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NMR Studies of Yeasts Grown on Substrates at УZeroФ Level Radioactivity.

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12 + 21-Cycloadditions of Allyl Cations**
However, not all 1,l-dialkylated, 2-unsubstituted allylic
systems react to form four-membered rings: under identical conditions reaction of l-chloro-3-methyl-2-butene
with
isobutene, 2-methyl-2-butene, and 2,3-dimethyl-2-butene
yields exclusively linear addition products12b1.
According to
molecular mechanics calc~lations[~1,
these acyclic adducts
are more stable than the isomeric cyclobutane derivatives.
On the other hand, the heat of formation of 3 is calc~lated~
to~ be
l 3.8 kcal/mol more negative than that of
the isomeric 6-chloro-2,4,5,5,6-pentamethyl-2-heptene;
gauche-interactions destabilize the linear isomer to a
greater extent than the cyclic system (gem-dialkyl effecd5').
Since steric effects should be similar in the corresponding
carbenium ions, the different behavior of 1 and l-chloro3-methyl-2-butene in addition reactions with tetramethylethylene 2 can be rationalized.
[2 21-Cycloadditions of this type can be expected if
they are electronically favored over [3+ 2]-cycloadditions
and if steric effects destabilize the linear adducts relative
to the four-membered rings.
By Herbert Klein, Gerhard Freyberger, and
Herbert Mayr*
Allyl cations are versatile building blocks for the construction of carbocycles. Hoffmann has reported the syntheses of six- and seven-membered ring systems via [2+4]and [3 + 4]-cycloadditions of allyl cations to 1,3-dienes"I.
We previously found that [3 2]-cycloadditions of allyl
cations to alkenes provide an easy access to cyclopentenesIZal,and now report [2 + 21-cycloadditions of allyl cations to afford cyclobutane derivatives.
When a mixture of 4-chloro-2-methyl-2-pentene 1 and
2,3-dimethyl-2-butene 2 is treated with zinc chloride-ether
at - 78 "C, the cyclobutane 3l3"Iis obtained in 72% yield1'].
Similarly, 4-chloro-2,4-dimethyl-2-pentene 4 and 1ethoxy-2-methyl-1-propene5 give 80% 613b1.In both cases
only one (probably the sterically favored trans) isomer is
detected by N M R spectroscopy. No linear intermediates
were detected. Lewis acid catalyzed rearrangements of 3
and 6 to give thermodynamically more stable five-membered rings, which would have to proceed via secondary
carbenium ions, were not observed. Treatment of 3 with
zinc chloride-ether at 20°C yields a complex mixture of
products, which contains 2-chloro-2,3-dimethylbutane; its
formation can be rationalized by [2 + 2]-cycloreversion of
the cyclobutylcarbinyl cation and addition of HCl to 2.
+
+
Received: September 22, 1982:
revised: October 27, 1982 [Z 154 IE]
German version: Angew. Chem. 95 (1983) 62
[I] H. M. R. Hoffmann, Angew. Chem. 85 (1973) 877; Angew. Chem. I n / . Ed.
Engl. 12 (1973) 819; H. M. R. Hoffmann, H. Vathke-Ernst, Chem. Ber.
114 (1981) 2208.
12) For experimental details see a) H. Klein, H. Mayr, Angew. Chem. 93
(1981) 1069: Angew. Chem. I n / . Ed. Engl. 20 (1981) 1027; b) H. Klein, A.
Erbe, H. Mayr, ibid. 94 (1982) 63 and 21 (1982) 82; Angew. Chem. Suppl.
1982, 105.
[3] a) 3-(l-Chloro-l-methylethyl)-l,l,2,2,4-pentamethylcyclobutane3 : 'HNMR(100 MH~,CCl~):6=0.81,0.88,0.96,1 . 0 8 ( 4 ~ 15H,
;
I-CH1.2-CH3,
the two middle signals are overlapped by the d of 4-CH3), 1.50 (s: 6 H ,
(CH,),CCI), 1.82-2.14 (m; 2H, 3-H, 4-H): "C-NMR (25 MHz, CDCId:
6= 13.71, 19.44, 20.63, 22.75, 25,54, 32.18, 32.64 (7q), 38.65 (S), 39.10 (d),
42.86 (s), 60.33 (d), 72.93 (s); b) 3-(l-chloro-l-methylethyl)-4-ethoxy1,1,2,2-tetrarnethylcyclobutane6 : 'H-NMR (60 MHz, CC14): 6=0.98 (s;
CH,), 1.00 (s; 2CH3 and right branch of the O-CH2CH, triplet), 1.13 (s,
CH,), 1.15 (t, J = 7 Hz; OCHKH,), 1.57 (s; C(CHJ)ZCI), 2.06 (d, J = 10
Hz; 3-H), 3.46 (q, J = 7 Hz; OCHz-CH,), 3.59 (d, J = 10 Hz; 4-H); "CNMR: 6=15.59, 18.93, 21.23, 23.60, 25.84, 32.67, 32.91 (7q), 37.40, 42.50
(2s), 59.30 (d), 65.73 (t), 71.71 (s), 81.42 (d).
141 N. L. Allinger, Y . Yuh, MM2 Molecular Mechanics, Program No. 395
QCPE, Indiana University, Bloornington 1980; N. L. Allinger, J. A m .
Chem. SOC.99 (1977) 8127.
IS] N. L. Allinger, V. Zalkow, J. Org. Chem. 25 (1960) 701; B. Capon, S . P.
McManos: Neighboring Group Participarion. Yol. I . Plenum Press, New
York 1976, p. 58ff.
1 (CH3)zCzCH-CH(CH3)Cl
F I H CH,
+
4 ( C H3) ZC=C H-C (CH3)zCl
C1H C H -
0 CH3
5 ( C H3) 2C =C HOC zH,
I
C 2%
2-Alkyl substituted allyl cations and alkenes undergo
[3 21-cycloadditions with formation of tertiary cyclopentyl cations['"]. We have now obtained four-membered rings
by reaction of alkenes with 1,l-dialkylated, 2-unsubstituted
allyl cations. Here [2 21-cycloadditions are preferred
since they give tertiary carbenium ions, whereas the corresponding [3 21-cycloadditions would afford secondary
carbenium ions.
+
NMR Studies of Yeasts Grown on Substrates at
"Zero" Level Radioactivity
+
+
CH3
H
c H3
C H,
H
3
C
~
R ZR4
[*I Priv.-Doz. Dr. H. Mayr, H. Klein, G . Freyberger
[**I
lnstitut fur Organische Chemie der Universitat Erlangen-Niirnberg
Henkestrasse 42, D-8520 Erlangen (Germany)
This work was supported by the Deutsche Forschungsgerneinschaft and
the Fonds der Chernischen Industrie.
Angew. Chem. I n / . Ed. Engl. 22 (1983) No. I
i
By Ricardo Basosi, Claudio Rossi, Enzo Tiezzi*, and
Gianni Valensin
The cultivation of different protein-rich microorganisms
which utilize hydrocarbons (petrochemicals) as sources of
carbon and energy has been frequently suggested'31.The
nutritional and biological value as well as the level of toxic
substances have still to be studied; the effect induced by a
prolonged alimentation on substrates which have less than
the natural level of I4C radioactivity has also still to be investigated.
Since many correlations have been found between the
N M R parameters of water and the physiological or pathological states of cells, as well as the conformations of cell
constituents". l0-''1 we have used water relaxation studies
.
[*] Prof. Dr. E. Tiezzi, R. Basosi, C . Rossi, G. Valensin
Institute of General Chemistry, University of Siena
Pian dei Mantellini 44, 1-64 100 Siena (Italy)
0 Verlag Chemie GmbH, 6940 Weinheim. 1983
0570-0833/83/010l-0049 $02.50/0
49
to investigate rapidly growing populations. The yeasts selected (Saccharomyces cerevisiae ATCC 9763, Candida
utilis CBS 621 and Saccharomycopsis lipolytica) can be easily grown on ethanol, glucose and n-paraffin substrates.
We used an ethanol substrate having various 14C levels to
ascertain whether a biological effect is apparent after
many generations. Moreover the use of ethanol yields a
cell biomass having a chemical composition quite similar
to that obtained with a glucose substrate.
Cell samples were centrifuged, washed three times, and
suspended in distilled water. N M R samples were prepared
by adding 0.7 mL DzO (99.75%) to 0.3 mL of the cellular
suspension. The ’H spin-lattice relaxation time ( T I )was
measured o n a Bruker WH-90 NMR spectrometer using
the (z-.t-n/2) pulse sequence[’o,’21.The relaxation data obtained from Candida utilis and Saccharomyces cerevisiae
with ethanol having various I4C levels are shown in Table
1. A concentration of 2 x 10’ cells per mL was used in each
case. The error in this value is relatively large (ca. 20%).
However, the dependence of TI on cell c~ncentration‘~’,
is
not sufficient to account for the observed changes in T , ,
suggesting a dependence o n the level of radioactivity of
the substrate, especially in the case of Candida utilis. Compared with cells grown on substrates having the “minimum” natural radioactivity, those cultivated on the same
substrate without radioactivity give rise to shortening of TI
whereas, in contrast, those grown on the substrate enriched
by radioactivity give rise to longer TI values. A similar
trend was shown by measurements on cultures at lower
growth levels and also o n lyophilized samples, but the effects were much less marked.
Table I. Spin lattice relaxation times of water protons in Succhuromyces cerevisiue ATCC 9763 and Cundidu utilis CBS 621 grown on ethanol at different
levels of radioactivity [a].
Source
of ethanol
Petrochemical
Fermentation
‘Tenriched
utilis
Succhuromyces
cerevisiae
0.2
19.0
6740.0
1.7i0.1
3.6+0.2
5.3 k 0.3
1.7 i 0 . 2
2.9 0.3
3.9k0.2
Candidu
[ 3 ] J. H. Litchtield, Ado. Appl. Microbiol. 22 (1977) 267.
[8] G . Valensin, N. Niccolai, Chem. Phys. Letters 79 (1981) 47.
[lo] P. E. Valensin, M. L. Bianchi Bandinelli, M. L. Di Cairano, G. Valensin,
E. Gaggelli, E. Tiezzi, Biophys. Chem. 14 (1981) 357 and references cited
therein.
[ I I] R. Basosi, E. Gaggelli, E. Tiezzi, G. Valensin, Proc. XXAmpere Congr. 20
(1978) 537.
[I21 R. M. De Vre, Prog. Biophys. Mol. Bio. 35 (1979) 109.
+
Table 2. Spin-lattice relaxation times of water protons in Succhuromycopsrs lipolyticu grown on different substrates [a].
TI
Substrate
151
4.6 f0.3
3.lf0.2
3.7+0.2
3.4 0.3
+
~~
[a] Samples contained (2k0.4) x 10’ cells/mL.
The results reported here, together with those obtained
on Candida boidinii”] grown o n different substrates, can be
interpreted by assuming that the level of radioactivity of
0 Verlag Chemie GmbH, 6940 Weinheim. 1983
C-Scrambling in the tert-Butyl Cation**
By G. K . Surya Prakash, Altaf Husain, and
George A . Olah*
Table 2 shows the spin-lattice relaxation data obtained
from Saccharumycopsis Iipolitica cultivated on different
carbon and energy sources. The longest TI value was
found for yeast cultivated o n a glucose substrate, a “natural” substrate, while the cultures grown on n-paraffins displayed relatively shorter TI values. Similar results were obtained from lyophilized samples of Candida boidinii grown
on both “natural” (glucose and xylose) and synthetic (methanol and ethanol) substrates[”].
50
Received: November 13, 1981 [Z 408 IE]
revised: October 22, 1982
German version: Angew. Chem. 95 (1983) 55
The complete manuscript of this communication appears in:
Angew. Chem. Suppl. 1983. 57-62
TI Is1
Radioactivity
[disintegrations
per min/g]
[a] Samples contained (2f0.4) x 10’ cells/mL
~~
the substrate causes a biological effect that is detectable by
measurement of the cell-water T I .To our knowledge, this
is the first time that “zero level” radioactivity is reported to
affect cell behavior. Previously, NMR studies of cell water,
besides other techniques, have been used to show biological damage resulting from radioactive
Since
ESR signals were not detected and the samples were carefully deoxygenated, paramagnetic impurities cannot account for the results. On the contrary, conformational
modifications of cell constituents and changes in water
content and in the molecular complexity of the sample
have been alternatively used to explain the NMR behavior
o f cell water[8~’0,’21.
As a consequence, the observed change
in T, could reflect any one of these phenomena or any
combination of them. However, high levels of radiation are
expected to cause cellular damage liberating interstitial
water, so that a change in the water content seems the most
proper explanation for the water TI behavior. It is important to underline that this effect becomes detectable only
in populations after many generations.
The ease of hydrogen scrambling in secondary and tertiary carbocations in superacid media is well documented“’. Saunders and Rosenfeld have
that tertpentyl and 1-methylcyclopentyl cations in SbF5/S02CIF
solutions undergo methyl- and methylene-hydrogen scrambling at 110- 140 “C, rendering all C and H atoms equivalent on the N M R time scale. The activation energy EA for
this process was f o ~ n d [ ~to. ~be] ca. 18-19 kcal/mol for
both systems. The scrambling was shown to occur via secondary cations and protonated cyclopropane intermediates rather than free primary cations.
A similar process is expected to occur in the tert-butyl
cation. Saunders et al. mention[2a1an attempt to observe
such a scrambling in the hexadeutero-tert-butyl cation at
100 “C,but were unable to observe any H I D exchange. We
now wish to report that scrambling does occur in the tertbutyl cation 1, but that the rate of exchange is too slow to
be observed by NMR line-broadening experiments.
[*] Prof. Dr. G. A. Olah, Dr. G . K. Surya Prakash, Dr. A. Husain
Hydrocarbon Research Institute and Department of Chemistry
University of Southern California
Los Angeles, CA. 90089 (USA)
[**I Stable Carbocations, Part 237. This work was supported by the National
Institutes of Health. G. A . Olah thanks the Alexander-von-Humboldt
Stiftung for a Senior US Scientist Award.-Part 236, see G . A. Olah, A.
L. Berrier, L. D. Field, G . K. S . Prakash, J . Am. Chem. Sot. 104 (1982)
1349.
0570-0833/83/0101-00S0 $02.50/0
Angew. Chem. Int. Ed. Engl. 22 (1983) No. I
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