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Novel Genetically Engineered Tetracenomycins.

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the imine. Other metal salts, like MgBr,, show similar effects,
but none as convincingly as zinc chloride dietherate. Typical
yields are in the range of 80% (Table 1 ) with best yields obtained
at -40°C in most cases, while optimal stereoselectivity was
always achieved at - 75 "C. Much lower optical and chemical
yields are obtained at temperatures above 0 "C.
Possible acid components are all soluble acids that are not too
strongly acidic. For example, trifluoroacetic acid already leads
to a rather uncertain mixture of by-products, while excellent
results were obtained with formic acid. Aromatic or heteroaromatic aldehydes do not form stable imino chelates, which differs
from our experiences concerning alkylated 1 -amino pyranoses.
Isocyanides have to be sufficiently nucleophilic, otherwise reaction times will be very long. Tests with (para-toluenesulfony1)methyl isocyanide, for instance, gave yields of less than 10 'h
after 10 days reaction time. THF and methanol are suitable
solvents for these reactions, whereas halogen-containing solvents strongly favor the P-3CR, making them as unsuitable as
nonpolar solvents (amine will not dissolve!) or solvents that
show a high viscosity at the temperatures employed. At - 75 "C.
reaction times including the time for the precondensation of the
imine are roughly six days. Other limitations were not found. All
compounds in Table 1 were made by this simple one-pot procedure without special adaptations. Syntheses of longer peptide
structures are currently under investigation as are preparations
of cyclopeptides. We are also developing a template for the
preparation of the analogous L derivatives. An important feature of the amine 2 is its ability to form dihydrooxazoles,
which should make cleaving the chiral template easier. We have
already been able to realize an acidolytic and a reductive
fragmentation process. However, since yields and overall reproductivity have yet to be optimized, this work will be reported
at a later date. One aspect herein, is the possible variation
of the amide group at C2 of 2 in order to get better fragmentation and also to improve the diastereoselectivity at higher temperatures.
[ I ] A. Strecker, Ann. Chem. Phurm 1850. 75. 27
[I] H. Bergs. C/iem.Zvnrruibi. 1933,27. I001 ; H . T. Bucherci-. W Steiner, Z . PruP/.
Cheni. 1934. 140, 291.
[3] H. Hellmann. G. Opitz. o-Artlinou/Pr/ir,rimg. Verlag Chemie. Weinhelm, 1960.
[4] M. Passerini. Gar:. Chim. fro/. 1921, 51 I f . 126.181; hid. 1926. 56, 826; M .
Passerini. G. Ragni. ihid. 1931, 61. 964.
[5] 1. Ugi, Isonrrrile Chemistr),. &1/.20, Academic Press. hew York, 1971: I. Ugi,
S. Lohberger. R. Karl in O n n p r ~ ~ h e n s rOrgunic
Svnth~stcE//iciencj. b'oi.20. (Eds.: B. M. Trost. C. H. Hcathcock). Pergamon,
Oxford 1991, p. 1083.
[6] A. Gautier, Ann. Clisni. Pharrn. 1867. 142.289: ihid. 1867. 144.114; Ann. Chrm.
P/IJJ.1869. 17. 103,203.
[7] I.Ugi. C. Steinbruckner, Anprii.. Chem. 1960. 72. 267: ihld 1962. 74, 9: Angeir.
Chem. Inr. Ed. Engi. 1962. 1, 6.
[8] I. Ugi. C. Steinbruckner, Chrm. Ber. 1961, 94, 2802.
[9] C. Steinbruckner, Dissertation, Universitit Miinchen. I961
1959, 71. 386.
[lo] 1 . Ugi. R. Meyer. U. Fetzer. C. Steinbruckner, A n g ~ w C/i<wn.
[ l l ] C. Steinbruckner, Chem. Bcr. 1961. 94, 2814.
[I21 I . Ugi, A . Domling. W. Hod, G f T Furh Luh. 1994. 3R. 140; Endeuiww 1994.
18. 115.
[I31 M. Bodanszky. M. A. Ondetti in P q h k Sj~nrhcsrs(Ed.: C i A. Olahj. Wiley
Interscience. New York 1966, p. 127.
[14] G. Neyer. .I.
Achatz. B. Danzer. I. Ugi. He/cror.ick 1990. 20. X63.
[15] 1. Ugi. G. Kaufhold. Jirsriis Liehips Ann. Chwi. 1967. 70Y. 1 1 .
[I61 F. Siglmuller, R. Herrmann. 1. Ugi. Tetruhedron 1986. 42. 5931.
[I71 R. Herrmann, G. Hubener. F. Siglmuller, I. Ugi. L r ~ b i gAnn.
~ Chwv. 1986,251.
[I 81 R. M. Williams. S~nthesirof Opfrc.u/li~
Ac/ii,i, a-Aminoucid.s. Vol 7. Pergamon,
Oxford. 1989: J. Mulzer, H.-J. Altenbach. M. Braun, K. Krohn. H . 4 . Reissig,
Organic Sxnrhrsrs Highlighrr. VCH, Weinheim. 1991. D. Seebach, R.
lmwinkelried, T. Weber, M o d Sxnrh. Mcrhod.c 1986, 4. U. Schollkopf, Purc
.4pp/. C h m . 1983, 55. 1799-1806: a-Amino Acid\. (Ed.: M. J.
O'Donnelj. Te/ruht&on 1988. 44. 5253-5614.
[I91 H. Kunz. W. Sager. A n g r i i . Chem. 1987. 99. 595: .4ngen Chrw h i . Ed. EnpI.
1987, 26. 557.
[20] H. Kunz. W. Pfrengle. J. Am. Chem. Six. 1988. 110.451. Tefruhi~dron1988.44.
1991. 1095.
[21] M. Goebel, I. Ugi, .S~nthe.~is
[22] S.Lehnhoff. Dissertation Technische Universitit Munchen. 1994.
[23] F. Micheel, A Klemer. Ads. Curho/zw/r. Chmi. 1961. i6. 95.
[24] W. Pfleiderer. E. Buhler, f h e r i i . Ber. 1966. 99.3022.
Novel Genetically Engineered Tetracenomycins""
E,xperimentuI Procedure
General procedure for the stereoselective U-4CR: To the amine 2 (10 mmol) and the
chosen aldehyde (10 mmol), dissolved in T H F or methanol (90 mL) at the chosen
temperature (standard value is - 7 5 'C). molecular sieves (4 A) (2.5 g) and catalyst
(ZnCI, in Et,O) (1 1 mmol) were added under an argon atmosphere. This mixture
was stirred for several hours until complete formation of the imine. Then the isocyanide (10 4 mmol). dissolved in precooled solvent ( 5 mL), and subsequently the
acid component (10.4 mmol), dissolved or suspended in precooled solvent ( 5 mL).
were added. The mixture was stirred at a constant temperature until no amine was
detectable by TLC. The reaction mixture was then filtered swiftly through a layer
of celite (1 cm) and this was additionally washed with dichloromethane (20 mL).
Under vigorous alirring a mixture of saturated aqueous sodium hydrogen carbonate
solution (15 mLj and methanol (65 mL) was poured into the reaction mixture. After
five minutes of additional stirring. the resulting suspension was filtered and the
solvent removed. The sometimes oily, often foamy residue was taken up in
dichloromethane (100 mL) and the remaining isocyanide was removed by extraction with aqueous tartaric acid (for rerl-butyl isocyanide) or by treatment with acetic
acid (for involatile isocyanides). This was followed by extraction with water, saturated sodium hydrogen carbonate, and water again (each 250 mL). The mixture was
then dried over sodium sulfate and the solvent evaporated. In most cases, after
several hours at high vacuum a crystalline foam was obtained, which was often
found to be already analytically pure. Otherwise it was purified by recrystallization
and/or chromatography
Received : December 14. 1994
Revised version: February 15, 1995 [27551 I€]
German version: Angrit,. Cham. 1995, 107. 1208
Keywords: asymmetric syntheses chiral auxiliaries
syntheses four-component reactions
Heinrich Decker, Sabine Haag, Gyorgyi Udvarnoki,
and Jiirgen Rohr*
Chemically untreatable diseases as well as increasing numbers
of bacteria,"] viruses, parasites, and cancer cells resistant to
chemotherapy demand innovative concepts to find new molecules, which might serve as lead structures for pharmaceutically
useful drugs. Natural products have proven to be a primary
basis for novel bioactive molecules. Since the pioneering experiments of Hopwood et al. in 1985, genetically engineered hybrid
microorganisms121with modified genes have also been discussed
as a possible source of new natural products.[3]Even though this
method has been highlighted as a major step forward in biotechn ~ l o g y , [ ~there
' ] are no other examples of genetically engineered
hybrid compounds except for some macrolide derivative^.'^] Re[*] Priv.-Doz. Dr. J. Rohr. Dipl.-lng. G. Udvarnoki
lnstitut fur Organische Chemie der Universitit
Tammannstrasse 2. D-37077 Gottingen (Germany).
Telefax: Int. code + (551)39-9660
e-mail : jrohr(a gwdgde
Dr. H. Decker, Dipl.-Biol. S. Haag
lnstitut fur Biologie I1 der Universitit Tiibingen (Germany)
This work was supported by the Deutsche Forschungsgemeinschaft and the
Fonds der Chemischen Industrie. We also thank DipLChem. R. Machinek for
the H,H-NOESY NMR spectrum. and Dr. J. Metzger (Universitdt Tiibingen)
for the electron spray. and Dr. G. Remberg (Universitit Gottingen) for all
other mass spectra.
only those derived from a hybrid polyketide synthase, but also
cently, however, Khosla et al. have proven the general concept
those that result from intercombining the polyketide biosyntheto be prosperous, since the interspecies exchange of polyketide
sis with the post-polyketide biosynthesis apparatus of the other
synthase genes of different producers of multicyclic aromatic
parent, for instance the enzymes of the carbohydrate biosynthepolyketides resulted in the production of several new metabosis, including the glycosyl transferase activities. Biosynthetic enl i t e ~ . [Consequently,
more and various types of hybrid prodzymes for middle or late biosynthetic steps, for example methylucts should be expected in the future. Polyketides occur in most
transferases and oxygenases, may be expected also to contribute
organisms (bacteria, fungi, plants) and are an extremely rich
by acting on the other strains intermediates or end products.
family of bioactive molecules (antibiotics, antitumor agents, imThe success of this approach depends mainly on the substrate
munosuppressants, etc.) . The biosynthetic pathways of many
specificity of the enzymes involved in the biosynthesis of the
polyketides have been elucidated and the analysis of the genetics
different polyketide compounds. To inspect this general possiof polyketide production is under intense investigation.[2b1Therebility, we introduced the entire tetracenomycin (tcm) gene clusfore, this group of natural products seems to be the most promising one to be approached by genetic engineering for the producter (pWHM3 with a 12.5 kb DNA insert = pWHM1026[*"])
and part of the elloramycin (elm)gene cluster (pKC505[sb1with
tion of novel hybrid compounds,[2'] as the current examples
a 25 kb DNA insert = 16F4['"]) into the urdamycin producer
Our strategy in this context was to combine genes of
Streptomyces,fradiae TU 271 7 by transformation.[*'] Most of the
two well-studied producers of related types of multicyclic, arometabolites in the fermentation broth of the two hybrid strains
matic polyketides in order to enhance the chance for detection of
were recognized as tetracenomycin-, elloramycin-, or urdamynovel hybrid products. The first results of our studies are three
cin-related natural products through their UV spectra (HPLC/
novel tetracenomycins isolated from two hybrid strains, which
diode array detector) .Iga]
Three of the compounds did not match
were made by transferring the tetracenomycin biosynthesis genes
with any known products and their subsequent isolation and
from Streptomyces glaucescens GLA. OI6] and elloramycin
structure elucidation revealed the presence of novel tetracenobiosynthesis genes from Streptomyces olivaceus Tii 2353,16' into
mycin derivatives, namely 6-hydroxytetracenomycin C (4) from
the urdamycin-producer Streptomyces fradiae Tii 271 7.l7]
Streptomyces,fradiae Tii2717(pWHM1026), and 3,8-didemethThe tetracenomycins (tetracenomycin C 1) and the related
elloramycins[61(elloramycin A 2) as well as the ~ r d a m y c i n s ~ ~ ]yltetracenomycin C (5) and 8-~-~-olivosyl-8-demethyltetracenomycin C (6) from Streptomyces fradiue Tii2717(16F4). The
(urdamycin A 3) are examples of biosynthetically well-studied
polyketide antibiotics. In addition, since the elloramycins and
structures could be determined from the analyses of the NMR
(Tables 1 and 2) and mass spectra by comparison with that of
urdamycins are glycosylated during the biosynthesis, the protetracenomycin C (1). The characteristic NMR features of 4 are
ducing strains contain. besides the biosynthetic genes coding for
the polyketide synthase, the genes necessary for the biosynthesis
the missing 6-H signal (Table 1) and the additional chelated OH
of the special carbohydrate moieties as well as for the glycosylagroup (6 =12.68; Table 2) in the ' H N M R spectrum, and the
quarternary C-6 in the I3C NMR spectrum (attached proton
tion of the polyketide-derived aglycon moieties. Thus, from a
test (APT); Table 1). The molecular formula C,,H,,O,, (488.4)
combination of the biosynthetic genes of both parent Streptofollows from the electron spray (ES) MS (m/z 556.5 (100%)
myces species, one could expect as possible hybrid products not
Table 1. I3C NMR Data of the novel tetracenomycin derivatives 4, 5, and 6 compared with that of 1 (relative to TMS, 125.7 MHz; S, multiplicity (APT)).
2, R1
= CH3, R2 =
4, R' = CH3, R2
@ = CH3
= H3COH3c
= OH, R3 = CH3 R4 = H
5, R' = H. R2 = H, R3 = H, R4 = H
1 14
4 la1
5 Lbl
190.8 s
99.7 d
175.3 s
57.2 q
70.3 d
85.2 s
194.1 s
141.2 s
120.7 d
129.0 s
108.4 d
158.3 s
56.6 q
129.6 s
167.5 s
52.8 q
137.9 s
21.1 q
121.0 s
168.0 s
109.8 s
197.9 s
83.6 s
190.6 s
100.0 d
174.9 s
57.4 q
71.0 d
84.0 s
196.7 s
106.3 s[d]
161.9 s
134.1 s
102.9 d
158.8 s
56.8 q
131.5 s
167.9 s
52.8 q
138.9 s
21.0 q
123.0 s
167.9 s
106.5 s[d]
199.5 s
190.8 s
98.0 d
187.0 s
74.4 d
86.0 s
196.3 s
141.8 s
120.6 d
129.7 s
112.0 d
158.0 s
190.3 s
100.0 d
175.0 s
57.3 q
70.5 d
19.2 s
193.9 s
141.1 s
121.3 d
129.1 s
112.1 d
156.0 s
129.9 s
168.0 s
52.9 q
139.5 s
21.1 q
120.8 s
170.2 s
109.6 s
199.5 s
82.5 s
131.0 s
167.9 s
52.8 q
138.4 s
21.0 q
322.0 s
168.0 s
110.2 s
197.9 s
83.6 s
[a] In [D,]acetone, the carbon atoms were further assigned through long-range C,H
couplings (2D-COLOC or HMBC). [b] In CD,OD, assignments in analogy to 1. [c]
Sugar moiety signals: 97.8 (d, C-l'), 39.6 (t. C-2'). 71.6 (d. C-3'), 78.0 (d, C-4), 73.3
(d. C-5'). 18.2 (9, C - 6 ) . [d] Assignments interchangeable.
mhH. 0-69451 Weinheim. 1995
$ 10.00i ,2510
Angew. Chem. Ini. Ed. Engl. 1995, 34, No. 10
Table 2. ' H N M R data of the novel tetracenomycin derivatives 4, 5, and 6 compared with that of I. ( relative to TMS, 500 MHz, 6, multiplicity).
5.61 s, br.
3.83 s
5.05 d[c]
4.95 d [c. d]
5.16 s[d, el
8.00 s
7.60 s
4.00 s
5.61 s
3x3 s
5.02 s
5.12 s[d. el
5.29 s[d, el
5.61 s, br.
3.81 s
5.04 s[c]
4.94 d [c. d]
5.11 s[d, el
7.99 s
12.68 s[d]
7.80 s
4.05 s
7.76 s
3.90 s
2.80 s
13.98 s[d]
5.73 s[d, el
3.95 s
2.78 s
13.92 s[d]
5.72 s[d, el
3.93 s
2.82 s
14.01 s[d]
5.76 s[d, el
5.57 dd[f]
1.75 ddd [g]
2.32 ddd[h]
3.71 m
4.25 s[d, i]
3.05 dd[j]
4.30 s[d, i]
3.66 dq [k]
1.32 d[l]
(7) in the urdamycin-producer Streptornyces frudiue TU 271 7
(pWHM1026) and S . fradiue Tii2717(16F4), respectively. Furthermore three novel compounds were produced by the hybrid
strains, namely a minor product 5, and two major products 4
and 6 (production ca. I0 mg L- I ) . 8-demethyltetracenomycin C
(7) has previously been isolated by Yue et al.['o"l from a mutant
of Streptomyces gluucescens impaired in the 8-O-methylation
step. The plasmid conferring tetracenomycin C (pWHMI 026)
production is well-characterized and all tern genes on this plasmid have been sequenced and analyzed.[lobl Since there is no
indication for an enzyme in the tcm cluster catalyzing the 6-hydroxylation of 1, this activity has to arise from an enzyme
present in Streptomyces ,fiadiue. Therefore, 4 could be formed
by one of the oxygenases of urdamycin biosynthesis through
which oxygens in the 12 and the 12b positions are introduced'"'
at carbons 4 and 6, respectively, of its initial decaketide. The
6-position of the tetracenomycins corresponds to the carbon 6
of its initial decaketide precursor (Scheme 1). 6-hydroxytetracenomycin (4) is the 1-analog of elloramycin F (6-hydroxyelloramycin A), known as a minor side product (production
0.01 mg L - I ) of the elloramycin-producer S . o/ivuceus.['21
[a] I n [D,]acetone. [b] I n CD,OD. [c] Broad, J = 7 Hz, not always observable. [d]
Broad. exchangeable with D,O. [el Assignments interchangeable. [f] J = 10. 2 Hz.
[g] J = 12. 12. 10 Hr. [h] J = 12. 5. 2 Hr. [i] Assignments interchangeable. [j] J = 9.
9 Hz. [k] J = 9. 6 Hz.[I] J = 6 Hz. [m] Not observeable because of H/D exchange.
[ M + 3Naj) as well as from the highly resolved molecular peak
of the EI-MS (488.0954). The molecular formula C,,H,,O,,
(443.3) and structure of 5 were derived from the ES-MS (m/z
466.5 (100%) [ M + Na]) and the N M R spectra, containing only
one OCH, group and the additional keto group at 6 =187.0.
The position of the remaining OCH, group at C-9 could be
deduced from its chemical shift in the N M R spectra (Tables 1
and 2) in comparison with that of the corresponding group in 1.
The compound is obtained after workup (pH 7) as the sodium
salt of the anion and has the 1,3-diketo structure, in which one
of the acidic 2-H atoms is removed. The compound resembles
3-demethylelloramycinone, a hydrolysis derivative of 2.[6a1The
N M R spectra of the third novel compound 6 show the structural elements of the 8-demethyltetracenomycin C aglycon plus
one D-olivose moiety. Its molecular formula C,,H,,O,, (588.5)
could be deduced from negative ion FAB-MS [m/z 587 (100 YO)
[M-H-] ). The assignment of the position of the sugar moiety
was complicated. since no helpful 3JC_H
coupling could be detected in the HMBC or COLOC spectrum. Although a significant upfield shift of C-4a in 6 (in comparison with the chemical
shifts of this carbon in 1, 4 and 5 , Table 1) seems to indicate a
variation of the molecule in this region, it could be unambigously concluded that 8 - 0 is the linkage position of the sugar moiety.
This was deduced from the ' H N M R spectrum (all other O H
groups are free, see Table 2), the UV data (the UV spectrum of
the compound at pH > 7 does not show the typical absorption
at 31 5 nm, caused from the 8-OH group, which is, for example,
observed in elloramycinone[6d1), and finally from the H,HNOESY N M R spectrum, in which a significant NOE effect is
observed inter alia between 1'-H and 7-H. The 8-glycosidic linkage follows from the coupling pattern of the 1' and 2'-Ha signals
in the ' H N M R spectrum, the absolute stereochemistry of the
sugar moiety from the [all,value of its liberated a-methylglycoside ( + 133, c = 0.08, EtOH, 21 oC).[9b1
We demonstrated the co-production of tetracenomycin C (1)
and its biosynthetic intermediate 8-demethyltetracenomycin C
Anjirw. C'hrrn Inr. Ed. Engl. 1995, 34. N o . 10
((3 VCH
Scheme 1. Decaketide precursors of 4 and 3.
The plasmid carrying some of the elloramycin biosynthetic
genes (16F4)[*'] is less well characterized than pWHMl026. It
confers resistance to elloramycin (2) and tetracenomycin C (l),
and introduction of 16F4 into Streptomyces,fiadiue Tii 2717 or
Streptomyces lividuns TK24 by transformation results in the
production of 8-demethyltetracenomycin C (7).["I In addition,
Streptomyces fradiae( 16F4) produced 3,8-didemethyltetracenomycin C (5) and 8-~-~-olivosyl-8-demethyltetracenomycin
C (6).
Compound 5, a minor side product (1 mg L- '), seems to be the
result of an incomplete aglycon biosynthesis in which 3-Umethylation did not occur. The occurrence of compound 5 can
be considered an indicator that the methylation sequence for the
can be
biosynthesis of 1, as outlined by Hutchinson et a1.,[6c.10a]
curtailed in some instances. In contrast, 6 is a true hybrid molecule, because the 2,6-dideoxy sugar olivose is normally only
produced for urdamycin biosynthesis; it occurs twice in urdamycin A (3). Elloramycin (2) bears a permethylated L-rhamnose
moiety, also linked at 8-0. The genes for the biosynthesis of the
permethylated L-rhamnose moiety are not present on 16F4.[8'1
Thus, the hybrid strain Streptomyces fradiue( 16F4) must use
glycosyl transferase activity to link the dideoxy sugar 8-Dolivose and the elloramycin polyketide backbone resulting in the
hybrid product 6 (Scheme 2). We cannot exclude the possibility
that there is a glycosyl transferase present on 16F4; therefore
this reaction may either be catalyzed by an enzyme present on
16F4 or by an enzyme of the urdamycin producer. That 6 can
only be obtained through transferring the genes into Streptomyces fradiae and not by simple microbial transformation[' 31
Verlqsgesellsrhaft m h H . 0-69451 WiJinhrim,1995
$ 10.00+ .25;i)
Scheme 2. PKS = Polyketide synthase: "DSS" = "deoxy sugar synthase". that is.
genes necessary <or the biosynthesis of the deoxysugar olivose from glucose:
GT = glycosyl transferase; Enz = enzyme.
was shown by a control experiment: Feeding of 7 to a growing
culture of the urdamycin-producing wild-type strain did not
yield any olivosyltetracenomycin (6). This indicates that the
glycosyltransferase from the urdamycin producer either does
not work on 7 but on a different biosynthetic intermediate, or
that there is an unidentified glycosyl transferase present on 16F4
that catalyzes the linkage of the sugar moiety."41
Our initial experiments indicate that co-expression of two
different polyketide pathways in one hybrid strain is a promising tool to create new natural products in reasonable yields
(10mgL-'). We have not yet detected any novel urdamycin
derivatives or novel types of polyketides derived from the activity of a hybrid polyketide ~ y n t h a s e . ' Therefore,
the expression
of engineered hybrid polyketide synthases in a heterologous
host as demonstrated by Khosla et al.t5] should be done to
obtain further hybrid compounds.
Experimental Procedure
Bacterial strains and plasmids: S./radiae TU 2717 was transformed with 16F4 and
pWMH1026 by protoplast fusion and protoplasts were regenerated on R2YE agar
plates [15]. Plasmid pWHM1026 carrying the entire ton cluster was described previously [gal. 16F4 consists of the cosmid vector pKC505 [8 b] that contains a 25 kb
DNA fragment isolated from the elloramycin-producer S . olivaceus TU 2353 [8c].
This plasmid contains all the genes necessary for the production of 7 [8c].
Cultivation of the recombinant strains: S . fradiue Tu2717(16F4) and S.frudiae
Tu2717(pWHM1026) were grown for 96 h in a production medium (2% soybean
meal. 2% glucose, pH 7.2 [7]) with 25 ~ 1 g m L - I apramycin and 10 pgmL-'
thiostrepton. respectively.
Isolation of the novel products: The culture WAS extracted twice with ethyl acetate
at pH 7 and evaporated to dryness. The ethyl acetate residue was disolved in CH,CI,
and further purified on silica gel saturated with oxalic acid (silica gel SI 60. Merck.
Germany). The silica gel column was washed with CH,CIZ and the compounds were
eluted with CH,CI,/MeOH (95:5, 9: 1. and 8:2). Fractions containing different
urdamycin and tetracenomycin metabolites were pooled, evaporated t o dryness,
and further purified on Sephudex LH 20 (MeOH). The final purification WAS performed by HPLC (preparative column, Nucleosil 100 C-18, particle size 10 pm),
elution with water/MeOH (gradient 4 0 % to 100% MeOH in 15 min).
Instruments: All NMR spectra were recorded on a Varian VXR 500 spectrometer
at a field strength of 11.7 Tesla, except the HMBC spectra and the NOESY spectrum
which were recorded on a Bruker AMX 300 at a field strength of7.1 Tesla. Details
see Tables 1 and 2. All mass spectra (except ES- MS. see below) were obtained on
a Finnigan MAT 95 spectrometer, for the FAB-MS 3-nitrobenzyl alcohol was used
as the matrix. The electron spray mass spectra (ES-MS) were recorded on a API 111
Taga 6000 E equipped with an ion spray source (Sciex, Thornhill, Canada).
[l] a) N . C . J. Strynadka, S. E. Jensen. K. Johns. H . Blanchard, M. Page, A.
Matagne, JLM. Frere, M. N . G. James, Nature 1994,368,657-660, and references therein; b) H . C. Neu, Science 1992. 257, 1064-1073, and references
therein; c) J. Travis, E. Culotta, R. Nowak, S. Kingman. R. Stone. ibid. 1994,
264. 360-367; d) L. L. Silver, K . A. Bostian, Antimicrob. Agenrs Chemother.
1993. 37, 377-383: e) V. Webb, J. Davies. ibid. 1993, 37, 2379-2384.
[2] a) C. R. Hutchinson, Med. Res. Rev. 1988,R. 557-567; b) L. Katr. S. Donadio,
Annu. Rev. Microbid. 1993. 47. 875-912; c) D. E. Cane, Science 1994. 263.
338-340, and references therein.
[3] a) D. A. Hopwood. F. Malpartida, H. M . Kieser. H. Ikeda. J. Duncan. I. Fujii,
B. A. Rudd. H. G. Floss, S. Omura, Nuture 1985,314, 642-644; b) S. Omura,
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Received: December 27, 1994 [Z7577IE]
German version. Angehi. Cheni. 1995. 107, 1214
Keywords: biosynthesis . gene technology . polyketides . tetracenom ycins
VCH Verlagsgesellschafi mbH. 0.69451 Weinheim, 1995
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