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Rapid Hydrolysis of RNA with a CuII Complex.

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~~~~
carried out in methanol as the solvent (Scheme 2 ) . Since we did
not consider it likely that the ketene was stable for 6 h, we
examined a solution prepared in [DJTHF by NMR spectroscopy. We found that not the ketene, but the dihydrooxazinone 8 is present in the solution,""] and is stable for at least 12 h
when air is excluded. This heterocycle is a cyclic imino anhydride, in other words an activated derivative, and reacts with
nucleophiles to give the desired homopeptides."
2
A (R' = Z-Ala, R2 = Me)
8
9
Scheme 2. Experiment used to identify the intermediate 8 formed from a peptidic
ketene A and methanolysis product 9 (m.p. 11 1 - 113 "C.DI.[ = - 46.9 (c = 1.06,
CHCI,)).
The method for the synthesis of homopeptides presented here
allows for an insertion of a p-amino acid at any stage of a
peptide synthesis.["] The active acyl derivative (A or dihydrooxazinone) involved reacts not only with amino acid esters and
peptides with an unprotected N-terminus, but also with other
nucleophiles. Experiments along these lines, for instance reactions with carbohydrates and 3-hydroxybutanoic acid oligornersr13]are in progress. The plethora of possibilities suggests
that the method may be used for constructing new types of
peptide-containing combinatorial libraries.[l4I
~
[l] C. N. C. Drey in Chemistry and Biochemistry of Amino Acids, (Ed.: G. C.
Barrettj, Chapman and Hall, London, 1985, pp. 25-54.
(21 W. P. Frankmolle, G. Kniibel, R. E. Moore, G . M. L. Patterson, J. Antibior.
1992, 45, 1458-1466.
131 The Organic Chemistry offl-Lacrams, (Ed.: G. I. Georg), VCH, New York,
1993.
[4] Recent comprehensive reviews: E. Juaristi, D. Quintana, J. Escalante,
Aldrichimica Acta 1994, 27, 3-11; D. C. Cole, Tetrahedron 1994, 50, 9517-
9582.
[5] a) F. Arndt, B. Eistert, W. Partale, Ber. Dtsch. Chem. Ces. 1927,60,1364-1370;
b) T. Ye, M. A. McKervey, Chem. Rev. 1994, 94, 1091-1160.
[6] There are numerous reports about the application of this reaction for the
homologation of u- to p-amino acids [4, 5bl. In almost all cases the enantiopurity of the products formed with retention of configuration was not determined by modern analytical techniques (NMR spectroscopy, gas chromatography) (K. BalenoviC, I. JambreSiC, 9. GaSpert, D. Cerar, Red. Trav. Chim.
Pays-Bas 1956, 75, 1252-1258; K. BalenoviC, D. FleS, I. Jambre%, Croat.
Chem. Actu 1956, 28, 303-305 and references therein). We used the following
amino acid derivatives in the Arndt-Eistert reaction (activation via the mixed
anhydrides): Z-Ala-OH, Z-Phe-OH, Boc-Phe-OH, Boc-tert-Leu-OH, BocOrn(Boc)-OH, Z-Phg-OH, Boc-Phg-OH. Analysis of the Mosher derivatives
showed that all reactions, except for that of phenylglycine (Phg), occur with
more than 98 O h retention of configuration. We tested milder activation methods with phenylglycine. Activation with isobutyl-2-isobutyloxy-l (ZH)-quinolinecarboxylate (IIDQ) afforded diazoketone (10% racemization, as with
CICO,Et), but reaction with N-hydroxysuccinimide/dicyclohexylcarbodiimide
(HOSu/DCC) or benzotriazol-1-yl-oxytripyrrolidinophosphoniumhexafluorophosphate (PyBOPj did not.
[7] The reaction could not be carried out with the hydrochloride of the amino acid
ester.
[8] For the preparation of the di- and tripeptides used here, see: H. G . Bossier, D.
Seebach, H d v . Chim. Acta 1994, 77, 1124- 1165.
[9] S. Abdalla, E. Bayer, H. Frank, Chromatographia 1987,23, 83-85; the enantiopurity of the p-amino acids could be determined by this method as well.
[lo] Compound 8 was unambiguously identified by 'H, "C, DEPT, COSY, and
HETCOR NMR spectroscopy. In an analogous experiment the diazoketone
from Z-alanine yielded Z-HomoAla-OMe when methanol was added after 6 h.
[ l l ] Dihydrooxazinones of this type were generated from various p-amino acid
derivatives by other methods and used as active esters (C. N. C. Drey, E.
Mtetwa, J. Chem. SOC.Perkin Truns. I 1982, 1587-1592 and references
therein).
[12] The method appears to be applicable to all appropriately protected amino acids
except for tryptophane and histidine.
1131 H.-M. Miiller, D. Seebach, Angew. Chem. 1993, IOS, 483-509; Angew. Chem.
Int. Ed. Engl. 1993, 32, 477-502.
[14] G. Jung, A. G. Beck-Sickinger, Angew. Chem. 1992, 104, 375-391; Angew.
Chem. Int. Ed. Engl. 1992, 31, 367-383.
[I51 Caution: The generation and the handling of diazomethane requires especial
caution: P. Lombardi, Clem. Ind. {London) Nov. 5 , 1990, 708. S . Moss, ibid.
Feb. 21, 1994, 122.
Experimental Procedure
Diazoketone3: Et,N (1.39 mL, 10.0 mmol) and CIC0,Et (953 pL, 10.0 mmol) were
added to a solution of Boc-Leu-Sar-Leu-OH (4.16 g, 10.0 mmol) in THF (50 mL,
distilled from Na/K) at - 15 'C under argon. After 15 min the resulting solution was
allowed to warm to 5 "C and then treated with a solution of CH,N, 1151 in ether,
until the yellow color of CH,N, persisted over a longer period of time. The mixture
was allowed to warm to room temperature and stirred for an additional 3 h. Excess
CH,N, was destroyed by vigorous stirring or by addition of a small amount of
HOAc. After aqueous workup by extraction with saturated NaHCO,, NH,CI, and
NaCl solutions, the organic solution was dried (MgSO,) and concentrated to dryness. Chromatography on silica gel (ethyl acetate/hexane 2/1) gave diazoketone 3 in
86% yield (3.76g. 8.60 mmol).
Protected homohexapeptide 7: A solution of silver benzoate (16.0 mg. 70.0 pmol) in
Et,N (215 pL. 1.54 mmol) was added to a solution of 3 (238 mg, 541 pmol) and
H-Ala-Sar-MeLeu-OBzl (528 mg, 3.40 mmol) in THF (10 mL) at -25 "C under
argon with exclusion of light. The reaction mixture was warmed to room temperature over 3 h, and some Et,O was added. After workup by extraction with HCI
(0.2 N, 2 x )and saturated NaCI, NaHCO,, and NaCl solutions, the organic solution
was dried (MgSO,) and concentrated to dryness. The residue was purified by chromatography on silica gel (ethyl acetate) to give the protected homopeptide 7
(257 mg, 326 pmol, 60%).
~
Received: September 14, 1994 [Z73121E]
German version: Angen. Chem. 1995, 107, 507
Keywords: p-amino acids . Arndt -Eistert reactions . dihydrooxazinones . homopeptides . peptide analogues
472
VCH Verlagsgesellschafr mbH. 0-69451 Weinheim, 1995
Rapid Hydrolysis of RNA with a Cut*
Complex**
Barry Linkletter and Jik Chin*
Over the years, many interesting artificial enzymes that hydrolyze the phosphate diester bonds of RNA have been reported. They include nonmetallic compounds"] as well as transition
metal complexes[21 and lanthanide complexes.[31 Sequencespecific hydrolysis of RNA has recently been achieved by using
various metal complexes covalently attached to deoxyoligori'1 Although these results are promising, the rebon~cleotides.'~.
activities of artificial RNases fall far short of natural RNases.
Typically, the half-life for artificial RNase-catalyzed hydrolysis
['I Prof. J. Chin, B. Linkletter
Department of Chemistry, McGill University
801 Sherbrooke Street West, Montreal, Quebec H3A 2K6 (Canada)
Telefax: Int. code + (514)398-3797
[**I Financial support was provided by the National Science and Engineering
Council of Canada and the U S . Army Research Office.
0570-0833/95/0404-0472 $10.00 i
.25/0
Angew. Chem. Int. Ed. Engl. 1995, 34, No. 4
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of RNA at neutral pH and 50°C ranges from hours to days.
Hence there is much current interest in increasing the reactivity
of artificial RNases as well as in understanding factors affecting
the reactivity of such catalysts. [C~(terpy)(OH,)]~(1) and
[C~(bpy)(0H,),]~'(2) are two of the most reactive transition
metal complexes reported to date for hydrolyzing the phosphate
diester bonds of RNA (terpy: terpyridyl; bpy: 2,2'bipyridyl) .I2. 41 Here we compare the reactivity of [Cu(neocuproine)(OH,)JZ+ (3) for hydrolyzing ApA to those of 1
and 2.
+
1
3
2
Copper complexes, 1,2, and 3 were freshly generated in solution from the corresponding chlorides. Cleavage of ApA to A,
Ap, and pA was monitored by high-pressure liquid chromatography (HPLC) . Optimal reactivity of the copper complexes is
reached at solution pH values close to the pK, of the copper-coordinated water molecules. The pKa values of the coordinated
water molecules in 1.2, and 3 are 8.2, 7.8, and 7.0, respectively,
indicating that the copper center in 3 is the most Lewis acidic.
The pseudo-first-order rate constants for 1, 2, and 3 (10 mM)
promoted cleavage of ApA (0.5 mM) at the respective optimal
pH values are 1.9 x
s-', 1.9 x
s - I , and 3 . 9 ~
1 0 - 3 s- 1 . The half-lives for 1 , 2, and 3 promoted cleavage of
ApA are 10 hours, 42 days, and 3 minutes, respectively. All of
the reaction solutions were buffered with N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonicacid (HEPES, 10 mM) and
kept at 25°C. A typical HPLC plot for 3 (10mM) promoted
cleavage of ApA (0.5 mM) at pH 7 and 20 "C is shown in Figure 1. The concentration of 2',3'-CAMP accumulates appreciably during the cleavage reaction, and it is subsequently converted to 3'-AMP and 2'-AMP in a ratio of about 6 to 1. It is evident
from the HPLC plot that the cleavage reaction occurs hydrolytically at the phosphate rather than oxidatively at the ribose ring.
B CD
A
The rate of cleavage of ApA is first order (Fig. 2) with respect
to the concentration of 3, clearly indicating that the cleavage
reaction is a mononuclear process (slope: 1.06; intercept:
-0.56; correlation coefficient: 0.994). Interestingly, 3 is over
200 times more effective than 1 and over 20000 times more effective than 2 for catalyzing ApA cleavage. A phosphate diester
coordinated to Co"' as a monodentate ligand hydrolyzes only
about lo2 times faster than the free phosphate diester.[61Hence
the dramatic difference in the reactivities of 1,2, and 3 is unlikely to be solely due to the difference in the Lewis acidities of the
metal complexes.
E
h
-2.8
-2.6
-2.4
-
-2.2
log [31
-2
-1.8
Fig. 2. Dependance of k,,, [s-'1 on the concentration of 3 [MI for hydrolysis of ApA
(0.1 mM) in 10 mM HEPES buffer (pH 7) at 2 0 T , a s a log/log plot.
At neutral pH, 2 dimerizesC7]with an equilibrium constant of
1 x 10' M-' [Eq. (a)], and the dimer is inactive for hydrolyzing
ApA. At pH 7.8, only about 8 % of 2 is in the monomeric form.
In contrast, 3 does not dimerize appreciably under the reaction
condition owing to the steric effect of the two methyl groups.
When the dimerization of 2 is accounted for, 3 is still about
1000 times more reactive than 2 for hydrolyzing ApA.
One possible mechanism that can account for the high reactivity of 3 involves chelation of the phosphate diester to the
copper complex resulting in double Lewis acid activation. In
general, a phosphate diester that is chelated to a metal center (4)
should hydrolyze more rapidly than a phosphate diester that is
singly coordinated to a metal center ( 5 ) . In the former case the
t= 440 s
4
2
4
6
8
t lmin I
10
12
Fig. I . Chromatograms for hydrolysis of ApA (0.1 mM) catalyzed by 3 (10 mM) in
the presence of 10 niM HEPES buffer (pH 7) at 20 'C (after 20,440, and 3000 s, and
after 12 h). The traces shows disappearance of ApA (E) and appearance of
adenosine (D) and 2'.3'-cAMP (B), which opens to 3'-AMP (A) and 2'-AMP (C).
Angew. Client. l n l . Ed. Engl. 1Y95, 34, N o . 4
- O-J
0 VCH
Verlagsgesellschaji mhH, 0.69451 Weinheim, 1995
5
-0 1
0570-0833/95/0404-0473$10.00
+ .25!0
473
COMMUNICATIONS
developing negative charge is stabilized by the cationic metal
center, while in the latter case the developing negative charge is
not stabilized by the metal. The metal center in 4 is providing
double Lewis acid activation, while the metal center in 5 is
providing single Lewis acid activation. We recently showed that
a dinuclear metal complex can rapidly cleave an RNA model
compound by providing double Lewis acid activation.['] The
two methyl groups in the neocuproine ligand should decrease
the 0 - C u - 0 bond angle in 3, which should facilitate the chelation of phosphate diesters. In octahedral Co"' complexes, the
0-Co-0 bond angles in chelated phosphates are significantly
smaller than the 90" angle found in regular octahedral complexes.I91
Among simple compounds, lanthanides and their complexes
are the most reactive for hydrolyzing nucleic acids.["] However,
enzymes use transition metals, Mgz+,or Ca2+ to cleave phosphate esters. Consequently, there is considerable interest in developing simple transition metal, Mg2+, or Ca2+ complexes
that efficiently hydrolyze the phosphate diester bonds of RNA.
Complex 3 represents by far the most reactive transition metal
complex reported to date for hydrolyzing RNA.
Experiinen tal Procedure
The copper chloride complexes [Cu(terpy)CI]CI, [Cu(bpy)CI,], and [Cu(neocuproine)CI,] were prepared by mixing the appropriate ligands with CuCI, in
methanol. All of the ligands and CuCI, are soluble in methanol, whereas the complexes precipitate out of the solvent. Cleavage of ApA was monitored by HPLC
(Hewlett-Packard 1090). I n a typical kinetic experiment, a solution of ApA
(0.5m~),3(10m~),andHEPES(10m~)
wasallowed toreactatpH 7.0and25"C.
Aliquots (50 pL) of the reaction mixture were quenched with 100 mM ethylenediaminetetraacetic acid (EDTA) (50 pL). The quenched solutions (10 pL) were injected onto a C-18 reversed-phase column (5 pm Hypersil maintained at 40'C) and
eluted for 5 min with NH,H,PO, (0.2 M at pH 5.5) followed by a 0-50% linear
gradient of NH,H2P0, (0.2 M at pH 5.5) and methanol/water (3:2) solutions over
10 min with a flow rate of 0.5 m l m i n - ' .
Received: September 24, 1994 [Z 7351 IE]
German version: Angew. Chem. 1995. 107. 529
Keywords : bioinorganic chemistry . copper compounds . nucleic
acids . RNA cleavage
[ I ] a) J. Smith, K. Ariga, E. V. Anslyn, J. Am. Chem. Soc. 1993. 115. 362; b) R.
Breslow, M. Labelle, hid. 1986, 108, 2655; c) B. Barbier, A. Brack. ibid. 1992.
114,351I . For hydrolysis of RNA model compounds see: d) M. W Gobel, J. W.
Bats, G. Durner, Angrn. Chem. 1992, 104, 211; Angen. Chem. I n l . Ed. Engl.
1992,31.207; e) V. Jubian, R. P. Dixon. A. Hamilton, J. Am. Chem. So(. 1992,
114, 1120.
[2] a) M. K. Stern, J. K. Bashkin, E. D. Sall, J. Am. Chem. Sor. 1990, 112, 5357;
b) Y. Matsumoto, M. Komiyama. J Chem. Soc. Chem. Commun. 1990, 1050;
c) R. Breslow, D. L. Huang, E. Anslyn, Proc. Narl. Acad. Sci. U S A 1989.86,
1146.
[3] a) J. R. Morrow, L. A. Buttrey, V. M. Shelton, K. A. Berback, J. Am. Chem.
Soc. 1992,114,1903;b) R. Breslow, D. L. Huang. Proc. Nnfl. Acud. Sci. USA
1991.88. 4080; c) H:J. Schneider. J. Rammo, R. Hettich, Angew. Chem. 1993,
105. 1773: Angew. Chem. I n ! . Ed. Engl. 1993, 32, 1116.
[4] J. K. Bashkin. E. I. Frolova, U. S . Sampath, J. Am. Chrtn. Soc. 1994.1f6.5981.
[5] D . Magda, R. A. Miller. J. L. Sessler. 8. Iverson, J. A m . Chem. SOC.1994. 116,
7439.
[6] P. Hendry, A.M. Sargeson, Inorg. Chem. 1990, 29, 92.
[7] R. L. Gustafson, A. E. Martell. J. Am. Chem. Soc. 1959,81, 525.
[XI a) D. Wahnon, R. C. Hynes, J. Chin,J. Chem. Soc. Chem. Commun. 1994,1441;
b) M. Wall, R. C. Hynes, J. Chin, Angew. Chem. 1993,105.1696; Angew. Chem.
Inr. Ed. Engl. 1993, 32. 1633.
[Y] a) B. Anderson, R. M. Milburn, J. M. Harrowfield, G. B. Robertson, A. M.
Sargeson, J . Am. Chem. Soc. 1977.99,2652; b) J. A. Connolly, M. Banaszczyk.
R. C. Hynes, J. Chin, Inorg. Chem. 1994. 33, 665.
1101 a)B. K. Takasaki, J. Chin, J A m . Chem. Soc. 1994, 116. 1121; b) 8.K.
Takasaki, J. Chin, ihid. 1993,115,9337; c) J. Sumaoka, S. Miyama, M. Komiyama, M. J. Chem. Soc. Chem. Commun. 1994,1155; d) M. Komiydma, K. Matsumura. Y Matsumoto, ibid. 1992, 640.
474
0 VCH
Vrrlagsgesellschaft mbH, 0-69451 Wernheim, 1995
Photo Electron Transfer Induced
Macrocyclization of N-Phthaloyl-w-aminocarboxylic Acids**
Axel G. Griesbeck,* Andreas Henz, K a r l Peters,
Eva-Maria Peters, and Hans Georg von Schnering
In the presence of strong electron donors such as arene carbonitriles,''] heteroarenes,['] and iminium cations,[31aliphatic
carboxylic acids and the respective anions can be photochemically decarboxylated. The carbon radicals produced undergo
fragmentation or (re)combination. Intramolecular versions of
these reactions yield complex products, for example spiroannulated heterocycles.[41During our investigations on the photochemistry of N-phthaloylamino acids['] we discovered a surprisingly efficient synthesis of systems containing medium-sized and
large rings.
The photochemical approach to macrocycles requires a
strong electron-donating substituent (e.g. thioether, enamine) in
the side chain of the N-alkylphthalimide. The phthalimide chromophore behaves as an electron-accepting group ("remote photocyclization") .I6] A disadvantage of this method is in all cases
the incorporation of the electron donor into the ring system
formed. An approach in which formation of the new 0 - C single
bond is combined with extrusion of the electron donor would be
promising.['] a-Aminocarboxylic acids could be suitable substrates; however, only the photochemical reactivity of a-amino
acids has been reported up to now.[81The corresponding Nphthaloyl derivatives are decarboxylated when irradiated at
300 nm. This reaction proceeds with high regioselectivity also in
the presence of other carboxyl groups in 1-or y-position. Even
prolonged photolysis does not cleave these remote substituents.
We have used this principle for the synthesis of deuteriumlabeled 8-and y-aminocarboxylic acids.[91A decisive factor for
the efficiency and regioselectivity might be formation of a intramolecular hydrogen bond. Presumably this reaction proceeds by a primary proton
to the electronically excited phthalimide group (Excited State Proton Transfer, ESPT'"])
followed by an electron transfer step. Since remote carboxyl
groups do not form this hydrogen bond, this initial proton
transfer step cannot occur.
Electron transfer even from remote positions is possible for
carboxylic acid anions when primary proton transfer is no
longer necessary. Irradiation of N-Pht-aspartic acid 1 (Pht =
phthaloyl) in acetone leads to N-Pht-8-amino-propionic acid 2
in quantitative yield. In the presence of ten equivalents of sodium carbonate, however, a 9: 1 mixture of N-ethylphthalimide 3
and the cyclized compound 4 was isolated (Scheme 1). Still decarboxylation from the a-position is considerably faster, as was
demonstrated for the glutamic acid derivative 5 a: irradiation
under nonionic conditions leads to N-Pht-y-aminobutyric acid 6
as the sole product. In the presence of sodium carbonate (ionic
conditions) the benzopyrrolizidine 7 a is formed in 76 % yield.
[*] Prof. Dr. A. G. Griesbeck,[+' Dipl.-Chem. A. Henz
['I
[**I
Institut fur Organische Chemie der Universitat
Am Hubland. D-97074 Wurzburg (Germany)
Dr. K. Peters, E.-M. Peters, Prof. Dr. H. G. von Schnering
Max-Pldnck-lnstitut fur Festkorperforschung
Heisenbergstrasse 1, D-70569 Stuttgart (Germany)
New address:
Institut fur Organische Chemie der Universitdt
Greinstrasse 4. D-50939 Koln (Germany)
Telefax: Int. code + (221)4705102
This work was supported by the Deutsche Forschungsgemeinschaft (DFG G r
881/7-1) and Degussa AG.
0570-0833/95/0404-0474 $ 10.00+ .25/0
Angew. Chem. I n l . Ed. Engl. 1995, 34, No. 4
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