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Hexa-tert-butyltribenzodecacyclenyl A Six-Stage Amphoteric Redox System.

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underlined sequences correspond to position 16-39 and 933-910 of the MeHNL
:
gene published) [6b] and cloned in the correct reading frame in the pQE4 expression
vector (Quiagen). The resulting expression plasmid pQE4-MeHNLwt was transformed in €. [ o h MI 5[pREP4] cells for overexpression (M15-MeHNL)
Takashi Kubo, Kagetoshi Yamamoto, Kazuhiro
An overnight culture (2 mL) of a single colony of M15-MeHNL in LB medium
Nakasuji,* Takeji Takui, and Ichiro Murata
containing ainpicillin (100 pgmL-') and kanamycin (25 pgmL-') was used to inoculate an 8 L culture that was then grown for 12 h at 37 ' C This culture was used
to inoculate a 100 L fermenter (Bioengineering). The Ml5-MeHNL cells were
Some organic compounds"' show amphoteric redox behavgrown <it 30 C for 1 h and expression of recombinant MeHNL was induced by
ior,
that is the numerical sum (E"") of oxidation potential (E"")
(IPTG) to a final concentration of 1 mM. The
adding isopropyl~~~-r~-th~oga~dctosId
E'"" = E"" +( - Ered),is very
and reduction potential (Fed),
cells were grown for an additional 3.5 h and concentrated to about 7 5 L by crosssmall. These compounds are oxidized or reduced easily by an
flow concentration (Maxisette system) with a 0.3 pm membrane (0.46 m') (Filtron).
To remove the remaining LB medium the cells were centrifuged for 10min at
electron transfer. Recently we synthesized the four-stage am10000 x x . The resulting pellet was resuspended in 2 L sodium acetate buffer
photeric redox hydrocarbons bearing two phenalenyl units,
(50 mM. pH 5.4). and the cells were disrupted by high-pressure homogenization
which are crucial for the amphoteric redox behavior. To study
(500 bar. 3 cycles; Rannie-APV MiniLab). To destroy chromosomal DNA the
the properties of higher amphoteric hydrocarbons, we designed
crude lysate (total amount of MeHNL 40000U. spec. activity 2 8 U m g - ' ) was
digested with benzonase (Merck) (final concentration of 500OUL-') for 1 h at
the tribenzodecacyclenyl la, which contains three phenalenyl
room temperature. After centrifugation at 130000 x g for 1 h MeHNL was enriched
units. Compound l a is expected to behave as a six-stage amphoto a spec. activity of' 12 U m g - ' by anion exchange chromatography. A Q-Sephteric redox compound (Scheme I)['] and to readily generate suArose FF 100!1200 column was equilibrated in 2 0 m u sodium acetate, pH 5.7
per-charged species such as the trication and trianion. We now
(buffer A ) and bound proteins were eluted with a 7200 mL linear gradient of 0- 1 M
NaCI in buffer A at a flow rate of
SO m L m i n - ' . McHNL eluted after 450 mL
Rz
salt gradient.
Enzyme-catalyzed preparation of (S)cyanohydrins The support (50 mg nitrocellulose) was soaked in 3 mL sodium citrate
buffer (0.02 M. pH 3.3) for 30 min After decanting. centrifuging
. . (30 min. 5700 xg).
+e
+e
+e
+e
and drying under high vacuum ( 5 h), a con13+
,(.)(2+)
- e 1+
-e
centrated solution of MeHNL (900UmL-'.
-e
iimounts as 111 Tables 1 and 2) was added
dropwise. After I5 min the enzyme-charged
support was centrifuged (at - S T , 30 min,
l a , A ' = R'= H
3650 x,g) and transferred to a flask. Diison
n
propyl ether ( 5 m l ) . substrates I or 3(0.3I b , R' = f B u , R2 = H
0.4 mmol) and HCN (100 pL. 2.6 mmol)
lc , R' = fBu, R2 = D
1'
were added. and the mixture stirred at room
temperature for the time given in Tables 1
Scheme 1. Six-stage amphoteric redox behavior of 1'.
and 2. The support was filtered off and
washed with diethyl ether The combined
filtrates were dried, and solvent and unchanged starting materials (la-g, 3a-f)
report the synthesis of lb, the hexa-tert-butyl derivative of la,
distilled off. Thus pure aldehyde cyanohydrins (S)-2a-j (Table 1) and ketone
and the properties of l b , l b 3 + , and lb3-.
cyanohydrins (S)-4a- f (Table 2) were isolated. To obtain compounds (S)-Zk-o and
The starting material, 2,5,8,11,14,17-hexa-tert-butyldecacy(S)-4g in pure form i t was necessary to convert them into acetates or trimethyl silyl
clene (2) /31 was formed directly by Friedel-Crafts alkylation of
ethers for the separation from unchanged starting materials. The yields obtained
correspond to those determined by ' H N M R spectroscopy (Tables 1 and 2).
decacyclene (Scheme 2) .L4I The bromination of 2 furnished an
Hexa-tevt-butyltribenzodecacyclenyl
A Six-Stage Amphoteric Redox System
+
Received: October 11, 1995 (Z8461IEI
German version: Angew. Chem. 1996. 108, 493-494
Keywords: asymmetric syntheses
matic catalysis lyases
-
.
(S)-cyanohydrins
enzy-
(11 F Effenberger. Angew. Cliem. 1994, 106. 1609-1619: Angew. Cliem. Inr. Ed.
EiigI. 1994. 33. 1555-1564.
[2] F Effenberger. B. Horsch, S . Forster, T. Ziegler. Terruhedron Lert. 1990, 31.
1249-1252.
131 a) U. Niedermeyer. M:R. Kula, Angew. Chem 1990, 102. 423-425; Angew.
Chern. In! E d €iig/. 1990,29,386-387: b) M.-R. Kula. U. Niedermeyer. I. M.
Stuertz (Degussa AG). EP-B 350908. 1990; DE-B 3823866. 1988 (Chem. Absrt-. 1990, / / 3 . 57462111
[4] a) N. Klempier, H. Griengl, M. Hayn, Terrahedron Lerr. 1993.34.4769-4772;
b) N. Klempier. U.Pichler. H Griengl, Tetrahedron Asymmetry 1995.6, 845848.
(51 a ) H. Wajant. H Bottinger. K.-W. Mundry, Biorechnol. Appl. Btochem. 1993,
18. 75 -82; b) H. Wajant, unpublished results.
(61 a) F. J P. DeC. Carvalho. Dissertation. University of California. Davis, 1981;
b) J. Hughes. F J P. DeC. Carvalho. M. A. Hughes, Arch. Biorhem. Bzopliys.
1994. 3it. 496- 502.
(71 a) H Wajant. S. Forster. H Bottinger, F. Effenberger, K. Pfirenmaier. Plant
Scr. ( L i i i w r i <X . Ire/. I 1995, 108, 1 - 11: b) H. Wajant. unpublished results.
[81 F. Effenberger. 1. Eichhorn. J. Roos. Tetrahedron Asimmerry 1995.6,271-282.
191 J Roos. Diplomarbeit. Universitit Stuttgart. 1995
1101 a) F Effenberger. B. Horsch. F. Weingart, T. Ziegler, S. Kiihner. Tetrahedron
Le/r. 1991.32. 2605-2608: b) J. Albrecht, I. Jansen. M.-R. Kula. Biorerhnol.
Appl. Blodlem 1993, 17.191 -203.
111) E. Menendez.. R. Brieva. F. Rebolledo. V. Gotor. J. Chem. Soc. Chem. tommun.
1995. 989 -990
Angew. C%em Inr. €11. Engl. 1996, 35, No. 4
isomeric mixture of the tribromides 3 in 76% yield,'51 which
were converted to tris(propionic acid) derivatives 7 by the usual
method (only one isomer is shown in Scheme 2). Friedel -Crafts
cyclization of 7 gave the triketones 8, which were reduced and
subsequently dehydrated to afford trihydro compounds
The dehydrogenation of 10 with p-benzoquinone o r p-chloranil
provided the neutral monoradical species lb' as an air-sensitive
brown solid (Scheme 2).
The ESR signal of lb' was observed, but lack of resolution
prevented the determination of the hyperfine coupling (hfc) constants. However, the ESR spectrum of the deuterated compound lc' gave hyperfine
from which two hfc constants were obtained, 1.43 G (6H), 0.47 G (6H). The hfc
[*] Prof. Dr. K. Nakasuji, T. Kubo, Prof. Dr. K. Yamamoto.
Prof. Dr 1. Murata"'
Department of Chemistry, Faculty of Science
Osaka University
Toyonaka, Osaka 560 (Japan)
Fax: Int. code +(6)850-5395
Prof. Dr. T. Takui
Department of Chemistry, Faculty of Science
Osaka City University
Sumiyoshi-ku. Osaka 558 (Japan)
['I Current address. Fukui Institute of Technology, Gakuen, Fukui 910 (Japan)
[**I This work was supported by a Grant-in-Aid for Scientific Research on Priority
Areas from the Ministry of Education, Science and Culture. Japan. We thank
Dr. Kay-Uwe Klabunde for preliminary efforts in this study.
VCH Verlagsgesellsrhuft mbH. 0-69451 Weinlieim, 1996
057U-0833196i3504-043Y $ 15.00+ 2 5 0
439
COMMUNICATIONS
t t
t t
2
6 , R = CH,CH(COOEl)p
f)G7 , R
4, R = C H ,
= CH,CH,COOH
s,
-
#$\$' $
0
= CH2Br
5, R
1,
d
0
8
I
I
I
+06
I
i
I
+02
i
I
I
4 6
-02
I
I
-10
l
I
-I 4
I
I
-1 8
W I
\'
1:
Fig. 1. Cyclic voltammogram of I b measured in dichloromethane with 0 1 M
nBu,NCIO, as supporting electrolyte at 0 ° C ; sweep rate 100 mVs-I.
++
/
t t
1b'
1b3-
Table 2. Cyclic voltammetric data [V vs. SCE] of I b and some related compounds
k,
1b3+
Ey
Scheme 2. Synthesis of I b . l b 3 + , and I b 3 - . Reaction conditions. a) tBuC1, AICI,,
CH,CI,. room temperature, 2 h, 74% b) Br,, CH,Cl,, 2 h, room temperature.
76%; c) nBuLi. CHJ, T H E - 7 8 T to room temperature, 8 1 % ; d) NBS. benzoyl
peroxide, benzene, reflux, 10 min, 96%: e) NaOEt. CH,(CO,Et),, benzene
+ethanol, room temperature, 1 d. 36%; f ) 1) aqueous KOH. EtOH, 2) 4~ HCI. 3)
IOO'C, 9 5 % , g ) 1) (COCI),, 2) AICI,. CH,CI,. -50°C. 4 h, 9 9 % ; h) NaBH,,
CH,CI, +ethanol. reflux, 1 d. 92%: i) cat. p-tolueneslufonic acid. benzene, reflux.
5 min, 9 0 % ; J) p-benzoquinone or p-chloranil, benzene. reflux, 10 min; k) conc.
D,SO, o r 3 equiv [(p-BrC,H,),N]'+[SbCl~]-; I) K mlrror, under vacuum. [D,]THF,
-78 C. 1 week.
.
constants of the hydrogen atoms H-2, H-9, and H-16 in l b
were determined by a computer simulation. Assignments of hfc
constants a H p to protons in the position p were based on a
HMO-McLachlan calculation (3. = 1.2)['1 of the spin density of
la'. The hfc constants and the calculated spin densities are listed
in Table 1 . The linear relationship between the hfc constants
Table 1 Hyperfine coupling constants zH,,and spin densities p,, for Ib. and ' H and
"C N M R chemical shifts (6) for l b 3 + and l b 3 - .
lb
[i
allp [Gl[al
1b3+
~ , [ b l 6('H) S('3C)[cl
1, 3. 8. 10. 15. 17(z')
1.43
0.055
2.9, 16(B1)
0.37 -0.012
4, 7. 11,14, 18, 21 ( a 2 )
5.6, 12. 13. 19, 20(/j2)
0.47 -0.009
5a, 5d. 12a. 12d, 19a. 19d(z3)
9.68
8.31
8.86
148.32
132.40
183.45
125.37
152.57
1b3-
6('H) 6("C)Icl
7.97
7.50
9.49
113.40
118.63
126.98
120.10
11427
[a] ESR measured in toluene at -30 'C. [b] Spin densities (McLachlan method, i.
=1.2). [c] Other "C N M R signals of l b 3 + (D,SO, at 60°C) and l b 3 - ([DJTHF
a t 2 5 'C):6 =133.54and 130.75(3a,7a. 10a. 14a. 17a.2la).respectively, 145.31 and
119.03 (5b. 5c, 12b, 12c. 19b. 19c). respectively; 125 63 and 129.58 (7b. 14b. 21b).
respectively: 13695 and 127.62 (7c. 14c. 21c), respectively; 41.77 and 37.16
(C(CH,),), respectively; 35.20 and 32.55 (C(CH,),), respectively.
stants and the theoretical values for the spin densities suggests
that the unpaired electron delocalization is not confined to one
phenalenyl unit but is operative over the entire C, symmetric
molecule.
The cyclic voltammogram of lb' measured in dichloromethane exhibited six reversible one-electron redox waves
(Fig. 1). The electrochemical data of l b and related hydrocarbons bearing two phenalenyl units are summarized in Table 2.
440
i
+1 0
t
-
i
Position
I
+I 4
0 VCH
Verlugsgesrllschuft mbH. 0.69451 Wrinhrbn, I996
E,"'
E?
Ib[d]
+0.87 +0.68 f0.27
+ 1 02 +0.56
PDPL[c]
iPr-IDPL[d]
+0.84 +0.62
EYd
EYd
EFd
E:""[b] E:""[b]
E:""[b]
-0.51
-0.43
-0.48
-0.89
-1.17
-1.07
-1.25
0.78
0.99
1.10
2.12
1.57
2.19
1.91
[a] Measured against the saturated calomel electrode (SCE) in dichloromethane
with 0 . 1 nBu,NCIO,
~
as supporting electrolyte at 0 T ; sweep rate 100 mVsC'.
[b] Er"' = E,"" +(- E;')). [c] PDPL = pentaleno[l,2,3-~,d:4,5,6-(.'d']diphenalene
[Id]. [d] IDPL = s-indaceno[l,2,3-c.d: 5,6,7-r'd']diphenalene [ l q
The sum of the redox potentials ,Sum of lb' is substantially
smaller than those reported so far for hydrocarbons. Such a
small EFm value can be explained in terms of the electronic
stability of three redox states of the phenalenyl unit owing to the
delocalization of the generated charges or the unpaired electron
over the molecules.
The trication l b 3 + was obtained as a dark green solution by
dissolving lb' in concentrated D,SO, or as dark green needles[g]
by treating lb' with three equivalents of tris(4-bromopheny1)aminium hexachloroantimonate. The trianion lb3- was
generated as a red purple solution[''] by the reaction of the
trihydro compound 10 with a potassium mirror in a sealed degassed tube at -78 "C for a week. The 'H and 13CN M R chemical shifts of l b 3 + and l b 3 - are given in Table 1. The total 13C
chemical shift change @A&) of the sp2 carbon atoms on going
from l b 3 + to lb3- is 1041.63 (or 173.61 per electron), which
supports the idea of the presence of the trication and the trianion, respectively.["] The shift changes (Ah,) are large for the C
atoms at positions X I , r 2 , and x 3 , which correspond to the x
positions of the phenalenyl,[121but small for the C atoms at
other positions except for the central benzene ring. This finding
suggests that the phenalenyl units contribute largely to the stability of both triply charged species.[13'
The most striking feature is that the signals of the protons at
the position b2 of lb3- unexpectedly appear at lower field than
the signals of the corresponding protons of l b 3 + , in spite of the
shielding effect caused by the three negative charges. The I3C
chemical shifts of the C atoms at position fl' reveal that the
electron density at the position b2 is slightly larger in lb3- than
in l b 3 + .These findings clearly suggest the effect of a diamagnetic ring current in l b 3 - . Ring currents of l b 3 + and l b 3 - were
calculated by London-McWeeny's method.[141For l b 3 + , the
phenalenyl unit causes a weakly diamagnetic effect and the
$ 15.00+ .25/0
0570-0~33~96/3504-0440
Angen. Chem. Int Ed. Engl. 1996. 35, No. 4
COMMUNICATIONS
trindenyl unit a paramagnetic effect. In contrast, l b 3 - exhibits
pronounced diamagnetic ring currents along the periphery of
the molecule.
Received: September 19, 1995 [Z8412IE]
German version: Angew. Chem. 1996, 108. 456-457
Keywords: hydrocarbons
tions
*
phenalenes
*
radicals
- redox reac-
[l] a) V. D.Parker. J. Am. Chem. Soc. 1976, 98, 98; b) K.Deuchert, S. Hunig,
Angeit.. Chem. 1978, 90, 927; Angex,. Chem. Int. Ed. Engl. 1978. 17, 875; c) K.
Nakasuji, K . Yoshida, 1. Murata. J. A m . Chem. Soc. 1982, 104. 1432, d) ibid.
1983. f05. 5136; e) Chcm. Leu. 1982. 969: f ) I. Murata, S. Sasaki, K. -U.
Klabunde. J. Toyoda. K. Nakasuji, Angebt.. Chem. 1991, 103, 198.: Angew.
Chrm. Itit. Ed. EngI. 1991, 30, 172; g) K.Takahashi, T. Suzuki, J. Am. Chem.
Soc. lY89, 1 I l . 5483.
[2] To our knowledge. only a few examples are known of compounds that exhibit
six-stage amphoteric redox behavior. See: J Heinze. Angew. Chem. 1981, 93,
186: Angeii, Chem. Int. Ed. Engl. 1981, 20. 202.
[3] The structure of 2 was confirmed unambigously by X-ray analysis. Yellow
needles of 2 suitable for X-ray structure analysis were grown by slow evaporation of a solution of 2 in CH,CI, and n-hexane. Details of the structure will be
reported in a separate paper.
(41 Decacyclene (Aldrich) was recrystallized from a xylene solution twice.
[5] Two regioisomeric tribromides could not be separated by column chromatography or recrystallization; however, this did not disrupt our synthesis, because
O
they were converted to a single symmetrical product in the final steps, 10 to Ib',
and 10 to lb3?
[6] Compounds 2 - 10 were fully Characterized spectroscopically ('H NMR, MS.
1R)
[7] Neutral monoradical species lb' and l c were stable in degassed toluene; their
ESR signals did not change for a week at room tempetrature.
[8] A. D. McLachlan. Mol. P h p . 1960, 3, 233.
C
191 Elemental analysis: found C 43.06, H 3.60%: calcd for C,,H,,(SbCI,),,
43.58. H 3.66Vo;m.p. >3OO"C; UV/Vis(H,SO,): imaX(&)
=775 (sh, 7760). 676
(sh. 26700), 601 (42500). 550 (sh, 37900), 494 (118000). 231 nm (81800).
[lo] UV;Vis ( T H F ) . am ax(^:) = 543 (56300)- 507 (52300), 421 (21000), 361 (37300).
333 (34300). 256 nm (39200).
[ i l l H. Spiesecke. W.G. Schneider. Tetrahedron Lett. 1986, 27, 937.
(121 In phenalenyl derivatives the positions 1, 3, 4, 6,7. and 9 are referred to as a
and 2, 5. and 8 as B. The shift changes (A&) are 51.8 at the a position and 4.9
at the [j position. I. Sethson, D. Johnels, U. Edlund, A. Sygula, J. Chem. Soc.
Perkin Tran,. 2 1990, 1339.
[I31 For super-charged mono- and polycyclic ions, see: K. Mullen, Cheni. Rev.
1984.84. 603: M.Rabinovitz. I. Willner, A. Minsky, Acc. Chem. Res. 1983. 16,
298.
[I41 F. London. J P17~.Radium. 1937. 8. 397; R. McWeeny, Mol. PhJs. 1958, 1,
311.
volving degenerate single proton transfer processes, which are
coupled to charge transfer and therefore to solvent reorientation, are currently available. Recently, in the conjugated porphyrin m ~ n o a n i o n [ ' an
~ ] intramolecular single proton transfer
that is characterized by a high activation barrier was observed.
In contrast, the barrier for proton transfer is very low in hydrogen-bonded complexes formed by acetic acid and pyridine at
low temperatures in organic solvents.['51 Here we introduce a
model system that lies between these two extremes : bis(4-fluorophenyl)[l,3-'5N,]triazene (1, Fig. 1) dissolved in an aprotic solvent together with a base such as trimethylamine (2). The NMR
experiments described below show that the intramolecular proton transfer in 1 occurs only in the presence of 2. Therefore,
2 acts as catalyst carrying the mobile proton of 1 from one
nitrogen site to the other, as illustrated in Figure l a .
F
N*-H*
N
N-CHs
'CHs
*M
-
2
@I,.
F
1
+2
11
-2
+211-2
I
N*
N I
N-H*
:*:
F
- 1 11.1
F
An Intramolecular Base-Catalyzed Proton
Transfer in 1,3-Bis(4-fluorophenyl)triazene
Ferdinand Mannle and Hans-Heinrich Limbach*
:I@,.
F
Proton transfer processes are elementary steps in many acidand base-catalyzed organic and biochemical reactions." '] Because of their complexity, simple model compounds are required if the proton motion is to be followed experimentally, for
example, by NMR spectroscopy or other fast reaction techniques. Model systems are also important for the theoretical
treatment of proton transfers." l o ] Whereas several model systems for double proton transfer within or between neutral molecules have been
only a few model systems in-
[*] Prof. Dr. H.-H. Limbach, Dip].-Chem. Dr. F. Mlnnle
Institut fur Orgdnische Chemie der Freien Universitat
Takustrasse 3. D-14195 Berlin (Germany)
Fax. Int. code +(30)838-5310
e-mail: limbdch(a chemkfu-berlin.de
[**I
This work was supported by the Deutsche Forschungsgemeinschdft and the
Fonds der Chemischen Industrie.
Angrn. Chem. In(. Ed. Engl. 1996, 35, No. 4
:J@:
F
Fig. 1. Proton transfer pathways for 1,3-bis(4-fluorophenyl)[l,?-'
'N,]triazene (1):
a) intramolecular proton transfer, catalyzed by trimethylamine: b) hypothetical
intramolecular uncatalyzed proton transfer; c) intermolecular double proton transfer in hypothetical cyclic dimers of 1.
The 'HNMR spectra of sealed samples of 1 in deuterated
ethyl methyl ether that are carefully prepared with well-established vacuum techniques to exclude water and other acid or
basic impuritites do not show any sign of proton mobility. The
partial room-temperature 'H NMR spectrum of such a sample
is shown in Figure 2a. The signal of the mobile proton of 1 is
split into a doublet by scalar coupling with a single I5N nucleus
= 93.5 Hz), which indicates that the mobile proton
of 1
is localized at a single nitrogen site. More precisely, potential
VCH Verlagsgrsell~chuf/mhH, 0-69451 Weinheim, 1996
0570-0833i96i3S04-0441 5 15.00f .25 0
441
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