close

Вход

Забыли?

вход по аккаунту

?

Imidazol-2-ylidene Generation of a Missing Carbene and Its Dication by NeutralizationЦReionizatin and Charge-Stripping Mass Spectrometry.

код для вставкиСкачать
COMMUNICATIONS
In conclusion, dyotropic p-lactone- y-lactone ring enlargement follows the nonconcerted mechanism B; the migratory aptitudes of the substituents are in the order z-donor>ndonor > o-donor substituent. Migration of z-donor (and
possibly also of o-donor) substituents proceeds via intermediate
Z-1, whose lifetime depends on carbenium stabilization. The
migration of n-donor (oxygen) substituents involves a bridged
oxonium species 2-3. In all rearrangements an inversion at C4
is observed without exception. The stereochemical course of the
reaction is better clarified,['I and its preparative value lies in the
stereocontrolled formation of three contiguous stereogenic centers in the y-lactones 2c,e-h.
Received: January 3. 1997 tZ9961 IE]
German version : Angew. Chem. 1997, 109, 1546- 1548
Keywords: dyotropy
rearrangements
.
lactones
*
reaction mechanisms
-
[l] M. T. Reetz, Angew. Chem. 1972,84, 161-162; Angew. Chem. In/. Ed. Engl.
1972, I t , 129-130, ibid 1979, 91, 185-192 and 1979, 18, 173-180; Adv.
Organornet. Chem. 1977, 16,33; Tetrahedron 1913,29,2189-2194.
[2] J. Mulzer, G Briintrup, Angew. Chem. 1979, 91, 840-841; Angew. Chem. Int.
Ed Engl. 1979,18,793-794.
[3] Review: D. J Coveney in Comprehensive Organic Synthesis (Eds.: B. M. Trost,
I. Fleming), Vol. 3 (Ed.: G. Pattenden), Pergamon Press, Oxford, 1991. p 777.
[4] T. H. Black, J. A. Fields, Synth. Commun. 1988, 18, 125-130.
[5] T. H. Black, S. H. Eisenheis, T. S. McDermott, S. L. Maluleka, Tetrahedron
1990, 46,2307-2316.
[6] The synthesis of the b-lactones proceeded by carhoxy-group activation with
phenylsulfonyl chloride as described [9,10]. The diastereomeric 8- and y-lactones were separated by HPLC under normal-phase conditions with a
Nucleosil (5-7 pm) stationary phase and n-hexanelethyl-acetate as eluent.
[7] For ring enlargement the b-lactones were dissolved in anhydrous diethyl ether
(10-i-10-2 mmolmL-') and treated with 1.0equiv of a 3.2 M solution of
MgBr, in diethyl ether with ice cooling and stirring. After 12-60h at
25-30°C the reactions were quenched with water, and the diastereomeric
y-lactones isolated from the ether phase [2]. Cross-check experiments showed
that :.-lactones 3a-e remained unchanged under the reaction conditions
[8] M. T. Reetz, A. Schmitz, X. Holdgriin, Tetrahedron Lett. 1989,30,5421-5424.
[9] J. Mulzer, A. Pointner. A. Chucholowski, G. Briintrup, .IChem. Soc. Chem.
Comm. 1919, 52-54.
[lo] W. Adam, J. Baeza, J.-C. Liu, .
I
Am. Chem. SOC.1972, 94, 2000-2006.
1111 X-ray crystal structure data for 1f: 0.6 x 0.6 x 0.6 mm', orthorhomhic, PcaZ,,
a =15.624,b = 6 . 4 3 4 , ~= 31.92A, V = 3 8 1 2 A 3 , Z = 8 , p = 1 . 1 7 9 g ~ m - ~ , a h sorption coefficient 0.077 mm-', 20 range 3.0-479, Mo,, (0.71073
w scan, T = 294 K. Of 3321 reflections 2892 were independent (because of a
split reflex profile only the data of type - h, k , + 1 were used for structure
determination and refinement), and 2385 observed [F>4u(F)]. Lorentzian and
polarization corrections were carried out, but no absorption corrections.
Siemens SHELXTL was used for direct methods, and anisotropic refinement was done by the full-matrix least-squares method; 451 parameters refined. The hydrogen atoms were fitted at calculated positions and coupled to
the corresponding atoms during refinement; a common isotropic displacement
coefficient was refined for all hydrogen atoms. R,,, = 0.1101, W R = 0.1020,
refinement against I, largest difference peak 0.33 e k 3 [14].
[12] E. L. Eliel, S. H. Wilen, Stereochemisrry qfOrganic Compormds, Wiley, New
York, 1996, p. 647-655.
[13] The relative configuration of 2f was determined by hydrogenolysis to 4, which
was characterized by X-ray crystallography [14].
[14] Crystallographic data (excluding structure factors) for 1f and 4 have
been deposited with the Cambridge Crystallographic Data Centre as supplementary publication no. CCDC-179178. Copies of the data can he ohtained free of charge on application to The Director, CCDC, 12Union
Road, Cambridge CBZIEZ, UK (fax: int. code +(1223)336-033; e-mail:
deposit@chemcrys.cam.ac.uk)
A),
+
+
Imidazol-Zylidene: Generation of a
Missing Carbene and Its Dication by
Neutralization- Reionization and
Charge-Stripping Mass Spectrometry**
Graham A. McGibbon, Christoph Heinemann,
David J. Lavorato, and Helmut Schwarz*
Dedicated to Professor Zvi Rappoport
on the occasion of his 60th birthday
There are many simple molecules that have been studied by
quantum chemical methods sufficiently often and thoroughly
enough that they take on an aura of familiarity but nevertheless
still manage to elude experimental observation. Among the
most famous of these is O=C=C=O.['l A more recent candidate is imidazol-2-ylidene 1, the parent compound of a carbene
family with members that proved stable enough to be isolated.r21
Although imidazolylidenes have been studied for over three
decades,r31the theoretical scrutiny of 1 has recently intensified.I4] While computational investigations have concentrated
on 1, to-date all the aminocarbenes[2,4d-f*
51 and related comp o u n d ~ [61~observed
~,
in condensed phases possess non-hydrogen substituents on the nitrogen atoms. Unsubstituted
aminocarbenes have been reported in the gas phase,"] for example the acyclic carbenes H,N-C-NH, and H,N-C-OH as well
as the heterocyclic tautomers of oxazole, pyridine, and thiazole
were prepared and characterized by using neutralization- reionization mass spectrometry (NRMS) .Is1 The latter method is particularly suitable for the identification of molecules that are
susceptible to intermolecular isomerization but relatively stable
toward unimolecular rearrangement since the experiments are
performed on solitary gaseous species. This approach may be a
route to 1 and to imidazol-4-ylidene 2, the 1,2-hydrogen shift
isomers of imidazole 3. Earlier computations predicted that
neutral 1 and 3 are separated by a substantial tautomerization
barrier as are their radical cations.r4h,91 Similarly, our hybrid
Hartree-Fock/density
functional
theory
calculations
(Be~ke3LYP/6-3lG**)['~~
indicate that a barrier of
36 kcalmol-' restricts the exothermic (50 kcalmol- ') isomerization 2 + 3 and that the apparently isoenergetic ions 2" and
3" are separated by a barrier of 56 kcalmol-'.r''l In light of
this, it would seem that 1 and 2 are targets suitable for generation and characterization by NRMS if precursor molecules can
be found (Scheme 1).
It was presumed that the ion 1'+ could be generated
analogously to previous gas phase syntheses of carbene radical
cations by dissociative electron ionization (EI) of an appropriately substituted imidazole. Indeed, the readily available imidazole-2-carboxaldehyde I seemed a good initial choice since it has
[*I Prof. Dr. H. Schwarz, C. Heinemann!+l Dr. G. A. McGibbon"+'
Institut fur Organische Chemie der Technischen Universitat
Strasse des 17. Juni 135, D-10623 Berlin (Germany)
Fax: Int. code +(30) 314 21102
Dr. D. J. Lavorato
Department of Chemistry, McMaster University
1228 Main Street West, Hamilton, Ontario, L8S 4M1 (Canada)
New Address: Hoechst AG
D-65926 Frankfurt am Main (Germany)
["I New Address: Barringer Research Ltd.
1730 Aimco Boulevard, Mississauga, Ontario, L4W 1V1 (Canada)
I'[
[**I
1478
0 VCH Verlagsgesellschaft mbH. 0-69451 Weinheim, 1997
The research at the TU Berlin was funded by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie. G. A. M. thanks NSERC
Canada for a Post Doctoral Fellowship and the Deutscher Akademischer
Anstauchdienst for a Research Scholarship Technical assistance from Prof.
J. K. Terlouw (McMaster) and Dr. D. Schroder (TUB) was appreciated.
0570-0833/97/36t3-1478 $ 1 7 . 5 0 i .SO10
Angew. Chem. Int. Ed. Engl. 1997, 36, No. 13/14
COMMUNICATIONS
Table 1. Data from the CID mass spectra of mjz 68 [C,H,N,]'+ ions generated
from I, 11, and imidazole [a]
Precursor
Spectrum
mi2
TS1/3
H
1: 29.0
68.8
66.7
l-+:0.0
3:
0.0
34: 14.0
TS213
H
85.0
69.6
2: 48.8
Z4: 13.6
Scheme I . Calculated (B3LYP/631G**) relative energies of imidazol-2-ylidene 1,
imidazole 3, and imidazol-4-ylidene2, their interconnecting transition states and the
respective radical cations.
a sizeable peak at m / z 68 (corresponding to [C,H,N,]'+) in its
70eV EI mass spectrum, which could correspond to 1" produced from the molecular ion following a formal 1,3-hydrogen
shift and decarbonylation (Scheme 2). The ionized dimethyl ester of imidazole-4,5-dicarboxylicacid I1 could generate 2 + by
H
42
40
39
38
29
21
26
25
24
16
15
14
13
12
I
CID
NRjCID
Intensity
24
39
10
7
1
8
5
21
32
12
8
3
8
7
3
1
0
1
3
3
1
1
45
17
10
5
8
3
5
1
0
0
1
1
1
I1
CID
NRiCID
Intensity
2
2
2
2
1
1
0
Imidazole
CID
NRjCID
Intensity
I
3
39
13
10
6
10
6
3
2
2
2
2
1
0
S2
1<.
I1
1
9
5
4
I
I
0
0
1
1
I
=
6
3
0
[a] Intensities based on peak heights normalized to X
metastable contnbutions have been omitted.
3
44
14
10
1
9
1
3
3
1
100. Peaks containing
H
(a) from precursor I is distinguished by an intense peak at m/z
42 (see Figure 1) that is attributed to the loss of acetylene and
thus taken as a strong indication of the carbene ion structure
l * +This
.
process has been shown to occur for the closely related
thiazol-2-ylidene radical
and we surmise that in this
case ionized diimide HN=C=NH'+ is the product ion. HowH
ever, attempts to demonstrate this experimentally are thwarted
l*
I
by the fact that isomeric CH,N, radical cations are very difficult
Scheme 2. Interpretation of the signal at mjz 68 in the EI (70 eV) mass spectrum of
to distinguish.['61 In any case, intense signals at m / z 42 are not
imidazole-2-carboxaldehyde I : 1" could form from the molecular ion by a formal
expected from 2 + and they are not present in the CID mass
1.3 hydrogen shift and decarbonylation.
spectrum of the imidazole radical cation. Indeed, the m/z 68 ions
from both I1 and imidazole are similar in the respect that there
is almost no signal to be seen at m / z 42 and in terms of the
the more complicated sequence of reactions shown in Scheme 3 .
relative abundance of the other fragments in their CID mass
Radical cations 3'+ are obtained simply by EI (70 eV) of imidaspectra; however, there are some notable differences between
zole. Ionized imidazole has been studied extensively by mass
the two spectra: a considerably enhanced ( 5 % of total CID intensity) frag0
H 1.'
ment peak at m/z 29 and a weak peak at
0
H
H
/I
1
ll
I
m/z 16 is observed only in the spectrum
/
of the ions from 11, which is an indica*r(
N\
H
tion that these ions have a structure different from 1'+ or 3'+.
-co
The existence of 1" and 2 + is further
H
H
endorsed by the CS mass spectral data in
Table 2 . Quite different intensity ratios
for the dications at m/z 68/67/66 are ob2*
II
served in all three spectra and in conScheme 3 Possible reaction sequence to 2'+ starting from the ionized dimethyl ester of imidazol-4,5-dicarboxylic
trast to 3'+ the CS peaks are much more
acid 11.
intense than the CID peaks for both of
spectrometry."
The tandem mass spectrometry experiments" described here were performed by using the VG Analytical ZAB-R instrument at McMaster
and the
modified VG ZAB/HF/AMD mass spectrometer at the TU
Berlin." 51
A careful comparison of the data from the collision-induced
dissociation (CID) mass spectra in Table 1 and the charge-stripping (CS) mass spectra in Table 2 reveals that two new m/z 68
[C,H,N,]'+ ions, distinguishable from imidazole do exist. At
first glance, the tabulated data from the CID mass spectra do
not seem greatly different, which is perhaps not surprising in
light of the structural similarity of the isomers and their complicated fragmentation pathways." Nevertheless, the spectrum
Angrw. Chem. int. Ed. Engi. 1997, 36. N o 13/14
Table 2. Data from the charge-stripping mass spectra of m/z 68 [C,H,N,]'+ ions
generated from I, 11, and imidazole [a].
Precursor
Spectrum
Dication ( m j z )
I
CS
NR/CS[b]
Intensity
68
67
66
CS-eff.[c]
100
5
2
0.60
100
40
10
0.48
I1
CS
NR/CS[b]
intensity
loo
9
2
1.11
100
39
5
1.02
Imidazole
CS
NR/CS[b]
Intensity
100
33
8
0 32
94
100
30
0.17
[a] Relative abundance are from peak heights. [b] NRjCS refers to dications
formed from neutralized ions (see text). [c] Charge-stripping efficiency as measured
by the peak height ratio of m/z 68 relative to that of the nearby M I Z 39 in the CID
mass spectrum.
a VCH firiagsgesellschaft mbH. 0-69451 Weinheim,1997
0570-0833/97/36l3-1479$17.50+ SO10
1479
COMMUNICATIONS
the new ions. The larger susceptibility of the ions 1" and
especially 2.+ to charge-stripping is typical for ylide ions as
compared to mundane isomer^."^] The differences in the CS
and CID mass spectra of the three isomers show that ions 1"
and 2" do not easily isomerize to 3 + .Given that the fragmentation mechanisms of even the well-known ion 3" are still largely speculative,[' 21 we avoid dwelling further on these differences.
Thus, the three isomers can be differentiated by CID and CS
mass spectrometry, therefore, it still remains to be demonstrated
that the corresponding neutral molecules are stable in NRMS
experiments.['*J
is consistent with the increased dissociation observed for the
dications produced by charge-stripping of the mjz 68 ions (see
the NRjCS mass spectra in Table 2). Taking this into account,
the CS efficiencies of the recovery ions and of the ions not
subjected to NR are comparable. These facts lead us to the
conclusion that a significant portion of the neutral molecules 1
and 2 are able to survive structurally intact during the microsecond transit time between the neutralization and reionization
cells in NRMS experiments.
In summary, the existence of imidazol-2-ylidene 1 and imidazol-4-ylidene 2, their radical cations, and dications have been
demonstrated through tandem mass spectrometry experiments.
Received: December 30, 1997 [29951 IE]
German version: Angew. Chem. 1997,109, 1572-1575
Keywords: carbenes
trometry
- cations .imidazolylidenes - mass spec-
[l] For a review, see: J. A. Berson, D. A. Birney, W. P. Dailey, J. F. Liebman in
Modern models of bonding and delocalization (Eds.: J. F. Liebman, A. Green-
Figure 1. NR mass spectra of the m / z 68 ions generated from (a) ionized imidazole2-carboxaldehyde I, (b) ionized precursor 11, and (c) ionized imidazole.
When the individual NR mass spectra of distinguishable isomers match their respective CID mass spectra in appearance
and contain observable recovery ion signals it is easy to conclude that the neutral molecules are stable. On this basis, the
existence of 1 is strongly suggested since its NR mass spectrum
(Figure 1 (a)) contains both the signature peak at mjz 42, and as
the base peak, the recovery signal at mjz 68. The evidence is less
clear-cut for 2 because the relative intensities of the peaks in the
NR mass spectrum are not quite the same as in the CID mass
spectrum. Note, however, that the m/z 12- 16 cluster patterns
are unique and the weak but observable dication peaks denoted
+ in Figure 1 are very informative. The relative abundance of
the dication at mjz 68 is once again most noticeable for 2'+ and
the largest peak that is attributed to a dication at mjz 67 is again
generated by Y+.To probe the existence of 2 and 1 in more
detail, we investigated the collision-induced dissociation behavior of the reionized species with mjz 68 (see Figure 1). This type
of experiment (NR/CID)" '1 requires a multisector mass spectrometer and high sensitivity.['* '']
To assist direct comparisons of the mjz 68 ions their NRjCID
and CID mass spectra have been tabulated together in Table 1.
An inspection quickly reveals the similarity of the NRjCID to
the CID mass spectra for the three sets of ions, especially in
regard to the key peaks at m/z 42 and 29. Scrutiny of the NR/
CID mass spectra reveals that the mjz 40 peaks are likely diminished because of extensive fragmentation into smaller ions,
which probably reflects a change to a higher internal energy
distribution in the radical cations themselves after NR.[7b1This
+
1480
8 VCH Verlagsgesellschafi mbH, 0.69451
Wemherm, 1997
berg) VCH, Weinheim, 1987. Recent attempts to identify C,O, through mass
spectrometry are summarized in: D. Schroder, H. Schwarz, Int. J. Mass Spectrom. Ion Processes 1995, 1461147, 183.
Since the first isolation of "stable" carbenes: A. J. Arduengo 111, R. L. Harlow,
M. Kline, 1 Am. Chem. Soc. 1991,113,361, the field has expanded rapidly and
a precis of the "incredible renaissance" in this area can be found in: M. Regitz,
Angen Chem. Int. Ed. Engi. 1996,35, 725; Angew. Chem. 1996, 108, 791.
Reviews: a) H.-W. Wanzlick, Angew. Chem. 1962, 74,129; Angen. Chem. Int.
Ed. Engl. 1962, I, 75; b) R. W. Hoffmann, ibid. 1968,80,823 and 1968, 7,154;
c) J. Hocker, R. Merten, fbid. 1972, 84, 1022 and 1972, 11, 964; d) M. Dries,
H. Grhtzmacher, ibid. 1996,108,900; and 1996,35, 829.
a) R. Gleiter, R. Hoffmann, J. Am. Cltem. Soc 1968, 90,5457; b) F. Geijo, F.
Lopez-Calahorra, S . Olivella, J: Heterocyclic Chem. 1984, 21, 1785; c) R.
Grigg, L. Wallace, J. 0 Morley, J. Chem. SOC.Perkin Trans 2 1990, 51;
d) D. A. Dixon, A. J. Arduengo 111, J. Phys. Chem. 1991, 95, 4180; e) A. J.
Arduengo 111, D. A. Dixon, D. A. Harlow, K. K. Kumashiro, C. Lee, W P.
Power, K. W. Zilm, J. Am. Chem. Soc. 1994,116,6361 ; f ) A.J. Arduengo 111,
H. Bock, H. Chen, M. Denk, D. A. Dixon, J. C. Green, W. A. Herrmann, N. L.
Jones, M. Wagner, R West, ibid. 1994,116,6641 ;g ) J. Cioslowski, I n t . J. Quant.
Chem., Quanf.Chem. Symp. 1993,27.309, h) C. Heinemann, W. Thiel, Chem.
Phys. Letters 1994, 217, 11; i) C . Heineinann, T. Miiller, Y. Apeloig, H.
Schwarz, J Am. Chem. Soc. 1996,118,2023; j) C. Boehme, G. Frenking, ibid.
1996, 118, 2039; k) R. R. Sauers, Telrahedron Lett. 1996, 37, 149; 1) A.J.
Arduengo 111, J. R. Goerlich, W. J. Marshall, Liebigs Ann./Receuil,1997, 305.
a) R. W Alder, P. R. Allen, S . J. Williams, J: Chem. SOC.Chem. Commun. 1995,
1267; b) A J. Arduengo 111, J. R. Goerlich, W. J. Marshall, J. Am. Chem. SOC.
1995,117,11027,~)
Methodender Organischen Chemie(Houben-WeyI),4thed.
1952-. Bd. E19b, 1989.
Nitrene: a) G . Boche, P. Andrews, K. Harms, M. Marsch, K. S . Rangappa, M.
Schimeczek, C. Willeke, J. Am. Chem. SOC.1996,118,4925, Fulvalene: b) T. A.
Taton, P. Chen, Angex. Chem. 1996, 108, 10980; Angew. Chem. Int. Ed. Engl.
1996,35, 1011.
a) G. A. McGibbon, C. A. Kingsmill, J. K. Terlouw, Chem Phys. Letters 1994,
222, 129;b) G.A. McGibbon, P. C. Burgers, J. K. Terlouw, Int. J: ,brass Specfrom. Ion Processes 1994, 136, 191; c) T. Wong, J. Warkentin, J. K. Terlouw,
ibid. 1990,115.33; d) D. J. Lavorato, J. K. Terlouw, T. Dargel, W. Koch, G . A.
McGibbon, H. Schwarz, 1 Am. Chem. Soc. 1996,118,11898; e) G.A. McGibbon, J. HruSak, D. J. Lavorato, H. Schwarz, J. K. Terlouw, Chem. Eur. J. 1997,
3, 232.
For a recent review, see: N. Goldberg, H. Schwarz, Acc. Chem. Res. 1994,27,
347.
V. Q . Nguyen, F. Turecek, J: Mass Spectrom. 1996, 31, 1173.
Gaussian 94 (Revision B.31, M. J. Frisch, G. W. Trucks, H. B. Schlegel,
P. M. W Gill, B. G. Johnson, M. A. Robb, J. R Cheeseman, T. A. Keith, G. A.
Petersson, J. A. Montgomery, K. Raghavachari, J. B. Foresman, J. Cioslowski,
B. B. Stefanov, A. Nanayakkara, M. Challacombe, C . Y. Peng, P. Y. Ayala, W.
Chen, M. W. Wong, J. L. Andres, E. S . Replogle, R. Gomperts, R. L. Martin,
D. J. Fox, J. S . Binkley, D. J. DeFrees, J. Baker, J. J. P. Stewart, M. Head-Gordon, C. Gonzales, J. A. Pople, Gaussian Inc., Pittsburgh PA, 1995.
Ill] According to our calculations Frank-Condon type neutralization of the radical cations at their optimized geometries results in vibrationally excited molecules 1-3, but theenergy deposited (9.9.8.7, and 11.9 kcalmol-', respectively)
is insufficient to enable their interconversion
[12] a) J. H. Bowie, R G. Cooks, S . 0. Lawesson, G. Schroll, Aust. J: Chem. 1967,
20, 1613; b) R. Hodges, M Grimmet, ibid. 1968, 21, 1086; c) K. J. Klebe, J. J.
0570-0833/97/3613-1480$17.50+.50/0
Angew. Chem. Int. Ed. Engl. 1997, 36, No. 13/14
COMMUNICATIONS
Houte, J. van Thuijl, Org. Muss Spectrom. 1971,5, 1101; d) J. van Thuijl, K. J.
Klebe. J. 3. Houte, ihid. 1972, 6, 1363; e) G. Bouchoux, Y Hoppilliard, ibid.
1977, 12, 196; f) J. Main-Bobo, S. Olesik, W Gase, T. Baer, A. A. Mommers,
J. L. Holmes. J. Am. Chem. Soc. 1986, 108, 106.
[13] K. L. Busch. G. L. Glish, S. A. McLuckey, Muss Spectromerry/Muss Spectronwtrj, Techniques and Applicutions of Tundem Muss Spectrometry, VCH,
New York, 1988.
[14] The VG Analytical ZAB-R mass spectrometer at McMaster University has a
three-sector BEE (B = magnet, E = electric sector) configuration, see: H. F.
van Garderen. P. J. A. Ruttink, P. C. Burgers, G . A. McGibbon, J. K. Terlouw,
Int. J. Muss Spectrom. Ion Processes 1992, 121, 159.
[15] The modified VG ZAB/HF/AMD at the TU Berlin is a four-sector instrument
with a BEBE configuration, see: a) R. Srinivas, D. Siilzle, T. Weiske, H.
Schwdrz. In1 J Muss Spectrom. Ion Proce.wes 1991, 107, 368; b) C. Schalley,
D Schroder. H. Schwarz, ihid. 1996, 153, 173.
[16] B. L. M. van Baar. Ph.D. Thesis. University of Utrecht, 1988, Chapter 1
1171 J. L. Holmes. F. P. Losring, J. K. Terlouw, P. C. Burgers, J. Am. Chem. SOC.
1982. 104, 2931
[18] In NRMS experiments 8 or 10 keV m/z 68 [C,H,NJ+ ions were mass-selected
(by B with the ZAB-R or B,E, with the ZAB/HF/AMD) and then subjected
to neutralizing collisions with N,N-dimethylaniline or xenon vapor present in
a small gas cell located in the field-free region. Afterward, any remaining ions
were removed from the beam by deflection with a positively charged electrode
so that only a beam of fast moving neutral species entered a second gas cell,
therein being (dissociatively) reionized by collisions with oxygen molecules.
For a NR mass spectrum, the survivor and fragment ions were analyzed by
using the next available sector (B2 in Berlin or El at McMaster). Alternatively,
when the recovery ions ( m / z 68) were selectively transmitted by the sector and
then passed through another oxygen-filledcollisioncell(70% transmission) the
resulting CID products could be analyzed by using the last sector. The NR/
CID mass spectrum so obtained is characteristic of only the species surviving
neutralization and reionization. Mass spectra of doubly charged ions (NR/CS)
were also obtained in the same manner. Comparative collision-induced dissociation (CID) and charge-stripping (CS) experiments were performed with the
deflector electrode switched off.
to the extreme importance of this class of enzymes in biochemistry and biotechnology, numerous attempts have been made to
design “artificial phosphodie~terases”.~~~
Independent of the
catalytic mechanism of these systems, highly sensitive assays are
needed for detecting phosphodiesterase activity. So far, UV/Visspectroscopical quantification of nitrophenols liberated from
the corresponding phosphodiesters or 31PNMR spectroscopy
were used in most cases. We found it worthwhile to design a
simple and much more sensitive fluorescence probe for detecting
phosphodiesterase activity. Due to the high sensitivity of fluorescence assays, we reasoned that with such probes phosphodiesterase activity might also be detected in complex mixtures of
biological or synthetic origin.
The mechanism of our reporter molecules is shown in
Scheme 1. In compound A, a fluorophore and a fluorescence
A
4
H~O
B
esterase
C
or
Fluorescence Reporters for
Phosphodiesterase Activity**
Albrecht Berkessel* and Rainer Riedl
Phosphodiesterases catalyze the hydrolysis of phosphodiester
Their most important biological substrates
bonds [Eq. (a)] .I1]
9
R-0-7-OH
R
R-0-7-0-R’
00
f
HO-R’
00
phosphodiesterase
or
b
“20
R-OH
+
(a)
9
HO-7-0-R’
OQ
Scheme 1 Mechanism of the fluorescence reporters
quencher are held in close proximity. The absorption spectrum
of the quencher and the emission spectrum of the fluorophore
should match as well as possible.141Upon irradiation of the
fluorophore, rapid energy transfer to the quencher takes place.
Therefore, this intact phosphodiester does not fluore~ce.‘~’
After hydrolysis of the phosphodiester group, the cleavage
products B and C (or B’ and C‘) can diffuse away from one
another, and the fluorescence is no longer quenched intramolec~ l a r l y . [We
~ ] synthesized two such fluorescence reporters, namely the phosphodiesters 1 and 2. In these compounds a naph-
are the nucleic acids. The manipulation of genetic material (“genetic engineering”) hinges on the sequence-specific cleavage of
DNA by the restriction enzymes,[21which are isolated from
natural sources and, consequently, invariable with respect to
their catalytic parameters and particularly their base-recognition properties (number and type of the bases recognized). Due
[‘I
[‘I
[**I
Prof. Dr A Berkessel,“’ Dipl.-Chem. R. Riedl
Organisch-chemisches Institut der Universitat
Im Neuenheimer Feld 270, D-69120 Heidelberg (Germany)
New addressInstitut fur Organische Chemie der Universitat
Greinstrasse 4. D-50939 Koln (Germany)
Fax: Int code +(221)470-5102
e-mail : berkessel(a uni-koeln.de
This work was supported by the Fonds der Chemischen Industrie
Angew. Chi,m. Int Ed Engl. 1997, 36, No. 13/14
8 VCH VerIagsgeseNschufi mbH. 0-69451
1
2
Wernheim. 1997
0570-0833/97/3613-14~f
8 17.5Nc 5010
1481
Документ
Категория
Без категории
Просмотров
1
Размер файла
492 Кб
Теги
mass, missing, imidazole, carbene, generation, neutralizationцreionizatin, ylidene, stripping, spectrometry, dication, charge
1/--страниц
Пожаловаться на содержимое документа