
enzyme. The enzyme contents of the fresh and the used
contact catalysts determined from Figure 5 are listed together with other values in Table 2.
These results clearly show that a loss of enzyme occurs
during the use of the catalyst. From measurements on used
catalysts it follows that the catalytic activity decreases proportionally with the enzyme content; accordingly, the loss
in activity must in the first place be attributed to a loss of
enzyme and not to a deactivation. The catalyst was used in
a continuousiy Operating fixed bed reactor with
rates
of S V 2 4 0 0 h - ' . The large initial drop in activity is most
(1570-0833/87/0505-~484$ 02.50/0
Angew. Cliem. Inr. Ed. Engl. 26 (1987) No. 5
fresh
A
-
Fig. 5. Comparison of the 1R spectra of fresh and used catalysts (cf. Fig. 2
and Table 2).
Table 2. Enzyme loss during the reaction (see Fig. 5 and text).
Catal.yst
Time
Lhl
Fresh contact I
Used
Used
-
Fresh contact I 1
Used
-
Enzyme content
[wt-%]
than 30 years, the simple species [AIMe2]@has not been
structurally characterized in the presence of neutral donor
ligands."] We now report that crown ethers stabilize this
cation and show the interesting features of its coordination
to [ 181crown-6 and to 115Jcrown-5.
We have recently shown that the [AICI2]@cation is generated in the reaction of AIC13,[21
EtA1C12,''.41and Et,AICI'"
with such crown ethers as [15]crown-5 and [18]crown-6.'"'
Indeed, crown ethers are not necessary: the [AIC12]@cation
results from the reaction of EtAIClz with two equivalents
of dimethoxyethane,"] or from the reaction of AICI, with
four equivalents of tetrahydrofuran in toluene.1x1The cationic species are of two structural types: those in which the
chlorine atoms are cis and those in which they are trans. I n
[AIC1,~[18]~rown-6]~,
the aluminum atom exists in an octahedral environment with cis chlorine ligar~ds,'~'
whereas in
[AICI2-[15]crown-5]@ the aluminum atom is in a pentagonal bipyramidal array with trans chlorine ligands.
The [A1Me2]@cation has been seen often in our attempts
to trap species related to ZieglerINatta catalysis with
crown ethers. In a typical experiment, one equivalent of
Cp2TiC12is allowed to react with two equivalents of AIMe,
in toluene in the presence of one equivalent of [ 181crown6.19' Mass balance indicates the reaction as shown in equation ( I ) .
Cp2TiCIZ+ 2AIMel
Enzyme remaining
19/01 Ial
+ [18]crown-6
[ A I M e l - ~ 1 8 ~ c r o w n - 6 ] ~ [ A f M ef
Z CCpZTiMe2
lZ]o
(1)
Cp=qS-C5H5
1
120
I20
9.5
7.2
7.0
74.5
72. I
10.3
6.8
66.1
Related reactions may be written for Cp,ZrCI? and
CpTiCI,, and [AIMe?.[ 1 8 ] ~ r o w n - 6 ]has
~ been obtained
from each of these.'"" [AIMe2-[15]crown-5]@has also been
isolated in accord with equation ( I ) with [15]crown-5 and
from the reaction of CoCI, with AIMe3 and [15]crown-S (in
toluene, molar ratio 1 : 4 : I).[' '1
[a] Referred to enzyme content of the fresh catalyst
probably due to the fact that protein is released from the
outer sheath of the carrier grain because of the high flow
rates and associated shearing.
c2
c1
Received: January 30, 1987;
revised: March 5 , 1987 [Z 2080 IE]
German version: Angew. Chem. 99 (1987) 493
c11
CAS Registry number:
u-chymotrypsin, 9004-07-3.
[I] G . M. Whitesides, C:H.
Wong, Angew. Chem 97 (1985) 617; Angew.
Chem In/. Ed. Engl. 24 (1985) 617.
[21 J. B. Jones, C. J. Sih. D. Perlman: Applicaiions oJBiochemica1 Sysrerns in
Oryunrc Chemirrrv. Purr I . Wiley, New York 1976, p. 116.
c 10
Stabilization of the IAIMe,]@ Cation by
Crown Ethers**
Bv Simon G. Bott. Abbas Ahanioour. S . David Morlev.
David A . Alwood. C. Mitchell Means. Anthony W. Coleman,
and Jerry L. Atwood*
Although alkylaluminum compounds have played a
commanding role in many areas of chemistry for more
2
['I
[**I
,
_ I ,
Prof. Dr. J. L. Atwood, Dr. S. G. Botf, Dr. A. Alvanipour,
S. I>. Morley, D. A. Atwood, C. M. Means, Dr. A. W. Coleman
Department of Chemistry. University of Alabama
TUSCalOOsa, A L 35487 (USA)
This work war supported by the U. S. National Science Foundation.
Anyrn. Cliem. I n ! Ed Enyl. 26 (1987) No. 5
Fig. I . Crystal structure of the cation ol'[AIMe2-[18]crown-6]'[AIMe2C12]" in
the crystal (12, 131.
The structure of the [AIMe2.[18]crown-6]@cation is
shown in Figure
The visually most surprising feature is that the crown ether exhibits its full symmetry configuration. (The six oxygen atoms are planar to within
0.29 A.) The aluminum atom is strongly coordinated to
O(2) at a distan:e
of 1.929(5) A, but also jnteracts with
O(1) at 2.181(5) A, and with O(3) at 2.435(5) A. The latter is
0 VCH Verlugsgesellschafr mbH. 0-6940 Weinheim. 1987
0570-0833/~?7/0505-0485$ 02 5 0 '0
485
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