AP562A - Novel streptogramins and a process for preparing streptogramins by mutasynthesis. - Google Patents

Novel streptogramins and a process for preparing streptogramins by mutasynthesis. Download PDF

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Publication number
AP562A
AP562A APAP/P/1995/000752A AP9500752A AP562A AP 562 A AP562 A AP 562A AP 9500752 A AP9500752 A AP 9500752A AP 562 A AP562 A AP 562A
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pristinamycin
dimethylamino
group
seq
strain
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APAP/P/1995/000752A
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AP9500752A0 (en
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Nathalie Bamas-Jacques
Jean-Claude Barriere
Veronique Blanc
Francis Blance
Joel Crouzet
Laurent Debussche
Gilles Dutruc-Rosset
Alain Famechon
Jean-Marc Paris
Denis Thibaut
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Rhone Poulenc Rorer Sa
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    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
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    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
    • C07D295/14—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
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    • C07C229/34—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton containing six-membered aromatic rings
    • C07C229/36—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton containing six-membered aromatic rings with at least one amino group and one carboxyl group bound to the same carbon atom of the carbon skeleton
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    • C07C229/40—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino groups bound to carbon atoms of at least one six-membered aromatic ring and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/42—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino groups bound to carbon atoms of at least one six-membered aromatic ring and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton with carboxyl groups linked to the six-membered aromatic ring, or to the condensed ring system containing that ring, by saturated carbon chains
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C233/00—Carboxylic acid amides
    • C07C233/01—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C233/12—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by halogen atoms or by nitro or nitroso groups
    • C07C233/15—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by halogen atoms or by nitro or nitroso groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by a carbon atom of a six-membered aromatic ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C233/00—Carboxylic acid amides
    • C07C233/01—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C233/16—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms
    • C07C233/24—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by a carbon atom of a six-membered aromatic ring
    • C07C233/25—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by a carbon atom of a six-membered aromatic ring having the carbon atom of the carboxamide group bound to a hydrogen atom or to a carbon atom of an acyclic saturated carbon skeleton
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    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
    • C07C323/50—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton
    • C07C323/62—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atom of at least one of the thio groups bound to a carbon atom of a six-membered aromatic ring of the carbon skeleton
    • C07C323/63—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atom of at least one of the thio groups bound to a carbon atom of a six-membered aromatic ring of the carbon skeleton the carbon skeleton being further substituted by nitrogen atoms, not being part of nitro or nitroso groups
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N15/76—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Actinomyces; for Streptomyces
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    • A61K38/00—Medicinal preparations containing peptides

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Abstract

The present invention relates to novel compounds which are related to the group

Description

NOVEL STREPTOGRAMINS AND A PROCESS FOR PREPARING
STREPTOGRAMINS BY MUTASYNTHESIS
.'j
The present invention relates principally to novel compounds which are related to the group B streptogramins, and to a process for preparing streptogramins by mutasynthesis. It also relates to novel genes which are involved in the biosynthesis of precursors of the group B streptogramins, and to their uses.
The streptogramins form a homogeneous group of 10 antibiotics consisting of an association of two types of chemically different molecules; on the one hand polyunsaturated macrolactones (group A components) and, on the other hand, depsipeptides (group B components). This group comprises numerous antibiotics which are known under different names according to their origin and includes pristinamycins, mikamycins and virginiamycins (Cocito 1979, 1983).
The A and B components have a synergistic antibacterial activity which can amount to 100 times that of the separate components and which, contrary to that of each component, is bactericidal (Cocito 1979). This activity is more particularly effective against Grampositive bacteria such as Staphylococci and Streptococci (Cocito 1979, Videau 1982). Components A and B inhibit protein synthesis by binding to the 50S subunit of ribosome (Cocito 1979; Di Giambattista et al., 1989).
While knowledge of the routes by which each of the components is biosynthesized still remains partial to date, earlier studies, presented in International Patent
Application PCT/FR93/0923, published as W094/08014, have made it possible to identify several proteins, and the corresponding structural genes, which are involved in the biosynthesis of the two types of component.
Two parts can be distinguished in the process for
AP/P/ 9 5 / 0 0 7 5 2
biosynthesizing group B streptogramins:
1) Biosynthesis of the precursors, or their analogues, of the macrocycle: 3-hydropicolinic acid, L-2-aminobutyric acid, 4-dimethylamino-L-phenylalanine,
L-pipecolic acid and L-phenylglycine.
2) Formation of the macrocycle from the precursors listed above, from L-threonine and from L-proline, or their analogues, possibly with subsequent modification of the peptide N-methylation, hydroxylation and oxidation type.
Patent Application W094/08014 relates, in particular, to the enzymes which catalyse incorporation of the precursors into the peptide chain of B streptogramins in the process of elongation, and also to their structural genes. These results have demonstrated the non-ribosomal character of the peptide synthesis leading to the type B components .
The present invention relates, more particularly, to novel compounds which are related to group B 20 streptogramins and, more precisely, to novel compounds of the pristinamycin I family (Figures 1 and 2), termed PI below, or of the virginiamycin S family (Figure 3).
The major constituent of the I pristinamycins (PI) is PIA (Figure 1), which represents approximately 94% 25 of the PI, with the remaining approximately 6% being represented by minor constituents of the depsipeptide (PIB to PIj) whose structures are depicted in Figure 2. PI results essentially from the condensation of amino acids, ’certain of which are essential for protein synthesis 30 (threonine and proline) and others of which are novel and themselves considered to be secondary metabolites (L-2-aminobutyric acid, 4-dimethylamino-L-phenylalanine (DMPAPA), L-pipecolic acid and L-phenylglycine for PIA) , and also of an aromatic precursor, 3-hydroxypicolinic acid.
The virginiamycin S derivatives result from condensation of the same acids as in the case of PI, apart
CM ir>
o co uo £
£ <
AP . Ο Ο 5 6 2 r- 3 from 4-DMPAPA, which is replaced by a phenylalanine (see Figure 3).
Production of these different compounds by biosynthesis therefore requires preliminary synthesis, by a producer strain, of the novel precursors identified above.
The present invention results specifically from a novel process for preparing streptogramins which employs, as a strain for producing streptogramins, a microorganism strain which is mutated so as to alter the biosynthesis of the precursors of the group B streptogramins. According to this process, the said mutant strain is cultured in a medium which is supplemented with a novel precursor which is different from the precursor whose biosynthesis is altered. Unexpectedly, this results in the production of novel compounds which are related to the group B streptogramins and which are of value in the therapeutic sphere .
More precisely, the present invention relates to novel compound of formula I:
* ί
ο ο
I wherein:
- R2 and R4 are independently a hydrogen atom or a methyl group;
- R3 is hydrogen or a hydroxyl group;
- X is a CO, CHOH or CH2 group; and
- Rj is
wherein, when
A, C, D and E are hydrogen, then B is
- a halogen atom, preferably fluorine,
- a monoalkylamino or dialkylamino group, in which the alkyl moiety or one or both of the alkyl moieties is preferably methyl or ethyl,
- an ether group, preferably an OR group, R being a substituted or unsubstituted alkyl or alkenyl group, preferably methyl, ethyl, trifluoromethyl or allyl,
- a thioether group, preferably an alkylthio group, more preferably a methylthio group,
- a C3 to C3 alkyl group, or
- a trihalogenomethyl group, preferably trifluoromethyl; and when A, B, D and E are hydrogen atom, then C is
- a halogen atom,
- an NRsR6 group with Rs and R6 independently being.
- hydrogen,
- a straight-chain or branched C3 to C4 alkyl group, with the proviso that when one of the
AP/P/ 9 5 / 0 0 7 5 2
AP . Ο Ο 5 6 2
- 5 substituents R5 or R6 is methyl then the other is ethyl,
- a alkyl cycloalkylmethyl group, in which the cycloalkyl group contains 3 to 4 carbon atoms,
- an optionally substituted C3 or C4 5 cycloalkyl group,
- a straight-chain or branched C3 or C4 alkenyl group, with the proviso that when one of the substituents Rs or Rfi is alkenyl, the other is not methyl or C3 to C6 cycloalkyl,
- a substituted or unsubstituted N-pyrrolidinyl group,
- an ether group, preferably an OR group with R being a substituted or unsubstituted alkyl or alkenyl group preferably methyl, ethyl, optionally substituted by a chlorine atom, trifluoromethyl or alkenyl, e.g. allyl,
- a thioether group, preferably an alkylthio group more preferably an alkylthio group wherein the alkyl group is a C3 to C3 alkyl group,
- an acyl group or alkoxy carbonyl group, preferably a COR group, more preferably a COR group in which R is a C3 to C3 alkyl group or a Cx to C3 alkoxy group,
- a Cj to C6 alkyl group which is straight-chain or branched, and preferably a methyl, isopropyl or tert25 butyl group,
- a alkylthio methyl group, preferably a CH2SR group wherein R is C3 to C3 alkyl,
- an aryl group, preferably phenyl, or
- a trihalogenomethyl group, preferably trifluoromethyl; and when A, D and E are hydrogen atom, then B is
- a halogen atom, preferably fluorine
- a monoalkylamino or dialkylamino group, preferably a monoalkyl amino group in which the alkyl group is methyl or ethyl or a dialkylamino group in which one or both alkyl groups are methyl or ethyl, t υ u / b ϋ /d/dtf
- an ether group, preferably an OR group in which R is a substituted or unsubstituted alkyl group, preferably methyl, ethyl or trifluoromethyl,
- a thioether group, preferably an alkylthio 5 group, more preferably an ethylthio group, or
- a Cj to C3 alkyl group, and C is
- a halogen atom, preferably fluorine,
- an amino, monoalkylamino or dialkylamino group, 10 preferably a monoalkylamino group in which the alkyl group is methyl or a dialkyl amino group in which one or both alkyl groups are methyl, with the proviso that B is not a bromine or chlorine atom or a substituted or unsubstituted allyl group,
- an ether group, preferably an OR group in which
R is an alkyl group, preferably methyl, ethyl or trifluoromethyl,
- a thioether group, preferably an alkylthio group, more preferably a methylthio group,
- a Cj to C8 alkyl group, or
- a trihalogenomethyl group, preferably trifluoromethyl; and when Β, E and D or a hydrogen atom, then A and C are methyl groups.
In this specification, including the accompanying claims, alkyl groups and moieties, unless otherwise specified, are straight- or branched- chain and contain from 1 to 4 carbon atoms.
The following may be more particularly mentioned as preferred compounds:
f-methylthio-de(4 f-dimethylamino)pristinamycin ζ-methylthio-de(4 f-dimethylamino)pristinamycin Ihz
5y-hydroxy-4 f-methylthio-de(4 fdimethylamino)pristinamycin I„,
AP/P/ 9 5 / 0 0 7 5 2 «ί !ife xAP.0 0 5 6 2
-74f-methyl-de (4f-dimethylamino)pristinamycin IA,
4f-methyl-de(4f-dimethylamino)pristinamycin I„,
4f-methoxy-de(4f-dimethylamino)pristinamycin IA, f-methoxycarbonyl-de(4 ξ5 dimethylamino)pristinamycin IA,
4f-chloro-de(4f-dimethylamino)pristinamycin IA, f-bromo-de(4 ζ-dimethylamino)pristinamycin IA,
4f-bromo-de(4ζ-dimethylamino)pristinamycin IH,
4f-iodo-de(4f-dimethylamino)pristinamycin IA,
4f-iodo-de(4f-dimethylamino)pristinamycin I„,
4f-trifluoromethyl-de(4f-dimethylamino)pristinamycin IA, f-trifluoromethyl-de(4 f-dimethylamino)pristinamycin IH,
4f-tert-butyl-de(4ζ-dimethylamino)-pristinamycin
Ia, f-isopropyl-de(4 f-dimethylamino)-pristinamycin Ia,
4f- isopropyl-de(4f-dimethylamino)-pristinamycin
IE, e-methylamino-de(4 f-dimethylamino)-pristinamycin Ia,
4e-methoxy-de(4ζ-dimethylamino)pristinamycin IA,
4e-methoxy-de(4ζ-dimethylamino)pristinamycin IH,
4e-fluoro 4f-methyl-de(4f-dimethylamino)pristinamycin IA,
4f-amino-de(4ζ-dimethylamino)pristinamycin IA, f-ethylamino-de(4 ζ-dimethylamino)-pristinamycin
Ia,
4f-diethylamino-de(4f-dimethylamino)pristinamycin IA, f-allylamino-de(4f-dimethylamino)-pristinamycin Ia, ζ-diallylamino-de(4 ζ-dimethylamino)35 pristinamycin IA, f-allylethylamino-de(4 f-dimethylamino)2 9 L 0 0 / S 6 /d/dV pristinamycin IA, f-ethylpropylamino-de(4 f-dimethylamino)pristinamycin IA, f-ethylisopropylamino-de(4 f-dimethylamino)5 pristinamycin IA,
4f-ethylcyclopropylmethylamino-de(4fdimethylamino)pristinamycin IA, ζ-(1-pyrrolidinyl)-de(4 f-dimethylamino)pristinamycin IA,
4f-trifluoromethoxy-de(4ζ-dimethylamino)pristinamycin IA> ( 4f-allyloxy-de(4f-dimethylamino)pristinamycin IA, f-ethoxy-de(4 f-dimethylamino) pristinamycin IA, f-ethylthio-de(4 f-dimethylamino)-pristinamycin
IA, f-methylthiomethyl-de(4 f-dimethylamino)pristinamycin IA,
4f-(2-chloroethoxy)-de(4ζ-dimethylamino)pristinamycin IA,
4f-acetyl-de(4f-dimethylamino)pristinamycin IA,
4f-ethyl-de(4f-dimethylamino)pristinamycin IA,
4f-ethyl-de(4f-dimethylamino)pristinamycin IH, < 4 e-dimethylamino-de(4 f-dimethylamino)χ-, pristinamycin IA,
4e-methylthio-de(4f-dimethylamino)- pristinamycin
Iaz
4e-ethoxy-de(4f-dimethylamino)pristinamycin IA. The present invention is also directed towards a 'process which is particularly useful for preparing the compounds of the general formula I.
More precisely, it relates to a process for preparing streptogramins, which process comprises culturing a streptogramin-producing microorganism strain having a genetic modification which affects the biosynthesis of a precursor of the group B streptogramins, cultured in an appropriate culture medium supplemented with a group B
AP/P/ 9 5 / 0 0 7 5 2
AP . Ο Ο 5 6 2
- 9 streptogramin precursor different from that whose biosynthesis is altered by the modification, and recovering the streptogramins therefrom.
The strains which are employed within the scope 5 of the present invention are therefore strains which produce streptogramins and which are mutated. The genetic modification(s) can be located either within one of the genes which is involved in the biosynthesis of the said precursors or outside the coding region, for example in the regions responsible for the expression and/or the transcriptional or post-transcriptional regulation of the said genes, or in a region belonging to the transcript containing the said genes.
According to one particular embodiment of the 15 invention, the mutant strains possess one or more genetic modifications within at least one of their genes which is/are involved in the biosynthesis of the group B streptogramin precursors.
Such genetic modification(s) alter(s) the 20 expression of the said gene(s) rendering the gene(s), involved in the biosynthesis of the precursors partially or totally incapable of expression as the natural enzyme(s) involved in the biosynthesis of the relevant group B ~ streptogramin precursor. The inability of the said gene(s) ** 25 to express the natural protein(s) may be manifested in any way, for example by the production of a protein which is inactive due to structural or conformational modifications, or by the absence or reduction of expression, or by the 'production of a protein having an altered enzymic activity, 30 or by the production of the natural protein at an attenuated level or in accordance with a desired mode of regulation. The result of any of these possible manifestations is an alteration of, or perhaps a blockage in, the synthesis of at least one of the group B streptogramin precursors.
The genes which are capable of being mutated
AP/P/ 9 5 / 0 0 7 5 2 c
C
Ο 25 within the scope of the present invention are preferably the genes which are involved in the biosynthesis of the following precursors: L-2-aminobutyric acid, 4dimethylamino-L-phenylalanine (DMPAPA), L-pipecolic acid, L-phenylglycine and/or 3-hydroxypicolinic acid (3-HPA).
These genes are more preferably the papA. papM. papB (SEQ ID No. 3), papC (SEQ ID No. 2), hpaA (SEQ ID No. Θ), snbF (SEQ ID No. 6) and pipA (SEQ ID No. 5) genes described below.
The papA and papM genes have already been described in Patent Application W094/08014. They are present on the cosmid pIBV2. The papA gene appears to correspond to a gene for biosynthesizing 4-amino-Lphenylalanine from chorismate. The 4-amino-L-phenylalanine is then dimethylated by the product of the papM gene, an Nmethyltransferase, in order to form 4-dimethylamino-Lphenylalanine, DMPAPA, which is then incorporated into pristinamycin IA. These two genes are more particularly involved, therefore, in the synthesis of the precursor termed DMPAPA.
The other genes, papB. papC. pipA. snbF and hpaA. have been identified and characterized within the scope of the present invention. They are grouped together with the snbA. papA and papM genes on a chromosomal region of approximately 10 kb (Figure 7) .
The homologies demonstrated for the PapB and PapC proteins, between the papA and papM genes, show that the corresponding proteins are also involved, jointly with the papA and papM proteins, in the biosynthesis of the DMPAPA precursor. These two novel genes were identified by subcloning which was carried out using cosmid pIBV2, described in Patent Application W094/08014, and a plasmid, pVRC900, which is derived from pIBV2 by means of a HindiII deletion and is also described in Patent Application W094/08014.
AP/P/ 95/00752
AP . Ο Ο 5 6 2 (
(
Ο 25
- 11 A comparison of the protein encoded by the papC gene with the protein sequences contained in the Genpro library shows a 27% homology with the region which is involved in the prephenate dehydrogenase activity of the bifunctional TyrA proteins of E. coli (Hudson and Davidson, 1984) and Erwinia herbicola (EMBL data library, 1991). This region of TyrA catalyses aromatization of the prephenate to form 4-hydroxyphenylpyruvate in the biosynthesis of tyrosine. A similar aromatization, which proceeds from 4deoxy-4-aminoprephenate and leads to 4-aminophenyl-pyruvate is very probably involved in the synthesis of DMPAPA. It would be catalysed by the PapC protein (SEQ ID No. 2).
PapB has a 24 to 30% homology with the region which is involved in the chorismate mutase activity of the TyrA and PheA bifunctional proteins of E. coli (Hudson and Davidson, 1984) and of the TyrA protein of Erwinia herbicola. This region catalyses isomerization of the chorismate to form prephenate in the biosynthesis of tyrosine and of phenylalanine. The PapB protein (SEQ ID No. 3) is probably involved in a similar isomerization which proceeds from 4-deoxy-4-aminochorismate and leads to 4deoxy-4-aminoprephenate in the synthesis of DMPAPA.
The pipA, snbF and hpaA genes have been located in the region which is contained between the snbA gene, which encodes 3-hydroxypicolinic acid AMP ligase and is described in Patent Application W094/08014, and the papA gene. They were located accurately by means of subcloning, which was carried out using the plasmid pVRC900 and the cosmid pIBV2, which are described in Patent Application W094/08014.
On comparing the protein encoded by the hpaA gene and the protein sequences contained in the Genpro library, a homology of from 30 to 40% was detected with a group of proteins which are probably involved (Thorson et al.. 1993) in the transamination of intermediates in the biosynthesis of various antibiotics (DnrJ, EryCl, TylB, StrS and PrgL).
AP/P/ 9 5 / 0 0 7 5 2
Synthesis of the 3-HPA precursor, which appears to derive from lysine by another route than that of cyclodeamination (see examples 1-2 and 2-1), probably requires a transamination step which can be catalysed by the product of this gene termed hpaA (SEQ ID No. 8) . Furthermore, the results of mutating this gene demonstrate unequivocally that it is involved in the synthesis of the 3-HPA precursor.
Comparison of the product encoded by the gene termed pipA with the protein sequences contained in the Genpro library shows a 30% homology with the ornithine cyclodeaminase of Agrobacterium tumefaciens (Schindler et al.. 1989) . This enzyme is involved in the final step of the catabolism of octopine; it converts L-ornithine into
L-proline by means of cyclodeamination. It has been demonstrated, by means of incorporating labelled lysine, that 4-oxopipecolic acid and 3-hydroxypicolinic acid, which are found both in PIA and in virginiamycin SI, is derived from lysine (Molinero et al.. 1989, Reed et al.. 1989).
Cyclodeamination of lysine, in a similar manner to that described for ornithine, would lead to the formation of pipecolic acid. Taking this hypothesis into account, this product was termed PipA (SEQ ID No. 5). The results of mutating the pipA gene, presented in the Examples below, demonstrate that it is involved solely in the synthesis of pipecolic acid. It is noted, in particular, that this mutation has no effect on the biosynthesis of 3-hydroxypicolinic acid, which is also derived from lysine and of which pipecolic acid could have been a precursor.
0 Finally, on comparing the product of the gene termed snbF with the protein sequences contained in the Genpro library, a 30 to 40% homology was noted with several hydroxylases of the cytochrome P450 type, which are involved in the biosynthesis of secondary metabolites (Omer et al.. 1990. Trower et al.. 1992). Several hydroxylations can be envisaged in the biosynthesis of the precursors of
AP/P/ 9 5 / 0 0 7 5 2
AP · Ο Ο 5 6 2
Si) ί
- 13 pristinamycin I, in particular in the biosynthesis of 3-HPA (hydroxylation of picolinic acid at the 3 position) and of 4-oxopipecolic acid (hydroxylation of pipecolic acid at the 4 position). The corresponding protein was termed SnbF (SEQ
ID No. 6).
The results of mutating the pjpA gene, with polar effects on the expression of the snbF gene, demonstrate the involvement of the snbF gene in the hydroxylation of the pipecolic acid residue of group B streptogramins. The expression of the snbF gene is thus altered by the expedient of effecting a genetic modification of the pjpA gene.
Preferably, the genetic modification(s) render(s) the said gene partially or totally incapable of expressing the natural protein.
Genetic modification should be understood to mean, more particularly, any suppression, substitution, deletion, or addition of one or more bases in the gene(s) under consideration. Such modifications may be obtained in vitro (on the isolated DNA) or in situ, for example, by means of genetic engineering techniques, or else by exposing the said microorganisms to a treatment using mutagenic agents. Examples of mutagenic agents are physical agents such as high-energy rays (X, γ, ultra violet, etc.
rays), or chemical agents which are able to react with different functional groups of the DNA bases, and, for example, akylating agents [ethyl methanesulphonate (EMS), N-methyl-N’-nitro-N-nitrosoguanidine, and N-nitroquinolineΊ-oxide (NQO)], bialkylating agents, intercalating agents, etc. Deletion is understood to mean any suppression of a part or all of the gene under consideration. This deletion can, in particular, be of a part of the region encoding the said proteins, and/or of all or part of the promoter region for transcription or translation, or else of the transcript.
The genetic modifications may also be obtained by
AP/F/ 9 5 / 0 0 7 5 2 means of gene disruption, for example using the protocol initially described by Rothstein [Meth. Enzymol. 101 (1983) 202] or, advantageously, by means of double homologous recombination. In this case, the integrity of the coding sequence will preferably be disrupted in order to permit, if need be, replacement, by means of homologous recombination, of the wild-type genomic sequence with a non-functional or mutant sequence.
According to another embodiment of the invention, 10 the genetic modifications can involve placing the gene(s) encoding the said proteins under the control of a ( regulatable promoter.
Mutant microorganism strains according to the present invention may be obtained from any microorganism which produces streptogramins (cf. Table V). According to one particular embodiment of the invention, the mutant strain is a strain which is derived from Streptomyces pristinaespiralis and, more particularly, from S. pristinaespiralis SP92.
Mutant strains which are preferred within the scope of the present invention and which may more particularly be mentioned are the strain SP92::pVRC508, in : which a mutation affects the biosynthesis of the DMPAPA precursor by disrupting the papA gene by means of simple θ 25 crossing over; and, more preferably, the strain SP212, in which a mutation affects the biosynthesis of the DMPAPA cursor by disrupting the papA gene by means of double homologous recombination. These strains no longer produce 'PI unless they are supplemented with the DMPAPA precursor. 30 Unexpectedly, when a novel precursor, which is different from DMPAPA and which is capable, in this case after being metabolized, of being incorporated by PI synthetase III (the SnbD protein which is responsible for incorporating Lproline and DMPAPA residues) is added to the production medium, these two strains then become able to produce novel I pristinamycins or virginiamycins, or else mainly to
APiPi 93/00752
AP. Ο Ο 5 6 2
- 15 produce a component which is normally a minor component of PI, in particular PIB (Figure 2).
Two other mutant strains have been prepared within the scope of the present invention. These are, respectively, the strain SP92pipA: :OamR, in which the pipA gene is disrupted by homologous recombination, and the strain SP92hpaA: :nam*. in which the hpaA gene is disrupted. While strain SP92pipA::DamR no longer produces PI under standard fermentation conditions, when its culture medium is augmented with L-pipecolic acid it strongly produces a B streptogramin component, which is a minor component among the B streptogramins produced by the unmutated strain, in which 4-oxopipecolic acid is replaced by L-pipecolic acid. Similarly, strain S. pristinaespiralis SP92hpaA::fiamR no longer produces PI under standard fermentation conditions, it is able to produce novel group B streptogramins in the presence of novel precursors.
By supplementing the medium for culturing mutant strains according to the invention with at least one novel precursor, it is possible to orient biosynthesis either towards novel streptogramins, or towards a minor form of the streptogramins, or else to favour formation of a particular streptogramin.
The precursors which are employed within the scope of the present invention can be derivatives or analogues of amino acids and, more particularly of phenylalanine, as well as organic acids and, in particular, alpha-ketocarboxylic acids and, more particularly, ’derivatives of phenylpyruvic acid.
Naturally, the novel precursor is such that it caters for the alteration or blockage, which is induced in accordance with the invention, within the biosynthesis of one of the natural precursors of the group B streptogramins and leads to the synthesis of streptogramins. According to one particular embodiment of the invention, this novel precursor is selected such that it is related to the
AP/P/ 9 5 / 0 0 7 52 r
c
O 25 precursor whose biosynthesis is altered. Thus, in the specific case of the mutant which is blocked in the biosynthesis of DMPAPA, the novel precursor is preferably a derivative of phenylalanine.
The following may, in particular, be cited as precursors which are suitable for the invention:
Phenylalanine, 4-dimethylaminophenylalanine,
4-methylaminophenylalanine, 4-aminophenylalanine,
4-diethylaminophenylalanine, 4-ethylaminophenylalanine, 4-methylthiophenylalanine, 4-methylphenylalanine,
4-methoxyphenylalanine, 4-trifluoromethoxyphenylalanine,
4-methoxycarbonylphenylalanine, 4-chlorophenylalanine,
4-bromophenylalanine, 4-iodophenylalanine,
4-1rifluoromethylphenylalanine, 4-tert-butylphenylalanine,
4-isopropylphenylalanine, 3-methylaminophenylalanine,
3-methoxyphenylalanine, 3-methylthiophenylalanine,
3-fluoro-4-methylphenylalanine, L-pipecolic acid, 4-tertbutylphenylpyruvic acid, 4-methylaminophenylpyruvic acid,
2- naphthylphenylalanine, 4 -fluorophenylalanine,
3- trifluorophenylalanine, 3-ethoxyphenylalanine,
2,4-dimethylphenylalanine, 3,4-dimethylphenylalanine,
3- methylphenylalanine, 4-phenylphenylalanine,
4- butylphenylalanine, 2-thienyl-3-alanine,
3-trifluoromethylphenylalanine, 3-hydroxyphenylalanine,
3-ethylaminophenylalanine, 4-allylaminophenylalanine,
3- ethylaminophenylalanine, 4-allylaminophenylalanine,
4- diallylaminophenylalanine,
4-allylethylaminophenylalanine,
4-ethylpropylaminophenylalanine,
4-ethylisopropylaminophenylalanine,
4-ethylcyclopropylmethylaminophenylalanine,
4-(1-pyrrolidinyl)phenylalanine, 4-O-allyltyrosine,
4-O-ethyltyrosine, 4-ethylthiophenylalanine,
-ethylthiomethylphenylalanine,
4-0-(2-chloroethyl) tyrosine, 4-acetylphenylalanine,
4-ethylphenylalanine, 3-dimethylaminophenylalanine,
AP/P/ 9 5/00752
AP. Ο Ο 5 6 2
- 17 3- ethoxyphenylalanine, 3-fluoro-4-methylphenylalanine and
4- aminomethylphenylalanine.
Among these precursors,
4-trifluoromethoxyphenylalanine,
3-methylaminophenylalanine, 3-methylthiophenylalanine,
3- fluoro-4-methylphenylalanine, 4-methylaminophenylpyruvic acid, 3-ethoxyphenylalanine, 4-allylaminophenylalanine,
4- diallylaminophenylalanine,
4-allylethylaminophenylalanine,
4-ethylpropylaminophenylalanine,
4-ethylisopropylaminophenylalanine,
4-ethylcyclopropylmethylaminophenylalanine,
4-(1-pyrrolidinyl) phenylalanine,
4-ethylthiomethylphenylalanine,
4-0-(2-chloroethyl)tyrosine, 3-dimethylaminophenylalanine and 3-ethylaminophenylalanine are novel and were prepared and characterized within the scope of the present invention. They are found to be particularly useful for preparing streptogramins according to the invention.
The process of the invention is particularly advantageous for preparing novel group B streptogramins or for favouring formation of particular streptogramins. As such, it is particularly useful for preparing PIB.
The present invention also relates to a nucleic acid sequence, selected from:
(a) all or part of One of the genes papC (SEQ ID
No. 2), papB (SEQ ID No. 3), pipA (SEQ ID No. 5), snbF (SEQ ID No. 6) and hpaA (SEQ ID No. 8) or a sequence 'complementary thereto;
(b) a nucleic acid sequence which hybridizes to all or part of a gene defined in (a) and (c) a nucleic acid sequence related to the sequences defined in (a) and/or (b) owing to the degeneracy of the genetic code.
The sequences, particularly sequences according to (b), preferably encode a polypeptide which is involved
AP/P/ 9 5 / 0 0 7 5 2
c.
Ο
Ο 25
- 18 in the biosynthesis of the streptogramins.
Still more preferably, the invention relates to the nucleotide sequences which are represented by the genes papC (SEQ ID No. 2), papB (SEQ ID No. 3), pipA (SEQ ID No.
5) , snbF (SEQ ID No. 6), and hpaA (SEQ ID No. 8).
The invention furthermore relates to any recombinant DNA which comprises a papC (SEQ ID No. 2), papB (SEQ ID No. 3), pipA (SEQ ID No. 5), snbF (SEQ ID No. 6) or hpaA (SEQ ID No. 8) gene.
Naturally, the nucleotide sequences defined above can be part of a vector of the expression vector type, which can be an autonomously replicating vector, an integrated vector or a suicide vector. The present invention is also directed to these vectors as well as to any use of a sequence according to the invention or of a corresponding vector, in particular for preparing metabolites of interest. It furthermore relates to any polypeptide which results from the expression of a claimed sequence.
The present invention also relates to any mutated
S. pristinaespiralis strain which possesses at least one genetic modification within one of the papC (SEQ ID No. 2), papB (SEQ ID No. 3), pipA (SEQ ID No. 5), snbF (SEQ ID No.
6) and hpaA (SEQ ID No. 8) genes, and, more preferably, to strains SP92pipA: :Dam* and SP92hpaA: :Dam*. as well as any S. pristinaespiralis strain, such as SP212, in which the papA gene is disrupted by means of double homologous recombination.
Combinations of a component of the group A streptogramins and of a compound of the general , according to the invention, are particularly advantageous in the therapeutic sphere. They are employed, in particular, for treating ailments which are due to Gram-positive bacteria (of the genera
Staphylococci, Streptococci, Pneumococci and Enterococci) and Gram-negative bacteria (of the genera Haemophilus, cxi
ΙΓ ro o
in σι &
Cu <
AP.0 0 5 6 2
- 19 ).
-i P
J i
J
Gonococci, Meningococci). Thus, the compounds according to the invention have a synergistic effect on the antibacterial action of pristinamycin IIB on Staphylococcus aureus IP8203 in mice in vivo, at oral doses which are principally between 30 mg/kg and 100 mg/kg, when they are combined in PI/PII proportions of the order of 30/70.
The present invention extends to any pharmaceutical composition which contains at least one compound of the general which is or is not combined with a group A streptogramin.
The examples appearing below are presented by way of illustrating the present invention and do not limit it.
LIST OF FIGURES.
Figure 1: Structure of pristinamycin IA.
Figure 2: Structure of the minor components of pristinamycin I.
Figure 3: Other examples of structures of B components of streptogramins.
Figure 4: Depiction of the Pstl-Xhol region of 2.9 kb.
Figure 5: Depiction of the Xhol-Pstl region of 4.5 kb.
Figure 6: Depiction of the Hindlll-Bcrlll region of 1.6 kb.
Figure 7: Depiction of the Bqlll-Xhol region of approximately 10 kb.
Figure 8: Depiction of plasmid pVRC415.
Figure 9: Depiction of plasmid pVRC420.
Figure 10: Depiction of plasmid pVRC411.
Figure 11: Depiction of plasmid pVRC421.
Figure 12: Depiction of plasmid pVRC414.
Figure 13: Strategy for constructing SP212. EXAMPLE 1: Sequencing and identification of genes
involved in the biosynthesis of pristinamycin I and its precursors.
AP/P' 95/00752
Identification, by means of sequencing, of the genes situated downstream and upstream of the gene which encodes the enzyme PapA and which is described in
W094/08014, as well as of a gene which is situated downstream of the gene which encodes the enzyme SnbA and which is also described in W094/08014.
This example describes how, using cosmid pIBV2, which is described in W094/08014 and which contains the structural genes for the enzymes PapA and PapM, which are involved in the synthesis of the 4-dimethylamino-Lphenylalanine (DMPAPA) precursor of pristinamycin I, and the structural gene for the enzyme SnbA, which is responsible for activating the aromatic precursor,
3-hydroxypicolinic acid (3-HPA), of pristinamycin I, it proved possible to identify, by sequencing around these genes and studying the corresponding mutants, other genes which are involved in the biosynthesis of the DMPAPA precursor or in the biosynthesis of other precursors of pristinamycin I.
With this aim in mind, subclonings were carried out using cosmid pIBV2 and plasmid pVRC900, which is derived from pIBV2 by means of a Hindlll deletion and which is also described in W094/08014.
This example illustrates how the nucleotide sequences of fragments situated downstream and upstream of the papA and snbA genes of S. pristinaespiralis can be obtained.
The techniques for cloning DNA fragments of interest in the M13mpl8 and 19 vectors (Messing et al.
1981) are standard techniques for cloning in Escherichia coli and are described in Maniatis et al. (1989).
1-1 Sequencing and analysis of the region downstream of the papA gene
In order to sequence this region, which is contained between the papA and papM genes, the Pstl-Pstl fragment of 1.5 kb, the Pstl-Xhol fragment of 0.7 kb, and the Xhol-Xhol fragment of 0.7 kb were subcloned into the
M13mpl8 and M13mpl9 vectors proceeding from plasmid pVRC900. The cloning sites were sequenced through by
AP/P/ 9 5 / 0 0 7 5 2
AP.00562
- 21 sequencing on double-stranded DNA using plasmids pVRC900 and pVRC409, which are described in W094/08014.
The clonings were carried out as follows. Approximately 2 /xg of plasmid pVRC900 were cut with restriction enzymes Pstl and/or Xhol (New England Biolands) under the conditions recommended by the supplier. The restriction fragments thus obtained were separated on a 0.8% agarose gel, and the 1.5 kb Pstl-Pstl. 0.7 kb PstlXhol and 0.7 kb Xhol-Xhol fragments of interest were isolated and purified using Geneclean (BiolOl, La Jolla, California). For each cloning, approximately 10 ng of ( M13mpl9 and/or M13mpl8, cut with Pstl and/or Xhol. were ligated to 100 ng of the fragment to be cloned under the conditions described by Maniatis et al. 1989. After transforming the strain TGI (K12, Δ(lac-pro) supE thi hsd AS F' traD36 proA‘B+ laclq lacZ Δ M15; Gibson, 1984) and selecting lysis plaques on an LB + X-gal + IPTG medium in accordance with the technique described by Maniatis et al. (1989), the phage carrying the desired fragments were isolated. The different inserts were sequenced by the chain termination reaction using, as the synthesis primer, the universal primer or synthetic oligonucleotides which were _·' complementary to a 20 nucleotide sequence of the insert to be sequenced. The reactions were carried out using w 25 fluorescent dideoxynucleotides (PRISM Ready Reaction
DyeDeoxy Terminator Cycle Sequencing Kit-Applied Biosystem) and analysed on a model 373 A Applied Biosystems DNA sequencer. The overlap between these different inserts was such that it was possible to establish the entire nucleotide sequence between the papA and papM genes (SEQ ID No. 1).
With the aid of this nucleotide sequence, it is possible to determine the open reading frames and thereby identify genes which are involved, in S. pristinaespiralis.
in the biosynthesis of PI or its precursors, as well as the polypeptides encoded by these genes.
AP/P/ 9 5 / 0 0 7 5 2 c
c
O 25
We looked for the presence of open reading frames within the 2.9 kb Pstl-Xhol fragment, which contains the nucleotide sequence between the papA and papM genes, making use of the fact that Streptomyces DNA displays a high percentage of G and C bases as well as a strong bias in the use of codons which make up the coding frames (Bibb et al. 1984). The method of Staden and McLachlan (1982) makes it possible to calculate the probability of coding frames in terms of the codon usage of Streptomyces genes which have already been sequenced and which are assembled in a data file which contains 19673 codons and which was obtained using the BISANCE (Dessen et al. 1990) computer server.
Using this method, it was possible to characterize four highly probable open reading frames within the 2.9 kb Pstl-Xhol fragment, which reading frames are depicted in the table below (TABLE I). They are designated frames 1 to 4 according to their position starting from the Pstl site. The length of each reading frame in bases, has been indicated, as has its position within the fragment (the Pstl site being situated at position 1); the number of amino acids in the encoded polypeptide has also been indicated for open reading frames 2 and 3. Frames 1, 3 and 4 are encoded by the same strand, while frame 2 is encoded by the complementary strand (Figure 4). Frames 1 and 4 correspond, respectively, to the C-terminal region of the PapA protein and to the N-terminal region of the PapM protein, which proteins were previously identified and described in application W094/08014.
AP/P/ 9 5 / 0 0 7 5 2
AP . Ο Ο 5 6 2
- 23 I
Frame number and/or gene name Position Number of nucleotides Number of amino acids
1 (PapA) 1-684 684 -
2 (PapC)(inv) 949-1836 888 296
3 (PapB) 1873-2259 387 129
4 (PapM) 2259-2887 629 -
TABLE I
Comparison of the product of frame 2 (TABLE I) with the protein sequences contained in the Genpro library shows a 27% homology with the region involved in the prephenate dehydrogenase activity of the bifunctional TyrA proteins of E. coli (Hudson and Davidson, 1984) and of Erwinia herbicola (EMBL data library, 1991). This region of
TyrA catalyses aromatization of prephenate to form
4-hydroxyphenylpyruvate in the biosynthesis of tyrosine. A similar aromatization, proceeding from 4-deoxy-4aminoprephenate and leading to 4-aminophenylpyruvate is very probably involved in the synthesis of DMPAPA. This reaction will be catalysed by the product of frame 2, termed PapC (SEQ ID No. 2).
Comparison of the product of frame 3 (TABLE I) with the protein sequences contained in the Genpro library shows a 24 to 30% homology with the region involved in the chorismate mutase activity of the bifunctional TyrA and
PheA proteins of E. coli (Hudson and Davidson, 1984) and of the TyrA protein of Erwinia herbicola. This region catalyses isomerization of chorismate to form prephenate in the biosynthesis of tyrosine and phenylalanine. A similar isomerization, proceeding from 4-deoxy-4-amino chorismate and leading to 4-deoxy-4-aminoprephenate, is very probably involved in the synthesis of DMPAPA. This reaction would be
AP/P/ 9 5 / 0 0 7 5 2
catalysed by the product of frame 3, termed PapB (SEQ ID
No. 3).
In the case of TyrA and PheA, the chorismate mutase and prephenate dehydratase, or prephenate dehydrogenase, activities are catalysed by the same protein. In S. pristinaespiralis. the chorismate mutase and prephenate dehydrogenase enzyme activities are catalysed by two separate proteins, i.e. PapB and PapC, respectively.
The homologies demonstrated for the PapB and PapC 10 proteins, between the papA and papM genes, demonstrate that these two proteins are involved, jointly with the PapA and PapM proteins, in the biosynthesis of the aromatic derivative DMPAPA. In the same way as for papA, disruption of the papB and papC genes should lead to the construction of S. pristinaespiralis strains which are incapable of producing PI but which are able, in the presence of novel precursors, to produce new PI which are modified at the level of the DMPAPA residue.
1-2. Sequencing and analysis of the region upstream of the papA gene
This region is contained between the snbA gene, which encodes 3-hydroxypicolinic acid AMP ligase and which is described in patent application W094/08014, and the papA gene .
The clonings were carried out as described in
Example 1-1, proceeding from plasmid pVRC900 and cosmid pIBV2, which are described in Patent Application W094/08014. The 1.3 kb Xhol-Xhol. 0.2 kb Xhol-Xhol. 3.3 kb Xhol-Xhol. 1.1 kb Hindlll-Pstl and 2.2 kb Pstl-Pstl fragments were subcloned into the M13mpl8 and M13mpl9 vectors. These different clonings made it possible to pass through all the cloning sites. The different inserts were sequenced as described in 1-1 using, as synthesis primer, the universal primer or synthetic oligonucleotides which were complementary to a 20 nucleotide sequence in the insert to be sequenced.
AP/P/ 9 5 / 0 0 7 5 2 ί 5 ι
I •1
I
I 10 f
c
O 25
AP . 0 0 5 6 2
- 25 The overlap between these different inserts enabled the entire nucleotide sequence which is present between the snbA and papA genes (SEQ ID No. 4) to be established.
On the basis of this nucleotide sequence, it is possible to determine the open reading frames and to identify genes which are involved, in S. pristinaespiralis. in the biosynthesis of precursors of PI, as well as the polypeptides encoded by these genes.
We have looked for the presence of open reading frames within the 4.5 kb Xhol-Pstl fragment, which contains the nucleotide sequence between the snbA and papA genes, as described in Example 1.1. Using this method, it was possible to characterize four highly probable open reading frames within the 4.5 kb Xhol-Pstl fragment, which frames L are depicted in the table below (TABLE II). They are f designated frames 1 to 4 in accordance with their position c starting from the Xhol site. Their length in bases, and their position within the fragment (the Xhol site being situated at position I) has been indicated for each fragment; the number of amino acids within the encoded polypeptide has also been indicated for open reading frames 2 and 3. Frames 2, 3 and 4 are encoded by the same strand, and frame 1 is encoded by the complementary strand (Figure 5). Frames 1 and 4 correspond, respectively, to the N-terminal regions of the SnbA and PapA proteins, which were previously identified and described in patent application W094/08014.
AP'D/ A C ( _ c
C 20
Frame number and/or gene name Position Number of nucleotides Number of amino acids
1 (SnbA)(inv) 1-329 329 -
2 (PipA) 607-1671 1065 355
3 (SnbF) 1800-2993 1194 398
4 (PapA) 3018-4496 1479 -
TABLE II
Comparison of the product of frame 2 (TABLE II) with the protein sequences contained in the Genpro library shows a 30% homology with ornithine cyclodeaminase of Agrobacterium tumefaciens (Schindler et al.. 1989) . This enzyme is involved in the final step in the catabolism of octopine; it converts L-ornithine into L-proline by means of cyclodeamination. Authors have demonstrated, by means of the incorporation of labelled lysine, that 4-oxopipecolic acid and 3-hydroxypicolinic acid, which are found both in PIA and in virginiamycin SI, derived from lysine (Molinero et al. . 1989; Reed et al., 1989). A reaction in which lysine was cyclodeaminated, similar to that described for ornithine, would lead to the formation of pipecolic acid. Taking this hypothesis into account, the product of frame 2 was termed PipA (SEQ ID No. 5). The results of mutating the pipA gene, presented in 2-1, demonstrate that the pipA gene is involved solely in the synthesis of pipecolic acid, since this mutation has no effect on the biosynthesis of 3-hydroxypicolinic acid, which is also derived from lysine and of which pipecolic acid could have been a precursor.
Comparison of the product of frame 3 (TABLE II) with the protein sequences contained in the Genpro library shows a 30 to 40% homology with several hydroxylases of the cytochrome P450 type, which hydroxylases are involved in
AP/P/ 9 5 / 0 0 7 5 2
1.0 r
c
O 25
AP .0 0 5 6 2
- 27 the biosynthesis of secondary metabolites (Omer et al., 1990, Trower et al., 1992) . Several hydroxylations can be envisaged in the biosynthesis of precursors of pristinamycin I, in particular in the biosynthesis of 3-HPA (hydroxylation of picolinic acid at the 3 position) and of 4-oxopipecolic acid (hydroxylation of pipecolic acid at the 4 position). The results of mutating the pipA gene, presented in 2-1-3, demonstrate that the product of frame 3 is involved in hydroxylation of the pipecolic acid residue of PIE. The corresponding gene has therefore been termed snbF, and the corresponding protein SnbF (SEQ ID No. 6).
1-3. Sequencing the region downstream of the snbA gene .
This region is included between the snbA gene, which encodes 3-hydroxypicolinic acid adenylate ligase, and the snbR gene, which encodes a membrane protein which is probably responsible for transport and for resistance to PI, with both genes having been described in Patent Application W094/08014. Sequencing of this region was carried out using a fragment which was isolated from cosmid pIBV2, as described in Example 1-1.
The 1.6 kb HindiII-BglII fragment was subcloned into the Ml3mpl8 and M13mpl9 vectors, proceeding from cosmid pIBV2. The insert was sequenced as described in 1-1, using, as synthesis primer, the universal primer or synthetic oligonucleotides which were complementary to a 20 nucleotide sequence of the insert to be sequenced. On the basis of the nucleotide sequence thus obtained (SEQ ID No.
7), it is possible to determine the open reading frames and to identify, in S. pristinaespiralis. genes which are involved in the biosynthesis of the precursors of PI, as well as the polypeptides encoded by these genes. We looked for the presence of open reading frames within the 1.6 kb Hindlll-Bglll fragment, which corresponds to the end of the snbA gene and its downstream region, as described in Example 1-1. A complete open coding frame, encoded by the
AP/P/ 9 5 / 0 0 7 5 2
same strand as the snbA gene (Figure 6), was detected. Relative to position 1, corresponding to the Hindlll site, this frame starts at nucleotide 249, i.e. 30 nucleotides after the end of the snbA gene, and terminates at nucleotide 1481. It is 1233 nucleotides in size, corresponding to a protein of 411 amino acids.
Comparison of the product of this open frame with the protein sequences contained in the Genpro library shows a 30 to 40% homology with a group of proteins which are probably involved (Thorson et al. 1993) in the transamination of intermediates in the biosynthesis of various antibiotics (DnrJ, EryCl, TylB, StrS and PrgL). Synthesis of the 3-HPA precursor, which appears to derive from lysine by a route other than cyclodeamination (see
Examples 1-2 and 2-1), could necessitate a transamination step which can be catalysed by the product of this frame 3, termed HpaA (SEQ ID No. 8). The results of mutating this gene, presented in 2-2, demonstrate unequivocally that this gene is involved in synthesis of the 3-HPA precursor and confirm our hypothesis.
The genes papB, papC. pipA. snbF and hpaA, which are described in the present invention, are grouped together with the snbA. papA and papM genes on a chromosomal region of approximately 10 kb (Figure 7). This confirms the presence of a cluster of genes which are involved in the biosynthesis of PI and its precursors. Studying regions upstream and downstream of this cluster should enable the other genes involved in the biosynthesis of PI precursors, in particular L-phenylglycine and
L-2-aminobutyric acid, to be identified.
AP/P/ 9 5 / 0 0 7 5 2
EXAMPLE 2: Construction of recombinant strains by means of disrupting identified genes.
This example illustrates how it is possible to demonstrate involvement of the genes described in Example 1 in the biosynthesis of pristinamycin precursors, and also
AP.0 0 5 6 2 ί
(
- 29 to construct S. pristinaespiralis strains which are able to produce novel pristinamycins. These strains are obtained by disrupting the genes which are involved in the biosynthesis of the residue which it is desired to replace, and the novel pristinamycins are produced by supplementing these mutants with novel precursors.
Strain SP92::pVRCC508, which is employed in the present invention to produce novel derivatives of PI by replacing the precursor DMPAPA with other molecules, is described in W094/08014. It is obtained by disrupting, by means of simple crossing over, the papA gene, which is involved in the biosynthesis of the precursor of DMPAPA and is thought to participate in an early step relating to the transamination of chorismate. This disruption has a polar character since, in this mutant, expression of the papM gene (W094/08014), which is situated 1.5 kb downstream of the papA gene and is involved in the double methylation of 4-amino-L-phenylalanine to form DMPAPA, is very reduced. Thus, assaying the activity of the SAM-dependant methylation enzyme for converting 4-amino-L-phenylalanine (PAPA) into DMPAPA indicates that mutant SP92::pVRC508 has an activity which is less than 5% of the activity of the wild-type strain.
In the present invention, this strain,
SP92::pVRC508, can be used, under appropriate fermentation conditions and supplementation conditions, to produce novel pristinamycins which are modified at the level of the DMPAPA residue, as will be explained in Example 3. Mutants having the same phenotype can be obtained by disrupting the paoB or papC genes described in the present invention.
Another type of S. pristinaespiralis strain, whose papA gene is disrupted and which possesses the same phenotype as strain SP92::pVRC508, was obtained in a similar manner by disrupting the papA gene by means of double crossing over. This construction was carried out starting with a 4.6 kb Sphl-Hindlll fragment, which
AP/P/ 95/00752
- 30 fragment was isolated from cosmid pIBV2 and contains the 3' region of the pipA gene, the entire snbF and papA genes and the 3' part of the papC gene. This fragment was cloned into the suicide vector pDH5, which vector is only able to 5 replicate in E. coli but carries a resistance marker which is expressed in Streptomyces (the gene for resistance to thiostrepton or to nohiheptide, tsr). This vector, pDH5, was developed by Wohlebben et al (1991 Nucleic Acid Res.
19, 727-731). A Bcll-Bcll deletion of 1.1 kb was then made 10 in the papA gene, and a 2.2 kb HindiII-HindiII fragment, carrying the amR gene (resistance to geneticin and to ft apramycin), was introduced after the cohesive ends had been filled in. The recombinant vector was termed pVRC414 and is depicted in Figure 12. After transforming the pristinamycin-producing strain with plasmid pVRC414, transformants which were resistant to geneticin and sensitive to thiostrepton were isolated and analysed. These clones are the result of a double homologous recombination between the S. pristinaespiralis DNA regions of plasmid pVRC414 and the corresponding chromosomal region of
S. pristinaespiralis. as described in Figure 13. One of these clones was termed SP212. Its phenotype is identical to that of strain SP92::pVRC508 as regards the absence of any production of PI and the ability of the strain to produce new antibiotics in the presence of novel precursors. Advantageously, this type of strain, which is obtained by double crossing over, is more stable than the strains which are obtained by simple crossing over.
2-1. Construction of a mutant of
S, pristinaespiralis SP92 whose pipA gene is disrupted.
This example illustrates how it is possible, by means of disrupting the pipA gene, to construct a strain of S. pristinaespiralis SP92 which no longer produces PI under standard fermentation conditions and which is able to produce new pristinamycins, which are modified at the level of the 4-oxopipecolic acid residue of PIA, when novel
AP/P/ 9 5 / 0 0 7 5 2 precursors are added to the fermentation.
It was constructed using a suicide vector, the vector pUC1318, which only replicates in E. coli. This vector does not carry any resistance marker which is expressed in Streptomyces. Its presence in the genome of Streptomyces can only be detected by colony hybridization.
2-1-1. Construction of plasmid pVRC420:
This example illustrates how it is possible to construct a plasmid which does not replicate in
S. pristinaespiralis SP92 and which can be employed to disrupt the pipA gene by means of double homologous recombination.
Plasmid pVRC420 was constructed in order to produce the chromosomal mutant of SP92 in which the pipA gene is disrupted, proceeding from cosmid pIBV2, which is described in W094/08014. Cosmid pIBV2 was cut with the restriction enzyme PstI and, after the fragments, thus generated, had been separated by electrophoresis on a 0.8% agarose gel, a 2.8 kb Pstl-Pstl fragment, containing the start of the snbA and snbF genes and the whole of the pipA gene, was isolated and purified using Geneclean (BiolOl, La Jolla, California). 50 ng of vector pUC1318, which had been linearized by digesting with Pstl. were ligated to 200 ng of the 2.8 kb fragment, as described in Example 1. A clone carrying the desired fragment was isolated following transformation of the strain TGI and selection on LB +
150 μg/ml ampicillin + X-gal + IPTG medium. The recombinant plasmid was termed pVRC415 (Figure 8). A cassette containing the am* gene, encoding resistance to apramycin or to geneticin (Kuhstoss et al., 1991), was then introduced into the unique Hindlll site of plasmid pVRC415, this site being situated 530 bp downstream of the start of the pipA gene. This construction was effected as follows. A
2.5 kb DNA fragment, containing the am* gene, the PermE promoter (Bibb et al.. 1985) and the first 158 amino acids of the gene for resistance to erythromycin , ermE. was
2^/00/56 /d/dV
AP.00562
- 32 isolated by means of a Sall-Bqlll double digestion of a plasmid which was derived from plasmids pIJ4026 (plasmid carrying the ermE gene under the control of the PermE promoter) and pHP45Dam*. After filling in the Sail and
Belli protruding 5' cohesive ends using Klenow enzyme in accordance with the protocol described by Maniatis et al., 1989, the fragment containing the am* gene was cloned into the Hindlll site of plasmid pVRC415, whose protruding 5' cohesive ends had also been filled in with Klenow enzyme as previously described. The recombinant plasmid thus obtained was designated pVRC420. Its restriction map is depicted in ( Figure 9.
2-1-2. Isolation of mutant SP92pjpA::Dam*. whose pjpA gene is disrupted by homologous recombination.
This example illustrates how the mutant of
S. pristinaespiralis SP92 whose pipA gene is disrupted was constructed.
This mutant was isolated by transforming strain SP92 with the suicide plasmid pVRC420.
The preparation of protoplasts, their transformation and extraction of the total DNA from the recombinant strains were all effected as described by
O Hopwood et al. (1985) .
The strain SP92 was cultured, at 30°C for
40 hours, in YEME medium (Hopwood et al.. 1985), 34% sucrose, 5 mM MgCl2 and 0.25% glycine. The mycelium was protoplasted in the presence of lysozyme, and 5 χ 1 ^g of pVRC420 were used to transform (by the method employing 'PEG) the protoplasts. After one night in which the protoplasts were regenerated on R2YE medium (D. Hopwood et al. 1985), the recombinants were selected by spreading on 3 ml of SNA medium (D. Hopwood et al. 1985) containing 1,500 /zg/ml geneticin.
100 clones which were resistant to geneticin were isolated from the 5 transformations that were carried out. These recombinants arise from integration, by means of
AP'P/ 9 5 / 0 0 7 5 2 simple or double homologous recombination between the pipA gene which is carried by the chromosome of strain SP92 and the parts of the pipA gene which are contained in the 5.3 kb fragment carried by the suicide plasmid pVRC420. In 5 order to select the recombinants which were obtained by double crossing over (that is which did not contain the pUC1318 part of plasmid pVRC420 in their genome), colony hybridizations were carried out on 90 clones using pUC19 labelled with [a-32P]dCTP as the probe, as described in 10 Maniatis et al (1989). 10 clones were selected which were resistant to geneticin but which did not hybridize the vector pUC19. The spores of the recombinants were isolated by streaking and growing on HT7 medium containing 10 μg/ml geneticin, and restreaked on the same medium in order to 15 obtain isolated colonies. In order to verify the position at which plasmid pVRC420 was integrated, various Southerns of the total DNA from several recombinant clones, purified as described by Hopwood et al. 1985, were carried out, with hybridization to the 2.8 kb Pstl-Pstl fragment, which was 20 used as a probe after having been labelled with [a32P]dCTP. The results confirm that these recombinants were obtained by double crossing over between vector pVRC420 and chromosome of strain SP92, resulting in replacement of 2.8 kb Pstl-Pstl fragment, containing the pipA gene, by 3 kb Pstl-Pstl fragment containing the pipA gene which is disrupted by introduction of the am” gene. One of these mutants was designated SP92pjpA::flam”.
2-1-3. Production of pristinamycins using mutant -SP92pjpA:: Dam”.
This example illustrates how it is established that the mutant of S. pristinaespiralis SP92 whose pipA gene is disrupted by integration of plasmid pVR420 on the one hand no longer produces PI under standard fermentation conditions and on the other hand exhibits a high level of production of a minor form of the B components of streptogramins in which 4-oxopipecolic acid is replaced by the the a 5.
AP/P' 95/00752
AP.00562
- 34 10 c
o
O 25 pipecolic acid.
Mutant SP92pipA::PamR. as well as strain SP92 in the role of a control strain, were cultured in liquid production medium. The fermentation was carried out as follows: 0.5 ml of a suspension of spores from the abovementioned strain are added, under sterile conditions, to 40 ml of inoculum medium in a 300 ml baffled Erlenmeyer flask. The inoculum medium is made up of 10 g/1 com steep, 15 g/1 sucrose, 10 g/1 (NH4)2SO4, 1 g/1 K2HPO4, 3 g/1 NaCl, 0.2 g/1 MgSO4-7H2O and 1.25 g/1 CaCO3. The pH is adjusted to 6.9 using sodium hydroxide solution before introducing the calcium carbonate. The Erlenmeyer flasks are shaken at 27°C for 44 h on a rotating shaker at a speed of 325 rpm. 2.5 ml of the previous culture, which is 44 hold, are added under sterile conditions to 30 ml of production medium in a 300 ml Erlenmeyer flask. The production medium is made up of 25 g/1 soya flour, 7.5 g/1 starch, 22.5 g/1 glucose,
3.5 g/1 fodder yeast, 0.5 g/1 zinc sulphate and 6 g/1 calcium carbonate. The pH is adjusted to 6.0 with hydrochloric acid before introducing the calcium carbonate. The Erlenmeyer flasks are shaken for 24, 28 and 32 hours at 27°C. At each time point, 10 g of must are weighed into a smooth Erlenmeyer flask to which 20 ml of mobile phase, consisting of 34% of acetonitrile and 66% of a solution of 0.1 M KH2PO4 (adjusted to pH 2.9 with concentrated H3PO4) are added for extracting the pristinamycins. After shaking, the whole is centrifuged and the pristinamycins contained in the supernatant are assayed by HPLC by means of •injecting 150 μΐ of the centrifugation supernatant onto a Nucleosil 5-C8 column of 4.6 x 150 mm, which is eluted with a mixture of 40% acetonitrile and 60% 0.1 M phosphate buffer, pH 2.9. The I pristinamycins are detected by means of their UV absorbance at 206 nm.
The results demonstrated that, under the fermentation conditions employed, mutant SP92pjpA::flamR did not produce PI at 24, 28 or 32 hrs of fermentation, while
AP/P/ 9 5 / 0 0 7 5 2 control strain SP92 produced a quantity of PI which was standard for the 3 times which were tested. The quantity of
PII which was produced remained the same for the two strains. Mutant SP92pipA::Dam* is definitely blocked at a step in the biosynthesis of PI. Fermentation complementation tests were carried out by adding different precursors of PI, separately or together, to the culture in production medium after 16 hours. The results of these complementations demonstrated that when 100 mg/1 pipecolic acid and 100 mg/1 DMPAPA are added simultaneously to the fermentation medium, the mutant produces what is normally a minor derivative of PI, i.e. PIE (which is produced by SP92 in a quantity which is less than 5%) at a level which is equivalent to the production of PIA by the control strain.
This production does not take place if the pipecolic acid and the DMPAPA are added separately. PIE differs from PIA (major component of PI) in the absence of the keto function in the 4 position on the pipecolic acid. The fact that mutant SP92pjpA:: Dam” can only be complemented by adding pipecolic acid and DMPAPA simultaneously indicates that the papA. and probably the papE and papM genes were disrupted by a polar effect of the construct. Thus, all these genes θ are situated downstream of pipA and are probably cotranscripts together with pjpA. Disruption of the latter o 25 therefore leads to disruption of the pap genes and, consequently, absence of DMPAPA synthesis. The fact that complementation of mutant SP92pipA::DamR with pipecolic acid results in the production of PIE and not PIA leads to two conclusions: the first is that construction of the PI cycle is achieved by incorporating pipecolic acid and not 4-oxopipecolic acid and that a hydroxylation generating the keto function in the 4 position then takes place subsequently. The second is that this hydroxylation is probably carried out by the enzyme SnbF whose structural gene is situated directly downstream of the pipA gene.
Thus, the obvious polarity of the disruption of the pipA
AP/P/ 9 5 / 0 0 7 5 2
AP . Ο Ο 5 6 2
- 36 gene on the pap genes probably involves a polar effect on the snbF gene, which is situated between pjpA and the pap genes, which is manifested in inhibition of the function of hydroxylation of the pipecolic acid residue of PIE to form 4-hydroxypipecolic acid, which is found in PIF and PIG (Figure 2) and then oxidized to 4-oxopipecolic acid in PIA.
Preparing a mutant of this nature made it possible to construct a strain of S. pristinaespiralis which is unable to produce PI except in the presence of the
PI precursors DMPAPA and pipecolic acid, using which it is able to produce, in a quantity equivalent to that of the ( starting strain, what is normally a minor derivative of PI within the pristinamycin mixture. Similarly, in the presence of novel precursors, or of a mixture of novel precursors and of precursors which are normally present in PI, this strain will be able to produce new pristinamycins which are modified in either DMPAPA or 4-oxopipecolic acid or in both these residues.
2-2. Construction of a mutant of
S. pristinaespiralis SP92 whose hpaA gene is disrupted.
This example illustrates how it is possible, by means of disrupting the hpaA gene, to construct a strain of D S. pristinaespiralis SP92 which no longer produces PI under
O standard fermentation conditions and which is able to produce new pristinamycins, which are modified at the level of the 3-HPA precursor, when novel precursors are added to the fermentation.
This mutant was constructed using a plasmid which does not replicate in S. pristinaespiralis SP92 and which can be used for disrupting the hpaA gene by means of double homologous recombination.
2-2-1. Construction of the suicide plasmid pVRC421
Plasmid pVRC421 was constructed using a suicide vector which, while only being able to replicate in E coli. carries a resistance marker which is expressed in
AP'P' 9 5 / 0 n 7 * 2 c
Ο
Ο 25
Streptomyces, i.e. the gene for resistance to thiostrepton or to nosiheptide, tsr. This vector, pDH5, was developed by Hillemann et al. (1991) .
Plasmid pVRC421 was constructed in order to produce the chromosomal mutant of SP92 whose hpaA gene is disrupted, making use of cosmid pIBV2, which is described in W094/08014. pIBV2 was digested with the restriction enzyme SphI and, after having separated the fragments, thus generated, by means of electrophoresis on a 0.6% agarose gel, a 4.8 kb SphI-SphI fragment, containing the whole of the hpaA gene and virtually the whole of the snbA gene, was isolated and purified using Geneclean as described above.
ng of the vector pDH5, linearized by digesting with SphI, were ligated to 200 ng of the 4.8 kb fragment, as subsequently described. A clone harbouring the desired fragment was isolated after transforming the strain TGI and selecting on LB + 150 μg/ml ampicillin + IPTG + X-gal medium. The recombinant plasmid was designated pVRC411 (Figure 10). A cassette containing the gene am”, encoding resistance to apramycin or to geneticin, was then introduced into the unique Pflml site of plasmid pVRC411, this site being situated 610 bp downstream of the start of the hpaA gene. This construct was produced as follows. A
2.2 kb DNA fragment, containing the am” gene, was isolated following digestion of the plasmid pHP45Qam”. containing the am” gene, with Hindlll. After filling in the HindiII protruding 5' cohesive ends using Klenow enzyme according to the protocol described by Maniatis et al. 1989, the .fragment containing the am” gene was cloned into the Pflml site of plasmid pVRC411, whose protruding 3' cohesive ends had been rendered blunt using the enzyme T4 polymerase as described in Maniatis et al. 1989. The recombinant plasmid thus obtained was termed pVRC421. Its restriction map is depicted in Figure 11.
2-2-2. Isolation of mutant SP92hpaA::Qam”. whose hpaA gene is disrupted by means of homologous
CXl a L 0 0 / S 6 /d/dV
AP . Ο Ο 5 6 2
- 38 recombination.
This example illustrates how the mutant of S. pristinaespiralis SP92 whose hpaA gene is disrupted was constructed.
This mutant was isolated by transforming strain
SP92 with the suicide plasmid pVRC421.
The protoplasts were prepared and transformed as described previously.
Strain SP92 was cultured, at 30°C for 40 hours, in YEME medium, 34% sucrose, 5 mM MgCl2, 0.25% glycine. The mycelium was protoplasted in the presence of lysozyme, and ( 5 χ 1 Mg of pVRC421 were employed for transforming (by the method using PEG) the protoplasts. After one night for regenerating the protoplasts on R2YE medium, the recombinants were selected by spreading on 3 ml of SNA medium containing 1,500 ^g/ml geneticin.
600 clones which were resistant to geneticin were isolated from the 5 transformations which were carried out. These recombinants result from integration by means of simple or double homologous recombination between the hpaA gene carried by the chromosome of strain SP92 and the 6 kb fragment of the suicide plasmid pVRC421. In order to select the recombinants obtained by double crossing over (that is, _ the clones which no longer contain, in their genome, the pDH5 moiety of plasmid pVRC421), the clones were subcultured on HT7 medium containing 400 ^g/ml thiostrepton. 6 clones which were resistant to geneticin but sensitive to thiostrepton were selected. The spores of ‘ the recombinants were selected by streaking and growth on
HT7 medium containing 10 Mg/ml geneticin, and restreaked on the same medium in order to obtain isolated colonies. In order to verify the position of integration of plasmid pVRC421, various Southerns of the total DNA from the 6 recombinant clones, purified as described by Hopwood et al.
1985, were carried out with hybridization to the 4.8 kb
SphI-SphI fragment, which was used as the probe after
AP/P/ 9 5 / 0 0 7 5 2 having been labelled with [a-32P]dCTP. The results confirm that these recombinants were obtained by double crossing over between the vector pVRC421 and the chromosome of the SP92 strain, resulting in replacement of the 4.8 kb
Sphl-SphI fragment, containing the hpaA gene, by a 6 kb Sphl-SphI fragment which contains the hpaA gene disrupted by the am gene. One of these mutants was designated SP92hpaA::QamR.
2-2-3. Production of pristinamycins by mutant 10 SP92hpaA::CamR.
This example illustrates how it is established C that the mutant of S. pristinaespiralis SP92 whose hpaA gene is disrupted by integration of plasmid pVR421 no longer produces PI under the standard fermentation conditions.
Mutant SP92hpaA::Dam”. and also strain SP92 in the role of control strain, were cultured in liquid production medium. The fermentation was carried out as described in Example 2-1-3, and the pristinamycins were then extracted and assayed as previously described. The results demonstrated that, under the fermentation conditions employed, mutant SP92hpaA::QamR did not produce ' PI, either at 24, 28 or 32 hrs of fermentation, whereas the control strain produced a quantity of PI which was standard for the 3 time points tested. The quantity of PII produced remained the same for the two strains. Mutant SP92hpaA: :0amR is definitely blocked at a step in the biosynthesis of PI. Complementary fermentation tests were carried out by adding different precursors of PI, separately or together, to the culture in production medium after 16 hours. When 100 mg/1 3-hydroxypicolinic acid are added to the fermentation medium, the mutant then produces PIA at a level which is equivalent to the production of PI by the control strain. The fact that mutant SP92hpaA::CamR can only be complemented by adding 3-hydroxypicolinic acid demonstrates that the hpaA gene is involved in the
AP/P/ 9 5 / 0 0 7 5 2
AP.00562
- 40 synthesis of this precursor.
Construction of this mutant made it possible to produce a strain of S. pristinaespiralis which is mutated as regards its production of PI but which, in the presence of the precursor 3-HPA, is capable of producing PI in a quantity equivalent to that produced by the starting strain. In the same way as in the preceding examples, it can be envisaged that it should be possible, using a mutant of this nature in the presence of novel precursors, to produce new pristinamycins which are modified at the level of the 3-hydroxypicolinic acid residue.
r
EXAMPLE 3: Production of compounds of the general by the mutant SP92::pVRC508.
This example illustrates how the mutant of 15 S, pristinaespiralis SP92 whose papA gene is disrupted by integration of plasmid pVRC508 is able to synthesize new streptogramins in the presence of precursors which are added to the production medium. These precursors can be derivatives of amino acids and, more particularly, of phenylalanine, but also of α-ketocarboxylic acids and, more particularly, of phenylpyruvic acid.
C The mutant SP92::pVRC508 was cultured in liquid —m. production medium. The fermentation was carried out as follows: 0.5 ml of a suspension of spores from the 25 previously mentioned strain is added, under sterile conditions, to 40 ml of inoculum medium in a 300 ml baffled Erlenmeyer flask. The inoculum medium is made up of 10 g/1 corn steep, 15 g/1 sucrose, 10 g/1 (NH4)2SO4, 1 g/1 K2HPO4, g/1 NaCl, 0.2 g/1 MgSO4-7H2O and 1.25 g/1 CaCO3. The pH is adjusted to 6.9 with sodium hydroxide solution before introducing the calcium carbonate. The Erlenmeyer flasks are shaken at 27°C for 44 h on a rotating shaker at a speed of 325 rpm. 2.5 ml of the previous culture, which is 44 h old, are added, under sterile conditions, to 30 ml of production medium in a 300 ml Erlenmeyer flask. The
AP/P/ 95/00752
production medium consists of 25 g/1 soya flour, 7.5 g/1 starch, 22.5 g/1 glucose, 3.5 g/1 fodder yeast, 0.5 g/1 zinc sulphate and 6 g/1 calcium carbonate. The pH is adjusted to 6.0 with hydrochloric acid before introducing the calcium carbonate. The Erlenmeyer flasks are shaken at 27°C on a rotating shaker at a speed of 325 rpm. After 16 h, 1 ml of a solution of one of the precursors listed in Table 3 (generally 5 or 10 g/1) is added to the culture.
The latter is terminated 8 or 24 h later. The volume of the must is measured immediately, and 2 volumes of mobile phase, consisting of 34% acetonitrile and 66% of a solution of 0.1 M KH2PO4 (adjusted to pH 2.9 with concentrated H3PO4) are added to it for extracting the pristinamycins. After shaking, the whole is centrifuged and the pristinamycins contained in the supernatant are extracted and purified as described in Example 4. They are also assayed by HPLC by means of injecting 150 μΐ of the centrifugation supernatant onto a Nucleosil 5-C8 4.6 x 150 mm column, which is eluted with a mixture of 40% acetonitrile and 60% 0.1 M phosphate buffer, pH 2.9. The new I pristinamycins are detected by means of their UV absorbance at 206 nm and, where appropriate, by means of their fluorescence emission (370 nm filter, excitation at 306 nm).
AP/P/ 9 5 / 0 0 7 5 2
PRECURSOR ORIGIN
phenylalanine Janssen
4-dimethylaminophenylalanine Example 33
4 - me t hy1aminopheny1a1anine Example 34-1
4-aminophenylalanine Janssen 22.794.96
4-diethylaminophenylalanine Example 33
4-ethylaminophenylalanine Example 33
4-methylthiophenylalanine Example 33
AP . 0 0 5 6 2
PRECURSOR ORIGIN
4-methylphenylalanine J.P.S101-312-4/ Example 33
4-methoxyphenylalanine Janssen 16.975.97
4-trifluoromethoxyphenylalanine Example 34-8
4-methoxycarbonylphenylalanine Example 33
4-chlorophenylalanine Janssen 15.728.14
4-bromophenylalanine Janssen 22.779.81
4 -iodophenylalanine Bachem F 1675
4-trifluoromethylphenylalanine P.C.R. Inc. 12 445-3
4 -tert-butylphenylalanine Example 35-1
4 -isopropylphenylalanine Example 36-1
3-methylaminophenylalanine Example 35-3
3-methoxyphenylalanine J.P.S. 101-313-2
3-methylthiophenylalanine Example 34-11
3 -fluoro-4-methylphenylalanine Example 34-5
4 -tert-butylphenylpyruvic acid Example 33
4-methylaminophenylpyruvic acid Example 34-4
2-napthylphenylalanine Bachem F 1865
4-fluorophenylalanine Bachem F 1535
3 - fluorophenylalanine Bachem F 2135
3-ethoxyphenylalanine Example 37-1
AP/P/ 9 5 / 0 0 7 5 2
PRECURSOR ORIGIN
2,4-dimethylphenylalanine Example 33
3,4-dimethylphenylalanine Example 33
3-methylphenylalanine Example 33
4-phenylphenylalanine Example 33
4-butylphenylalanine Example 36-3
2-thienyl-3-alanine Aldrich 28.728.8
3-trifluoromethylphenylalanine Example 33
3 -hydroxyphenylalanine Aldrich T 9.039.5
3-ethylaminophenylalanine Example 35-6
4-aminomethylphenylalanine Example 33
4-allylaminophenylalanine Example 38-2
4-diallylaminophenylalanine Example 38-1
4-allylethylaminophenylalanine Example 39-4
4-ethylpropylaminophenylalanine Example 39-6
4-ethylisopropylaminophenylalanine Example 39-1
4-ethylcyclopropylmethylaminophenylalanine Example 39-8
4-(1-pyrrolidinyl)phenylalanine Example 40-1
4-O-allyltyrosine Example 33
4-O-ethyltyrosine Example 33
4-ethylthiophenylalanine Example 33
4-ethylthiomethylphenylalanine Example 41-1
4-0-(2-chloroethyl)tyrosine Example 42-1
AP/P/ 95/00752
AP . Ο Ο 5 6 2
PRECURSOR ORIGIN
4-acetylphenylalanine Example 33
4-ethylphenylalanine Example 33
3-dimethylaminophenylalanine Example 35-10
TABLE III
The following table (TABLE IV) indicates the relative retention times of the new PI which are produced, taking PIA as the reference. The absolute retention times were determined at 25°C in the HPLC system described above; they vary slightly from one injection to another and also in accordance with temperature.
Precursor t„ (relative retention time) of the new PI (Neo PI)
Neo PIA Neo PIH Other neo PI
4-methylaminophenylalanine 0.85
4-aminophenylalanine 0.64
4-methylthiophenylalanine 1.93 2.73 1.63
4-methylphenylalanine 1.77 2.65
4-methoxyphenylalanine 1.46
4-methoxycarbonylphenylalanine 1.49
4-chlorophenylalanine 2.04
4-bromophenylalanine 2.16
4 - iodophenylalanine 2.42
4 -trifluoromethylphenylalanine 2.56 3.74
4 -tert-butylphenylalanine 3.34
4 -isopropylphenylalanine 2.80 4.35
3-methylaminophenylalanine 1.15
9/00/56 IdldXl
3-methoxyphenylalanine 1.49 2.04
3-fluoro-4methylphenylalanine 2.93
4 -tert-butylphenylpyruvic acid 3.34
4-methylaminophenylpyruvic acid 0.85
4-ethylaminophenylalanine 0.94
4-diethylaminophenylalanine 0.61
4-allylaminophenylalanine 1.83
4-diallylaminophenylalanine 2.64
4-allylethylaminophenylalanine 2.4
4-ethylpropylaminophenylalanine 1.06
4-ethylisopropylaminophenylalanine 0.89
4-ethylcyclopropylmethylaminophenylalanine 1.1
4-(1-pyrrolidinyl)phenylalanine 2.0
4-O-trifluoromethyltyrosine 2.42
4-0-allyltyrosine 2.62
4-O-ethyltyrosine 2.2
4-ethylthiophenylalanine 1.96
4-methylthiomethylphenylalanine 1.98
4-0-(2-chloroethyl)tyrosine 2.45
4-acetylphenylalanine 1.61
4-ethylphenylalanine 1.86 2.40
3-dimethylaminophenylalanine 1.49
3 -methylthiophenylalanine 1.93
3-O-ethyltyrosine 1.78
AP/P/ 95/007^2
TABLE IV
AP . Ο Ο 5 6 2
- 46 The new PI, with a tR of 4.35, for 4-isopropylphenylalanine corresponds to a neo PIE which is described in Example 14.
The new PI, with a tR of 1.63, for 5 4-methylthiophenylalanine corresponds to a 5y-hydroxy neo
PIH, which is described in Example 5.
The mutant SP92::pVRC50S was otherwise fermented in the presence of 4-dimethylaminophenylalanine. Under these conditions of complementation, mutant SP92::pVRC508 produces a quantity of IA pristinamycins which is equivalent to that produced by strain SP92.
EXAMPLE 4: Preparation of pristinamycin IB [4frne t hyl amino-de (4f-dimethylamino) pristinamycin IA] and of 4f-amino-de(4f-dimethylamino)pristinamycin IA
4.1: Preparation of pristinamycin IB [4f-methylamino-de)4fdimethylamino)pristinamycin IA]
The strain SP92::pVRC508 is cultured in production medium, using 60 Erlenmeyer flasks as described in Example 3, with 1 ml of a 10 g/1 aqueous solution of (R,S)-4-methylaminophenylalanine, synthesized as in Example
34-1, being added at 16 h. At the end of 40 h of culture, the 1.8 litres of must recovered from the 60 Erlenmeyer flasks are extracted with 2 volumes of a mixture consisting of 66% 100 mM phosphate buffer, pH 2.9, and 34% acetonitrile, and then centrifuged. The supernatant is extracted with 2 times 0.5 volumes of dichloromethane. The chloromethylene phases are washed with water and then combined, dried over sodium sulphate and evaporated. The dry extract is taken up in 20 ml of dichloromethane and injected onto a silica (30 g) column which is mounted in dichlormethane and is successively eluted with plateaus of from 0 to 10% methanol in dichloromethane. The fractions containing pristinamycin IB are combined and evaporated.
The dry residue is taken up in 6 ml of a mixture of 65% water and 35% acetonitrile and injected onto a semiAP/P/ 9 5 / 0 0 7 5 2
preparative Nucleosil 7μ C8 10x250 mm column (Macherey
Nagel), which is eluted with a mixture of 65% 100 mM phosphate buffer, pH 2.9, and 35% acetonitrile. The fractions containing pristinamycin IB are combined and extracted with one volume of dichloromethane. The organic phase is washed with water, dried on sodium sulphate and then evaporated. 52 mg of pristinamycin IB are obtained.
NMR spectrum. XH (4 00 MHz, CDC13, δ in ppm, ref. TMS) : 0.71 (dd, J=16 and 6 Hz, 1H, 5 β2) , 0.92 (t, J=7.5 Hz, 3H: CH3 2 γ) , from 1.10 to 1.40 (mt, 2H: 3 β2 and 3 γ2) , 1.34 (d, J=7.5 Hz, 3H: CH3 1 γ) , from 1.50 to 1.85 (mt, 3H: 3 γχ and CH2 2 /3), 2.03 (mt, 1H, 3 0J , 2.22 (mt, 1H, 5 δ2) , 2.33 (broad d, J=16 Hz, 1H: 5 δχ) , 2.40 (d, J=16 Hz, 1H: 5 /3X) , 2.82 (mt, 1H: 5 e2) , 2.81 (s, 3H: 4 NCH3 in the para position of the phenyl), 2.90 (dd, J=12 and
Hz, 1H: 4 β2) , 3.29 (s, 3H: 4 NCH3) from 3.20 to 3.45 and 3.60 (2 mts, 1H each: CH2 3 δ), 3.40 (t, J=12 Hz, 1H: 4 β2), 4.57 (dd, J=7 and 8 Hz, 1H, 3 a), 4.75 (broad dd, J=13 and 7 Hz, 1H: 5 ex) , 4.83 (mt, 1H: 2a), 4.89 (broad d,
J=10 Hz, 1H: la), 5.24 (dd, J=12 and 4 Hz, 1H: 4 a), 5.32 (broad d, J=6 Hz, 1H: 5 a), 5.89 (d, J=9 Hz, 1H: 6 a), 5.90 (broad q, J = 7.5 Hz, 1H: 1/3), 6.53 (d, J=9 Hz, 1H: NH 2),
6.53 (d, J=8 Hz, 2H: 4e), 7.03 (d, J=8 Hz, 2H: 4δ), from 7.10 to 7.35 (mt, 5H: aromatic H 6), 7.46 (mt, 2H: l'Hs and l'H«), 7.85 (dd, J=5.5 and 2 Hz, 1H: l'H6), 8.44 (d, J=10
Hz, 1H: NH 1), 8.76 (d, J=9 Hz, 1H: NH 6), 11.63 (s,
1H: OH).
4.2: Preparation of 4f-amino-de(4fdimethylamino)pristinamycin Ιλ
Strain SP92::pVRC508 is cultured in production medium, using 60 Erlenmeyer flasks as described in Example 3, with 1 ml of a 5 g/1 aqueous solution of (S)-4aminophenylalanine being added at 16 h. At the end of 40 h of culture, the 1.8 litres of must recovered from the 60
Erlenmeyer flasks are extracted with 2 volumes of a mixture
AP . Ο Ο 5 6 2
- 48 consisting of 66% 100 mM phosphate buffer, pH 2.9 and 34% acetonitrile, and then centrifuged. The supernatant is extracted with 2 times 0.5 volumes of dichloromethane. The chloromethylene phases are washed with water and then combined, dried over sodium sulphate and evaporated. The dry extract is taken up in 20 ml of dichloromethane and injected onto a silica (30 g) column which is mounted in dichloromethane and is eluted successively with plateaus of from 0 to 10% methanol in dichloromethane. The fractions containing the new derivative of pristinamycin IA are combined and evaporated. The dry residue is taken up in 6 ml of a mixture consisting of 65% water and 35% acetonitrile and injected onto a semi-preparative Nucleosil Ίμ C8 10x250 mm column (Macherey Nagel), which is eluted with a mixture consisting of 65% 100 mM phosphate buffer, pH 2.9, and 35% acetonitrile. The fractions containing the new pristinamycin are combined and extracted with one volume of dichloromethane. The organic phase is washed with water, dried over sodium sulphate and then evaporated. 5 mg of 4f-amino-de(4f-dimethylamino)pristinamycin IA are obtained.
NMR spectrum: XH (400 MHz, CDC13, δ in ppm, ref. TMS): 0.72 (dd, J=16 and 5..5 Hz, 1H, 5 β2) , 0.90 (t,
J=7.5 Hz, 3H: CH3 2 γ) , from 1.10 to 1.40 (mt, 2H: 3 β2 and
3 γ2) , 1.33 (d, J=7.5 Hz, 3H: CH3 1 γ) , from 1.50 to 1.85 (mt, 3H: 3 and CH2 2 β) , 2.02 (mt, 1H, 3 &) , 2.19 (mt,
1H, 5 δ2) , 2.33 (broad d, J=16 Hz, 1H: 5 δ3) , 2.42 (d,
J=16 Hz, 1H: 5 β2) , 2.81 (dt, J=13 and 4 Hz, 1H: 5 e2) ,
2.90 (dd, J=12 and 4 Hz, 1H: 4 β2) , 3.24 (s, 3H: NCH3 4), from 3.20 to 3.40 and 3.54 (2 mts, 1H each: CH2 3 δ), 3.30 (t, J=12 Hz, 1H: 4 β2) , 3.72 (unres. comp. , 2H: ArNH2) , 4.54 (dd, J=7.5 and 7 Hz, 1H, 3 a), 4.73 (broad dd, J=13 and 8 Hz, 1H: 5 e3) , 4.82 (rmt, 1H: 2a), 4.89 (broad d,
J=10 Hz, 1H: la), 5.22 (dd, J=12 and 4 Hz, 1H: 4 a), 5.32 (broad d, J=5.5 Hz, 1H: 5 a), 5.89 (mt, 2H: 6 a and 1/3),
6.51 (d, J=9.5 Hz, 1H: NH 2) 6.61 (d J=8, 2H: 4e), 6.98 (d,
AP/P/ 9 5 / 0 0 7 5 2
J=8 Hz, 2H: 4δ), from 7.15 to 7.35 (mt, 5H: aromatic H 6),
7.45 (dd, J=8.5 and 1.5 Hz, 1H: 1'HJ, 7.48 (dd, J=8.5 and
Hz, 1H: 1'HJ, 7.82 (dd, J=4 and 1.5 Hz, 1H: l'H6), 8.43 (d, J=10 Hz, 1H: NH 1), 8.76 (d, J=9.5 Hz, 1H: NH 6), 11.63 (S, 1H: OH).
Example 5: Preparation of 4f-methylthio-de(4fdimethylamino)pristinamycin Ix, of 4f-methylthio-de(4fdimethylamino)pristinamycin IB and of 5-y-hydroxy-4fmethylthio-de (4f-dimethylamino) pristinamycin IH
Strain SP92::pVRC508 is cultured in production medium using 60 Erlenmeyer flasks, as described in Example 3, with 1 ml of a 10 g/1 solution of (R, S)-4-methylthiophenylalanine, synthesized as in Example 33, in 0. IN sodium hydroxide solution being added at 16 h.
At the end of 40 h of culture, the 1.8 litres of must recovered from the 60 Erlenmeyer flasks are extracted with 2 volumes of a mixture consisting of 66% 100 mM phosphate buffer, pH 2.9, and 34% acetonitrile, and then centrifuged. The supernatant is extracted with 2 times 0.5 volumes of dichloromethane. The chloromethylene phases are washed with water and then combined, dried over sodium sulphate and evaporated. The dry extract is taken up in 20 ml of dichloromethane and injected onto a silica (30 g) column which is mounted in dichloromethane and is eluted successively with plateaus of from 0 to 10% methanol in dichloromethane. The fractions containing the new derivative of pristinamycin Ix are combined and evaporated. 65 mg of dry residue are obtained. This is taken up in 6 ml of a mixture consisting of 60% water and 40% acetonitrile and injected in two batches onto a semi-preparative
Nucleosil 7μ C8 10x250 mm column (Macherey Nagel), which is eluted with a mixture consisting of 55% 100 mM phosphate buffer, pH 2.9, and 45% acetonitrile. The fractions
AP.00562
20 containing the new pristinamycin are combined and extracted with one volume of dichloromethane. The organic phase is washed with water, dried over sodium sulphate and then evaporated. 45 mg of 4ζ-methylthio-de(4f5 dimethylamino)pristinamycin IA are obtained.
NMR spectrum: 1H (400 MHz, CDC13, δ in ppm, ref. TMS) : 0.68 (dd, J=16 and 5.5 Hz, 1H 5 β2) , 0.93 (t, J=7.5 Hz, 3H: CH3, 2 γ) , 1.13 (mt, 1H: 3 02) , from 1.25 to 1.40 (mt, 1H: 3 γ2) , 1.33 (d, J=7.5 Hz, 3H-. CH3 1 7), from 1.55 to 1.85 (mt, 3H: 3 y, and CH2 2 β) , 2.02 (mt, 1H, 3 β2) ,
2.18 (mt, 1H, 5 δ2) , 2.38 (broad d, J=16.5 Hz, IH: 5 δχ) ,
2.46 (s, 3H: SCH3), 2.48 (d, J=16 Hz, 1H, 5 /3J , 2.85 (dt, J=13.5 and 4 Hz, 1H: 5 e2) , 3.00 (dd, J=12 and 5 Hz, IH: 4 β2) , 3.23 (s, 3H: NCH3, 4), 3.37 (t, J=12 Hz, 1H: 4 ,
3.37 and 3.58 (2 mts, 1H each: CH2 3 δ), 4.55 (t, J=7.5 Hz,
1H, 3 a), 4.ΊΊ (broad dd, J=13.5 and 8 Hz, 1H: 5 ej , 4.86 (mt, 1H: 2a), 4.89 (dd, J=10 and 1.5 Hz, 1H: la), 5.30 (broad d, J=5.5 Hz, 1H: 5a), 5.32 (dd, J=12 and 5 Hz, 1H:
a), 5.90 (d, J=9.5 Hz, 1H: 6 a), 5.92 (dq, J=7.5 and 1/0), 6.55 (d, J=9.5 Hz, 1H: NH 2), 7.13 (d,
4δ), from 7.15 to 7.35 (mt, 5H: aromatic H 6),
7.19 (d, J=8 Hz, 2H: 4e), 7.45 (mt, 2H: l'H4 and H5) , 7.76 (t, J=5 Hz, l'Hs), 8.42 (d, J=10 Hz, 1H: NH 1), 8.76 (d, J=9.5 Hz, 1H: NH 6), 11.65 (s, 1H: OH).
Using the fractions derived from the silica column described above which contain the novel derivative of pristinamycin IH, 10 mg of 4f-methylthio-de(4fdimethylamino)pristinamycin IH are isolated by means of 'semi-preparative column chromatography as described above but bringing the proportion of acetonitrile in the eluent phase to 50%.
NMR spectrum: XH (400 MHz, CDC13, δ in ppm, ref. TMS): 0.32 (mt, IH, 5 β2) , 0.93 (t, J=7.5 Hz, 3H: CH3 2 γ) , from 1.20 to 1.3 5 (mt, 2H: 3 β2 and 3 y2) , 1.30 (d,
J=7.5 Hz, 3H: CH3 1 7), from 1.35 to 2.05 (mt, 9H: 3γ3β2 - CH2 2 β - CH2 5 δ - CH2 5y and 5 β2) , 2.44 (dt, J=13.5
1.5 Hz, IH: J=8 Hz, 2H;
AP/P/ 9 5 / 0 0 7 5 2 and 1.5 Hz, 1H: 5 e2) , 2.49 (s,3H: SCH3) , 2.99 (dd, J=12 and 5 Hz, 1H: 4 β2) , 3.09 (dd, J=12.5 and 12 Hz, 1H: 4 ,
3.54 and 3.64 (2 mts, 1H each: CH2 3 δ), 4.17 (dd, J=7 and e, from
Hz, 1H: 3 a), 4.49 (broad d, J=13.5 Hz: 1H;
4.70 to 4.80 (mt, 3H: 2a - 5 a and 4 a), 4.84 (dd, J=10 and
I. 5 Hz, 1H: la), 5.51 (d, J=7 Hz, 1H: 6 a), 5.73 (mt, 1H: 1β), 6.65 (d, J=9.5 Hz, 1H: NH 2), 7.10 (d, J=8 Hz, 2H:
4δ), 7.22 (d, J=8 Hz, 2H: 4e), from 7.20 to 7.40 (mt, 7H: aromatic H 6 = 1' H4 and l'Hs), 7.87 (d, J=4 Hz, 1H: l'H6),
8.55 (unres. comp., 1H: NH 6), 8.55 (d, J=10 Hz, 1H: NH 1),
II. 70 (s, 1H: OH) .
Using the fractions derived from the silica column described above which contain the novel derivative of pristinamycin I, 3 mg of 5y-hydroxy-4f-methylthio-de(4ζ15 dimethylamino)pristinamycin IH are isolated by carrying out semi-preparative column chromatography as described above and maintaining the proportion of acetonitrile in the eluent phase at 45%.
NMR spectrum: *H (400 MHz, CDC13, δ in ppm, ref.
TMS): a markedly preponderant isomer is observed: the -OH in the 5 γ position in an axial position. 0.37 (d mt,
J=16 Hz, 1H, 5 β2) , 0.93 (t, J=7.5 Hz, 3H: CH3 2 7), from 1.20 to 1.45 (mt, 2H: 3 β2 and 3 γ2) 1.31 (d, J=7.5 Hz, 3H: CH3 1 7), from 1.40 to 1.85 (mt, 5H: 3 γ3 - CH2 2 β and CH2
5 δ), 1.98 (mt, 1H, 3 βγϊ , 2.17 (d, J=16 Hz, 1H: 5 /?x) ,
2.50 (s, 3H: SCH3) , 2.77 (dt, J=13.5 and 2 Hz, 1H: 5 e2) , 2.99 (dd, J=12 and 4 Hz, 1H: 4 β2) , 3.11 (t, J=12 Hz, 1H:
βχ, from 3.45 to 3.70 (mt, 2H: CH2 3 δ), 3.73 (mt, 1H: 5 γ in an equatorial position), 4.13 (t, J=7 Hz, 1H, 3 a),
4.37 (broad d, J=13.5 Hz, 1H: 5 ex) , from 4,75 to 4.95 (mt, 3H: 2a, 4 a and 5 a), 4.89 (dd, J=10 and 1 Hz, 1H: la),
5.70 (d, J=8 Hz, 1H: 6 a), 5.80 (dq, J=7.5 and 1 Hz, 1H:
1β), 6.37 (d, J=5 Hz, 1H: NH 4), 6.71 (d, J=10 Hz, 1H: NH
2), 7.10 (d, J=8 Hz, 2H: 4δ), 7.22 (d, J=8 Hz, 2H: 4 e), from 7.20 to 7.40 (mt, 5H: aromatic H 6), 7.43 (dd, J=8.5 and 1.5 Hz, 1H: l'H4), 7.46 (dd, J=8.5 and 4 Hz, 1H: l'Hs),
APIPI 9 5 / 0 0 7 5 2
AP. Ο Ο 5 6 2
- 52 7.89 (dd, J=4 and 1.5 Hz, IH: l'H6), 8.55 (d, J=1O Hz, IH: NH 1), 9.15 (d, J=8 Hz, IH: NH 6), 11.70 (s, IH: OH).
EXAMPLE 6: Preparation of 4f-methyl-de(4fdimethylamino)pristinamycin IA and of 4f-methyl-de(4f5 dimethylamino)pristinamycin IH
Strain SP92::pVRC508 is cultured in production medium, using 60 Erlenmeyer flasks, as described in Example 3, with 1 ml of a 5 g/1 solution of (R,S)-4methylphenylalanine in 0.1 N sodium hydroxide solution 10 being added at 16 h. At the end of 40 h of culture, the
1.8 litres of must recovered from the 60 Erlenmeyer flasks are extracted with 2 volumes of a mixture consisting of 66% 100 mM phosphate buffer, pH 2.9, and 34% acetonitrile, and then centrifuged. The supernatant is extracted with 2 times
0.5 volumes of dichloromethane. The chloromethylene phases are washed with water and then combined, dried over sodium sulphate and evaporated. The dry extract is taken up in 20 ml of dichloromethane and injected onto a silica (30 g) column which is mounted in dichloromethane and is eluted successively with plateaus of from 0 to 10% methanol in dichloromethane. The fractions containing the new derivative of pristinamycin IA are combined and evaporated. 49 mg of dry residue are obtained. This residue is taken up in 6 ml of a mixture consisting of 60% water and 40% acetonitrile and injected, in two batches, onto a semipreparative Nucleosil Ίμ C8 10x250 mm column (Macherey Nagel), which is eluted with a mixture consisting of 55%
100 mM phosphate buffer, pH 2.9, and 45% acetonitrile. The fractions containing the new pristinamycin are combined and extracted with one volume of dichloromethane. The organic
AP/P/ 9 5 / 0 0 7 5 2
phase is washed with water, dried over sodium sulphate and then evaporated. 44 mg of 4f-methyl-de(4fdimethylamino)pristinamycin IA are obtained.
NMR spectrum: JH (400 MHz, CDC13, δ in ppm, ref.
TMS): 0.52 (dd, J=16 and 6 Hz, IH, 5 β2) , 0.93 (t,
J=7.5 Hz, 3H: CH3 2 γ) , 1.15 (mt, IH: 3 β2} , from 1.20 to 1.40 (mt, IH: 3 γ2) , 1.35 (d, J=7.5 Hz, 3H: CH3 1 γ), from 1.50 to 1.85 (mt, 3H: 3 and CH2 2/3), 2.04 (mt, IH, 3 /3,) , 2.18 (mt, IH, 5 δ2) , from 2.25 to 2.45 (mt, 2H: 5 δ, and 5 /3,), 2.36 (s, 3H: ArCH3) , 2.83 (dt, J=13 and 4 Hz,
IH: 5 e2) , 2.99 (dd, J=13 and 4 Hz, IH: 4 β2) , 3.28 (s, 3H: NCH34), 3.31 and 3,59 (2 mts, IH each: CH2 3 δ), 3.40 (t, J=13 Hz, IH: 4 /3,), 4.59 (t, J=7.5 Hz, IH, 3 a), 4.74 (broad dd, J=13 and 7 Hz, IH: 5 c,) , 4.85 (mt, IH: 2a),
4.89 (broad d, J=10 Hz, IH: la), from 5.25 to 5.35 (mt, 2H:
a and 4 a), from 5.85 to 5.95 (mt, 2H: 6 a and 1/3) , 6.52 (d, J=9.5 Hz, IH: NH 2), 7.14 (AB limit, J=9 Hz, 4H: 4δ and 4c), from 7.15 to 7.35 (mt, 5H: aromatic H 6), 7.50 (mt,
2H: l'H4 and l'Hs), 7.81 (dd, J=4 and 2Hz, IH: l'H6), 8.41 (d, J=10 HZ, IH: NH 1), 8.74 (d, J=9 Hz, IH: NH 6), 11.63 (S, 1H:OH).
Using the fractions derived from the silica column described above which contain the new derivative of pristinamycin IH, 21 mg of 4^-methyl-de(4f25 dimethylamino)pristinamycin I„ (mass spectrometry:
M+H*=810) are isolated by carrying out semi-preparative column chromatography as described above.
AP/P/ 9 5 / 0 0 7 5 2
EXAMPLE 7: Preparation of 4f-methoxy-de(4fdime thy lamino) pristinamycin Ix.
Strain SP92::pVRC508 is cultured in production medium using 12 Erlenmeyer flasks, as described in Example 3, with 1 ml of a 5 g/1 solution of (RS)-4methoxyphenylalanine in 0.1 N sodium hydroxide solution being added at 16 h. At the end of 40 h of culture, the 35 0.35 litres of must recovered from the 12 Erlenmeyer flasks
AP. Ο Ο 5 6 2
- 54 is extracted with 2 volumes of a mixture consisting of 66% 100 mM phosphate buffer, pH 2.9, and 34% acetonitrile, and then centrifuged. The supernatant is extracted with 2 times 0.5 volumes of dichloromethane. The chloromethylene phases are washed with water and then combined, dried over sodium sulphate and evaporated. The dry extract is taken up in 20 ml of dichloromethane and injected onto a silica (30 g) column which is mounted in dichloromethane and is eluted successively with plateaus of from 0 to 10% methanol in dichloromethane. The fractions containing the new derivative of pristinamycin IA are combined and evaporated. 14 mg of dry residue are obtained. This residue is taken up in 3 ml of a mixture consisting of 60% water and 40% acetonitrile and injected onto a semi-preparative Nucleosil
7μ C8 10x250 mm column (Machery Nagel), which is eluted with a mixture consisting of 60% 100 mM phosphate buffer, pH 2.9, and 40% acetonitrile. The fractions containing the new pristinamycin are combined and extracted with one volume of dichloromethane. The organic phase is washed with water, dried over sodium sulphate and then evaporated.
mg of 4Γ-methoxy-de(4ζ-dimethylamino)pristinamycin IA are obtained.
NMR spectrum: *H (400 MHz, CDC13, d in ppm, ref. TMS): 0.63 (dd, J=16 and 5.5 Hz, 1H, 5 02) , 0.96 (t,
J=7.5 Hz, 3H: CH3 2 γ) , 1.17 (mt, 1H: 3 02), from 1.30 to 1.45 (mt, 1H: 3 γ2) , 1.38 (d, J=7.5 Hz, 3H: CH3 1 γ) from
1.55 to 1.85 (mt, 3H: 3 γχ and CH2 2 0) , 2.05 (mt, 1H, 3 03) , 2.20 (mt, 1H, 5 δ2) , 2.40 (broad d, J=16 Hz, 1H: 5 δ3) ,
2.47 (d, J=16 Hz, 1H: 5 0J , 2.88 (dt, J=13 and 4 Hz, 1H: 5 €2) , 2.99 (dd, J=12.5 and 5 Hz, 1H: 4 02) , 3.30 (s, 3H: NCH3 4), 3.32 and 3.60 (2 mts, 1H each: CH2 3 δ), 3.40 (t,
J=12.5 Hz, 1H: 4 03) , 3.80 (s, 3H: 0CH3) , 4.60 (t,
J=7.5 Hz, 1H, 3a), 4.80 (broad dd, J=13 and 8.5 Hz, 1H: 5 e3) , 4.88 (mt, 1H: 2a), 4.92 (broad d, J=10 Hz, 1H: la),
5.31 (dd, J=12.5 and 5 Hz, 1H: 4 a), 5.34 (broad d, J=5.5 Hz, 1H: 5 a), 5.90 (d, J=9 Hz, 1H: 6 a), 5.93 (broad q,
AP/P/ 9 5 / 0 0 7 5 2
I
J=7.5 Hz, 1H: 1/?) , 6.54 (d, J=9 Hz, 1H: NH 2), 6.87 (d,
J=8 Hz, 2H: 4e), 7.16 (d, J=8 Hz, 2H: 4δ), from 7.15 to
7.40 (mt, 5H: aromatic H 6), 7.50 (mt, 2H: l'Hs and l'HJ,
7.80 (dd, J=4 and 2.5 Hz, 1H: l'Hs), 8.43 (d, J=10 Hz, 1H:
NH 1), 8.78 (d, J=9 Hz, 1H: NH 6), 11.65 (s, 1H:OH).
c c
o 20
EXAMPLE 8: Preparation of 4f-methoxycarbonylde(4f-dimethylamino)pristinamycin IA.
Strain SP92 : .-pVRC508 is cultured in production medium using 60 Erlenmeyer flasks, as described in Example 3, with 1 ml of a 10 g/1 solution of (R,S)-4methoxycarbonylphenylalanine, synthesized as in Example 33, being added at 16 h. At the end of 24 h of culture, the
1.8 litres of must recovered from the 60 Erlenmeyer flasks are extracted with 2 volumes of a mixture consisting of 66% 100 mM phosphate buffer, pH 2.9, and 34% acetonitrile, and then centrifuged. The supernatant is extracted with 2 times 0.5 volumes of dichloromethane. The chloromethylene phases are washed with water and then combined, dried over sodium sulphate and evaporated. The dry extract is taken up in 20 ml of dichloromethane and injected onto a silica (30 g) column which is mounted in dichloromethane and is eluted successively with plateaus of from 0 to 10% methanol in dichloromethane. The fractions containing the new derivative of pristinamycin Ix are combined and evaporated.
*14 mg of dry residue are obtained. This residue is taken up in 3 ml of a mixture consisting of 60% water and 40% acetonitrile and injected onto a semi-preparative Nucleosil 7μ C8 10x250 mm column (Macherey Nagel), which is eluted with a mixture consisting of 55% 100 mM phosphate buffer, pH 2.9, and 45% acetonitrile. The fractions containing the new pristinamycin are combined and extracted with one volume of dichloromethane. The organic phase is washed with
AP/P/ 9 5 / 0 0 7 5 2
AP . 0 0 5 6 2
- 56 water, dried over sodium sulphate and then evaporated. 9 mg of 4 f-methoxycarbonyl-de (4f-dimethylamino) pristinamycin IA are obtained.
NMR spectrum: 1H (400 MHz, CDC13, δ in ppm, ref.
TMS): 0.70 (dd, J=16 and 6 Hz, IH, 5 β2) , 0.93 (t,
J=7.5 Hz, 3H: CH3 2 γ), 1.08 (mt, IH: 3 β2) , from 1.30 to 1.40 (mt, IH: 3 γ2), 1.33 (d, J=7.5 Hz, 3H: CH3 1 γ) from
1.55 to 1.85 (mt, 3H: 3 γ3 and CH2 2 β) , 2.02 (mt, IH, 3 ft), 2.13 (mt, IH, 5 δ2) , 2.40 (broad d, J=16.5 Hz, IH: 5 δ3), 2.48 (d, J=16 Hz, IH, 5 β2) , 2.89 (dt, J=14.5 and
4.5 Hz, IH: 5 e2) , 3.10 (dd, J=13.5 and 6 Hz, IH: 4 β2} ,
3.24 (s, 3H: NCH3 4), 3.38 and 3.61 (2 mts, IH each: CH2 3 δ), 3.47 (t, J=13.5 Hz, IH: 4 0,), 3.96 (s, 3H: COOCH3) ,
4.55 (t, J=7.5 Hz, IH, 3 a), 4.78 (broad dd, J=14.5 and
8 Hz, IH: 5 ej , 4.86 (mt, IH: 2a), 4.89 (broad d, J=10 Hz,
IH: la), 5.33 (broad d, J=6 Hz, IH: 5 a), 5.42 (dd, J=13.5 and 6 Hz, IH: 4 a), 5.92 (d, (J=9.5 Hz) and mt, IH each: 6 a and 10 respectively), 6.52 (d, J=10 Hz, IH: NH 2), from 7.15 to 7.35 (mt, 5H: aromatic H 6), 7.28 (d, J=8 Hz, 2H:
46) , 7.43 (dd, J=9 and 1.5 Hz, IH: l'H4), 7.47 (dd, J=9 and
Hz, IK: l'Hs), 7.66 (d, J=5 and 1.5 Hz, IH: l'H6), 7.98 (d, J=8 Hz, 2H: 4e), 8.38 (d, J=10 Hz, IH: NH 1), 8.76 (d, J=9.5 Hz, IH: NH 6), 11.70 (s, IH: OH).
EXAMPLE 9: Preparation of 4f-chloro-de(4f25 dimethylamino) pristinamycin IA.
Strain SP92::pVRC508 is cultured in production medium using 60 Erlenmeyer flasks, as described in Example 3, with 1 ml of a 10 g/1 solution of (R,S)-4chlorophenylalanine in 0.1 N sodium hydroxide solution 30 being added at 16 h. At the end of 40 h of culture, the
1.8 litres of must recovered from the 60 Erlenmeyer flasks are extracted with 2 volumes of a mixture consisting of 66% 100 mM phosphate buffer, pH 2.9, and 34% acetonitrile, and then centrifuged. The supernatant is extracted with 2 times
0.5 volumes of dichloromethane. The chloromethylene phases
AP/P/ 9 5 / 0 0 7 5 2 are washed with water and then combined, dried over sodium sulphate and evaporated. The dry extract is taken up in 20 ml of dichloromethane and injected onto a silica (30 g) column which is mounted in dichloromethane and eluted successively with plateaus of from 0 to 10% methanol in dichloromethane. The fractions containing the new derivative of pristinamycin IA are combined and evaporated. The dry residue is taken up in 3 ml of a mixture consisting of 60% water and 40% acetonitrile and injected onto a semi10 preparative Nucleosil 7μ C8 10x250 mm column (Macherey Nagel), which is eluted with a mixture consisting of 60%
100 mM phosphate buffer, pH 2.9, and 40% acetonitrile. The fractions containing the new pristinamycin are combined and extracted with one volume of dichloromethane. The organic phase is washed with water, dried over sodium sulphate and then evaporated. 1 mg of 4ζ-chloro-de(4fdimethylamino)pristinamycin IA is obtained.
NMR spectrum: XH (400 MHz, CDC13, 6 in ppm, ref. TMS): 0.93 (t, J=7.5 Hz, 3H: CH3 2 γ) , 0.95 (dd, J=16 and
5 Hz, 1H, 5 β2) , 1.09 (mt, 1H: 3 /32), from 1.20 to 1.40 (mt, 1H: 3 γ2), 1.35 (d, J=7.5 Hz, 3H: CH3 1 γ) from 1.50 to 1.85 (mt, 3H: 3 γ, and CH2 2 β), 2.02 (mt, 1H, 3 /8J ,
2.17 (mt, 1H, 5 δ2) , 2.43. (broad d, J=16 Hz, 1H: 5 δ3) ,
2.59 (d, J=16 Hz, 1H: 5 /3J , 2.90 (dt, J=13.5 and 4 Hz, 1H:
5 c2) , 3.04 (dd, J=13 and 6 Hz, 1H: 4 β2) , 3.21 (s, 3H: 4
NCH3) , 3.36 (t, J=13 Hz, 1H: 4 /3J , 3.39 and 3.59 (2 mts,
1H each: CH2 3 δ), 4.53 (t, J=7.5 Hz, 1H, 3 a), 4.76 (broad dd, J=13.5 and 8 Hz, 1H: 5 e3) , 4.86 (mt, 1H: 2a), 4.87 (broad d, J=10 Hz, 1H: la), 5.38 (mt, 2H: 5 a and 4 a),
5.93 (mt, 2H: 6 a and 1/3), 6.52 (d, J=10 Hz, 1H: NH 2),
7.12 (d, J=8 Hz, 2H: 4δ) from 7.15 to 7.35 (mt, 7H: aromatic H 6 and 4e), 7.38 (dd, J=9 and 4.5 Hz, 1H:1'HS), 7.43 (broad d, J=9 Hz, 1H: 1'HJ, 7.68 (dd, J=4.5 and 1 Hz, 1H: l'H6), 8.36 (d, J=10 Hz, 1H: NH 1), 8.75 (d, J=9 Hz,
1H: NH 6), 11.65 (s, 1H:OH).
AP/P/ 9 5 / 0 0 7 5 2
AP . Ο Ο 5 6 2
- 58 EXAMPLE 10: Preparation of 4f-bromo-de(4fdimethylamino)pristinamycin IA and of 4f-bromo-de(4fdimethylamino)pristinamycin IH.
Strain SP92::pVRC508 is cultured in production 5 medium using 60 Erlenmeyer flasks, as described in Example
3, with 1 ml of a 10 g/1 solution of (R,S)-4bromophenylalanine in 0.1 N sodium hydroxide solution being added at 16 h. At the end of 40 h of culture, the
1.8 litres of must recovered from the 60 Erlenmeyer flasks 10 are extracted with 2 volumes of a mixture consisting of 66%
100 mM phosphate buffer, pH 2.9, and 34% acetonitrile, and then centrifuged. The supernatant is extracted with 2 times 0.5 volumes of dichloromethane. The chloromethylene phases are washed with water and then combined, dried over sodium sulphate and evaporated. The dry extract is taken up in 20 ml of dichloromethane and injected onto a silica (30 g) column which is mounted in dichloromethane and is eluted successively with plateaus of from 0 to 10% methanol in dichloromethane. The fractions containing the new derivative of pristinamycin IA are combined and evaporated. The dry residue is taken up in 6 ml of a mixture consisting of 60% water and 40% acetonitrile and injected in two batches onto a semi-preparative Nucleosil Ίμ C8 10x250 mm column (Macherey Nagel), which is eluted with a mixture consisting of 60% 100 mM phosphate buffer, pH 2.9, and 40% acetonitrile. The fractions containing the new pristinamycin are combined and extracted with one volume of dichloromethane. The organic phase is washed with water, dried over sodium sulphate and then evaporated. 6 mg of 4f30 bromo-de(4f-dimethylamino)pristinamycin IA are obtained.
NMR spectrum: *H (4 00 MHz, CDC13, δ in ppm, ref.
AP/P/ 9 5 / 0 0 7 5 2
TMS) : 0.93 (J=7.5 Hz, 3H: CH3 2 γ) , 0. 95 (dd, J=16 and
5 Hz ·, 1H, 5 β2) , 1 .10 (mt, 1H: 3 )32) , 1.35 (d, J=7.5 Hz,
3H: CH3 1 γ) 1.36 (mt, 1H: 3 γ2) , from 1.50 to 1.85 (mt,
3H, 3 7j and CH2 2 )3), 2.02 (mt, 1H, 3 /3J , 2.18 (mt, 1H:
δ2) , 2.43 (broad d , J=16 Hz, 1H: 5 δ2) , 2.59 (d, J=16 Hz,
1H: 5 β2) , 2.90 (dt, J=13 and 4 Hz, 1H: 5 €2) , 3.02 (dd,
J=13 and 5.5 Hz, 1H: 4 02) , 3.21 (s, 3H: 4 NCH3) , 3.33 (dd, J=13-ll Hz, 1H: 4 0J , 3.39 and 3.59 (2 mts, 1H each: CH2 3 δ), 4.53 (t, J=7.5 Hz, 1H, 3 a), 4.76 (broad dd, J=13 and
7 Hz, 1H: 5 e3) , 4.86 (mt, 1H, 2a), 4.89 (d broad, J=10 Hz,
1H: la), 5.37 (broad d, J=5 Hz, 1H: 5 a), (dd, J=ll and
5.5 Hz, 1H: 4 a), 5.92 (mt, 2H: 6 a and 1/3) , 6.56 (d,
J=9.5 Hz, 1H: NH 2), 7.08 (d, J=8 Hz, 2H: 46), from 7.15 to 7.35 (mt, aromatic H 6), 7.40 (mt, 4H: 1'H4 - 1 'H5 and 4e),
7.70 (broad d, J=5 Hz, 1H: l'H6), 8.40 (d, J=10 Hz, 1H: NH
1), 8.77 (d, J=9 Hz, 1H: NH 6), 11.68 (s, 1H: OH).
Using the fractions derived from the silica column described above which contain the new derivative of pristinamycin I„, 3 mg of 4f-bromo-de(4ζ15 dimethylamino)pristinamycin IH (mass spectrometry:
M+H*=874) are isolated by carrying out semi-preparative column chromatography as described above.
EXAMPLE 11: Preparation of 4f-iodo-de(4fdimethylamino)pristinamycin IA and of -iodo-de(4f20 dimethylamino)pristinamycin IB.
Strain SP92::pVRC508 is cultured in production medium using 60 Erlenmeyer flasks, as described in Example 3, with 1 ml of a 10 g/1 solution of (RS)-4iodophenylalanine in sodium hydroxide solution being added at 16 h. At the end of 40 h of culture, the 1.8 litres of must recovered from the 60 Erlenmeyer flasks are extracted with 2 volumes of a mixture consisting of 66% 100 mM •phosphate buffer, pH 2.9, and 34% acetonitrile, and then centrifuged. The supernatant is extracted with 2 times 0.5 volumes of dichloromethane. The chloromethylene phases are washed with water and then combined, dried on sodium sulphate and evaporated. The dry extract is taken up in 20 ml of dichloromethane and injected onto a silica (30 g) column which is mounted in dichloromethane and eluted successively with plateaus of from 0 to 10% methanol in
AP/P/ 95/00752
AP . Ο Ο 5 6 2
- 60 dichloromethane. The fractions containing the new derivative of pristinamycin IA are combined and evaporated. The dry residue is taken up in 6 ml of a mixture consisting of 60% water and 40% acetonitrile and injected in two batches onto a semi-preparative Nucleosil 7μ C8 10x250 mm column (Macherey Nagel), which is eluted with a mixture consisting of 60% 100 mM phosphate buffer, pH 2.9, and 40% acetonitrile. The fractions containing the new pristinamycin are combined and extracted with one volume of dichloromethane. The organic phase is washed with water, dried over sodium sulphate and then evaporated. 12 mg of 4f-iodo-de(4ζ-dimethylamino)pristinamycin IA are obtained.
NMR spectrum: XH (4 00 MHz, CDC13, δ in ppm, ref. TMS): 0.93 (J=7.5 Hz, 3H: CH3 2 γ), 0.95 (dd, J=16 and
5.5 Hz, IH: 5 β2) , 1.10 (mt, IH: 3 β2) , 1.35 (d, J=7.5 Hz, 3H: CH3 1 γ) , 1.38 (mt, IH: 3 γ2) , from 1.55 to 1.85 (mt,
3H, 3 γ3 and CH2 2 β) , 2.02 (mt, IH, 3 βχ) , 2.17 (mt, IH: 5 δ2) , 2.43 (broad d, J=16.5 Hz, IH: 5 δ3) , 2.60 (d, J=16 Hz, IH: 5 ^), 2.89 (dt, J=14 and 4.5 Hz, IH: 5 £2) , 3.02 (dd,
J=13 and 5.5 Hz, IH: 4 β2) , 3.21 (s, 3H: NCH3 4), 3.31 (dd,
J=13 and 11 Hz, IK: 4 β2) , 3.39 and 3.59 (2 mts, IH each:
CH2 3 δ), 4.53 (t, J=7.5 Hz, IH, 3 a), 4.75 (broad dd, J=14 and 8 Hz, IH: 5 ej , 4.83- (mt, IH: 2a), 4.88 (broad d,
J=10 Hz, IH: la), 5.37 (broad d, J=5.5 Hz, IH: 5 a), 5.39 (dd, J=ll and 5.5 Hz, IH: 4 a), 5.92 (mt, 2H: 6 a and 1β) , 6.54 (d, J=9.5 Hz, IH: NH 2), 6.94 (d, J=7.5 Hz, 2H: 4δ), from 7.15 to 7.50 (mt, 5H: aromatic H 6), 7.36 (dd, J=9 and 4 Hz, IH: l'Hs) , 7.43 (broad d, J=9 Hz, IH: l'H«) , 7.62 (d, ' j=7.5 Hz, 2H: 4f) , 7.68 (d, J=4 Hz, IH: l'HJ , 8.38 (d,
J=10 Hz, IH: NH 1), 8.76 (d, J=9 Hz, IH: NH 6), 11.60 (s,
IH: OH).
Using the fractions derived from the silica column described above which contain the new derivative of pristinamycin IH, 6 mg of 4f-iodo-de(4f35 dimethylamino)pristinamycin IH (mass spectrometry:
M+H’=922) are isolated by carrying out semi-preparative
AP/P/ 9 5 / 0 0 7 5 2
AP . Ο Ο 5 6 2
- 61 column chromatography as described above.
EXAMPLE 12 Preparation of 4f-trifluoromethylde(4f-dimethylamino)pristinamycin IA and of 4ftrifluoromethyl-de(4f-dimethylamino)pristinamycin Ia.
*5 Strain SP92 : :pVRC508 is cultured in production medium using 60 Erlenmeyer flasks, as described in Example 3, with 1 ml of a 5 g/1 solution of (S)-4trifluoromethylphenylalanine in 0.1 N sodium hydroxide solution being added at 16 h. At the end of 40 h of culture, the 1.8 litres of must recovered from the 60
Erlenmeyer flasks are extracted with 2 volumes of a mixture consisting of 66% 100 mM phosphate buffer, pH 2.9, and 34% acetonitrile, and then centrifuged. The supernatant is extracted with 2 times 0.5 volumes of dichloromethane. The chloromethylene phases are washed with water and then combined, dried on sodium sulphate and evaporated. The dry extract is taken up in 20 ml of dichloromethane and injected onto a silica (30 g) column which is mounted in dichloromethane and eluted successively with plateaus of from 0 to 10% methanol in dichloromethane. The fractions containing the new derivative of pristinamycin ΙΛ are combined and evaporated. The dry residue is taken up. in 3 ml of a mixture consisting of 60% water and 40% acetonitrile and injected onto a semi-preparative Nucleosil
Ίμ C8 10x250 mm column (Macherey Nagel), which is eluted with a mixture consisting of 55% 100 mM phosphate buffer, pH 2.9, and 45% acetonitrile. The fractions containing the ’ new pristinamycin are combined and extracted with one volume of dichloromethane. The organic phase is washed with water, dried over sodium sulphate and then evaporated. 5 mg of 4f-trifluoromethyl-de(4f-dimethylamino)pristinamycin IA are obtained.
NMR spectrum: XH (400 MHz, CDC13, δ in ppm, ref. TMS) : 0.86 (dd, J=16 and 5.5 Hz, IH, 5 β2) , 0.91 (t, J=7.5
Hz, 3H: CH3 2γ) , 1.13 (mt, IH: 3 β2) , 1.31 (d, J=7.5 Hz,
AP/P/ 9 5 / 0 0 7 5 2
3H: CH3 1 7) 1.42 (mt, IH: 3 γ2) , from 1.55 to 1.80 (mt,
3H: 3 7X and CH2 2 /3), 2.02 (mt, IH, 3 /3J , 2.15 (mt, IH, 5 δ2), 2.40 (broad d, J=16.5 Hz, IH: 5 δ2) , 2.55 (d, J=16 Hz, IH: 5 /3j) , 2.88 (dt, J=14 and 4 Hz, IH: 5 e2) , 3.18 (s, 3H:
NCH3 4), 3.20 and 3.31 (2 dd, respectively J=13 and 6 Hz and J=13 and 10 Hz, IH each: 4 β2 and 4 /31), 3.42 and 3.60 (2 mts, IH each: CH2 3 δ), 4.50 (t, J=7.5 Hz, IH, 3 a}, 4.73 (broad dd, J=14 and 7.5 Hz, IH: 5 ex) , 4.83 (mt, IH: 2a), 4.91 (broad d, J=10 Hz, IH: la), 5.40 (broad d, J=5.5
Hz, IH: 5 a), 5.55 (dd, J=10 and 6 Hz, IH: 4 a), 5.87 (d,
J=9 .5 Hz, IH: 6 a) , 5.90 (broad q, J=7.5 Hz, IH: 1/3) , 6.68
(d, J=9.5 Hz, IH: NH 2), from 7.15 to 7.40 (mt, 9 H:
4δ- aromatic H 6 - l'Hs and l'H4) , 7 .52 (d, J=8 Hz, 2H: 4e) ,
7.6 8 (d, J=4 and 1 .5 Hz, IH: l'H6) , 8.43 (d, J=10 Hz, IH:
NH 1), 8.76 (d, J=9.5 Hz, IH: NH 6), 11.70 (s, IH: OH).
Using the fractions derived from the silica column described above which contain the new derivative of pristinamycin IH, 4 mg of f-trifluoromethyl-de(4fdimethylamino)pristinamycin IH (mass spectrometry:
M+H*=864) are isolated by carrying out semi-preparative column chromatography as described above.
EXAMPLE 13: Preparation of 4f-tert-butyl-de(4fdimethylamino)pristinamycin IA.
Strain SP92::pVRC508 is cultured in production medium using 60 Erlenmeyer flasks, as described in Example 3, with 1 ml of a 5 g/1 solution of (R,S)-4-tertbutylphenylalanine, synthesized as in Example 35-1, in 0.1 N sodium hydroxide solution being added at 16 h. At the end of 40 h of culture, the 1.8 litres of must recovered from the 60 Erlenmeyers are extracted with 2 volumes of a mixture consisting of 66% 100 mM phosphate buffer, pH 2.9, and 34% acetonitrile, and then centrifuged. The supernatant is extracted with 2 times 0.5 volumes of dichloromethane. The chloromethylene phases are washed with water and then combined, dried on sodium sulphate and evaporated. The dry
AP/P/ 9 5 / 0 0 7 5 2
AP.00562
- 63 extract is taken up in 20 ml of dichloromethane and injected onto a silica (30 g) column which is mounted in dichloromethane and eluted successively with plateaus of from 0 to 10% methanol in dichloromethane. The fractions containing the new derivative of pristinamycin IA are combined and evaporated. The dry residue is taken up in 7 ml of a mixture consisting of 60% water and 40% acetonitrile and injected in 2 batches onto a semipreparative Nucleosil Ίμ C8 10x250 mm column (Macherey
Nagel), which is eluted with a mixture consisting of 55% λ 100 mM phosphate buffer, pH 2.9, and 45% acetonitrile. The fractions containing the new pristinamycin are combined and extracted with one volume of dichloromethane. The organic phase is washed with water, dried over sodium sulphate and then evaporated. 30 mg of 4f-tert-butyl-de(4fdimethylamino)pristinamycin IA are obtained.
NMR spectrum: 1H (4 00 MHz, CDC13, δ in ppm, ref. TMS): 0.21 (dd, J=16 and 5.5 Hz, 1H, 5 β2) , 0.91 (t, J=7.5 Hz, 3H: CH3 2 γ), 1.17 (mt, 1H: 3 β2) , from 1.20 to 1.40 (mt, 1H: 3 γ2) , 1.33 (s, 9H: CH3 of tert-butyl), 1.35 (d, J=7.5 Hz, 3H: CH3 1 γ) , from 1.50 to 1.85 (mt, 3H: 3 γ, and r-j CH2 2 β) , 2.04 (mt, 1H, 3 β/ , 2.13 (mt, 1H, 5 δ2) , 2.30 <mt, 2H: 5 δ, and 5 βχ) , 2.80 (dt, J=13 and 4 Hz, 1H: 5 e2) ,
3.00 (dd, J=12 and 4 Hz, 1H: 4 β2) , 3.29 (s, 3H: NCH34) ,
3.31 and 3.59 (2 mts, 1H each: CH2 3 δ), 3.40 (t, J=12 Hz,
1H: 4 /3,), 4.57 (t, J=7.5 Hz, 1H, 3 a), 4.74 (broad dd,
J-13 and 7 Hz, 1H: 5 €,), 4.85 (mt, 1H: 2a), 4.90 (broad d, J=10 Hz, 1H: la), 5.21 (broad d, J=5.5 Hz, 1H: 5 a), 5.25 (dd, J-12 and 4 Hz, 1H: 4 a), 5.87(d, J=9 Hz, 1H: 6 a),
5.92 (broad q, J=7.5 Hz, 1H: 1/3), 6.56 (d, J=9.5 Hz, 1H: NH
2), from 7.10 to 7.30 (mt, 5H: aromatic H 6), 7.28 (d,
J=7.5 Hz, 2H: 4δ), 7.38 (d, J=7.5 Hz, 2H: 4e), 7.49 (broad d, J=8.5 Hz, 1H: l'Hj, 7.53 (dd, J=8.5 and 4 Hz, 1H: l'Hs), 7.86 (d, J=4 Hz, 1H: l'HJ, 8.45 (d, J=10 Hz, 1H: NH
1), 8.74 (d, J=9 Hz, 1H: NH 6), 11.65 (s, 1H:OH).
AP/P/ 9 5 / 0 0 7 5 2
EXAMPLE 14: Preparation of 4f-isopropyl-de(4fdimethylamino)pristinamycin IA and of 4f-isopropyl-de(4fdimethylamino)pristinamycin IB.
Strain SP92::pVRC508 is cultured in production 5 medium using 60 Erlenmeyer flasks, as described in Example
3, with 1 ml of a 10 g/1 solution of (R,S)-4isopropylphenylalanine, synthesized as in Example 36-1, in 0.1 N sodium hydroxide solution being added at 16 h. At the end of 40 h of culture, the 1.8 litres of must recovered from the 60 Erlenmeyer flasks are extracted with 2 volumes of a mixture consisting of 66% 100 mM phosphate buffer, ft pH 2.9, and 34% acetonitrile, and then centrifuged. The supernatant is extracted with 2 times 0.5 volumes of dichloromethane. The chloromethylene phases are washed with water and then combined, dried over sodium sulphate and evaporated. The dry extract is taken up in 20 ml of dichloromethane and injected onto a silica (30 g) column which is mounted in dichloromethane and eluted successively with plateaus of from 0 to 10% methanol in dichloromethane.
The fractions containing the new derivative of pristinamycin IA are combined and evaporated. 61 mg of the dry residue are obtained. This residue is taken up in 9 ml of a mixture consisting of 60% water and 40% acetonitrile and injected in 3 batches onto a semi-preparative Nucleosil
7μ C8 10x250 mm column (Macherey Nagel), which is eluted with a mixture consisting of 55% 100 mM phosphate buffer, pH 2.9, and 45% acetonitrile. The fractions containing the new pristinamycin are combined and extracted with one •volume of dichloromethane. The organic phase is washed with water, dried over sodium sulphate and then evaporated.
mg of 4 f-isopropyl-de (4 f-dimethylamino) pristinamycin IA are obtained.
NMR spectrum: :H (250 MHz, CDC13, δ in ppm, ref. TMS): 0.31 (dd, J=16 and 5.5 Hz, 1H, 5 β2), 0.91 (t, J=7.5
Hz, 3H: CH3 2 7), from 1.00 to 1.45 (mt, 2H: 3 β2 and 3 γ2) , 1.25 (d, J=7.5 Hz, 6H: CH3 of isopropyl), 1.35 (d,
AP/P/ 9 5 / 0 0 7 5 2
AP.00562
J=7.5 Hz, 3H: CH3 1 γ) , from 1.50 to 1.85 (mt, 3H: 3 y1 and
CH2 2 β) , from 1.95 to 2.20 (mt, 2H, 3 β and 5 δ2) , 2.30 (mt, 2H: 5 δ3 and 5 β2) , 2.80 (dt, J=13 and 4 Hz, 1H: 5 e2) ,
2.88 (mt, 1H: CH of isopropyl), 2.98 (dd, J=12 and 4 Hz,
1H: 4 β2) , 3.30 (s, 3H: NCH3 4), 3.32 and 3.55 (2 mts, 1H each: CH2 3 δ), 3.38 (t, J=12 Hz, 1H: 4 03) , 4.55 (t, J=7.5 Hz, 1H, 3 a), 4.72 (broad dd, J=13 and 7 Hz, 1H: 5 ej ,
4.85 (mt, 1H: 2a), 4.88 (broad d, J=10 Hz, 1H: la), 5.21 (broad d, J=5.5 Hz, 1H: 5a), 5.25 (dd, J=12 and 4 Hz, 1H:
4a), 5.87 (d, J=9 Hz, 1H: 6 a), 5.90 (broad q, J=7.5 Hz,
ΙΗ: 10), 6.50 (d, J=9.5 Hz, 1H: NH 2), from 7.05 to 7.35 (mt, 9H: aromatic H 6 - 4e and 4δ), 7.50 (mt, 2H: 1'Hs and l'HJ, 7.86 (dd, J=4 and 1.5 Hz, 1H: l'H6), 8.40 (d, J=10 Hz, 1H: NH 1), 8.72 (d, J=9 Hz, 1H: NH 6), 11.60 <s,
1H: OH).
Using the same fractions derived from the silica column described above, which fractions also contain the new derivative of pristinamycin IE, 5 mg of f-isopropylde(4f-dimethylamino)pristinamycin IE are isolated by carrying out semi-preparative column chromatography as described above.
NMR spectrum: XH (4 00 MHz, CDC13, δ in ppm, ref. TMS): 0.20 (mt, 1H, 5 02) , 0.92 (t, J=7.5 Hz, 3H: CH3 2 γ) , from 1.15 to 1.40 (mt, 2H: 3 β2 and 3 γ2) , 1.24 (d,
J=7.5 Hz, 6H: CH3 of isopropyl), 1.34 (d, J=7.5 Hz, 3H: CH3 1 γ) , from 1.35 to 2.05 (mt, 9H: 3 γ3 - 3 β1 - CH2 2 β - CH2 5 δ - CH2 5 γ and 5ft), 2.45 (dt, J=13 and 1.5 Hz, 1H:
5cx) , 2.89 (mt, 1H: ArCH), 3.09 (dd, J=14 and 7 Hz, 1H: 4 ‘ β2), 3.17 (s, 3H: NCH3 4), 3.25 (dd, J=14 and 9 Hz, 1H: 4
03) , 3.32 and 3.52 (2 mts, 1H each: CH2 3 δ), 4.55 (mt, 2H:
a and 5 c3) , 4.80 (mt, 1H: 2a), 4.89 (dd, J=10 and 1.5
Hz, 1H: la), 4.90 (mt, 1H: 5 a), 5.35 (dd, J=9 and 7 Hz,
1H: 4 a), 5.60 (d, J=8 Hz, 1H: 6 a), 5.89 (dq, J=7.5 and
1.5 Hz, 1H: 1/3), 6.65 (d, J=9.5 Hz, 1H: NH 2), 7.08 (d,
J=8 Hz, 2H: 4δ), 7.14 (d, J=8 Hz, 2H: 4c), from 7.20 to
7.40 (mt, 7H: aromatic H 6 - l'H< and l'Hs), 7.77 (broad d,
AP/P/ 9 5 / 0 0 7 5 2
J=4 Hz, 1H: l'Hj), 8.46 (d, J=10 Hz, 1H: NH 1), 8.48 (d,
J=8 Hz, 1H: NH 6), 11.70 (s, 1H: OH).
EXAMPLE 15: Preparation of 4e-methylamino-de(4fdimethylamino)pristinamycin IA.
Strain SP92::pVRC508 is cultured in production medium using 60 Erlenmeyer flasks, as described in Example 3, with 1 ml of a 10 g/1 solution of (R,S)-3methylaminophenylalanine, synthesized as in Example 35-3, in water being added at 16 h. At the end of 40 h of culture, the 1.8 litres of must recovered from the 60
Erlenmeyer flasks are extracted with 2 volumes of a mixture consisting of 66% of 100 mM phosphate buffer, pH 2.9, and 34% acetonitrile, and then centrifuged. The supernatant is extracted with 2 times 0.5 volumes of dichloromethane. The chloromethylene phases are washed with water and then combined, dried over sodium sulphate and evaporated. The dry extract is taken up in 20 ml of dichloromethane and injected onto a silica (30 g) column which is mounted in dichloromethane and is eluted successively with plateaus of from 0 to 10% methanol in dichloromethane. The fractions containing the new derivative of pristinamycin IA are combined and evaporated. 19 mg of dry residue are obtained. This residue is taken up in 3 ml of a mixture consisting of 60% water and 40% acetonitrile and injected onto a semi25 preparative Nucleosil 7μ C8 10x250 mm column (Macherey Nagel), which is eluted with a mixture consisting of 55%
100 mM phosphate buffer, pH 2.9, and 45% acetonitrile. The fractions containing the new pristinamycin are combined and extracted with one volume of dichloromethane. The organic phase is washed with water, dried over sodium sulphate and then evaporated. 8 mg of 4e-methylamino-de(4fdimethylamino)pristinamycin IA are obtained.
NMR spectrum: XH (4 00 MHz, CDC13, δ in ppm, ref. TMS): 0.93 (t, J=7.5 Hz, 3H: CH3 2 γ), 1.00 (dd, J=16 and 6
Hz, 1H, 5 β2] , 1.17 (mt, 1H: 3 β2) , from 1.25 to 1.40 (mt,
AP/P/ 9 5 / 0 0 7 5 2
AP. Ο Ο 5 6 2
- 67 2Η: 3 γ2), 1.35 (d, J=7.5 Hz, 3Η: CH3 1 γ) , from 1.55 to 1.80 (mt, 3Η: 3 γ3 and CH2 2 β) , 2.03 (mt, IH, 3 , 2.23 (mt, IH, 5 δ2) , 2.39 (broad d, J=16 Hz, IH: 5 δ3) , 2.52 (d,
J=16 Hz, IH: 5 β/> , 2.78 (s, 3H: ArNCH3 4), 2.85 (dt, J=13 and 4 Hz, IH: 5 e2) , 2.99 (dd, J=13 and 4.5 Hz, IH: 4 β2) ,
3.23 (s, 3H: NCH3 4), 3.25 (t, J=13 Hz, IH: 40J , 3.38 and
3.58 (2 mts, IH each: CH2 3 δ), 4.05 (unres. comp., IH: ArNH), 4.58 (dd, J=6.5 and 7.5 Hz, IH, 3a), 4.76 (broad dd, J=13 and 8 Hz, IH: 5 e3) , 4.85 (mt, IH: 2a), 4.87 (broad d, J=10 Hz, IH: la), 5.35 (dd, J=13 and 4.5 Hz, IH:
a), 5.38 (broad d, J=6 Hz, IH: 5 a), 5.90 (d, J=9.5 Hz, IH: 6 a), 5.91 (mt, IH: 1/2), 6.36 (broad s, IH: H 2 of the aromatic moiety at position 4), from 6.45 to 6.55 (mt, 2H: H4 and H6 of the aromatic moiety in position 4), 6.53 (d,
J=10 Hz, IH: NH 2), 7.12 (t, J=8 Hz, IH: H 5 of the aromatic moiety in position 4), from 7.15 to 7.45 (mt, 5H: aromatic H 6), 7.35 (mt, 2H: 1' H« and 1' Hs) , 7.75 (t,
J=3 Hz, IH: 1' H6) , 8.40 (d, J=10 Hz, IH: NH 1), 8.78 (d, J=9.5 Hz, IH: NH 6), 11.60 (s, IH: OH).
EXAMPLE 16: Preparation of 4e-methoxy-de(4fdimethylamino)pristinamycin IA and of 4c-methoxy-de(4fdimethylamino)pristinamycin IE.
Strain SP92::pVRC508 is cultured in production medium using 60 Erlenmeyer flasks, as described in Example
3, with 1 ml of a 5 g/1 solution of (S)-3methoxyphenylalanine in 0.1N sodium hydroxide solution being added at 16 h. At the end of 40 h of culture, the
1.8 litres of must recovered from the 60 Erlenmeyer flasks are extracted with 2 volumes of a mixture consisting of 66%
100 mM phosphate buffer, pH 2.9, and 34% acetonitrile, and then centrifuged. The supernatant is extracted with 2 times 0.5 volumes of dichloromethane. The chloromethylene phases are washed with water and then combined, dried over sodium sulphate and evaporated. The dry extract is taken up in
20 ml of dichloromethane and injected onto a silica (30 g)
AP/P/ 9 5 / 0 0 7 5 2 column which is mounted in dichloromethane and eluted successively with plateaus of from 0 to 10% methanol in dichloromethane. The fractions containing the new derivative of pristinamycin IA are combined and evaporated.
41 mg of dry residue are obtained. This residue is taken up in 6 ml of a mixture consisting of 60% water and 40% acetonitrile and injected in 2 batches onto a semipreparative Nucleosil 7μ C8 10x250 mm column (Macherey Nagel), which is eluted with a mixture consisting of 55%
100 mM phosphate buffer, pH 2.9, and 45% acetonitrile. The fractions containing the new pristinamycin are combined and extracted with one volume of dichloromethane. The organic phase is washed with water, dried over sodium sulphate and then evaporated. 28 mg of 4e-methoxy-de(4f15 dimethylamino)pristinamycin IA are obtained.
NMR spectrum: :H (400 MHz, CDC13, δ in ppm, ref.
TMS): 0.52 (dd, J=16 ana 5.5 Hz, 1H, 5 β2) , 0.90 (t, J=7.5
Hz, 3H: CH3 2 γ), from 1.10 to 1.34 (mt, 2H: 3 β2 and 3 y2)
1.34 (d, J=7.5 Hz, 3H: CH3 ly), from 1.50 to 1.80 (mt, 3H:
20 3 Ύι and CH2 2 β), 2.40 (mt, 1H, 3 0X) , 2.20 (mt, 1H, 5 δ2) ,
2.35 (broad d, J=16 Hz, 1H: 5 δχ) , 2.38 (d, J=16 Hz, 1H: 5 β2) , 2.83 (dt, J=13 and 4 Hz, 1H: 5 e2) , 2.97 (dd, J=12 and 4 Hz, 1H: 4 β2) , 3.28 (s, 3H: NCH3 4), 3.28 and 3.56 (2 mts, 1H each: CH2 3 δ), 3.40 (t, J=12 Hz, 1H: 4 β2) , 3.80 (s, 3H: OCH3) , 4.58 (t, J=7.5 Hz, 1H, 3 or), 4.76 (broad dd, J=13 and 8 Hz, 1H: 5 e3) , 4.85 (mt, 1H: 2a), 4.90 (broad d, J=10 Hz, 1H: la): 5.27 (dd, J=12 and 4 Hz, 1H: 4 a), 5.30 (broad d, J=5.5 Hz, 1H: 5 a), 5.89 (d, J=9.5 Hz, 1H: 6 a), 5.91 (broad q, J=7.5 Hz, ΙΗ: 1β), 6.51 (d, J=10 Hz, 1H: NH
2), from 6.80 to 6.90 (mt, 3H: H 2 - H 4 and H 6 of the aromatic moiety in position 4), from 7.15 to 7.40 (mt, 6H: H 5 of the aromatic moiety in position 4 and aromatic H 6), 7.45 (broad d, J=9 Hz, 1H: 1'HJ, 7.50 (dd, J=9 and 4 Hz, 1H:1'HS), 7.80 (broad d, J = 4 Hz, 1H: 1'HJ, 8.40 (d,
J=10 Hz, 1H: NH 1), 8.73 (d, J=9.5 Hz, 1H: NH 6), 11.62 (s,
1H: OH).
AP/P/ 9 5 / 0 0 7 5 2
AP.00562
- 69 Using the fractions derived from the silica column described above which contain the new derivative of pristinamycin IH, 7 mg of 4e-methoxy-de(4ζdimethylamino)pristinamycin IH (mass spectrometry: M+H* =
826, are isolated by carrying out semi-preparative column chromatography as described above.
EXAMPLE 17: Preparation of 4e-fluoro-4f-methylde(4f-dimethylamino)pristinamycin IA.
C Strain SP92::pVRC508 is cultured in production medium using 60 Erlenmeyer flasks, as described in Example 3, with 1 ml of a 10 g/1 solution of (R,S)-3-fluoro-4methylphenylalanine, synthesized as in Example 34-5, in 0. IN sodium hydroxide solution being added at 16 h. At the end of 40 h of culture, the 1.8 litres of must recovered from the 60 Erlenmeyer flasks are extracted with 2 volumes of a mixture consisting of 66% 100 mM phosphate buffer, pH 2.9, and 34% acetonitrile, and then centrifuged. The supernatant is extracted with 2 times 0.5 volumes of dichloromethane. The chloromethylene phases are washed with water and then combined, dried over sodium sulphate and evaporated. The dry extract is taken up in 20 ml of dichloromethane and injected onto a silica (30 g) column which is mounted in dichloromethane and eluted successively with plateaus of from 0 to 10% methanol in dichloromethane.
The fractions containing the new derivative of pristinamycin IA are combined and evaporated. 15 mg of dry residue are obtained. This residue is taken up in 3 ml of a mixture consisting of 60% water and 40% acetonitrile and injected onto a semi-preparative Nucleosil 7μ C8 10x250 mm (Macherey Nagel) column, which is eluted with a mixture consisting of 55% 100 mM phosphate buffer, pH 2.9, and 45% acetonitrile. The fractions containing the new pristinamycin are combined and extracted with one volume of
AP/P/ 9 5 / 0 0 7 5 2 dichloromethane. The organic phase is washed with water, dried over sodium sulphate and then evaporated. 9 mg of 4efluoro-4 f-methyl-de (4 f-dimethylamino) pristinamycin IA are obtained.
NMR spectrum: 1H (4 00 MHz, CDC13, δ in ppm, ref.
TMS): 0.60 (dd, J=16 and 5.5 Hz, 1H, 5 02) , 0.91 (t, J=7.5 Hz, 3H: CH3 2 γ) , 1.12 (mt, 1H: 3 02) , from 1.25 to 1.35 (mt, 1H: 3 γ2) , 1.33 (d, J=7.5 Hz, 3H: CH3 1 γ) , from 1.50 to 1.85 (mt, 3H: 3 γ3 and CH2 2 0), 2.02 (mt, 1H, 3 03) ,
2.13 (mt, 1H, 5 δ2) , 2.27 (s, 3H: ArCH3) , 2.36 (broad d,
J=16 Hz, 1H: 5 δ3) , 2.45 (d, J=16 Hz, 1H: 5 0J , 2,85 (dt, J=13 and 4.5 Hz, 1H: 5 e2) , 2.97 (dd, J=12.5 and 4.5 Hz,
1H: 4 02) , 3.23 (s, 3H: NCH3 4 ), 3.30 and 3.56 (2 mts, 1H each: CH2 3 δ), 3.37 (t, J=12.5 Hz, 1H: 4 03) , 4.55 (t,
J=7.5 Hz, 1H, 3a), 4.75 (broad dd, J=13 and 8 Hz, 1H: 5 ej , 4.83 (mt, 1H: 2a), 4.89 (broad d, J=10 Hz, 1H: la),
5.29 (dd, J=12.5 and 4.5 Hz, 1H: 4 a), 5.32 (broad d, J=5.5
Hz, 1H: 5 a), 5.89 (d J=9.5 Hz, 1H: 6 a), 5.92 (mt, 1H:
10), 6.49 (d, J=10 Hz, 1H: NH 2), 6.90 (mt, 2H: H 2 and H 6 of the aromatic moiety in position 4), 7.11 (t, J=8 Hz, 1H:
H 5 of the aromatic moiety in position 4), from 7.10 to
7.30 (mt, 5H: aromatic H 6), 7.43 (dd, J=8.5 and 1 Hz, 1H: l'H«), 7.49 (dd, J=8.5 and 4.5 Hz, 1H: l'Hs), 7.75 (dd,
J=4.5 and 1 Hz, 1H: l'H6), 8.48 (d, J=10 Hz, 1H: NH 1),
8.70 (d, J=9.5 Hz, 1H: NH 6), 11.60 (s, 1H: OH).
AP/P/ 9 5 / 0 0 7 5 2
EXAMPLE 18: Preparation of 4J-ethylamino-de(4fdimethylamino) pristinamycin IA
Strain SP92::pVRC508 is cultured in production medium using 50 erlenmeyer flasks, as described in Example
3, with 1 ml of a 20 g/1 solution of (R,S)-4ethylaminophenylalanine dihydrochloride, synthesized as in Example 33, in 0. IN sodium hydroxide solution being added at I6h. At the end of 40h of culture, the 1.5 litres of must recovered from the 50 erlenmeyer flasks are extracted with 2 volumes of a mixture of 66% 100 mM phosphate buffer,
AP. 00562
- 71 pH 2.9, and 34% acetonitrile, and then centrifuged. The supernatant is extracted with 2 times 0.5 volumes of dichloromethane. The chloromethylene phases are washed with water and then combined, dried over sodium sulphate and evaporated. The dry extract is taken up in 20 ml of dichloromethane and injected onto a silica column (30 g) which is mounted in dichloromethane and eluted successively with plateaus of from 0 to 10% methanol in dichloromethane. The fractions containing 4f-ethylamino-de(4f10 dimethylamino)pristinamycin IA are combined and evaporated. The dry residue is taken up in 7 ml of a mixture consisting of 65% water and 35% acetonitrile and injected onto a semipreparative Nucleosil 7μ C8 10x250 mm (Macherey Nagel) column, which is eluted with a mixture consisting of 60%
100 mM phosphate buffer, pH 2.9, and 40% acetonitrile. The fractions containing 4f-ethylamino-de(4fdimethylamino)pristinamycin IA are combined and extracted with one volume of dichloromethane. The organic phase is washed with water, dried over sodium sulphate and then evaporated. 10 mg of 4^-ethylamino-de(4fdimethylamino)pristinamycin IA are obtained.
NMR spectrum. :H (400 MHz, CDC13, δ in ppm, ref. TMS) : 0.72 (dd, J = 16 and 6 Hz, 1H: 1H of the CH2 in 5 β) ; 0.90 (t, J = 7.5 Hz, 3H: CH3 in 2 γ) ; 1.15 (mt, 1H: 1H of the CH2 in 3 β); from 1.20 to 1.40 (mt, 1H: 1H of the CH2 in 3 γ); 1.27 (t, J = 7.5 Hz, 3H: CH3 of the ethyl); 1.33 (d, J = 7 Hz, 3H: CH3 in 1 γ) ; from 1.50 to 1.65 (mt, 1H: the other H of the CH2 in 3 γ) ; 1.60 and 1.74 (2 mts, 1H each: CH2 in 2 0); 2.02 (mt, 1H: the other H of CH2 in 3
β); 2.21 and 2.33 (respectively, mt and broad d, J = 16.5 Hz, 1H each: CH2 in 5 δ) ,- 2.40 (d, J = 16 Hz, 1H: the other H of the CH2 in 5 β); 2.82 (dt, J = 13 and 4.5 Hz, 1H: 1H of the CH2 in 5 e); 2.89 (dd, J = 12 and 4 Hz, 1H: 1H of the CH2 in 4 β) ; 3.10 (mt, 2H: NCH2 of the ethyl); from
3.20 to 3.35 (mt, 1H: 1H of the CH2 in 3 δ); 3.26 (s, 3H:
NCH3) ; 3.31 (t, J = 12 Hz, 1H: the other H of the CH2 in 4
AP/P/ 95/00752
β) ; 3.54 (mt, 1Η: the other H of the CH2 in 3 δ); 3.67 (unres. comp., 1H: NH); 4.56 (dd, J = 6.5 and 7 Hz, 1H: 3
a) ; 4.75 (broad dd, J = 13 and 8 Hz, 1H: the other H of the
CH2 in 5 e); 4.84 (mt, 1H: 2 a); 4.90 (broad d, J = 10 Hz,
1H : 1 a) ; 5.24 (dd, J = 12 and 4 Hz, 1H: 4 a); 5.32 (broad d, J = 6 Hz, 1H: 5 a); 5.88 (d, J = 9.5 Hz, 1H : 6 a); 5.90 (mt, 1H : 1 β); 6.52 (d, J = 8 Hz, 3H : NH in 2 and aromatic H in 4 e) ; 7.00 (d, J = 8 Hz, 2H : aromatic H in 4 δ); from 7.10 to 7.35 (mt, 5H: aromatic H in 6); 7.46 (limiting AB, 2H: l'H« and 1'Hs) ; 7.84 (dd, J = 4 and 1 Hz, 1H: l'H6); 8.45 (d, J = 10 Hz, 1H: NH in 1); 8.77 (d, J =
C 9.5 Hz, 1H: NH in 6); 11.65 (s, 1H: OH).
EXAMPLE 19: Preparation of 4f-diethylamino-de(4fdimethylamino)pristinamycin IA
Strain SP92::pVRC508 is cultured in production medium using 50 erlenmeyer flasks, as described in Example 3, with 1 ml of a 20 g/1 solution of (R,S)-4diethylaminophenylalanine dihydrochloride, synthesized as in Example 33, in 0.1N sodium hydroxide solution being added at 16h. At the end of 40h of culture, the 1.5 litres of must recovered from the 50 erlenmeyer flasks are extracted with 2 volumes of a mixture of 66% 100. mM phosphate buffer, pH 2.9, and 34% acetonitrile, and then centrifuged. The supernatant is extracted with 2 times 0.5 volumes of dichloromethane. The chloromethylene phases are washed with water and then combined, dried over sodium sulphate and evaporated. The dry extract is taken up in ’ 20 ml of dichloromethane and injected onto a silica column (30 g) which is mounted in dichloromethane and eluted successively with plateaus of from 0 to 10% methanol in dichloromethane. The fractions containing 4f-diethylaminode(4ζ-dimethylamino)pristinamycin IA are combined and evaporated. The dry residue is taken up in 7 ml of a mixture consisting of 60% water and 40% acetonitrile and injected in two portions onto a semi-preparative Nucleosil
AP/P/ 95/00752
AP.00562
- 73 7μ C8 10x250 mm (Macherey Nagel) column, which is eluted with a mixture consisting of 68% 100 mM phosphate buffer, pH 2.9, and 32% acetonitrile. The fractions containing 4fdiethylamino-de(4f-dimethylamino)pristinamycin IA are combined and extracted with one volume of dichloromethane. The organic phase is washed with water, dried over sodium sulphate and then evaporated. 50 mg of 4f-diethylaminode(4f-dimethylamino)pristinamycin IA are obtained.
NMR spectrum. *H (4 00 MHz, CDC13, δ in ppm, ref.
TMS) : 0.65 (dd, J = 16 and 6 Hz, 1H: 1H of the CH2 in 5 β) ;
0.90 (t, J = 7.5 Hz, 3H: CH3 in 2 γ); 1.14 (t, J = 7 Hz,
6H: CH3 of the ethyl); 1.15 (mt, 1H: 1H of the CH2 in 3 β) ;
1.26 (mt, 1H: 1H of the CH2 in 3 γ); 1.32 (d, J = 6.5 Hz, 3H: CH3 in 1 γ); 1.55 (mt, 1H: the other H of the CH2 in 3
γ) ; 1.63 and 1.75 (2 mts, 1H each: CH2 in 2 β): 2.02 (mt, 1H: the other H of the CH2 in 3 β}; 2.22 and 2.31 (respectively, mt and broad d, J = 16.5 Hz, 1H each: CH2 in 5 δ); 2.37(d, J=16 Hz, 1H: the other H of the CH2 in 5 β) ; 2.80 (dt, J = 13 and 4.5 Hz, 1H: 1H of the CH2 in 5 e) ;
2.89 (dd, J = 12.5 and 4 Hz, 1H : 1H of the CH2 in 4 β) ; from 3.20 to 3.40 (mt, 6H: NCH2 of the ethyl - 1H of the CH2 in 3 δ and the other H of the CH2 in 4 β) ; 3.27 (s, 3H: NCH3) ; 3.55 (mt, 1H: the other H of the CH2 in 3 δ); 4.58 (dd, J = 8 and 6 Hz, 1H: 3 a); 4.76 (broad dd, J = 13 and
7.5 Hz, 1H: the other H of the CH2 in 5 e); 4.84 (mt, 1H: 2
a); 4.89 (dd, J = 10 and 1 Hz, 1H: 1 a) ; 5.21 (dd, J =
12.5 and 4 Hz, 1H: 4 a); 5.28 (broad d, J = 6 Hz, 1H : 5
a); 5.87 (d, J = 9.5 Hz, 1H: 6 a); 5.90 (mt, 1H: 1 β); 6.52 (d, J = 9.5 Hz, 1H : NH in 2); 6.60 (d, J = 8 Hz, 2H:
aromatic H in 4 e); 7,02 (d, J = 8 Hz, 2H: aromatic H in 4
δ); from 7.10 to 7.35 (mt, 5H: aromatic H in 6); 7.46 (limiting AB, 2H: l'H« and l'Hs); 7.88 (dd, J = 4.5 and 2.5 Hz, 1H: l'H6); 8.43 (d, J = 10 Hz, 1H: NH in 1); 8.76 (d, J = 9.5 Hz, 1H: NH in 6); 11.62 (s, 1H: OH).
AP/P/ 95/00752
EXAMPLE 20: Preparation of 4f-diallylamino-de(4f74 dimethylamino)pristinamycin IA
Strain SP92::pVRC508 is cultured in production medium using 94 erlenmeyer flasks, as described in Example 3, with 1 ml of a 20 g/1 solution of (R,S)-4_5 diallylaminophenylalanine dihydrochloride, synthesized as in Example 38-1, in water being added at 16h. At the end of 40h of culture, the 2.8 litres of must recovered from the 94 erlenmeyer flasks are extracted with 2 volumes of a mixture consisting of 66% 100 mM phosphate buffer, pH 2.9, and 34% acetonitrile, and then centrifuged. The supernatant is extracted with 2 times 0.5 volumes of dichloromethane. The chloromethylene phases are washed with water and then combined, dried over sodium sulphate and evaporated. The dry extract is taken up in 20 ml of dichloromethane and injected onto a silica (30 g) column which is mounted in dichloromethane and eluted successively with plateaus of from 0 to 10% methanol in dichloromethane. The fractions containing 4 f-diallylatnino-de (4 fdimethylamino)pristinamycin IA are combined and evaporated.
The dry residue is taken up in 7 ml of a mixture consisting of 60% water and 40% acetonitrile and injected onto a semipreparative Nucleosil 7μ C8 10x250 mm (Machery Nagel) column, which is eluted with a mixture consisting of 52%
100 mM phosphate buffer, pH 2.9, and 48% acetonitrile. The fractions containing 4f-diallylamino-de(4fdimethylamino)pristinamycin IA are combined and extracted with one volume of dichloromethane. The organic phase is washed with water, dried over sodium sulphate and then evaporated. 15 mg of 4f-diallylamino-de(4f30 dimethylamino)pristinamycin IA are obtained.
NMR spectrum. XH (400 MHz, CDC13, δ in ppm, ref.TMS): 0.55 (dd, J = 16 and 6 Hz, IH : IH of the CH2 in 5 β); 0.93 (t, J = 7.5 Hz, 3H: CH3 in 2 γ); 1.18 (mt, IH:
IH of the CH2 in 3 β) ; 1.25 (mt, IH : IH of the CH2 in 3
γ) ; 1.34 (d, J = 6.5 Hz, 3H: CH3 in 1 γ) ; 1.59 (mt, IH: the other H of the CH2 in 3 γ) ; 1.68 and 1.78 (2 mts, IH each:
&PIPI 95/00752
AP .00562
- 75 CH2 in 2 β) ; 2.04 (mt, IH: the other H of CH2 in 3 β) ; 2.25 and 2.34 (respectively, mt and broad d, J = 16.5 Hz, IH each: CH2 in 5 δ); 2.40 (d, J = 16 Hz, IH: the other H of the CH2 in 5/3); 2.83 (dt, J = 13 and 4.5 Hz, IH: IH of the ,5 CH2 in 5 c); 2.92 (dd, J = 12 and 4 Hz, IH: IH of the CH2 in 4 β); from 3.20 to 3.30 (mt, IH: IH of the CH2 in 3 δ) ; 3.29 (s, 3H: NCH3) ; 3.33 (t, J = 12 Hz, IH: the other H of the CH2 in 4 β) ; 3.57 (mt, IH: the other H of the CH2 in 3 δ); 3.93 (limiting AB, 4H: NCH2 of the allyl); 4.60 (dd, J = 8 and 6.5 Hz, IH: 3 a); 4.78 (broad dd, J = 13 and 7.5
Hz, IH: the other H of the CH2 in 5 e); 4.87 (mt, IH: 2a); 4.92 (dd, J = 10 and 1 Hz, IH: 1 a); from 5.10 to 5.25 (mt, 5H: 4 a and =CH2 of the allyl); 5.28 (broad d, J = 6 Hz,
IH: 5 a); 5.85 (mt, 2H: CH= of the allyl); 5.92 (d, J = 9.5
Hz, IH: 6 a); 5.94 (mt, IH : 1/3); 6.54 (d, J = 10 Hz, IH:
NH in 2); 6.65 (d, J = 8 Hz, 2H : aromatic H in 4 e); 7.05 (d, J = 8 Hz, 2H: aromatic H in 4 δ); from 7.10 to 7.35 (mt, 5H: aromatic H in 6); 7.51 (limiting AB, 2H: l'H4 and l'H5); 7.88 (dd, J = 4 and 2 Hz, IH: l'H6); 8.43 (d, J = 10
Hz, IH: NH in 1); 8.77 (d, J = 9.5 Hz, IH: NH in 6); 11.65 (S, IH : OH) .
EXAMPLE 21: Preparation of -allylethylaminode(4f-dimethylamino)pristinamycin IA
Strain SP92::pVRC508 is cultured in production medium using 26 erlenmeyer flasks, as described in Example 3, with 1 ml of a 20 g/1 solution of (R,S)-4allylethylaminophenylalanine dihydrochloride, synthesized as in Example 39-4, in 0.1N sodium hydroxide solution being added at 16h. At the end of 40h of culture, the 0.78 litre of must recovered from the 26 erlenmeyer flasks is extracted with 2 volumes of a mixture consisting of 66% 100 mM phosphate buffer, pH 2.9, and 34% acetonitrile, and then centrifuged. The supernatant is extracted with 2 times 0.5 volumes of dichloromethane. The chloromethylene phases are washed with water and then combined, dried over sodium
AP/P/ 9 5 / 0 0 7 5 2
- 76 sulphate and evaporated. The dry extract is taken up in ml of dichloromethane and injected onto a silica (30 g) column which is mounted in dichloromethane and eluted successively with plateaus of from 0 to 10% methanol in dichloromethane. The fractions containing 4fallylethylamino-de (4 f-dimethylamino)pristinamycin IA are combined and evaporated. The dry residue is taken up in 7 ml of a mixture consisting of 60% of water and 40% acetonitrile and injected onto a semi-preparative Nucleosil
7μ C8 10x250 mm (Macherey Nagel) column, which is eluted with a mixture consisting of 52% 100 mM phosphate buffer, pH 2.9, and 48% of acetonitrile. The fractions containing 4f-allylethylamino-de(4f-dimethylamino)pristinamycin IA are combined and extracted with one volume of dichloromethane.
The organic phase is washed with water, dried over sodium sulphate and then evaporated. 20 mg of 4f-allylethylaminode (4 f-dimethylamino) pristinamycin IA are obtained.
NMR spectrum. TH (400 MHz, CDC13, δ in ppm, ref. TMS) : 0.58 (dd, J = 16 and 6 Hz, 1H: 1H of CH2 in 5 β) ;
0.91 (t, J = 7.5 Hz, 3H: CH3 in 2 γ); 1.16 (t, J = 7 Hz,
3H: CH3 of the ethyl); 1.16 (mt, 1H: 1H of the CH2 in 3 β) ; 1.25 (mt, 1H: 1H of CH2 in 3 γ); 1.32 (d, J = 6.5 Hz, 3H:
CH3 in 1 γ) ; 1.54 (mt, 1H: the other H of the CH2 in 3 γ) ;
1.63 and 1.75 (2 mts, 1H each: CH2 in 2 β) ; 2.02 (mt, 1H:
the other H of the CH2 in 3 β}; 2.23 and 2.31 (respectively, mt and broad d, J = 16.5 Hz, 1H each: CH2 in 5 δ) ; 2.37 (d, J = 16 Hz, 1H: the other H of the CH2 in 5 β) ; 2.80 (dt, J = 13 and 4.5 Hz, 1H : 1H of CH2 in 5 c) ;
2.87 (dd, J = 12 and 4 Hz, 1H: 1H of the CH2 in 4 β) ; from
3.15 to 3.30 (mt, 1H: 1H of the CH2 in 3 δ); 3.26 (s, 3H:
NCH3) ; 3.30 (t, J = 12 Hz, 1H: the other H of CH2 in 4 β) ; 3.36 (mt, 2H: NCH2 of the ethyl); 3.54 (mt, 1H: the other H of the CH2 in 3 δ); 3.90 (limiting AB, 2H: NCH2 of the allyl); 4.57 (dd, J = 8 and 6 Hz, 1H: 3 a); 4.76 (broad dd,
5 J = 13 and 7.5 Hz, 1H: the other H of the CH2 in 5 e); 4.84 (mt, 1H: 2 a); 4.89 (dd, J = 10 and 1 Hz, 1H: 1 a); from
AP/P/ 9 5 / 0 0 7 5 2
AP . Ο Ο 5 6 2
- 77 5.05 to 5.20 (mt, 3Η: 4 a and =CH2 of the allyl); 5.27 (broad d, J = 6 Hz, IH : 5 a); 5.83 (mt, IH: CH= of the allyl); 5.88 (d, J = 9.5 Hz, IH: 6 a); 5.91 (mt, IH: 1 β) ; 6.50 (d, J = 10 Hz, IH: NH in 2); 6.60 (d, J = 8 Hz, 2H:
aromatic H in 4 e); 7.02 (d, J = 8 Hz, 2H: aromatic H in 4 δ); from 7.10 to 7.35 (mt, 5H: aromatic H in 6); 7.47 (limiting AB, 2H: l'H4 and l'Hs); 7.88 (dd, J = 4 and 2 Hz, IH: l'He); 8.41 (d, J = 10 Hz, IH: NH in 1); 8.75 (d, J =
9.5 Hz, IH: NH in 6); 11.62 (s, IH: OH).
EXAMPLE 22: Preparation of the -ethylpropylamino-de(4f-dimethylamino)pristinamycin IA
Strain SP92::pVRC508 is cultured in production medium using 60 erlenmeyer flasks, as described in Example 3, with 1 ml of a 20 g/1 solution of (R,S)-415 ethylpropylaminophenylalanine dihydrochloride, synthesized as in Example 39-6, in 0. IN sodium hydroxide solution being added at 16h. At the end of 40h of culture, the 1.8 litre of must recovered from the 60 erlenmeyer flasks is extracted with 2 volumes of a mixture consisting of 66% 100 mM phosphate buffer, pH 2.9, and 34% acetonitrile, and then centrifuged. The supernatant is extracted with 2 times 0.5 volumes of dichloromethane. The chloromethylene phases are washed with water and then combined, dried over sodium sulphate and evaporated. The dry extract is taken up in
20 ml of dichloromethane and injected onto a silica (30 g) column which is mounted in dichloromethane and eluted successively with plateaus of from 0 to 10% methanol in ' dichloromethane. The fractions containing 4fethylpropylamino-de(4f-dimethylamino)pristinamycin IA are combined and evaporated. The dry residue is taken up in 7 ml of a mixture consisting of 60% of water and 40% acetonitrile and injected onto a semi-preparative Nucleosil Ίμ C8 10x250 mm (Macherey Nagel) column, which is eluted with a mixture consisting of 63% 100 mM phosphate buffer, pH 2.9, and 37% of acetonitrile. The fractions containing
AP/P/ 9 5 / 0 0 7 5 2
4f-ethylpropylamino-de(4f-dimethylamino)pristinamycin IA are combined and extracted with one volume of dichloromethane. The organic phase is washed with water, dried over sodium sulphate and then evaporated. 16 mg of
4f-ethylpropylamino-de(4ζ-dimethylamino)pristinamycin IA are obtained.
NMR spectrum. 1H (400 MHz, CDC13, δ in ppm, ref. TMS): 0.67 (dd, J = 16 and 6 Hz, IH: IH of the CH2 in 5 0) ; 0.91 (t, J = 7.5 Hz, 3H: CH3 in 2 γ); 0.95 (t, J = 7.5 Hz,
3H: CH3 of propyl); 1.14 (t, J = 7 Hz, 3H : CH3 of the ethyl); 1.15 (mt, IH: IH of the CH2 in 3 0) ; 1.25 (mt, IH: IH of the CH2 in 3 γ); 1.33 (d, J = 7 Hz, 3H : CH3 in 1 γ); from 1.45 to 1.65 (mt, 3H: the other H of the CH2 in 3 γ and CH2 propyl); 1.63 and 1.75 (2 mts, IH each: CH2 in 2
0); 2.02 (mt, IH: the other H of the CH2 in 3 0); 2.23 and
2.33 (respectively, mt and broad d, J = 16.5 Hz, IH each: CH2 in 5 δ); 2.37 (d, J = 16 Hz, IH: the other H of the CH2 in 5 0); 2.80 (dt, J = 13 and 5 Hz, IH: IH of the CH2 in 5
e); 2.89 (dd, J = 12 and 4 Hz, IH: IH of the CH2 in 4 0) ;
from 3.10 to 3.25 (mt, 3H: IH of the CH2 in 3 δ and NCH2 of the propyl); 3.26 (s, 3H: NCH3) ; from 3.25 to 3.40 (mt, 2H: NCH2 of the ethyl); 3.34 (t, J = 12 Hz, IH: the other H of the CH2 in 4 0); 3.54 (mt, IH: the other H of the CH2 in 3 δ); 4.57 (dd, J = 7.5 and 6 Hz, IH: 3a); 4.76 (broad dd, J = 13 and 7.5 Hz, IH: the other H of the CH2 in 5 e); 4.84 (mt, IH: 2 a); 4.89 (dd, J = 10 and 1 Hz, IH : la); 5.21 (dd, J = 12 and 4 Hz, IH: 4 a); 5.28 (broad d, J = 6 Hz,
IH: 5 a); 5.88 (d, J = 9.5 Hz, IH: 6 a); 5.91 (mt, IH: 1 0); 6.48 (d, J = 10 Hz, IH: NH in 2); 6.60 (d, J = 8 Hz,
2H: aromatic H in 4 e); 7.03 (d, J = 8 Hz, 2H: aromatic H in 4 δ); from 7.10 to 7.35 (mt, 5H: aromatic H in 6); 7.47 (limiting AB, 2H: l'H, and l'Hs); 7.89 (mt, IH: l'H6) ; 8.42 (d, J = 10 Hz, IH : NH in 1); 8.76 (d, J = 9.5 Hz, IH: NH in 6); 11.62 (s, IH: OH).
AP/P/ 9 5 / 0 0 7 5 2
EXAMPLE 23: Preparation of the 4f-trifluoroAP. Ο Ο 5 6 2
methoxy-de(4 J-dimethylamino)pristinamycin IA
Strain SP92::pVRC508 is cultured in production medium using 60 erlenmeyer flasks, as described in Example 3, with 1 ml of a 20 g/1 solution of (R,S)-4-O5 trifluoromethyltyrosine hydrochloride, synthesized as in Example 34-8, in water being added at 16h. At the end of 40h of culture, the 1.8 litres of must recovered from the 60 erlenmeyer flasks is extracted with 2 volumes of a mixture consisting of 66% 100.mM phosphate buffer, pH 2.9, and 34% acetonitrile, and then centrifuged. The supernatant is extracted with 2 volumes of dichloromethane. The chloromethylene phases are washed with water and then combined, dried over sodium sulphate and evaporated. The dry extract is taken up in dichloromethane and injected onto a silica (30 g) column which is mounted in dichloromethane and eluted successively with plateaus of from 0 to 10% methanol in dichloromethane. The fractions containing 4f-trifluoromethoxy-de(4fdimethylamino)pristinamycin IA are combined and evaporated.
The dry residue is taken up in 7 ml of a mixture consisting of 60% of water and 40% acetonitrile and injected in two portions onto a semi-preparative Nucleosil 7μ C8 10x250 mm (Macherey Nagel) column, which is eluted with a mixture consisting of 60% 100 mM phosphate buffer, pH 2.9, and 40% of acetonitrile. The fractions containing 4ftrifluoromethoxy-de(4f-dimethylamino)pristinamycin IA are combined and extracted with one volume of dichloromethane. •The organic phase is washed with water, dried over sodium sulphate and then evaporated. 46.5 mg of 4f30 trifluoromethoxy-de(4f-dimethylamino)pristinamycin IA are obtained.
NMR spectrum. :H (400 MHz, CDC13, δ in ppm, ref. TMS): 0.77 (dd, J = 16 and 5.5 Hz, 1H: 1H of the CH2 in 5 /0); 0.92 (t, J = 7.5 Hz, 3H: CH3 in 2 γ) ; 1.08 (mt, 1H: 1H of the CH2 in 3 β) ; from 1.30 to 1.40 (mt, 1H: 1H of the CH2 in 3 γ) ; 1.33 (d, J = 7 Hz, 3H: CH3 in 1 γ) ; from 1.55
AP/P/ 9 5 / 0 0 7 52 to 1.70 (mt, IH: the other H of the CH2 in 3 γ); 1.65 and
1.76 (2 mts, IH each: CH2 in 2 /3); 2.02 (mt, IH: the other
H of the CH2 in 3 /3); 2.11 and 2.40 (respectively, mt and broad d, J = 16.5 Hz, IH each: CH2 in 5 6); 2.54 (d, J = 16 _5 Hz, IH: the other H of the CH2 in 5 /3); 2.88 (dt, J = 13 and 4 Hz, IH: IH of the CH2 in 5 ¢); 3.08 (dd, J = 12 and 5 Hz, IH: IH of the CH2 in 4 β', ; 3.22 (s, 3H: NCHJ ; from
3.30 to 3.45 (mt, IH: IH of the CH2 in 3 6); 3.39 (t, J =
Hz, IH: the other H of the CH2 in 4 β) ; 3.59 (mt, IH:
the other H of the CH2 in 3 δ); 4.53 (t, J = 7.5 Hz, IH : 3 a); 4.75 (broad dd, J = 13 and 8 Hz, IH: the other H of the CH2 in 5 e); 4.85 (mt, IH: 2 a); 4.89 (dd, J = 10 and 1.5 Hz, IH: 1 at); 5.35 (broad d, J = 5.5 Hz, IH: 5 a); 5.41 (dd, J = 12 and 5 Hz, IH: 4 a); 5.92 (d, J = 10 Hz, IH : 6
a); 5.93 (mt, IH: 1 /3); 6.53 (d, J = 9.5 Hz, IH: NH in 2); from 7.15 to 7.35 (mt, 5H: aromatic H in 6); 7.16 (d, J = 8 Hz, 2H: aromatic H in 4 e); 7.26 (d, J = 8 Hz, 2H: aromatic H in 4 δ); 7.37 (dd, J = 8.5 and 4 Hz, IH: l'Hs); 7.42 (dd, J = 8.5 and 1.5 Hz, IH: 1'HJ; 7.70 (dd, J = 4 and 1.5 Hz,
IH: l'H6); 8.37 (d, J = 10 Hz, IH: NH in 1); 8.75 (d, J =
Hz, IH: NH in 6); 11.66 (s, IH: OH).
EXAMPLE 24: Preparation of 4f-allyloxy-de(4fdimethylamino) pristinamycin Ix
Strain SP92::pVRC508 is cultured in production.
medium using 90 erlenmeyer flasks, as described in Example 3, with 1 ml of a 20 g/1 solution of (S)-4-0-allyltyrosine hydrochloride, synthesized as in Example 33, in 0.1N hydrochloric acid being added at 16h. At the end of 40h of culture, the 2.7 litres of must recovered from the 90 erlenmeyer flasks is extracted with 2 volumes of a mixture consisting of 66% 100 mM phosphate buffer, pH 2.9, and 34% acetonitrile, and then centrifuged. The supernatant is extracted with 2 times 0.5 volumes of dichloromethane. The chloromethylene phases are washed with water and then combined, dried over sodium sulphate and evaporated. The
AP/P/ 9 5 / 0 0 7 5 2
AP . 0 0 5 6 2
- 81 dry extract is taken up in 20 ml of dichloromethane and injected onto a silica (30 g) column which is mounted in dichloromethane and eluted successively with plateaus of from 0 to 10% methanol in dichloromethane. The fractions _5 containing 4f-allyloxy-de(4f-dimethylamino)pristinamycin IA are combined and evaporated. The dry residue is taken up in 7 ml of a mixture consisting of 60% of water and 40% acetonitrile and injected onto a semi-preparative Nucleosil 7μ C8 10x250 mm (Macherey Nagel) column, which is eluted with a mixture consisting of 52% 100 mM phosphate buffer, pH 2.9, and 48% of acetonitrile. The fractions containing 4f-allyloxy-de(4ζ-dimethylamino)pristinamycin IA are combined and extracted with one volume of dichloromethane. The organic phase is washed with water, dried over sodium sulphate and then evaporated. 29 mg of 4f-allyloxy-de(4fdimethylamino)pristinamycin IA are obtained.
NMR spectrum. :H (400 MHz, CDC13, δ in ppm, ref. TMS) :0.63 (dd, J = 16 and 6 Hz, ΙΗ: 1H of CH2 in 5 β) ; 0.91 (t, J = 7.5 Hz, 3H: CH3 in 2 7); 1.13 (mt, ΙΗ: 1H of CH2 in
3 β) ; 1.29 (mt, 1H: 1H of CH2 in 3 7); 1.33 (d, J = 6.5 Hz, 3H: CH3 in 1 7); 1.57 (mt, 1H: the other H of the CH2 in 3 7); 165 and 1.74 (2 mts, 1H each: CH2 in 2 β); 2.02 (mt,
1H: the other H of the CH2 in 3 β) ; 2.14 and 2.34 (respectively, mt and broad d, J = 16.5 Hz, 1H each: CH2 in
5 δ); 2.43 (d, J = 16 Hz, 1H: the other H of the CH2 in 5 β) ; 2.85 (dt, J = 13 and 4 Hz, ΙΗ: 1H of the CH2 in 5 c) ; 2.95 (dd, J = 12 and 4 Hz, ΙΗ: 1H of the CH2 in 4 β) ; 3.25 (s, 3H: NCH3) ; 3.33 (mt, ΙΗ: 1H of the CH2 in 3 δ): 3.36 (t, J = 12 Hz, 1H: the other H of the CH2 in 4 β) ; 3.56 (mt, 1H: the other H of the CH2 in 3 δ); 4.51 (limiting AB, 2H: OCH2 of the allyl); 4.56 (t, J = 7.5 Hz, 1H: 3 a); 4.75 (broad dd, J = 13 and 8 Hz, 1H: the other H of the CH2 in 5 e); 4.84 (mt, 1H: 2 a); 4.88 (dd, J = 10 and 1 Hz, 1H: 1 Of); 5.27 (dd, J = 12 and 4 Hz, 1H: 4 a); 5.32 (broad d, J =
6 Hz, 1H: 5 a}; 5.30 and 5.40 (respectively, mt and dd, J = 17 and 1.5 Hz, 1H each: =CH2 of the allyl); 5.89 (d, J =
AP/P/ 9 5 / 0 0 7 5 2
Γ'
o. 20 θ
9.5 Hz, IH: 6 a); 5.91 (mt, IH: 1 )3) ; 6.02 (mt, IH: CH= of the allyl); 6.50 (d, J = 10 Hz, IH: NH in 2); 6.85 (d, J = 8 Hz, 2H: aromatic H in 4 e) ; 7.12 (d, J = 8 Hz, 2H: aromatic H in 4 δ); from 7.10 to 7.35 (mt, 5H: aromatic H in 6); 7.45 (dd, J = 8.5 and 1.5 Hz, IH: 1Ή<) ; 7.57 (dd, J = 8.5 and 4 Hz, IH: l'Hs) ; 7.77 (dd, J = 4 and 1.5 Hz, IH: l'Hj); 8.41 (d, J = 10 Hz, IH: NH in 1); 8.74 (d, J = 9.5 Hz, IH: NH in 6); 11.63 (s, IH: OH).
EXAMPLE 25: Preparation of 4f-ethoxy-de(4fdimethylamino)pristinamycin IA
Strain SP92::pVRC508 is cultured in production medium using 90 erlenmeyer flasks, as described in Example 3, with 1 ml of a 20 g/1 solution of (S)-4-O-ethyltyrosine hydrochloride, synthesized as in Example 33, in 0. IN hydrochloric acid being added at 16h. At the end of 4Oh of culture, the 2.7 litres of must recovered from the 90 erlenmeyer flasks is extracted with 2 volumes of a mixture consisting of 66% 100 mM phosphate buffer, pH 2.9, and 34% acetonitrile, and then centrifuged. The supernatant is extracted with 2 times 0.5 volumes of dichloromethane. The chloromethylene phases are washed with water and then combined, dried over sodium sulphate and evaporated. The dry extract is taken up in 20 ml of dichloromethane and injected onto a silica (30 g) column which is mounted in dichloromethane and eluted successively with plateaus of .from 0 to 10% methanol in dichloromethane. The fractions containing 4f-ethoxy-de(4f-dimethylamino)pristinamycin IA are combined and evaporated. The dry residue is taken up in 7 ml of a mixture consisting of 60% of water and 40% acetonitrile and injected onto a semi-preparative Nucleosil 7μ C8 10x250 mm (Macherey Nagel) column, which is eluted with a mixture consisting of 52% 100 mM phosphate buffer, pH 2.9, and 48% of acetonitrile. The fractions containing 4f-ethoxy-de(4f-dimethylamino)pristinamycin IA are combined
AP/P/ 95/00752
AP . 0 0 5 6 2
- 83 and extracted with one volume of dichloromethane. The organic phase is washed with water, dried over sodium sulphate and then evaporated. 29 mg of 4f-ethoxy-de(4fdimethylamino)pristinamycin IA are obtained.
NMR spectrum. 3H (400 MHz, CDC13, δ in ppm, ref.
TMS): 0.64 (dd, J = 16 and 5.5 Hz, IH: IH of the CH2 in 5 /3); 0.90 (t, J = 7.5 Hz, 3H: CH3 in 2 γ) ; 1.12 (mt, IH: IH of the CH2 in 3 /3); 1.25 (mt, IH: IH of the CH2 in 3 γ) ;
1.33 (d, J = 7 Hz, 3H: CH3 in 1 γ) ; 1.42 (t, J = 7 Hz, 3H:
CH3 of the ethyl); 1.57 (mt, IH: the other H of the CH2 in 3 γ) ; 1.63 and 1.74 (2 mts, IH each: CH2 in 2 /3); 2.02 (mt, IH: the other H of the CH2 in 3 /3); 2.16 and 2.35 (respectively mt and broad d, J = 16.5 Hz, IH each: CH2 in
δ); 2.43 (d, J = 16 Hz, IH: the other H of the CH2 in 5 /3) ; 2.83 (dt, J = 13 and 4 Hz, IH: IH of the CH2 in 5 c);
2.93 (dd, J = 12 and 4 Hz, IH: IH of the CH2 in 4 /3) ; from 3.15 to 3.30 (mt, IH: IH of the CH2 in 3 δ); 3.24 (s, 3H: NCH3) ; 3.3 5 (t, J = 12 Hz, IH: the other H of the CH2 in 4 /3); 3.55 (mt, IH: the other H of the CH2 in 3 δ); 3.95 (limiting AB, 2H: OCH2 of the ethyl); 4.56 (dd, J = 7.5 and
Hz, IH: 3 a); 4.75 (broad dd, J = 13 and 8 Hz, IH: the other H of the CH2 in 5 e); 4.84 (mt, IH: 2 a); 4.87 (dd, J = 10 and 1 Hz, IH: 1 a); 5.26 (dd, J = 12 and 4 Hz, IH: 4 a); 5.32 (broad d, J » 5.5 Hz, IH: 5 a); 5.88 (d, J = 10
Hz, IH: 6 a); 5.92 (mt, IH: 1 /3); 6.48 (d, J = 10 Hz, IH:
NH in 2); 6.83 (d, J = 8 Hz, 2H: aromatic H in 4 e); 7.10 (d, J = 8 Hz, 2H: aromatic H in 4 δ); from 7.10 to 7.35 (mt, 5H: aromatic H in 6); 7.44 (dd, J = 8.5 and 1.5 Hz,
IH: l'H4); 7.57 (dd, J = 8.5 and 4.5 Hz, IH: l'H5); 7.77 (dd, J = 4.5 and 1.5 Hz, IH: l'He); 8.38 (d, J = 10 Hz, IH: NH in 1); 8.75 (d, J = 10 Hz, IH : NH in 6); 11.60 (s, IH: OH) .
EXAMPLE 26: Preparation of 4f-(2-chloro-ethoxy)de(4f-dimethylamino)pristinamycin IA
Strain SP92::pVRC508 is cultured in production
AP/P/ 9 5 / 0 0 7 5 2 medium using 60 erlenmeyer flasks, as described in Example 3, with 1 ml of a 20 g/1 solution of (S)-4-0-(2chloroethyl) tyrosine hydrochloride, synthesized as in Example 42-1, in water being added at 16h. At the end of .5 40h of culture, the 1.8 litres of must recovered from the erlenmeyer flasks is extracted with 2 volumes of a mixture consisting of 66% 100 mM phosphate buffer, pH 2.9, and 34% acetonitrile, and then centrifuged. The supernatant is extracted with 2 times 0.5 volumes of dichloromethane.
The chloromethylene phases are washed with water and then combined, dried over sodium sulphate and evaporated. The dry extract is taken up in 20 ml of dichloromethane and injected onto a silica (30 g) column which is mounted in dichloromethane and eluted successively with plateaus of from 0 to 10% methanol in dichloromethane. The fractions containing 4f-(2-chloroethoxy) -de(4f-dimethylamino)pristinamycin IA are combined and evaporated. The dry residue is taken up in 7 ml of a mixture consisting of 60% of water and 40% acetonitrile and injected onto a semi20 preparative Nucleosil 7μ C8 10x250 mm (Macherey Nagel) column, which is eluted with a mixture consisting of 60%
100 mM phosphate buffer, pH 2.9, and 40% of acetonitrile. The fractions containing 4f-(2-chloroethoxy)-de(4fdimethylamino)pristinamycin IA are combined and extracted with one volume of dichloromethane. The organic phase is. washed with water, dried over sodium sulphate and then evaporated. 3.2 mg of 4f-(2-chloroethoxy)-de(4f-dimethyl.amino)pristinamycin IA are obtained.
NMR spectrum. *H (400 MHz, CDC13, δ in ppm, ref.
TMS): 0.66 (dd, J = 16 and 5.5 Hz, IH: IH of the CH2 in 5 β) ; 0.91 (t, J = 7.5 Hz, 3H: CH3 in 2 γ) ; 1.13 (mt, IH: IH of the CH2 in 3 β) ; 1.28 (mt, IH: IH of the CH2 in 3 γ) ;
1.33 (d, J = 7 Hz, 3H: CH3 in 1 γ) ; 1.57 (mt, IH: the other H of the CH2 in 3 γ) ; 1.66 and 1.76 (2 mts, IH each: CH2 in
2 β); 2.02 (mt, IH: the other H of the CH2 in 3 β); 2.16 and 2.37 (respectively, mt and broad d, J = 16.5 Hz, IH
AP/P/ 9 5 / 0 0 7 5 2
AP · ο Ο 5 6 2
- 85 each: CH2 in 5 δ) : 2.47 (d, J = 16 Hz, 1H: the other H of the CH2 in 5 β) ; 2.86 (dt, J = 13 and 4 Hz, 1H: 1H of the CH2 in 5 c); 2.95 (dd, J = 12 and 4 Hz, 1H: 1H of the CH2 in 4 β) ; 3.23 (s, 3H: NCH3) ; 3.32 (mt, 1H: 1H of the CH2 in .5 3 δ) ; 3.3 7 (t, J = 12 Hz, 1H: the other H of the CH2 in 4
β) ; 3.57 (mt, 1H: the other H of the CH2 in 3 δ); 3.82 (t,
J = 6 Hz, 2H: CHjCl); 4.19 (limiting AB, 2H: OCH2 of the ethyl); 4.55 (dd, J = 7.5 and 7 Hz, 1H: 3 a); 4.15 (broad dd, J = 13 and 8 Hz, 1H: the other H of the CH2 in 5 e);
4.84 (mt, 1H: 2 a); 4.87 (broad d, J = 10 Hz, 1H: 1 a);
5.28 (dd, J = 12 and 4 Hz, 1H: 4 a); 5.32 (broad d, J = 5.5 Hz, 1H : 5a); 5.88 (d, J = 10 Hz, 1H: 6a); 5.90 (mt, 1H :
β) ; 6.50 (d, J = 10 HZ, 1H: NH in 2); 6.86 (d, J = 8 Hz, 2H: aromatic H in 4 e); 7.13 (d, J = 8 Hz, 2H: aromatic H in 4 δ); from 7.10 to 7.35 (mt, 5H: aromatic H in 6); 7.45 (limiting AB, 2H: l'H, and l'Hs); 7.75 (dd, J = 4 and 2 Hz, 1H: l'Hj); 8.38 (d, J = 10 Hz, 1H: NH in 1); 8.74 (d, J =
Hz, 1H: NH in 6); 11.62 (s, 1H: OH).
EXAMPLE 27: Preparation of 4f-acetyl-de(4f20 dimethylamino)pristinamycin IA
Strain SP92::pVRC508 is cultured in production medium using 60 erlenmeyer flasks, as described in Example G 3, with 1 ml of a 20 g/1 solution of (S)-4acetylphenylalanine, synthesized as in Example 33, in 0.1N sodium hydroxide solution being added at 16h. At the end of 40h of culture, the 1.8 litres of must recovered from the 60 erlenmeyer flasks is extracted with 2 volumes of a mixture consisting of 66% 100 mM phosphate buffer, pH 2.9, and 34% acetonitrile, and then centrifuged. The supernatant is extracted with 2 times 0.5 volumes of dichloromethane. The chloromethylene phases are washed with water and then combined, dried over sodium sulphate and evaporated. The dry extract is taken up in 20 ml of dichloromethane and injected onto a silica (30 g) column which is mounted in dichloromethane and eluted successively with plateaus of
AP/P/ 9 5 / 0 0 7 5 2
from 0 to 10% methanol in dichloromethane. The fractions containing 4f-acetyl-de(4f-dimethylamino)pristinamycin IA are combined and evaporated. The dry residue is taken up in 7 ml of a mixture consisting of 60% of water and 40%
-5 acetonitrile and injected onto a semi-preparative Nucleosil Ίμ C8 10x250 mm (Macherey Nagel) column, which is eluted with a mixture consisting of 60% 100 mM phosphate buffer, pH 2.9, and 40% of acetonitrile. The fractions containing 4ζ-acetyl-de(4ζ-dimethylamino)pristinamycin IA are combined and extracted with one volume of dichloromethane. The organic phase is washed with water, dried over sodium sulphate and then evaporated. 4.2 mg of 4f-acetyl-de(4fdimethylamino)pristinamycin IA are obtained.
NMR spectrum. *H (400 MHz, CDC13, δ in ppm, ref.
TMS): 0.73 (dd, J = 16 and 6 Hz, 1H: 1H of the CH2 in 5 β) ; 0.93 (t, J = 7.5 Hz, 3H: CH3 in 2 γ) ; 1.12 (mt, 1H: 1H of the CH2 in 3 β); from 1.25 to 1.45 (mt, 1H: 1H of the CH2 in 3 7); 1.33 (d, J = 7 Hz, 3H: CH3 in 1 7); 1.62 (mt, 1H: the other H of the CH2 in 3 7); from 1.60 to 1.85 (mt, 2H:
CH2 in 2 β); 2.02 (mt, 1H: the other H of the CH2 in 3 β) ; 2.20 and 2.42 (respectively, mt and broad d, J = 16.5 Hz,
1H each: CH2 in 5 δ); 2.52 (d, J = 16 Hz, 1H: the other H of the CH2 in 5 β) ; 2.60 (s, 3H: ArCOCHj ; 2.88 (dt, J = 13 and 4.5 Hz, 1H: 1H of CH2 in 5 e); 3.13 (dd, J = 13.5 and
5.5 Hz, 1H: 1H of the CH2 in 4 β) ; 3.21 (s, 3H: NCH3) ; from
3.30 to 3.50 (mt, 1H: the other H of the CH2 in 4 β); from
3.30 to 3.50 and 3.63 (2 mts, 1H each: CH2 in 3 δ); 4.53 (t, J = 7.5 Hz, 1H: 3a); 4.75 (broad dd, J = 13 and 8 Hz,
1H: the other H of the CH2 in 5 e) ; 4.84 (mt, 1H: 2 a);
4.88 (dd, J = 10 and 1 Hz, 1H: la); 5.35 (broad d, J = 6
Hz, 1H: 5 a); 5.43 (dd, J = 10. 5 and 4 Hz, 1H: 4 a); 5.90 (d, J = 9.5 Hz, 1H: 6 a); 5.92 (mt, 1H: 1 β) ; 6.56 (d, J =
9.5 Hz, 1H: NH in 2); from 7.10 to 7.35 (mt, 5H: aromatic H in 6); 7.28 (d, J = 8 Hz, 2H: aromatic H in 4 δ); 7.38 (dd,
5 J = 8.5 and 2 Hz, 1H: l'Hj; 7.4 2 (dd, J = 8.5 and 4.5 Hz,
1H: l'H5); 7.66 (dd, J = 4.5 and 2 Hz, 1H : l'H«) ; 7.88 (d,
AP/P/ 9 5 / 0 0 7 5 2
AP . Ο Ο 5 6 2
J = 8 Hz, 2Η: aromatic Η in 4 e); 8.38 (d, J = 10 Hz, 1H: NH in 1); 8.74 (d, J = 9.5 Hz, 1H: NH in 6); 11.65 <s, 1H: OH) .
EXAMPLE 28: Preparation of 4c -dimethylamino5 de (4dimethylamino)pristinamycin IA.
Strain SP92::pVRC508 is cultured in production medium using 60 erlenmeyer flasks, as described in Example 3, with 1 ml of a 20 g/1 solution of (R,S)-3dimethylaminophenylalanine dihydrochloride, synthesized as 10 in Example 35-10, in 0. IN sodium hydroxide solution being added at 16h. At the end of 40h of culture, the 1.8 litres of must recovered from the 60 erlenmeyer flasks is extracted with 2 volumes of a mixture consisting of 66% 100 mM phosphate buffer, pH 2.9, and 34% acetonitrile, and then centrifuged. The supernatant is extracted with 2 times 0.5 volumes of dichloromethane. The chloromethylene phases are washed with water and then combined, dried over sodium sulphate and evaporated. The dry extract is taken up in 20 ml of dichloromethane and injected onto a silica (30 g) column which is mounted in dichloromethane and eluted successively with plateaus of from 0 to 10% methanol in dichloromethane. The fractions containing 4e-dimethylaminode (4 ζ-dimethylamino)pristinamycin IA are combined and evaporated. The dry residue is taken up in 3 ml of a mixture consisting of 60% of water and 40% acetonitrile and injected onto a semi-preparative Nucleosil Ίμ C8 10x250 mm (Macherey Nagel) column, which is eluted with a mixture consisting of 57% 100 mM phosphate buffer, pH 2.9, and 43% of acetonitrile. The fractions containing 4e-dimethylamino30 de (4 ξ-dimethylamino)pristinamycin IA are combined and extracted with one volume of dichloromethane. The organic phase is washed with water, dried over sodium sulphate and then evaporated. 1.1 mg of 4e-dimethylamino-de(4f-dimethylamino) pristinamycin IA are obtained.
NMR spectrum. JH (400 MHz, CDC13, δ in ppm, ref.
TMS): 0.63 (dd, J = 16 and 5 Hz, ΙΗ: 1H of the CH2 in 5 β);
AP/P/ 95/00752
0.91 (t, J = 7.5 Hz, 3H: CH3 in 2 γ); 1.13 (mt, ΙΗ: 1H of the CH2 in 3 0) ; from 1.20 to 1.35 (mt, ΙΗ: 1H of the CH2 in 3 γ); 1.32 (d, J = 6.5 Hz, 3H: CH3 in 1 γ); 1.57 (mt,
1H: the other H of the CH2 in 3 γ) ; 1.63 and 1.76 (2 mts,
1H each: CH2 in 2 0); 2.02 (mt, 1H: the other H of the CH2 in 3 0); 2.08 and 2.31 (respectively, mt and broad d, J =
16.5 Hz, 1H each: CH2 in 5 δ): 2.35 (d, J = 16 Hz, 1H: the other H of the CH2 in 5 0) ; 2.81 (dt, J = 13 and 4 Hz, 1H: 1H of the CH2 in 5 e); 2.90 (s, 6H: N(CH3)2); 2.97 (dd, J = 12 and 4 Hz, ΙΗ: 1H of the CH2 in 4 0); from 3.20 to 3.30 (mt, ΙΗ: 1H of the CH2 in 3 δ); 3.28 (s, 3H: NCH3) ; 3.37 (t, J = 12 Hz, 1H: the other H of the CH2 in 4 0); 3.57 (mt, 1H: the other H of the CH2 in 3 δ); 4.58 (t, J = 7.5 Hz, 1H : 3a); 4.74 (broad dd, J = 13 and 8 Hz, 1H: the other H of the CH2 in 5 c); 4.86 (mt, 1H: 2 a); 4.89 (broad d, J = 10 Hz, 1H: 1 a); 5.27 (dd, J = 12 and 4 Hz, 1H: 4
а) ; 5.29 (broad d, J = 5 Hz, 1H : 5 a); 5.89 (d, J = 9.5 Hz, 1H: 6 a); 5.90 (mt, 1H: 1 0); 6.50 (d, J = 10 Hz, 1H:
NH in 2); from 6.50 to 6.70 (mt, 3H: aromatic Hs in the ortho and in the para positions with respect to the dimethylamino); from 7.15 to 7.35 (mt, 5H: aromatic Hs in
б) ; 7.20 (t, J = 8 Hz, 1H: aromatic H in the meta position with respect to the dimethylamino); 7.43 (l-imiting AB, 2H: l'H« and l'Hs) ; 7.82 (mt, 1H: l'H6) ; 8.38 (d, J = 10 Hz, 1H: NH in 1); 8.73 (d, J = 9.5 Hz, 1H: NH in 6); 11.61 (s, 1H: OH) .
EXAMPLE 29: Preparation of 4c-methylthio-de(4fdimethylamino)pristinamycin IA
Strain SP92::pVRC508 is cultured in production medium using 56 erlenmeyer flasks, as described in Example 3, with 1 ml of a 20 g/1 solution of (R,S)-3methylthiophenylalanine hydrochloride, synthesized as in Example 34-11, in 0. IN sodium hydroxide solution being added at 16h. At the end of 40h of culture, the 1.68 litres of must recovered from the 56 erlenmeyer flasks is extracted with 2 volumes of a mixture consisting of 66% 100
AP/P/ 9 5 / 0 0 7 52
AP . Ο Ο 5 6 2
- 89 mM phosphate buffer, pH 2.9, and 34% acetonitrile, and then centrifuged. The supernatant is extracted with 2 times 0.5 volumes of dichloromethane. The chloromethylene phases are washed with water and then combined, dried over sodium sulphate and evaporated. The dry extract is taken up in 20 ml of dichloromethane and injected onto a silica (30 g) column which is mounted in dichloromethane and eluted successively with plateaus of from 0 to 10% methanol in dichloromethane. The fractions containing the novel derivative of pristinamycin IA are combined and evaporated. ¢-. The dry residue is taken up in 7 ml of a mixture consisting of 54% of water and 46% acetonitrile and injected onto a semi-preparative Nucleosil 7μ C8 10x250 mm (Macherey Nagel) column, which is eluted with a mixture consisting of 55%
100 mM phosphate buffer, pH 2.9, and 45% of acetonitrile.
The fractions containing the novel pristinamycin are combined and extracted with one volume of dichloromethane. The organic phase is washed with water, dried over sodium sulphate and then evaporated. 20 mg of 4e-methylthio-de(4f20 dimethylamino)pristinamycin IA are obtained.
NMR spectrum. XH (4 00 MHz, CDC13, δ in ppm, ref.
TMS): 0.56 (dd, J = 16 and 5.5 Hz, ΙΗ: 1H of the CH2 in 5 β) ; 0.90 (t, J = 7.5 Hz, 3H: CH3 in 2 γ); 1.13 (mt, ΙΗ: 1H θ of the CH2 in 3 β); 1.28 (mt, ΙΗ: 1H of the CH2 in 3 γ);
1.32 (d, J - 6.5 Hz, 3H: CH3 in 1 γ) ; 1.58 (mt, 1H: the other H of the CH2 in 3 γ) ; 1.62 and 1.74 (2 mts, 1H each: CH2 in 2 β) ; 2.02 (mt, 1H: the other H of the CH2 in 3 β) ; 2.25 and 2.35 (respectively, mt and broad d, J = 16.5 Hz,
1H each: CH2 in 5 δ); 2.39 (d, J = 16 Hz, 1H: the other H of the CH2 in 5 β) ; 2.43 (s, 3H: SCH3) ; 2.82 (dt, J = 13 and 4 Hz, ΙΗ: 1H of the CH2 in 5 e); 2.98 (dd, J = 12 and
4.5 Hz, ΙΗ: 1H of the CH2 in 4 β) ; 3.26 (s, 3H: NCH3) ; 3.30 (t, J = 12 Hz ΙΗ: 1H of CH2 in 3 δ); 3.38 (mt, 1H: the other H of the CH2 in 4 β} ; 3.57 (mt, 1H: the other H of the CH2 in 3 δ); 4.56 (t, J = 7.5 Hz, 1H: 3 a); 4.74 (broad dd, J = 13 and 8 Hz, 1H: the other H of the CH2 in 5 c);
AP/P/ 9 5 / 0 0 7 5 2
4.84 (mt, IH: 2 a); 4.89 (dd, J = 10 and 1 Hz, IH: 1 a); 5.29 (dd, J = 12 and 4.5 Hz, IH : 4a); 5.32 (broad d, J =
5.5 Hz, IH : 5 a); 5.88 (d, J = 9.5 Hz, IH: 6 a); 5.90 (mt, IH: 1 β) ; 6.51 (d, J = 10 Hz, IH: NH in 2); 6.99 (broad d,
J = 8 Hz, IH: aromatic H in the para position with respect to the methylthio); 7.10 and 7.15 (respectively, broad s and broad d, J = 8 Hz, IH each: aromatic Hs in the ortho position with respect to the methylthio); from 7.15 to 7.35 (mt, 6H: aromatic Hs in 6 and aromatic Hs in the meta position with respect to the methylthio); 7.43 (broad d, J
= 8 Hz, IH: l'H«) ; 7.52 (dd, J = 8 and 4 Hz, IH: l'H5) ;
7.79 (broad d, J = 4 Hz, IH: l'Hj; 8.38 (d, J = 10 Hz,
NH in 1); 8.73 (d, J = 9.5 Hz, IH: NH in 6) ; 11.62 (s,
OH) .
EXAMPLE 30: Preparation of 4e-ethoxy-de(4fdimethylamino)pristinamycin IA.
Strain SP92::pVRC508 is cultured in production medium using 60 erlenmeyer flasks, as described in Example 3, with 1 ml of a 20 g/1 solution of (S)-3-O-ethyltyrosine hydrochloride, synthesized as in Example 37-1, in 0.2N sodium hydroxide solution being added at 16h. At the end of 40h of culture, the 1.8 litres of must recovered from the 60 erlenmeyer flasks is extracted with 2 volumes of a mixture consisting of 66% 100 mM phosphate buffer, pH 2.9, and 34% acetonitrile, and then centrifuged. The supernatant is extracted with 2 times 0.5 volumes of dichloromethane. The chloromethylene phases are washed with water and then combined, dried over sodium sulphate and evaporated. The dry extract is taken up in 20 ml of dichloromethane and injected onto a silica (30 g) column which is mounted in dichloromethane and eluted successively with plateaus of from 0 to 10% methanol in dichloromethane. The fractions containing the novel derivative of pristinamycin IA are combined and evaporated. 19 mg of dry residue are obtained.
The latter is taken up in 3 ml of a mixture consisting of 60% of water and 40% acetonitrile and injected onto a semiAP/P/ 9 5 / 0 0 7 5 2
AP.00562
- 91 preparative Nucleosil Ίμ C8 10x250 mm (Macherey Nagel) column, which is eluted with a mixture consisting of 60%
100 mM phosphate buffer, pH 2.9, and 40% of acetonitrile. The fractions containing the novel pristinamycin are combined and extracted with one volume of dichloromethane. The organic phase is washed with water, dried over sodium sulphate and then evaporated. 15.8 mg of 4e-O-ethoxy-de (4ζ dimethylamino)pristinamycin Ix are obtained.
NMR spectrum. *H (4 00 MHz, CDCl3, δ in ppm, ref.
TMS): 0.55 (dd, J = 16 and 5.5 Hz, IH: IH of the CH2 in 5 ζ β) ; 0.90 (t, J = 7.5 Hz, 3H: CH3 in 2 γ) ; 1.12 (mt, IH: IH of the CH2 in 3 β) ; 1.20 (mt, IH: IH of the CH2 in 3 γ) ;
1.31 (d, J = 6.5 Hz, 3H: CH3 in 1 γ) ; 1.49 (t, J = 7 Hz,
3H: CH3 of the ethyl); 1.54 (mt, IH: the other H of the CH2 in 3 γ); 1.63 and 1.73 (2 mts, IH each: CH2 in 2 β); 2.02 (mt, IH: the other H of the CH2 in 3 β); 2.22 and 2.33 (respectively, mt and broad d, J = 16.5 Hz, IH each: CH2 in 5 δ); 2.46 (d, J = 16 Hz, IH: the other H of the CH2 in 5 β); 2.83 (dt, J = 13 and 4 Hz, IH: IH of the CH2 in 5 e);
2.95 (dd, J = 12 and 4 Hz, IH: IH of the CH2 in 4 β); 3.22 (mt, IH: IH of the CH2 in 3 δ); 3.27 (s, 3H: NCH3) ; 3.39 (t, J = 12 Hz, IH: the other H of the CH2 in 4 β) ; 3.53 (mt, IH: the other H of the CH2 in 3 δ); 3.93 and 4.03 (2 φ mts, IH each: 0CH2 of the ethyl); 4.56 (dd, J = 7 and 5.5
Hz, IH: 3 a); 4.75 (broad dd, J = 13 and 8 Hz, IH: the other H of the CH2 in 5 e) ; 4.82 (mt, IH: 2 a); 4.88 (dd, J = 10 and 1 Hz, IH: 1 a); 5.23 (dd, J = 12 and 4 Hz, IH: 4 a); 5.23 (broad d, J = 5.5 Hz, IH: 5 a); 5.87 (d, J = 9.5 Hz, IH: 6 a); 5.92 (mt, IH: 1 β); 6.47 (d, J = 10 Hz, IH:
NH in 2); 6.80 (mt, 3H: aromatic H in the ortho and in the para positions with respect to the ethoxy); from 7.10 to 7.35 (mt, 6H: aromatic Hs in 6 and aromatic Hs in the meta position with respect to the ethoxy); 7.43 (dd, J = 8 and 1 Hz, IH: l’H<); 7.50 (dd, J = 8 and 4 Hz, IH: l'Hs); 7.77 (dd, J = 4 and 1 Hz, IH: l'H6); 8.38 (d, J = 10 Hz, IH: NH in 1); 8.70 (d, J = 9.5 Hz, IH: NH in 6); 11.60 (s, IH:
AP/P/ 95/00752
OH) .
EXAMPLE 31: Preparation of 4f-ethylthio-de (4fdimethylamino)pristinamycin IA
Strain SP92::pVRC508 is cultured in production medium using 2 erlenmeyer flasks, as described in Example
3, with 1 ml of a 20 g/1 solution of (S)-4ethylthiophenylalanine hydrochloride, synthesized as in Example 33, in 0.1N sodium hydroxide solution being added at 16h. At the end of 40h of culture, the 60 ml of must recovered from the 2 erlenmeyer flasks is extracted with 2 volumes of a mixture consisting of 66% 100 mM phosphate
G buffer, pH 2.9, and 34% acetonitrile, and then centrifuged.
The supernatant is extracted with 2 times 0.5 volumes of dichloromethane. The chloromethylene phases are washed with water and then combined, dried over sodium sulphate and evaporated. The dry extract is taken up in 20 ml of dichloromethane and injected onto a silica (30 g) column which is mounted in dichloromethane and eluted successively with plateaus of from 0 to 10% methanol in dichloromethane.
The fractions containing 4f-ethylthio-de(4f-dimethylamino)pristinamycin IA are combined and evaporated. The dry residue is taken up in 7 ml of a mixture consisting of 60% of water and 40% acetonitrile and injected onto a semi_ preparative Nucleosil 7μ C8 10x250 mm (Macherey Nagel) *** 25 column, which is eluted with a mixture consisting of 52%
100 mM phosphate buffer, pH 2.9, and 48% of acetonitrile. The fractions containing 4f-ethylthio-de(4ζ-dimethylamino)pristinamycin IA are combined and extracted with one volume of dichloromethane. The organic phase is washed with water, dried over sodium sulphate and then evaporated. ? mg of 4/ethylthio-de(4/-dimethylamino)pristinamycin IA are obtained.
NMR spectrum. XH (400 MHz, CDC13, δ in ppm) :
0.68 (dd, J = 16 and 6 Hz, 1H: 1H of the CH2 in 5 /3); 0.92 (t, J = 7.5 Hz, 3H: CH3 in 2 γ) ; from 1.10 to 1.40 (mt, 5H:
1H of the CH2 in 3 β and 1H of the CH2 in 3 γ and CH3 of the
AP/P/ 9 5 / 0 0 7 5 2
AP . Ο Ο 5 6 2
- 93 ethyl); 1.32 (d, J = 7 Hz, 3H: CH3 in 1 γ) ; from 1.45 to 1.85 (mt, 3H: the other H of the CH2 in 3 γ and CH2 in 2 0) ; 2.02 (mt, 1H: the other H of the CH2 in 3 0); 2.18 and 2.37 (respectively, mt and broad d, J = 16.5 Hz, 1H each:
CH2 in 5 δ) ; 2.45 (broad d, J ~ 16 Hz, 1H: the other H of the CH2 in 5 0); 2.85 (dt, J = 13 and 4 Hz, ΙΗ: 1H of the CH2 in 5 c); 2.90 (mt, 2H: ArSCH2 ethyl); 2.98 (dd, J = 12 and 4 Hz, ΙΗ: 1H of the CH2 in 4 0); 3.25 (s, 3H: NCHJ ;
3.35 (mt, ΙΗ: 1H of the CH2 in 3 δ); 3.39 (t, J = 12 Hz,
1H: the other H of the CH2 in 4 0); 3.57 (mt, 1H: the other
H of the CH2 in 3 δ); 4.55 (t, J = 7.5 Hz, 1H: 3a); 4.75 (broad dd, J = 13 and 7.5 Hz, 1H, : the other H of the CH2 in 5 c); 4.85 (mt, 1H: 2 a); 4.89 (dd, J = 10 and 1 Hz, 1H: 1 a); from 5.25 to 5.40 (mt, 2H: 5 a and 4 a); 5.88 (d, J =
9.5 Hz, 1H: 6 a); 5.91 (mt, 1H: 1 0); 6.55 (d, J = 9.5 Hz,
1H: NH in 2); 7.10 (d, J = 8 Hz, 2H: aromatic Hs in 4 δ);
from 7.10 to 7.35 (mt, 7H: aromatic Hs in 6 and 4 e); 7.44 (limiting AB, 2H: l'H« and l'Hs) ; 7.74 (mt, 1H: l'H6); 8.38 (d, J = 10 Hz, 1H: NH in 1); 8.75 (d, J = 9.5 Hz, 1H: NH in
6); 11.62 (s, 1H: OH).
EXAMPLE 32j Preparation of 4f-ethyl-de(4fdimethylamino)pristinamycin Ix
Strain SP92::pVRC508 is cultured in production medium using 2 erlenmeyer flasks, as described in Example
3, with 1 ml of a 20 g/1 solution of (R,S)-4ethylphenylalanine, synthesized as in Example 33, in 0.1N sodium hydroxide solution being added at 16h. At the end of 40h of culture, the 60 ml of must recovered from the 2 erlenmeyer flasks is extracted with 2 volumes of a mixture consisting of 66% 100 mM phosphate buffer, pH 2.9, and 34% acetonitrile, and then centrifuged. The supernatant is extracted 2 times with dichloromethane. The chloromethylene phases are washed with water and then combined, dried over sodium sulphate and evaporated. The dry extract is taken up in 20 ml of dichloromethane and injected onto a silica
AP/P/ 95/00752 * »
- 94 (30 g) column which is mounted in dichloromethane and eluted successively with plateaus of from 0 to 10% methanol in dichloromethane. The fractions containing 4f-ethylde (4 f -dimethylamino) pristinamycin IA are combined and evaporated. The dry residue is taken up in 7 ml of a mixture consisting of 52% of water and 48% acetonitrile and injected onto a semi-preparative Nucleosil Ίμ C8 10x250 mm (Macherey Nagel) column, which is eluted with a mixture consisting of 52% 100 mM phosphate buffer, pH 2.9, and 48% of acetonitrile. The fractions containing 4f-ethyl-de(4fdimethylamino)pristinamycin IA are combined and extracted with one volume of dichloromethane. The organic phase is washed with water, dried over sodium sulphate and then evaporated. 0.50 mg of 4f-ethyl-de(4f-dimethylamino)15 pristinamycin IA are obtained.
NMR spectrum. *H (400 MHz, CDC13, δ in ppm, ref.
TMS) : 0.42 (dd, J = 16 and 5.5 Hz, 1H: 1H of the CH2 in 5 /3); 0.92 (t, J = 7.5 Hz, 3H: CH3 in 2 γ) ; from 1.10 to 1.40 (mt, 2H: 1H of the CH2 in 3 β and 1H of the CH2 in 3 γ) ;
1.23 (t, J = 7.5 Hz, 3H: CH3 of the ethyl); 1.35 (d, J = 7 Hz, 3H: CH3 in 1 γ); from 1.45 to 1.85 (mt, 3H: the other H of the CH2 in 3 γ and CH2 in 2 /3); 2.02 (mt, 1H: the other H of the CH2 in 3 /3); 2.15 and from 2.25 to 2.40 (2 mts, 1H each: CH2 in 5 δ); from 2.25 to 2.40 (mt, 1H: the other H of the CH2 in 5 /3); 2.60 (q, J = 7.5 Hz, 2H: ArCH2 of the ethyl); 2.83 (dt, J = 13 and 4 Hz, 1H: 1H of the CH2 in 5
e); 2.98 (dd, J = 12 and 4 Hz, 1H: 1H of the CH2 in 4 /3) ; from 3.25 to 3.3 5 (mt, 1H: 1H of the CH2 in 3 δ); 3.27 (s, 3H: NCH3) ; 3.3 9 (t, J = 12 Hz, 1H: the other H of the CH2 in 4 /3); 3.59 (mt, 1H: the other H of the CH2 in 3 δ); 4.58 (dd, J = 7 and 6.5 Hz, 1H: 3 a); 4.75 (broad dd, J = 13 and 8 Hz, 1H: the other H of the CH2 in 5 e) ; 4.87 (mt, 1H: 2 a); 4.89 (dd, J = 10 and 1 Hz, 1H: 1 a); 5.24 (broad d, J =
5.5 Hz, 1H: 5 a); 5.29 (dd, J = 12 and 4 Hz, 1H: 4 a); 5.88 (d, J = 10 Hz, 1H: 6 a); 5.92 (mt, 1H: 1 /3); 6.73 (d, J =
Hz, 1H: NH in 2); from 7.10 to 7.35 (mt, 9H: aromatic Hs
5/00/56 /d/dV
AP. Ο Ο 5 6 2
- 95 in 6 - 4 e and 4 δ); 7.44 (dd, J = 8.5 and 1.5 Hz, IH: l'HJ; 7.50 (dd, J = 8.5 and 4.5 Hz, IH: l'Hs); 7.80 (dd, J = 4.5 and 1.5 Hz, IH: l'H6) ; 8.38 (d, J = 10 Hz, IH: NH in 1); 8.75 (d, J = 10 Hz, IH: NH in 6); 11.66 (s, IH: OH).
Using the same fractions derived from the silica column described above, which fractions also contain the novel pristinamycin IH derivative, 0.3 mg of f-ethyl-de (4fdimethylamino)pristinamycin IH is isolated by carrying out semi-preparative column chromatography as described above. £tr·, 10 NMR spectrum. XH (400 MHz, CDC13, δ in ppm): 0.04 (mt IH: IH Of the CH2 in 5 β) ; 0.92 (t, J = 7.5 Hz, 3H: CH3 in 2 γ); from 1.10 to 1.40 (mt, 2H: IH of the CH2 in 5 δ and IH of the CH2 in 5 γ) ; 1.18 (t, J = 7.5 Hz, 3H: CH3 of the ethyl); 1.30 (d, J = 6.5 Hz, 3H: CH3 in 1 γ); from 1.45 15 to 1.85 (mt, 7H: the other H of the CH2 in 5 γ - the other
H of the CH2 in 5 δ - IH of the CH2 in 3 β - CH2 in 3 γ and
CH2 in 2 β); 1.81 (broad d, J = 13 Hz, IH: the other H of the CH2 in 5 β) ; 2.02 (mt, IH: the other H of the CH2 in 3
β) ; 2.40 (dt, J = 13 and 4 Hz, IH: IH of the CH2 in 5 e) ;
2.65 (q, J = 7.5 Hz, 2H: ArCH2 of the ethyl); 2.97 and 3.09 (respectively, dd and t, J = 12 and 5 Hz and J = 12 Hz, IH each: CH2 in 4 β) ; 3.50 and 3.60 (2 mts, IH each: CH2 in 3 G δ); 4.13 (dd, J = 8 and 5 Hz, IH: 3 a); 4.49 (broad d, J =
Hz, IH: the other H of the CH2 in 5 e) ; 4.70 (mt, 2H: 5 25 a and 4 a); 4.77 (mt, IH: 2 a); 4.83 (dd, J = 10 and 1 Hz, IH: 1 Of); 5.50 (d, J = 7 Hz, IH: 6 a); 5.74 (mt, IH: 1 β); 6.09 (d, J = 4 HZ, IH: NH in 4); 6.72 (mf, IH: NH in 2); 7.07 (d, J= 8 Hz, 2H: aromatic Hs in 4 e) ; 7.15 (d, J = 8 Hz, 2H: aromatic Hs in 4 δ); from 7.15 to 7.35 (mt, 5H:
aromatic Hs in 6); 7.40 (dd, J = 8 and 1 Hz, IH: l'HJ;
7.45 (dd, J = 8 and 4 Hz, IH: l'Hs) ; 7.92 (dd, J = 4 and 1 Hz, IH: l'Hj); 8.40 (mf, IH: NH in 6); 8.50 (d, J = 10 Hz, IH: NH in 1); 11.72 (s, IH: OH).
AP/P/ 9 5 / 0 0 7 5 2
EXAMPLE 33: Preparation of derivatives of phenylalanine and of phenylpyruvic acid which have already been described.
Phenylalanine, and its derivatives
4-methoxyphenylalanine, 4-bromophenylalanine,
4-chlorophenylalanine, 4-iodophenylalanine,
4-trifluoromethylphenylalanine, 4-aminophenylalanine and 3-methoxyphenylalanine, which are employed in this work, are commercially available.
The following derivatives of phenylalanine can be 10 prepared in accordance with methods described in the literature .
(RS) -4-dimethylaminophenylalanine
D.F. Elliott, A.T. Fuller, C.R. Harrington,
J. Chem. Soc., 1948, 85-89.
(RS)-4-diethylaminophenylalanine
Moldaver B.L., Pushkareva Z.V., Zhur. Obshchei Khim., 31, 1560-1569 (1961); C.A. 1961, 22226f.; J.A.
Stock, J. Chem. Soc, 1959, 90-97 (RS)-4-ethylaminophenylalanine
F. Bergel, J.A. Stock, J. Chem. Soc, 1959, 90-97.
(RS)-4-phenylphenylalanine
J.V. Braun, J. Nelles, Berichte, 66B, 1933, 14641470.
(RS)-4-methylphenylalanine
R.R., Herr, T. Enjoki, J.P. Dailey, J. Am. Chem.
Soc, 1957, 79, 4229-4231.
(RS)-4-methylthiophenylalanine and (R,S)-4ethylthiophenylalanine
R.L. Colescott, R.R. Herr, J.P. Dailey 30 J. Am. Chem. Soc, 1957, 79, 4232-4235.
(RS)-4-methoxycarbonylphenylalanine
H. Cleland, J. Org. Chem., 1969, 34, 747.
(RS)-2,4-dimethylphenylalanine
R.R., Herr, T. Enjoki, J.P. Dailey,
J. Am. Chem. Soc, 1957, 79, 4229-4231.
(RS)-3,4-dimethylphenylalanine
AP/P/ 9 5 / 0 0 7 52
AP. Ο Ο 5 6 2
- 97 R.R., Herr, Τ. Enjoki, J.P. Dailey,
J. Am. Chem. Soc, 1957, 79, 4229-4231.
(RS) -3-trif luoromethylphenylalanine hydrochloride R. Filler and H. Novar. J. Org. Chem, 1960, 25,
733-736.
Γ ί
-Μ
(S)-4-aminomethylphenylalanine
G.E. Stokker, W.F. Hoffman and C.F. Homnick,
J. Org. Chem., 1993, 58, 5015-5017.
(R,S)-3-methylphenylalanine
J.H. Burckhalter, V.C. Stephens, J.A.C.S. 1951,
73, 56-58.
(R,S)-4-acetylphenylalanine
J.I. Degaw et coll., J. Med. Che., 1969, 11, 225227 (S)-4-O-allyltyroeine
A. Loffet, H. Zang, Int. J. Pept. Protein. Res.,
1993, 42, 346 (S) -4-O-ethyltyrosine
Y. Sasaki et coll., Chem. Pharm, Bull., 1982, 30,
4435 (RS)-4-ethylphenylalanine
A. Zhuze et coll., Coll., Czech. Chem. Comm.,
1965, 62, 2648
4-tert-butylphenylpyruvic acid can be prepared in 25 accordance with R. Breslow, J.W. Canary, M. Varney,
S.T. Waddell and D. Yang, J. Am. Chem. Soc., 1990, 112, 5212-5219.
AP/P/ 95/00752
The other derivatives of phenylalanine were prepared in accordance with Examples 34 to 42 which are given below. In these examples, flash chromatography was carried out under a mean nitrogen pressure of 50 kPa using a silica of granule size 40-53 μτη, in accordance with Still et al.. J. Org. Chem., 43 . 2923, (1978).
- 98 EXAMPLE 34: Preparation of derivatives of phenylalanine and of a derivative of phenylpyruvic acid using method A.
Ο 10 ©
34-1 (RS)-4-methylaminophenylalanine, dihvdrochloride ml of 12 N hydrochloric acid are added to 3.70 g of methyl N-acetyl-4-methylaminophenylalaninate, and the mixture is heated to reflux, while stirring, for 8 h. After one night at room temperature, the reaction medium is concentrated to dryness under reduced pressure (50 kPa), and the residue is taken ύρ in a mixture of 50 ml of toluene and 50 ml of ethanol, and this mixture is concentrated once again. After drying in a desiccator under reduced pressure (2.6 kPa), 4.18 g (100%) of (RS)-4methylaminophenylalanine dihydrochloride are obtained in the form of a hygroscopic light beige solid which melts at 158°C.
34-2: Methyl (RS)-N-acetvl-4methylaminophenylalaninate
0.4 g of 10% palladium on charcoal, and then 50 ml of absolute ethanol, are added to 4 g of methyl 4methylamino-2-acetamidocinnamate which is placed under a nitrogen atmosphere in an autoclave. The mixture is placed
AP/P/ 9 5 / 0 0 7 5 2
AP. Ο Ο 5 6 2
- 99 under a pressure of 5.5 bar of hydrogen and heated at 50°C for 15 h with stirring. After stabilizing the temperature at 26°C, and returning the pressure to atmospheric, the medium is filtered through Clarcel®, washed with ethanol .5 and then concentrated to dryness under reduced pressure (2.6 kPa). This results in 3.73 g of methyl N-acetyl-4methylaminophenylalaninate in the form of white crystals which melt at 118°C.
34-3: Methvl 4-methvlamino-2-acetamidocinnamate
5.75 g of methyl 2-acetamidoacrylate, 0.185 g of
F palladium acetate, 8.1 g of tetrabutylammonium chloride and
6.03 g of sodium hydrogen carbonate are added to a 3-necked flask which is placed under nitrogen, and then 6.5 g of 4-iodo-N-methylalanine, in solution in 200 ml of DMF, are added to this mixture. The mixture is heated at 82°C for 16 h 30 min and then, after having been cooled down, is poured into 1000 ml of distilled water. The medium is extracted with 250 ml of CH2C12 and the organic phase is separated off; the aqueous phase is then washed twice with
250 ml of CH2C12. The organic phases are combined, dried over sodium sulphate, filtered and concentrated under reduced pressure (50 kPa) at 70°C to yield a brown oil which is purified by flash chromatography (eluent,
AcOEt/cyclohexane and then pure AcOEt).
In this way, 4 g of methyl 4-methylamino-2acetamidocinnamate is obtained in the form of a yellow solid (Merck Silica 5719, Rf = 0.48), which is employed in this form.
N-Methyl-p-iodoanaline can be prepared in accordance with: S. Krishnamurthy, Tetrahedron Letters, 33, 3315-3318, 1982.
34-4: 4-methvlaminophenvlpyruvic acid
2.4 g of methyl 4-methylamino-2acetamidocinnamate and 32 ml of 12 N hydrochloric acid are placed in a round-bottomed flask. The mixture is heated to reflux for 3 h and then cooled down and washed twice with
AP/P/ 9 5 / 0 0 7 5 2
100
ml of diethyl ether. The aqueous phase is cooled down to
-10°C and the precipitate which is obtained is filtered and then rinsed with a minimum of cold hydrochloric acid. The solid which is obtained is dried in a desiccator under reduced pressure in order to yield 1.1 g of 4methylaminophenylpyruvic acid in the form of a light beige solid which melts at 210°C.
4 -5: (R,S)-3-Fluoro-4-methvlphenvlalanine hydrochloride
0.6 g of (R,S)-3-fluoro-4-methylphenylalanine hydrochloride is obtained in the form of white crystals which melt at a temperature greater than 260°C by proceeding as in Example 34-1 but using 1.6 g of methyl Nacetyl(3 -fluoro-4-methyl)phenylalaninate.
34-6 : Methyl (R.S)-N-acetvl-(3-fluoro-4methyl)phenylalaninate
1.6 g of methyl N-acetyl-(3-fluoro-4methyl)phenylalaninate are obtained in the form of a colourless oil (Merck Silica 5719, Rf =0.46; eluent
CH2Cl2/AcOEt 50/50), by proceeding as in Example 34-2 but using 1.9 g of methyl (4-methyl-3-fluoro)-2 acetamidocinnamate and 0.2 g of 10% palladium on charcoal in 230 ml of ethanol.
34-7: Methyl (3-fluoro-4-methyl)-225 acetamidocinnamate
2.6 g of methyl (3-fluoro-4-methyl)-2acetamidocinnamate are obtained in the form of a white solid which melts at 163°C by proceeding as in Example 34-3 'but using 3.6 g of methyl 2-acetamidoacrylate, 0.12 g of 30 palladium acetate, 5.2 g of tetrabutylammonium chloride,
3.8 g of sodium hydrogen carbonate and 4 g of 2-fluoro-4bromotoluene in solution in 120 ml of anhydrous DMF.
4 - 8: (R,S)-4-Trifluoromethoxvphenylalanine hydrochloride or (R.S)-O-trifluoromethvltyrosine hydrochloride
AP/P/ 9 5 / 0 0 7 5 2
1.5 g of (R,S)-4-trifluoromethoxyphenylalanine
AP . 0 0 5 6 2
- 101 hydrochloride are obtained in the form of white crystals which melt at 260°C by proceeding as in Example 34-1 but using 3 g of methyl N-acetyl-(4trifluoromethoxy)phenylalaninate and 30 ml of 12 N 5 hydrochloric acid.
34-9: Methvl (R.S)-N-acetvl-(4trifluoromethoxy)phenylalaninate g of methyl N-acetyl-(4-trifluoroethoxy)phenylalaninate are obtained in the form of a white solid which melts at 80°C by proceeding as in Example 34-2 but using 3.1 g of methyl (4-trifluoromethoxy)-2acetamidocinnamate and 0.3 g of 10% palladium on charcoal in 50 ml of ethanol.
34-10: Methyl 4-trifluoromethoxy-215 acetamidocinnamate
3.1 g of methyl (4-trifluoromethoxy)-2acetamidocinnamate are obtained in the form of a white solid which melts at 135°C by proceeding as in Example 34-3 but using 4.3 g of methyl 2-acetamido acrylate, 0.14 g of palladium acetate, 6.1 g of tetrabutyl-mmonium chloride,
4.6 g of sodium hydrogen carbonate and 5 g of
Q 4-trifluoromethoxybromobenzene in solution in 150 ml of anhydrous DMF.
© 3 4 -11: (R.S)-3-Methvlthiophenvlalanine hydrochloride
1.38 g of (R,S)-3-methylthiophenylalanine hydrochloride are obtained in the form of white crystals which melt at 190°C by proceeding as in Example 34-1 but using 3.3 g of methyl N-acetyl-3-methylthiophenylalaninate and 40 ml of 12 N hydrochloric acid.
34-12: Methvl (RS)-N-acetvl-3methvlthiophenvlalaninate
3.72 g of methyl 3-methylthio-2acetamidocinnamate, dissolved in 100 ml of methanol, and
30 ml of tetrahydrofuran are placed in a round-bottomed flask, and 1.4 g of magnesium are then added. After
AP/P/ 9 5 / 0 0 7 5 2
102 γλ reacting for 20 min, the mixture is cooled in an ice bath and a further 1.4 g of magnesium are then added. The mixture is stirred at room temperature for 18 h and then poured into 1.4 1 of distilled water and 300 ml of CH2C12;
.5 this mixture is then filtered through Clarcel®. The aqueous phase is adjusted to pH 6 by adding 12 N hydrochloric acid and then separated off and washed with 100 ml of CH2C12.
The organic phases are collected, dried over magnesium sulphate, filtered and then concentrated to dryness under reduced pressure in order to yield 3.42 g of methyl Nacetyl-3-methylthiophenylalaninate in the form of a colourless oil (Merck Silica 5719, Rf=0.5; AcOEt).
34-13: Methyl 3-methylthio-2-acetamidocinnamate 4.8 g of methyl (3-methylthio)-215 acetamidocinnamate are obtained in the form of a white solid which melts at 139°C by proceeding as in Example 34-3 but using 5.6 g of methyl 2-acetamidoacrylate, 0.18 g of palladium acetate, 8.2 g of tetrabutylammonium chloride, 5.86 g of sodium hydrogen carbonate and 6.5 g of 3-iodo-120 methylthiobenzene dissolved in 160 ml of anhydrous DMF.
-14: 3-Iodomethvlthiobenzene ml of distilled water and 20 ml of 12 N hydrochloric acid are placed, with stirring, in a threenecked flask, and 10 ml of 3-methylthioaniline are then added using a dropping funnel. The mixture is warmed to ensure dissolution and is then cooled down to 5°C. 5.86 g of sodium nitrite dissolved in 15 ml of water are subsequently added slowly, using a dropping funnel, while maintaining the temperature between 5 and 8°C. 20 min after having completed the addition, 13.57 g of potassium iodide dissolved in 15 ml of water are added over a period of 10 min and the mixture is then stirred at room temperature for 15 h. The oil which forms is separated from the aqueous phase by decantation, and an aqueous solution of sodium thiosulphate is then added to it. The aqueous phase is decanted and the product is extracted with 100 ml of
AP/P/ 9 5 / 0 0 7 5 2
AP. Ο Ο 5 6 2
- 103 dichloromethane. The organic phase is washed with 100 ml of water, and the aqueous phase is adjusted to pH 9 with concentrated sodium hydroxide solution, and then separated off. The organic phase is washed with 2 times 100 ml of water, separated off, dried over magnesium *5 sulphate, filtered and then concentrated to dryness under reduced pressure (50 kPa) at 40°C. The resulting product is purified by flash chromatography (eluent, cyclohexane) in order to yield 13 g of 3-iodo-1-methylthiobenzene in the form of a yellow liquid (Merck Silica 5719,
Rf=0.8/cyclohexane).
EXAMPLE 35: Preparation of derivatives of phenylalanine using method B.
o o
AP/P/ 95/00752
5-1: (RS)-4-tert-butvlphenvlalanine 25 g of diethyl 4-(tert-butyl)benzyl acetamidomalonate and 250 ml of 37% hydrochloric acid are added to a three-necked flask which is surmounted by a condenser. The mixture is stirred and heated to reflux
104 until there is no further evolution of gas. After the reaction medium has been cooled down, the precipitate which is obtained is filtered and then recrystallized in acetonitrile to yield 25.6 g of (R,S)-4-tert5 butylphenylalanine hydrochloride in the form of a white solid which melts at 234°C.
35-2: Diethyl 4-(tertbutvl) benzvlacetamidomalonate g of 4-(tert-butyl)benzyl bromide, 50 ml of anhydrous toluene and 3.1 g of sodium hydride in 80% suspension in oil are added to a three-necked flask which is surmounted by a condenser, followed by 21.8 g of diethyl acetamidomalonate. The mixture is heated at 110°C for 17 h. After it has been cooled down, 15 ml of absolute ethanol, then 15 ml of 50% ethanol and then 50 ml of water are added slowly to it using a dropping funnel. The organic phase is decanted and the aqueous phase is washed with 3 times 50 ml of diethyl ether. The organic phases are combined, washed with water and then dried over sodium sulphate. Following filtration and concentration under reduced pressure, the product is crystallized in petroleum ether in order to yield 25 g of diethyl 4-(tert-butyl)benzylacetamidomalonate in the form of a white solid which melts at 80°C.
35-3 : (R.S)-3-Methvlaminophenvlalanine dihydrochloride
1.03 g of a yellow-beige solid are obtained by proceeding as in Example 35-1 but using 1.17 g of diethyl 3-methylaminobenzylacetamidomalonate and 20 ml of 12 N hydrochloric acid. This yellow-beige solid is dissolved in
20 ml of absolute ethanol, and 0.4 g of animal charcoal is added to this solution. The solution is filtered through Clarcel and then filtered and concentrated under reduced pressure (50 kPa) . The same procedure is repeated starting with 1 g of animal charcoal, and the solid which is obtained is triturated in 20 ml of ether. Following filtration and drying under reduced pressure (2.7 kPa) at
AP/P/ 9 5 / 0 0 7 5 2
AP . 0 0 5 6 2
- 105 50°C, 0.65 g of (R,S)-3-methylaminophenylalanine dihydrochloride is obtained in the form of a white powder which melts at a temperature approaching 135°C (decomposition).
35-4: Diethyl 3-methvlaminobenzvlacetamidomalonate
3.11 ml of acetic anhydride are placed in a three-necked flask which is maintained under a nitrogen atmosphere. 1.51 ml of formic acid are subsequently added within 3 min at 0°C, and the mixture is then heated at 50°C for 2 hours. The mixture is allowed to return to room temperature, while shaking for 3 h 20 min, and 4 ml of anhydrous THF are added under nitrogen; the mixture is then cooled to -20°C. A solution of 4 g of diethyl
3-aminobenzylacetamidomalonate in a mixture of 15 ml of anhydrous THF and 15 ml of anhydrous dichloromethane is added within 10 min. Stirring is continued for 1 h 10 min at -20°C and then for 16 h at 20°C. The reaction mixture is concentrated to dryness under reduced pressure (50 kPa) at
30°C and then co-evaporated with 30 ml of anhydrous toluene in order to yield a white solid, which is dissolved in a mixture of 10 ml of anhydrous THF and 20 ml of anhydrous 1,2-dichloroethane, which solution is then placed in a three-necked flask under nitrogen.
The medium is cooled down to -5°C, and 1.55 ml of borane-dimethyl sulphide complex (2M solution in THF) are then added within 10 min. The mixture is allowed to return to room temperature, and the solution is heated to reflux for 3 h and then stirred at room temperature for 15 h. The reaction medium is cooled to 0°C, and 10 ml of MeOH are then added within 25 min. The mixture is stirred for 45 min at 0°C and then for 30 min at room temperature. It is then cooled to 0°C and HCI gas is bubbled in until a pH of 2 is reached. The mixture is heated at reflux for 1 h and is then concentrated to dryness under reduced pressure at 30°C in order to yield 5 g of a product which is taken up in
AP/P/ 9 5 / 0 0 7 5 2
106 ml of an aqueous solution of NaHCO3 and 30 ml of CH2C12. The organic phase is decanted and the aqueous phase is washed with 20 ml of water. The organic phases are pooled, dried over magnesium sulphate, filtered and then concentrated to dryness under reduced pressure (2.6 kPa) in order to yield 3.43 g of a yellow oil, which is purified by flash chromatography (eluent, AcOEt/cyclohexane 50/50). After drying under reduced pressure (2.7 kPa) at 20°C,
1.18 g of diethyl 3-methylaminobenzylacetamidomalonate are thus obtained in the form of a light beige solid which C' melts at 122°C.
35- 5: Diethyl 3-aminobenzvlacetamidomalonate Diethyl 3-aminobenzylacetamidomalonate can be prepared as described in:
T.S. Osdene, D.N. Ward, W.H. Chapman and
H. Rakoff, J. Am. Chem. Soc., 81, 1959, 3100-3102.
5-6: (R,S)-3-Ethvlaminophenvlalanine dihvdrochloride
1.7 g of (R,S)-3-ethylaminophenylalanine 20 dihydrochloride are obtained in the form of a hygroscopic light beige solid, which contains 10 molar % of (R,S)-3o diethylaminophenylalanine dihydrochloride, by proceeding as in Example 34-1 but using 2 g of ethyl (R,S)-N-acetyl-3ethylaminophenylalaninate and 30 ml of 12N hydrochloric acid.
5 -7: (R, S)-N-acetvl-3-ethvlaminophenylalaninate 3 g of ethyl (R,S)-N-acetyl-3-aminophenylalaninate, 40 ml of ethanol and 14 g of Raney nickel, which has previously been washed with distilled water and ethanol, are placed in a round-bottomed flask under a nitrogen atmosphere. The mixture is heated to reflux for 19 h, cooled down, filtered through Clarcel®, and then concentrated to dryness under reduced pressure (50 kPa) in order to yield 3.07 g of a colourless oil, which is purified by flash chromatography (eluent, AcOet) in order to yield 2.1 g of ethyl (R,S)-N-acetyl-3AP/P/ 9 5 / 0 0 7 5 2
AP . Ο Ο 5 6 2
- 107 ethylaminophenylalaninate in the form of a colourless oil (Merck Silica 5719, Rf=0.6: AcOEt) which contains 10% ethyl (R,S)-N-acetyl-3-diethylaminophenylalaninate.
35-8: Ethyl (R.S)-N-acetyl-3-aminophenvlalaninate 5 25 g of a mixture of ethyl (R,S)-N-acetyl-3nitrophenylalaninate (75 mol %/mol) and diethyl 3-nitrobenzylacetamidomalonate (25 mol %/mol) are placed under nitrogen in an autoclave. 2.5 g of 10% palladium on charcoal and then 200 ml of dichloromethane are added. The mixture is placed under a hydrogen pressure of 9 bar and then stirred at 18°C for 4 h. After returning the pressure to atmospheric, the reaction medium is filtered through Clarcel®, washed with dichloromethane and then concentrated to dryness under reduced pressure (50 kPa) in order to yield a solid, which is recrystallized in 450 ml of distilled water under reflux and in the presence of 4 g of 3S animal charcoal. Following hot filtration through Clarcel®, the mixture is left to crystallize at 4°C, with the crystals being filtered and then dried in order to yield 9.9 g of ethyl (R,S)-N-acetyl-3-aminophenylalaninate in the form of a light beige solid which melts at 106°C and which contains 5% of diethyl
3-aminobenzylacetamidomalonate.
35-9: Ethyl (R,S)-N-acetvl-3-nitrophenvlalaninate and diethyl 3-nitrobenzvlacetamidomalonate
600 ml of absolute ethanol and then 7.9 g of sodium are placed, under a nitrogen atmosphere, in a threenecked flask which is surmounted by a condenser. Once dissolution is complete, 74.5 g of diethyl acetamidomalonate and then 60 g of 4-nitrobenzyl chloride in 200 ml of anhydrous ethanol are added. The mixture is heated to reflux for 16 h 30 min. After cooling, the reaction medium is concentrated under reduced pressure (50 kPa) and then taken up in a mixture of 500 ml of CH2C12 and 500 ml of water. The pH is adjusted to 7 by adding 0.5N sulphuric acid, and the organic phase is then separated off
AP/P/ 95/00752
- 108 and the aqueous phase is washed with 2 times 200 ml of CH2C12. The organic phases are pooled, washed with 200 ml of water saturated with sodium bicarbonate, separated off and then dried over magnesium sulphate. Following .5 filtration and concentration under reduced pressure (50 kPa), the product is recrystallized in 600 ml of ethanol at reflux in order to yield, after crystallizing at ambient temperature, filtering and drying, 70.4 g of diethyl 3-nitrobenzylacetamido- malonate in the form of white crystals which melt at 156°C. The mother liquors are concentrated and then purified by flash chromatography (eluent, AcOEt) in order to yield 25.6 g of a mixture of ethyl N-acetyl-3-nitrophenylalaninate (75 mol %/mol) and diethyl 3-nitrobenzylacetamidomalonate (25 mol %/mol) in the form of a light beige solid, which is used in this form in the following step.
5-10: (RS,)-3-Dimethvlaminophenvlalanine dihvdrochloride
A solid is obtained, after evaporation, by proceeding as in Example 35-1 but using 0.72 g of ethyl (RS)-N-acetyl-3-dimethylaminophenylalaninate and 8.6 ml of 10N hydrochloric acid; the solid is subsequently triturated in 50 ml of acetone, filtered and then dried under reduced
O pressure (2.7 kPa) at 40°C. 0.68 g (93%) of (RS)-325 dimethylaminophenylalanine dihydrochloride is obtained in the form of a white solid which melts in the region of 120°C (decomposition).
35-11: Ethyl (RS)-N-acetvl-3 dimethvlaminophenvlalaninate
4 g of ethyl (RS)-N-acetyl-3aminophenylalaninate, prepared as described in Example 358, in 15 ml of DMF are placed in a three-necked flask under a nitrogen atmosphere, and 5.5 ml of triethylamine, and then 2.5 ml of methyl iodide and 4 ml of dichloromethane, are added while maintaining the temperature in the region of 30°C using an icebath. The mixture is then warmed at
AP/P/ 95/00752
AP.00562
- 109 35°C for 18h. 1 ml of methyl iodide dissolved in 1 ml of DMF is then added slowly while maintaining the temperature in the region of 30°C; 2.2 ml of triethylamine are then added and the mixture is subsequently warmed for a further
5h at 35°C. The mixture is brought to room temperature and then extracted with 100 ml of ethyl acetate and 150 ml of distilled water. The aqueous phase is separated off after settling and then rewashed with 2 times 70 ml of ethyl acetate. The organic phases are combined, washed with 2 times 80 ml of distilled water and then with 50 ml of distilled water which is saturated with NaCl. The organic phase is separated off after settling, dried over magnesium sulphate, filtered and then concentrated to dryness under reduced pressure in order to yield 2.4 g of a product which is purified by flash chromatography (dichloromethane, MeOH 90/10). 0.72 g (16%) of ethyl (RS)-3-N-acetyl-3dimethylamino phenylalaninate is thus obtained in the form of yellow crystals.
EXAMPLE 36: Preparation of derivatives of 20 phenylalanine using method C.
6 -1: (R,S)-4-Isopropylphenylalanine 7 g of red phosphorus and 8 g of
4-(isopropylbenzylidene)-2-methyl-5-oxazolone, in 45 ml of acetic anhydride, are placed in a three-necked flask, and then 35 ml of 57% hydriodic acid are added slowly, with stirring, using a dropping funnel. Once the addition is
AP/P/ 9 5 / 0 0 7 5 2
- 110 complete, the mixture is heated to reflux for 3 h 30 min and then left at room temperature for 3 days. The reaction mixture is filtered and the solid which is obtained is rinsed twice with 10 ml of acetic acid on each occasion, .5 and the filtrate is then concentrated to dryness under reduced pressure. The residue which is obtained is taken up in 100 ml of distilled water, and this solution is concentrated to dryness under reduced pressure in order to yield a solid which is taken up in 50 ml of distilled water; this solution is then extracted with 3 times 50 ml of diethyl ether after 0.5 g of sodium sulphite have been added. The ether is separated off and the aqueous phase is placed under reduced pressure in order to eliminate traces of diethyl ether. 2 g of animal charcoal are added to the aqueous phase, which is heated at 40-50°C, and then filtered through Clarcel®; rinsing then takes place with a minimum of water. The pH is adjusted to 5 by adding 32% ammonia at 4°C. The precipitate which is obtained is filtered in the cold, rinsed with 2 times 10 ml of water, with 10 ml of ethanol and then with 2 times 10 ml of ether in order to yield, after drying under reduced pressure at 20°C, 3.97 g of (R,S)-4-isopropylphenylalanine in the form of a white solid which melts at a temperature greater than 260°C. (See also Journal of the Takeda Research
Laboratories, vol. 43; nos. 3/4, Dec. 1984, pp 53-76).
36-2: 4-(Isopropylbenzvlidene)-2-methvl-5oxazolone
18.52 g of N-acetylglycine, 10.6 g of sodium acetate, 20 ml of 4-isopropylbenzaldehyde and 57 ml of acetic anhydride are placed in a round-bottomed flask which is provided with a condenser. The mixture is stirred for 30 min and then stirred for 1 h at 110°C and subsequently for 15 h at room temperature. The reaction medium is poured into 600 ml of water and 400 ml of petroleum ether which has previously been heated to 50°C. The organic phase is separated off and the aqueous phase is washed with 2 times
AP/P/ 9 5 / 0 0 7 5 2
AP. Ο Ο 5 6 2
- Ill 150 ml of petroleum ether.
The organic phases are combined, dried over magnesium sulphate, filtered and concentrated under reduced pressure until the volume is 100 ml and a precipitate is .5 obtained. The latter is filtered and washed with 2 times 50 ml of pentane in order to yield 8.2 g of 4-(isopropylbenzylidene)-2-methyl-5-oxazolone in the form of a yellow solid which melts at 77°C.
6 -3: (R.S)-4-Butvlphenvlalanine
0.35 g of* (R,S)-4-butylphenylalanine is obtained in the form of a light beige solid which melts at a temperature greater than 260° by proceeding as in Example 36-1 but using 1.49 g of red phosphorus, 1.8 g of 4 -(butylbenzylidene)-2-methyl-5-oxazolone, in 9.23 ml of acetic anhydride, and 7.39 ml of 57% hydriodic acid.
36- 4: 4-(Butylbenzylidene)-2-methyl-5-oxazolone 1.89 g of 4-(butylbenzylidene)-2-methyl-5oxazolone are obtained in the form of a yellow solid which melts at 74°C by proceeding as in Example 36-2 but using
8.43 g of N-acetylglycine, 4.92 g of sodium acetate, 9.8 g of 4-butylbenzaldehyde and 26 ml of acetic anhydride.
EXAMPLE 37: Preparation of a derivative of phenylalanine using method D.
37- 1: (R,S)-3-Ethoxvphenvlalanine hydrochloride 25 (or (R,S)-3-0-ethvltvrosine hydrochloride) g of (R,S)-N-tert-butoxycarbonyl-3ethoxyphenylalanine, dissolved in 3.6 ml of hydrochloric dioxane, is placed in a round-bottomed flask, and the mixture is then stirred at room temperature for 5 h. The 30 precipitate which forms is filtered, rinsed with dioxane and then ether, and then dried under reduced pressure (2.7 kPa) at 40°C to yield 0.65 g of (R,S)-3ethoxyphenylalanine hydrochloride in the form of a white solid which melts at 200°C.
7-2: (R,S)-N-tert-Butoxvcarbonvl-3 AP/P/ 9 5 / 0 0 7 5 2
112 ethoxyphenvlalanine
1.33 g of ethyl (R,S)-N-tert-butoxycarbonyl-3ethoxyphenylalaninate, dissolved in 8 ml of methanol, are placed in a round-bottomed flask, and 8 ml of IN sodium .5 hydroxide solution are then added. After the mixture has been stirred at room temperature for 18 h, it is evaporated under reduced pressure and then acidified with 8.56 ml of IN hydrochloric acid. The product is extracted with 2 times 10 ml of ethyl acetate, and the organic phases are pooled, washed with 2 times 10 ml of water, dried, filtered and then concentrated to dryness under reduced pressure to yield 1 g of (R,S)-N-tert-butoxycarbonyl-3ethoxyphenylalanine in the form of a yellow oil (Merck Silica 5719, R£=0.7, eluent: toluene 80/MeOH
10/diethylamine 10).
7-3: (R,S)-N-tert-Butoxvcarbonvl-3ethoxyphenvlalaninate
1.5 g of (R,S)-N-tert-butoxycarbonyl-3-tyrosine, dissolved in 7.5 ml of dry DMF, are placed in a three20 necked flask under a nitrogen atmosphere, and 0.508 g of sodium hydride, as a 50% dispersion in oil, is then added. After the mixture has been stirred at room temperature for 2 h, 0.86 ml of iodoethane is added and the mixture is then stirred at room temperature for 4 h. The medium is filtered and the resulting solid is washed with 3 times 10 ml of water and then 2 times 10 ml of petroleum ether to yield, .after drying under reduced pressure (2.7 kPa) at 30°C,
1.33 g of ethyl (R,S)-N-tert-butoxycarbonyl-3 ethoxyphenylalaninate in the form of a white solid.
0 3 7-4: (R,S)-N-tert-Butoxvcarbonvl-3-tvrosine g of (R,S)-3-tyrosine, dissolved in 180 ml of dioxane, are placed, with stirring, in a three-necked flask, and 99 ml of IN sodium hydroxide solution, followed by 26 g of di-tert-butyl dicarbonate, dissolved in 160 ml of dioxane, are then added. After the mixture has been
AP/P/ 95/00752
AP · Ο Ο '5 6 2
- 113 stirred for 36 h, it is concentrated under reduced pressure at 30°C and the residue is taken up in 100 ml of distilled water; this solution is acidified to pH 5 with IN hydrochloric acid and then extracted with 2 times 200 ml of •5 ethyl acetate. The organic phase is dried over magnesium sulphate, filtered and then concentrated to dryness under reduced pressure at 30°C to yield 30 g of (R,S)-N-tertbutoxycarbonyl-3-tyrosine in the form of a white solid (Merck Silica 5719, Rf=0.25, eluent: toluene 80, MeOH 10, diethylamine 10).
c
EXAMPLE 38: Preparation of derivatives of phenylalanine using method E.
8 -1: (RS)-4-Diallvlaminophenvlalanine dihydrochloride
A solid is obtained, after evaporation, by proceeding as in Example 35-1 but using 5.8 g of diethyl 4diallylaminobenzylacetamido malonate and 48 ml of 10N hydrochloric acid; the solid is then triturated in 50 ml of acetone, filtered, then triturated in 10 ml of dichloromethane, filtered and then rinsed with 3 times ml of ethyl ether. After drying under reduced pressure (2.7 kPa) at 40°C, 4.4l’g of (RS)-4J diallylaminophenylalanine dihydrochloride are obtained in the form of an off-white solid which melts in the region of
135°C (decomposition).
8 -2: (RS)-4-Allvlaminophenvlalanine dihydrochloride
A solid is obtained, after evaporation, by proceeding as in Example 35-1 but using 3.27 g of diethyl
4-allylaminobenzylacetamidomalonate and 30 ml of 10N hydrochloric acid; the solid is then triturated in 50 ml of acetone, filtered and then dried under reduced pressure (2.7 kPa) at 40°C. 2.3 g of (RS)-4-allylaminophenylalanine dihydrochloride are obtained in the form of a white solid which melts in the region of 134°C (decomposition).
AP/P/ 95/00752
- 114 38-3: Diethyl 4-diallvlaminobenzvlacetamidomalonate and diethyl 4-allvlaminobenzvlacetamidomalonate g of diethyl 4-aminobenzylacetamidomalonate dissolved in 150 ml of DMF are placed in a three-necked ^5 flask which is surmounted with a dropping funnel and maintained under a nitrogen atmosphere. 6.57 ml of allyl bromide, and then 10.76 ml of triethylamine, are added slowly, at room temperature and while stirring. After stirring for 19h, a further 1.31 ml of allylbromide and
2.15 ml of triethylamine are then added and the mixture is stirred for 26h. The reaction medium is poured onto 1.5 1 of distilled water and this mixture is extracted with 1 1 of ethyl acetate. The aqueous phase is separated off after settling and washed with 2 times 500 ml of ethyl acetate.
The organic phases are combined, washed with 500 ml of distilled water and then with 500 ml of water which is saturated with sodium chloride, separated off, dried over magnesium sulphate, filtered and then concentrated to dryness in order to yield a chestnut oil; this oil is purified by flash chromatography (eluant, CH2Cl290/AcOEt 10) in order to yield 6.66 g of diethyl 4diallylaminobenzylacetamidomalonate in the form of a beige solid which melts at 94-96°C (Rf = 0.6, AcOEt 50/ cyclohexane 50) and 3.49 g of diethyl 4-allylaminobenzyl25 acetamidomalonate in the form of a beige solid which melts at 104-106°C (Rf = 0.45 AcOEt 50/cyclohexane 50).
The diethyl 4-aminobenzylacetamidomalonate can be prepared as described in J.B. Burckhalter, VC Stephens, J. Am. Chem. Soc. 56., 1951, 73.
AP/P/ 95/00752
AP . Ο Ο 5 6 2
- 115 EXAMPLE 39: Preparation of derivatives of phenylalanine using method F
9 -1: (RS)-4 -ethylisopropylphenvlalanine dihvdrochloride
A solid is obtained, after evaporation, by proceeding as in Example 35-1 but using 2.9 g of diethyl 4ethylisopropylbenzylacetamidomalonate and 24.6 ml of ION hydrochloric acid; the solid is then triturated in 20 ml of .acetone, filtered and then dried under reduced pressure 10 (2.7 kPa) at 40°C. 2 g of (RS)-4ethylisopropylaminophenylalanine dihydrochloride are obtained in the form of a white solid which melts in the region of 147°C (decomposition).
AP/P/ 9 5 / 0 0 7 5 2
116
39-2 : Diethyl 4-ethvlisopropylaminobenzvlacetamidomalonate g of diethyl 4-ethylaminobenzylacetamidomalonate in 70 ml of THF are placed in a three-necked flask which is maintained under a nitrogen atmosphere; 6.4 ml of 2-iodopropane, and then 8.4 ml of 1,5diazabicyclo(4.3.0]non-5-ene are added and the mixture is then heated at 60°C for 24h. 2.13 ml of 2 - iodopropane, and then 8.4 ml of 1,5-diazabicyclo[4.3.0]non-5-ene, are subsequently added and the mixture is then heated for a further 24h at 60°C. The mixture is brought to room temperature and then extracted with 50 ml of dichloromethane and 50 ml of distilled water. The aqueous phase is separated off after settling and then rewashed with 2 times 30 ml of dichloromethane. The organic phases are combined, washed with 60 ml of distilled water and then with 50 ml of distilled water which is saturated with NaCl. The organic phase is separated off after settling, dried over magnesium sulphate, filtered and then concentrated to dryness under reduced pressure in order to yield 16.2 g of a product which is purified by flash chromatography (dichloromethane, MeOH 90/10). This results in 4.59 g of a product which is recrystallized in 45 ml of cyclohexane in order to yield 3.44 g of diethyl 425 ethylisopropylaminobenzylacetamidomalonate in the form of white crystals which melt at 80°C.
39-3: Diethyl 4-ethvlaminobenzylacetamidomalonate Diethyl 4-ethylaminobenzylacetamidomalonate can be prepared by proceeding as in Example 35-7 but using 22 g of diethyl 4-aminobenzylacetamidomalonate, 500 ml of ethanol and 70 g of Raney nickel. This results in 23.8 g of diethyl 4-ethylaminobenzylacetamidomalonate in the form of an off-white solid which melts at 136°C.
9 -4: (RS)-4-Allvlethylaminophenylalanine dihydrochloride
AP/P/ 9 5 / 0 0 7 5 2
A solid is obtained, after evaporation, by
AP . 0 0 5 6 2
- 117 proceeding as in Example 35-1 but using 4.54 g of diethyl 4-allylethylbenzylacetamidomalonate and 37.9 ml of ION hydrochloric acid; the solid is then dried under reduced pressure (2.7 kPa) at 40°C. 3.67 g of (RS)-4_5 allylethylaminophenylalanine dihydrochloride are obtained in the form of a brown solid which melts in the region of 130°C (decomposition).
39-5: Diethyl 4-allvlethvlaminobenzvlacetamidomalonate
5.6 g of a solid are obtained, after purification by flash chromatography (eluant, CH2Cl2/AcOET 90-10 by volume), by proceeding as in Example 39-2 but using 8 g of diethyl 4-ethylaminobenzylacetamidomalonate, 4 ml of allyl bromide and 5.82 ml of 1,5-diazabicyclo[4.3.0]non-5-ene in
50 ml of THF; the solid is then recrystallized in 35 ml of cyclohexane. This results in 5.43 g of diethyl 4allylethylaminobenzylacetamidomalonate in the form of a white solid which melts at 86°C.
9 - 6: (RS)-4-Ethylpropvlaminophenvlalanine dihvdrochloride
A solid is obtained, after evaporation, by proceeding as in Example 35-1 but using 2.5 g of diethyl 4ethylpropylaminobenzy.lacetamidomalonate and 21 ml of 10N hydrochloric acid;. The solid is then dried under reduced pressure (2.7 kPa) at 40°C. 2 g (97%) of (RS)-4ethylpropylaminophenylalanine dihydrochloride are obtained in the form of a white solid which melts in the region of 147°C (decomposition).
39-7: Diethyl 4-ethvlpropylaminobenzvlacetamido30 malonate
2.8 g of a solid are obtained, after reacting for 36 hours and then purifying by flash chromatography (eluant, CH2Cl2/MeOH 97-3 by volume), by proceeding as in Example 39-2 but using 10 g of diethyl 4-ethylaminobenzyl35 acetamidomalonate, 5.6 ml of 1-iodopropane and 7.2 ml of
1,5-diazabicyclo[4.3.0]non-5-ene in 70 ml of THF; the solid
AP/P/ 9 5 / 0 0 7 5 2
118 r-.
L.
is then recrystallized in 26 ml of cyclohexane. This results in 2.9 g of diethyl 4-ethylpropylaminobenzylacetamidomalonate in the form of a white solid which melts at 84-86°C.
3 9-8: (RS)-4-Ethvlcvclopropvlmethvlaminophenvlalanine dihvdrochloride
A solid is obtained, after reacting for 3 days and then evaporating, by proceeding as in Example 35-1 but using 3 g of diethyl 4-ethylcyclopropylmethyl10 aminobenzylacetamidomalonate and 25 ml of ION hydrochloric acid; the solid is then triturated in 40 ml of acetone, filtered and then dried under reduced pressure (2.7 kPa) at 40°C. 2.24 g of (RS)-4ethylcyclopropylmethylaminophenylalanine dihydrochloride are obtained in the form of a white solid which melts in the region of 140°C (decomposition).
39-9: Diethvl 4-ethvlcvclopropylmethvlaminobenzvlacetamidomalonate
By proceeding as in Example 39-2, but using 8 g of diethyl 4-ethylaminobenzylacetamidomalonate, 2.6 ml of bromomethylcyclopropane and 2.97 ml of 1,5diazabicyclo[4.3.0]non-5-ene in 50 ml of THF, 3.3 g of diethyl 4-ethylcycl.opropylmethylaminobenzylacetamidomalonate are obtained, after reacting for 3 days and then purifying by flash chromatography (eluant CH2Cl2/AcOEt 9010 by volume), in the form of a white solid which melts at 112-114°C.
C\J
AP/P/ 9 5 / 0 0 7 5
EXAMPLE 40: Preparation of derivatives of phenylalanine using method G
AP . Ο Ο 5 6 2
- 119 -
40-1: (RS) -4-(1-Pyrrolidinyl)phenylalanine dihvdrochloride
A solid is obtained, after evaporation, by proceeding as in Example 35-1 but using 1.5 g of diethyl 4(1-pyrrolidinyl) benzylacetamidomalonate and 40 ml of 5N hydrochloric acid; the solid is then triturated in 15 ml of acetone, filtered and then dried under reduced pressure (2.7 kPa) at 40°C. 0.6 g of (RS)-4-(lpyrrolidinyl)phenylanaline dihydrochloride is obtained in the form of an off-white solid.
40-2: Diethyl 4-(1 -pyrrolidinyl)benzylacetamidomalonate
4 g of diethyl 4-(1-pyrrolyl)benzylacetamidomalonate, dissolved in 100 ml of MeOH, and 1 g of 10% palladium on charcoal are placed in an autoclave. After having purged the autoclave 3 times with nitrogen, the product is hydrogenated at 19°C under a pressure of 14 bars of hydrogen. After stirring for 25 hours, the hydrogenation is stopped and the product is filtered through Clarcel® and rinsed with dichloromethane; the solution is then
AP/P/ 9 5 / 0 0 7 5 2
120 concentrated under reduced pressure in order to yield 3.85 g of a solid which is triturated in a mixture of 50 ml of heptane and 10 ml of ethyl ether. The resulting solid is filtered, dried and then purified by flash chromatography (eluant CH2Cl2/acetone 90/10 by volume) in order to yield 1.6 g of diethyl 4-(1-pyrrolidinyl)benzylacetamidomalonate in the form of a white solid which melts at 132°C.
40-3: Diethyl 4-(1-pyrrolyl)benzvlacetamidomalonate
4,6 g of diethyl 4-aminobenzylacetamidomalonate in 104 ml of acetic acid are placed in a three-necked flask which is maintained under nitrogen. 7.02 g of sodium acetate are added, followed by 1.87 ml of 2,5dimethoxytetrahydrofuran. The mixture is heated at 65°C for lh 15 min, then cooled down and extracted with 100 ml of dichloromethane and 100 ml of distilled water. The aqueous phase is separated off after settling and then washed with 3 times 100 ml of dichloromethane. The organic phases are combined, washed with 100 ml of water and then with 100 ml of a saturated solution of NaCl, separated off after settling and then dried over magnesium sulphate; the phases are filtered and then evaporated to dryness under reduced pressure (50 kPa) in order to yield 6.2 g of a solid which is purifed by flash chromatography (eluent CH2Cl2/acetone
75/25 by volume). This results in 3.57 g of diethyl 4-(1pyrrolyl)benzylacetamidomalonate in the form of a beige solid which melts at 110°C.
AP/P/ 95/00752
EXAMPLE 41: Preparation of derivatives of phenylalanine using method H
0 41-1: (RS)-4-Ethvlthiomethvlphenvlalanine
300 ml of anhydrous methanol are placed in a three-necked flask which is maintained under nitrogen; subsequently, 1.72 g of sodium methoxide, and then 5.55 ml of ethyl mercaptan, are added while stirring. The solution is concentrated under reduced pressure at 40°C in order to
AP.00562
- 121 yield 8.5 g of the sodium salt or ethyl mercaptan, which is dissolved in 100 ml of anhydrous THF. 3.6 g of (RS)-4chloromethylphenylalanine are added at room temperature and the mixture is then heated to reflux for 18h. The solvent is evaporated under reduced pressure at 4 0°C and the residue is taken up in 100 ml of distilled water. The turbid solution which is obtained is acidified with 5 ml of acetic acid. The resulting precipitate is filtered, rinsed with distilled water and then dried at 60°C under reduced pressure in order to yield 3.6 g of a solid which is •s purified by flash chromatography (eluant AcOEt 60, AcOH 12, water 10). This results in 256 mg of (RS)-4ethylthiomethylphenylalanine in the form of a white solid which melts at 251°C.
The (RS)-4-chloromethylphenylalanine can be obtained by analogy with (S)-4-chloromethylphenylalanine as described in: R.Gonzalez-Muniz, F. Cornille, F. Bergeron,
D. Ficheux, J. Pothier, C. Durieux and B. Roques, Int. J. Pept. Protein. Res., 1991, 37 (41), 331-340.
EXAMPLE 42: Preparation of derivatives of phenylalanine using method I
42-1 -. (S) -4-0- (2-Chloroethvl) tyrosine hydrochloride g of (S)-N-tert-butoxycarbonyl-4-O-(225 chloroethyl) tyrosine, dissolved in 50 ml of hydrochloric dioxane, are placed in a round-bottomed flask. After having been stirred for 28h, the mixture is concentrated to dryness under reduced pressure. The resulting residue is taken up in 50 ml of ether and this solution is then stirred and filtered. The resulting solid is washed with 2 times 25 ml of ether and then dried under reduced pressure in order to yield 1.58 g of (S)-4-0-(2-chloroethyl)tyrosine hydrochloride in the form of a white solid which melts at 260°C.
25/00/56 /d/dV
122
2 -2: (S)-N-tert-Butoxvcarbonvl-4-0-(2chloroethvl) tyrosine g of (S)-N-tert-butoxycarbonyltyrosine, dissolved in 140 ml of DMF, are placed in a three-necked flask under a nitrogen atmosphere. 4.8 g of 50% sodium hydride in oil are added slowly using a spatula. 16.87 g of 1-tosyl-2-chloroethanol are added after the mixture has been stirred for 2h at room temperature. 2.4 g of 50% sodium hydride in oil, and a further 8.4 ml of l-tosyl-210 chloroethanol, are added after the mixture has been stirred for 2 days. The same procedure is carried out after 24h and the stirring is continued for a further 24h. The reaction is stopped by adding 100 ml of distilled water, and the reaction mixture is concentrated to dryness under reduced pressure. The residue which is obtained is taken up in
100 ml of distilled water and then extracted with 3 times 100 ml of ethyl acetate. The aqueous phase is separated off after settling and acidified to pH3 with 50 ml of IN HCl, and the product is extracted with 3 times 100 ml of ethyl acetate. The organic phases are combined, washed with 2 times 50 ml of water, separated off, dried over magnesium sulphate, filtered and then concentrated to dryness under reduced pressure in order to yield 13.51 g of (S)-N-tertbutoxycarbonyl-4-O-(2-chloroethyl)tyrosine in the form of a chestnut oil (Rf = 0.5, toluene 70%/methanol
20%/diethylamine 10%), which is used as such in the following step.
AP/P/ 95/007^2
AP . Ο Ο 5 6 2
- 123 TABLE V
MICROORGANISMS ANTIBIOTICS
FUNGI
Micromonospora sp. Vernamycins
STREPTOMYCES
S. alborectus Virginiamycins F1370 A, B
S. conqanensis (ATCC13528)
S. diastaticus Plauracins,
S. qraminofasciens Streptogramins Streptogramins Viridogrisein
S. qriseus (NRRL2426)
S. qriseoviridus (Etamycin) Griseoviridin
S. crriseoviridus (FERMP3562) Neoviridogriseins Etamycins Vernamycins Mikamycins
S. lavendulae
S. loidensis (ATCC11415)
S. mitakaensis (ATCC15297)
S. olivaceus (ATCC12019) Synergistins (PA 114 A, B) Ostreogrycins Pristinamycins Virginiamycins (Staphylomycins)
S. ostreocrriseus (ATCC27455)
S. pristinaespiralis (ATCC25486)
S. virqiniae (ATCC13161)
ACTINOMYCETES
A. auranticolor (ATCC31011) Plauracins
A. azureus (ATCC31157) Plauracins
A. dacrhestanicus Etamycin A-2315 A,B,C
A. philippinensis
Actinoplanes sp. (ATCC3302) A15104
Actinoplanes sp. A17002 A,B,C,F
Actinomadura flava Madumycins
AP/P/ 9 5 / 0 0 7 5 2
124
Abbreviations employed:
C 10 c
o
AcOEt
DNA
AMP
HPLC dCTP
DMF
DMPAPA
HCI
HT7
3-HPA
IPTG kb
LB
MeOH
MMPAPA
NaOH
PAPA
PEG
P I
P II bp
SAM
TE
THF
Tris
UV
X-gal
YEME ethyl acetate deoxyribonucleic acid adenosine 5'-monophosphate high-performance liquid chromatography deoxycytosine 5'-triphosphate dimethylformamide
4-dimethylamino-L-phenylalanine hydrochloric acid
Hickey Tresner solid medium
3- hydroxypicolinic acid i sopropy 1 - 0 - D -1 hioga 1 ac t opyranos ide kilobase
Luria broth (rich growth medium for E, coli) methanol
4- methylamino-L-phenylalanine sodium hydroxide
4- amino-L-phenylalanine polyethylene glycol pristinamycin I pristinamycin II base pair
5- adenosylmethionine mM Tris-HCl buffer, 1 mM EDTA, pH 7.5 tetrahydrofuran
2-amino-2-(hydroxymethyl)-1,3-propanediol ultraviolet rays
5-bromo-4-chloro-3-indoyl-0-D-galactoside yeast extract-malt extract medium (rich growth medium for Streptomyces)
AP/P/ 9 5 / 0 0 7 5 2
00562
- 125 Bibliography;
c
Q
Bibb M. J., Findlay P.R. and Johnson M.W. (1984)
Gene, 30: 157-166.
Bibb M.J., Janssen G.R., and Ward J.M. (1985) Gene,
38: 215-226.
Cocito C.G. (1979) Microbiol. Rev., 43: 145-198.
Cocito C.G. (1983) In Antibiotics, 6: (Ed. F.E. Hahn), 296-332.
Dessen P.C., Fondrat C., Valencien C. and Mugnier C. (1990) Compl. Appl. in Biosciences, 6: 355-356.
Di Giambattista Μ., Chinali G. and Cocito C.G. (1989) J. Antim. Chemother., 24: 485-507
Gibson T.J. (1984) Ph.D. thesis, Cambridge University, England.
Hilleman D., Pulher A. and Wohlleben W. (1991) Nucl. Acids Res., 19: 727-731.
Hopwood D.A., Bibb M.J., Chater K.F., Kieser T.,
Bruton C.J., Kieser H.M., Lydiate D.J., Smith C.P., Ward J.M. and Scrempf H. (1985) A laboratory manual., The John Innes Foundation, Norwich, England.
Hudson G.S. and Davidson B.E. (1984) J. Mol. Biol., 180: 1023-1051.
Kuhstoss S., Richardson M.A., and Rao R.N. (1991) Gene 97: 143-146.
Maniatis T., Fritsch E.F. and Sambrook J. (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor, Ν. Y.,
Messing J., Crea R. and Seeburg P.H. (1981) Nucleic Acid Res. 9: 309.
Molinero A.A., Kingston D.G.I. and Reed J.W. (1989)
J. Nat. Prod., 52: 99-108.
Omer C.A., Lenstra R., Litle P.J., Dean J., Tepperman J.M., Leto K.J., Romesser J.A., and O'Keefe D.P.
(1990) J. Bact. 172: 3335-3345.
Reed J.W., Purvis M.B., Kingston D.G.I., Biot A., and
AP/P/ 9 5 / 0 0 7 5 2
1161-1165. Nucleic Acids
- 126 Gossele F. (1989) J. Org. Chem. 54:
Staden R. and McLachlan A.D. (1982)
Res., 10: 141-156.
Schindler U., Sans N., and Schroder J. (1989) J. -5 171: 847-854.
Thorson J.S., Lo S.F., and Liu H-W (1993)
J. Am. Chem. Soc. 115: 6993-6994.
Videau D. (1982) Path. Biol., 30: 529-534.
Pact.
AP/P/ 9 5 / 0 0 7 5 2
SEQUENCE LISTING (1) GENERAL INFORMATION:
(i) (ii)
AP.0 0 5 6 2
- 127 APPLICANT:
(A) NAME: RHONE-POULENC RORER S.A.
(B) STREET: 20, avenue Raymond ARON (C) CITY: ANTONY (E) COUNTRY: FRANCE (F) POSTAL CODE: 92165
TITLE OF INVENTION: NOVEL STREPTOGRAMINS AND
PROCESS FOR PREPARING STREPTOGRAMINS BY MUTASYNTHESIS.
(iii) (iv)
NUMBER OF SEQUENCES: 8
COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Tape
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Release #1.0, Version
(OEB)
(2) INFORMATION FOR SEQ ID NO: 1:
AP/P/ 9 5 / 0 0 7 5 2 (i)
SEQUENCE CHARACTERISTICS:
20 (A) LENGTH: 2888 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
25 (iii) HYPOTHETICAL: no
(iii) ANTISENSE: no
(vi) ORIGINAL SOURCE:
128 (A) ORGANISM: Streptomyces pristinaespiralis (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
10 20 30 40 SO 60
CTGCAGTTCC CCGGGGCCAC CGTGCTCAGC TCCTCACCCG AACGGTTCCT GCGCATCGGC
70 80 90 100 110 120
GCGGACGGCT GGGCGGAGTC CAAACCCATC AAGGGCACCC GCCCCCGCGG CGCCGGCCCC
130 140 15C 160 170 180
GCCCAGGACG CCGCCGTCAA GGCCTCCCTC GCCGCGGCCG AGAAGGACCG CAGCGAGAAC
190 200 210 220 230 240
CTGATGATCG TCGACCTGGT CCGCAACGAC CTCGGCCAGG TCTGCGACAT CGGCTCCGTC
250 260 270 280 290 300
CACGTACCGG GCCTGTTCGA GGTGGAGACC TACGCCACCG TCCACCAGCT CGTCAGCACG
310 320 330 340 350 360
GTCCGCGGCC GCCTGGCGGC CGACGTCTCC CGCCCCCGCG CGGTACGGGC CGCCTTCCCC
95/00752
a.
CL <
AP. Ο Ο 5 6 2
- 129 -
370 GGCGGGTCGA 380 TGACCGGCGC 390 GCCCAAGGTC 400 CGCACCATGC 410 AGTTCATCGA 420 CCGGCTCGAG
430 440 450 460 470 480
5 AAGGGCCCGC GCGGCGTGTA CTCGGGCGCG CTC-GC-CTACT TCGCCCTCAG CGGCGCGGCC
. 490 500 510 520 530 540
GACCTCAGCA TCGTCATCCG CACCATCGTC GCCACCGAGG AGGCCGCCAC CATCGGCGTG
10 550 560 570 S80 590 600
GGCGGCGCCG TCGTCGCCCT GTCCGACCCC GACGACGAGG TCCGCGAAAT GCTCCTCAAG
610 620 630 640 650 660
GCGCAGACCA CCCTCGCCGC CCTGCGCCAG GCACACGCGG GCGCCACCGC CTCGGACCGT
15
670 680 690 700 710 720
GAACTCCTGG CCGGCAGCCT GCGGTGACCC ACCCACCGCC CCACCCCGGC CACCGCAACC
730 740 750 760 770 780
20 CCGGCTCACC CCCGGGGCGG CCGCGCGCGG TGCCGCCCGG CGC-CCGACCC GGCGACGGGT
790 800 810 820 830 840
CCGCTCGCGG ACCGGGTGAC GC-ACCCGGCG GCGGGGCCGG CGGCGC-GCCC- GGACC-TGGGC
25 850 8 60 870 880 890 900
CGGGACGTGG GCCCGGCGTC CCCGGCGACC GGCACGGCGG CGGGCCCGGA CGTGGGCCCG
910 920 930 940 950 960
GCGTGCCCGG CGACCGGCAC GGTGGCGGGG CGGC-GCGGGG GACGGTCAGT GCAGGGCGGT
30
970 980 990 1000 1010 1020
GAACATCCGC GCGCACAGCC GTTCCAGCTC CGCGCCGTGC TCGCCCAGCA CACCGCGCAG
1030 1040 1050 1060 1070 1080
35 TTCGGCGAAC AGGGCGGCGA ACGTCTCCTC GTCGCCCCTC TCGACGGCCT GCCCCAGCCG
1090 1100 1110 1120 1130 1140
CACCAGGCCG CGGCCCAGCG CCTGCCGCGC GGCCGGCGCG CCGGGGTTGG CGGCCTGGAT
40 1150 1160 1170 1180 1190 1200
GTCGAAATAC ACCTCCGGCG TCCCGCCGGC GATCCGGGCC AGCAGCGCCA GCATCGCCAG
1210 1220 1230 1240 1250 1260
ATGCGGCGGC GGGGCACTGT CCCGCAGCGC CCCCACGTCC ACCGACAGCT CACCCAGGCC
45
1270 1280 1290 1300 1310 1320
CAGCCCGAAG GCCAGCACCG CGGCATGCGT GGCGGCCTGC TGCGCGGCGG TCAGCTCGTC
1330 1340 1350 1360 1370 1380
50 GTGCCGCCGC GCCGGCATCT CCACCACCCG GGCCCCCCAC CCGGCCACCA GCTCCACCAG
1390 1400 1410 1420 1430 1440
GGCCCGCACA CCGGGCCCGT CGGTGACCAC CACCGCCGCC ACCGGCCGCC CCTGAAGACC
55 1450 14 60 1470 1480 1490 1500
CAGCGAGGGG GCGAACATCG GGTTCAGCCC CACCGCCTGC AGCCCCGGCG CCGCCTCACG
1S10 1520 1530 1540 1SS0 1S60
60 CAGCCGCCCG GCGATCCGGC TCTTGACCGA CAAGGTGTCC GCGAGCACCG CACCGGGCCG
1S70 1S80 1590 1600 1610 1620
CATCACCCCC GCCAGCACCT CCACCGCCTC CCACGCCACC GGCTCCGGCA CCGCCAGCAC
1630 1640 16S0 1660 1670 1680
65 CACCACGTCC GCCGCCGCCA GCGCCGCGAC CGCCTCCGGC CCCGGCCGCC GCACATCACC
1690 1700 1710 1720 1730 1740
ύ b L U 0 / S 6 /d/dV
130
GGCCACCACC CGCACCCCGT CCGCCGCACC GGCCCCGGCC ACGTCCAGCC AGGTCACCGC
1750 1760 1770 1780 1790 1800
CACCCCCGAA CGCACCAGCC AGTGGCTGAA CATGCGGCCC ACCGCACCGG CCCCGCCCAC
1810 1820 1830 1840 1850 1860
CACCACACAA CGCCCGAACA CCGAACCACC CCTCATCCGC GTTCCCGATC CCCCCGGTAC
1870 1880 1890 1900 1910 1920
GGAGGAAGAA CCATGACCCC GCCCGCCATC CCCGCCGCCC CGCCCGCCAC CGGGCCCGCC
1930 1940 1950 1960 1970 1980
CCCGCCACCG ACCCCCTCGA CGCGCTGCGC GCCCGCCTGG ACGCCGCGGA CC-CCC-CCCTG
1990 2000 2010 2020 2030 2040
CTGGACGCCG TCCGCACACG CCTGGACATC TGCCTGCGCA TCGGCGAGTA CAAGCGCCTC
2050 2060 2070 2080 2090 2100
CACCAGGTGC CGATGATGCA GCCCCACCGG ATCGCCCAGG TCCACGCCAA CC-CCC-CCCGC
2110 2120 2130 2140 2150 2160
TACGCCGCCG ACCACGGCAT CGACCCCGCC TTCCTGCGCA CCCTGTACGA CACC-ATCATC
2170 2180 2190 2200 2210 2220
ACCGAGACCT GCCGCCTCGA GGACGAGTGG ATCGCCTCCG GCGGCGCCCC CGTCCCCACG
2230 2240 2250 2260 2270 2280
CCCGTGCACG CGTCCGCGTC CGCGCGGGGG GCCGTGTCGT C-ACCGCCGCC GCACCCACCC
2290 2300 2310 2320 2330 2340
TCGCCCAGGC GCTGGACGAG GCCACCGGGC AGCTGACCGG CGCCGGGATC ACCGCCC-ACG
2350 2360 2370 2380 2390 2400
CCGCCCGGGC CGACACCCGG CTGCTGGCCG CCCACGCCTG CCAGGTCGCC CCGGGGGACC
2410 2420 2430 2440 2450 2460
TCGACACCTG CCTGGCCGGC CCGGTGCCGC CCCGGTTCTG GCACTACGTC CGGCGCCGTC
2470 2480 2490 2S00 2510 2520
TGACCCGCGA ACCCGCCGAA CGCATCGTCG GCCACGCCTA CTTCATGGGC CACCGCTTCG
2530 2540 2550 2560 2570 2580
ACCTGGCCCC CGGCGTCTTC GTCCCCAAAC CCGAGACCGA GGAGATCACC CGGGACGCCA
2590 2600 2610 2620 2630 2640
TCGCCCGCCT GGAGGCCCTC GTCCGCCGCG GCACCACCGC ACCCCTGGTC GTCGACCTGT
2650 2660 2670 2680 2690 2700
GCGCCGGACC GGGCACCATG GCCGTCACCC TGGCCCGCCA CGTACCGGCC GCCCGCGTCC
2710 2720 2730 2740 2750 2760
TGGGCATCGA ACTCTCCCAG GCCGCCGCCC GCGCCGCCCG GCGCAACGCC CGCGGCACCG
2770 2780 2790 2800 2810 2820
GCGCCCGCAT CGTGCAGGGC GACGCCCGCG ACGCCTTCCC CGAACTGAGC GGCACCGTCG
2830 2840 2850 2860 2870 2880
ACCTCGTCGT CACCAACCCG CCCTACATCC CCATCGGACT GCGCACCTCC GCACCCGAAG
TGCTCGAG
AP . Ο Ο 5 6 2
- 131 (3) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 888 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: no (iv) ANTISENSE: no (vi) ORIGINAL SOURCE:
(A) ORGANISM: Strectomyces pristinaespiralis (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2
ATG AGG GGT GGT TCG GTG TTC GGG CGT TGT C-TC- GTG GTG GGC C-C-C- GCC GGT GCG 54
Ket Arg Gly Gly Ser Val Phe Gly Arg Cys Val Val Val C-ly Glv Ala Gly Ala 18
GTG GGC CGC ATG TTC AGC CAC TGG CTG GTG CC-T TCG GGG GTG GCG GTG ACC TGG 108
Val Gly Arg Met Phe Ser His Trp Leu Val Arc Ser C-ly Val Ai c. Val Thr Trp 36
CTG GAC GTG GCC GGG GCC GGT GCG GCG GAC GGG GTG CGG GTG GTG GCC GGT GAT 162
Leu Asp Val Ala Gly Ala Gly Ala Ala Asp Gly Val Arc Val Val Ala Gly Asp 54
GTG CGG CGG CCG GGG CCG GAG GCG GTC GCG GCG CTG GCG GCG GCG GAC GTG GTG 216
Val Arg Arg Pro Gly Pro Glu Ala Val Ala Ala Leu Ala Ala Ala Asp Val Val 72
GTG CTG GCG GTG CCG GAG CCG GTG GCG TGG GAG GCG GTG GAG GTG CTG GCG GGG 270
Val Leu Ala Val Pro Glu Pro Val Ala Trp Glu Ala Val Glu Val Leu Ala Gly 90
GTG ATG CGG CCC GGT GCG GTG CTC GCG GAC ACC TTG TCG GTC AAG AGC CGG ATC 324
Val Met Arg Pro Gly Ala Val Leu Ala Asp Thr Leu Ser Val Lys Ser Arg Xie 108
GCC GGG CGG CTG CGT GAG GCG GCG CCG GGG CTG CAG GCG GTG GGG CTG AAC CCG 378
Ala Gly Arg Leu Arg Glu Ala Ala Pro Gly Leu Gin Ala Val Gly Leu Asn Pro 126
ATG TTC GCC CCC TCG CTG GGT CTT CAG GGG CGG CCG GTG GCG GCG GTG GTG GTC 432
Met Phe Ala Pro Ser Leu Gly Leu Gin Gly Arg Pro Val Ala Ala Val Val Val 144
ACC GAC GGG CCC GGT GTG CGG GCC CTG GTG GAG CTG GTG GCC GGG TGG GGG GCC 486
Thr Asp Gly Pro Gly Val Arg Ala Leu Val Glu Leu Val Ala Gly Trp Gly Ala 162
CGG GTG GTG GAG ATG CCG GCG CGG CGG CAC GAC GAG CTG ACC GCC GCG CAG CAG 540
Arg Val Val Glu Met Pro Ala Arg Arg His Asp Glu Leu Thr Ala Ala Gin Gin 180
GCC GCC ACG CAT GCC GCG GTG CTG GCC TTC GGG CTG GGC CTG GGT GAG CTG TCG 594
Al a Ala Thr ills Ala Ala Val Leu Ala Phe Gly Leu Gly Leu Gly Glu Leu Ser 198
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GTG GAC GTG GGG GCG CTG CGG GAC AGT GCC CCG CCG CCG CAT CTG GCG ATG CTG 648
Val Asp Val Gly Ala Leu Arg Asp Ser Ala Pro Pro Pro His Leu Ala Met Leu 216
GCG CTG CTG GCC CGG ATC GCC GGC GGG ACG CCG GAG GTG TAT TTC GAC ATC CAG 702
Ala Leu Leu Ala Arg Ile Al a Gly Gly Thr Pro Glu Val Tyr Phe Asp Ile Gin 234
GCC GCC AAC CCC GGC GCG CCG GCC GCG CGG CAG GCG CTG GGC CGC GGC CTG GTG 756
'Ala Ala Asn Pro Gly Ala Tro Ala Ala Arg Gin Ala Leu Gly Arg Gly Leu Val 252
CGG CTG GGG CAG GCC GTC GAG AGG GGC GAC GAG GAG ACG TTC GCC GCC CTG TTC 810
Arg Leu Gly Gin Ala Val Glu Arg Gly A.sp Glu Glu Thr Phe Ala Ala Leu Phe 270
GCC GAA CTG CGC GGT GTG CTG GGC GAG CAC GGC GCG GAG CTG GAA CGG CTG TGC 864
Ala C-lu Leu Arg Gly Val Leu Gly Glu His Gly Ala Glu Leu Glu Arg Leu cys 288
GCG CGG ATG TTC ACC C-CC CTG CAC 888
Ai c. Arg Ket Phe Thr Ala Leu Kis 2S6
(4) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 387 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: no (iii) ANTISENSE: no (vi) ORIGINAL SOURCE:
(A) ORGANISM: Streptomyces pristinaespiralis (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3 ©J
LO o
o
LO
DL
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ATG ACC CCG CCC GCC ATC CCC GCC GCC CCG CCC GCC ACC GGG CCC GCC CCC GCC 54
Met Thr Pro Pro Ala Ile Pro Ala Ala Pro Pro Ala Thr Gly Pro Ala Ala Ala 18
ACC GAC CCC CTC GAC GCG CTG CGC GCC CGC CTG GAC GCC GCG GAC GCC GCC CTG 108
Thr Asp Pro Leu Asp Ala Leu Arg Ala Arg Leu Asp Ala Ala Asp Ala Ala Leu 36
CTG GAC GCC GTC CGC ACA CGC CTG GAC ATC TGC CTG CGC ATC GGC GAG TAC AAG 162
Leu Asp Ala Val Arg Thr Arg Leu Asp Ile cys Leu Arg Ile Gly Glu Tyr Lys 54
CGC CTC CAC CAG GTG CCG ATG ATG CAG CCC CAC CGG ATC GCC CAG GTC CAC GCC 216
Arg Leu His Gin Val Pro Met Met Gin Pro His Arg Ile Al a Gin Val His Al a 72
AP . Ο Ο 5 6 2
- 133 -
AAC Asn GCC Ala GCC Ala CGC Arg TAC Tyr GCC Ala GCC Ala GAC Asp CAC His GGC ATC GAC CCC Pro GCC Ala TTC Phe CTG Leu CGC Arg ACC Thr 270 50
Gly lie Asp
CTG TAC GAC ACG ATC ATC ACC GAG ACC TGC CGC CTC GAG GAC GAG TGG ATC GCC 324
Leu Tyr Asp Thr lie He Thr Glu Thr cys Arg Leu Glu Asp Glu Trp He Ala ιοε
TCC GGC GGC GCC CCC GTC CCC ACG CCC GTG CAC GCC TCC GCG TCC GCG CGG GGG 37e
Ser Gly Gly Ala Pro Val Pro Thr Pro Val His Ala Ser Ala Ser Ala Arg Gly 126
GCC GTG TCG »& '
Ala Val Ser 125 c
(5) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4496 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: no (iii) (vi)
ANTISENSE: no
ORIGINAL SOURCE:
(A) ORGANISM: Streptomyces pristinaespiralis (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4
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10 20 30 40 50 60
CTCGAGCAGG TGCCCCACCT CGGCGGCACG GTGCGCGGGC AGCGCGAACA CCGGCAGCGC
70 80 90 100 110 120
GCCCAGACGG AACAGCGCGA AGCACACCGC GACGAACTCG GGCGTGTTCG GCAGCTGCAC
130 140 150 160 170 180
CAGCACCCGC TCGCCGGCGC CGATCCCGCG CGCCGCGAAC CCCGCCGCCA GCCGGTCGCA
190 200 210 220 230 240
CCAGCGGTCC AGGGCACGGT AGGTGACACG GGAGCACCCG TCCGCGCCGA CCAGCGCCTC
2S0 260 270 280 290 300
CCGCTCGCCG TACTGCTCCG CCCAGCGGCC CAGCAGCATG CCCAGCGGCT CGCCCCGCCA
310 320 330 340 350 360
GTAGCCGGCC GCCCGGTACT TCGCGGCCAC ATCCTCGGGC CAGGGAACGC ATCCGTCCAG
370 380 390 400 410 420
134
CATCGTTGGT CCTTTCCGGC TTCGTCCTCG CGTCC-CGCCC AGTGTCGGCA GCGCCGTTGA
430 CACGCCGCTG 440 ATGCGCCGCG 450 CCCGCGCGCC 460 GCCGCTCCGT 470 CAGGAGCCGA 480 TCAGGGCGGC
490 GTCAGCCGGG 500 CCGGACAGGA 510 TGCCGCCCAC S20 GGGGCCCGGC 530 ACACCGGGCC S40 GCGGCGACAC-
S50 CGGGCCGGCG 560 ACCCGCAGGC 570 CGACACCACG 580 CAC GC-ACG AG 590 AAGAAACAAC 600 ACAAGGGGAG
610 CACCCGATGG 620 AGACCTGGGT 630 CCTGGGCCGG 640 CGCC-ACGTCG 650 CCGAGGTGGT 660 GGCCGCCGTC
670 GGCCGCGACG 680 AACTCATGCG 690 CCGCATCATC 700 C-ACCC-CCTCA 710 CCC-GCGGACT 720 GGCCGAGATC
730 GGCCGCGGCG 740 AGCGGCACCT 750 C-TCCCCGCTG 760 CGCGC-CGGAC 770 TGGAACGCAG 780 CC-AACCCGTG
790 CCCGGCATCT 800 GGGAATGGAT 810 GCCGCACCGC 820 GAACCCGGCG 830 ACCACATCAC 840 CCTCAAGACC
850 GTCGGCTACA 860 GCCCCGCCAA 870 CCCCGGCCGC 880 TTCGGCCTGC 890 CGACCATCCT 900 C-GGCACCGTC
910 GCCCGCTACG 920 ACGACACCAC 930 CC-GCGCCCTG 940 ACCGCCCTGA 950 TGGACGGCGT 960 GCTGCTCACC
970 GCCCTGCGCA 980 CCGGCGCCGC 990 CTCCGCCGTC 1000 GCCTCCCGCC 1010 TGCTGGCCCG 1020 CCCCGACAGC
1030 CACACCCTGG 1040 GACTGATCGG 1050 CACCGGCGCC 1060 CAGGCCGTCA 1070 CCCAACTGCA 1080 CGCCCTGTCC
1090 CTGGTACTGC 1100 CCCTGCAACG 1110 GGCCCTGGTG 1120 TGGGACACCG 1130 ACCCCGCCCA 1140 CCGGGAAAGC
1150 TTCGCCCGGC 1160 GCGCCGCGTT 1170 CACCGGCGTC 1180 AGCGTCGAGA 1190 TCGCCGAGCC 1200 CGCCCGGATC
1210 GCCGCCGAGG 1220 CCGACGTCAT 1230 CTCCACCGCC 1240 ACCTCGGTAG 1250 CCGTCGGCCA 1260 GGGCCCGGTC
1270 CTGCCCGACA 1280 CCGGCGTCCG 1290 CGAGCACCTG 1300 CACATCAACG 1310 CCGTCGGCGC 1320 GGACCTCGTC
1330 GGCAAGACGG 1340 AACTGCCGCT 13S0 CGGCCTGCTC 1360 GAGCC-GGCGT 1370 TCGTCACCGC 1380 CGACCACCCC
1390 GAGCAGGCGC 1400 TGCGCGAGGG 1410 CGAGTGCCAG 1420 CAACTCTCCG 1430 CCGACCGGCT 1440 CGGCCCGCAG
14S0 CTGGCCCACC 1460 TGTGCGCCGA 1470 CCCGGCGGCC 1480 GCCGCCGGCC 1490 GGCAGGACAC 1500 CCTGAGCGTC
1510 TTCGACTCCA 1520 CCGGCTTCGC 1S30 CTTCGAGGAC 1540 GCCCTGGCGA 1550 TGGAAGTGTT 1560 CCTCGAGGCC
1570 GCCGCCGAAC 1580 GGGACCTGGG 1590 CATCCGGGTG 1600 GGCATCGAAC 1610 ACCACCCCGG 1620 CGACGCCCTG
1630 GACCCCTACG 1640 CCCTCCAGCC 1650 CCTGCCCCTG 1660 CCCCTGGCCG 1670 CCCCCGCCCA 1680 CTGACCCCCC
1690 CCTTTTTTCG 1700 GGACCCCCGC 1710 TCTTTTTCGA 1720 GACCCCCGCC 1730 CGGCCGGCCC 1740 GGCCCTCCTC
ΑΡ/Ρ/ 9 5 / 0 0 7 5 2
AP. Ο Ο 5 6 2
- 135 -
1750 CCGCCGGCCC 1760 CCATGCCCGG 1770 CCGGGCCGGG 1780 GCACCCACGA 1790 CGCCCTCGCG 1800 AGGAGAGAGA
1610 TGCCCCCCAC 1820 CCCCCGGCCC 1830 ACCACCGACG 1840 ACGC-CC-GCCC- 1850 TGAACTGCTC 1860 GCCTGGCTGC
1870 GCGAGATGCG 1880 CCACCACCAC 1890 CCCGTCCACG 1900 AGGACGAATA 1910 CGGTGCCTTC 1920 CACGTCTTCC
1930 GGCACGCCGA 1940 CGTCCTCACC 1950 GTCGCCTCCG 1960 ACCCCGGCGT 1970 CTACTCCTCC 1980 CAGCTCAGCC
1990 GGCTACGGCC 2000 CGGCTCCCAG 2010 GCGTTGAGCG 2020 AACAGATCCT 2030 GTCGGTCATC 2040 GACCCGCCGA
2050 TGCACCGCAC 2060 CCTGCGCCGC 2070 CTGGTCAGCC 2080 AGC-CCTTCAC 2090 CCCCCGCACC 2100 GTCGCCGACC
2110 TCGAACCACG 2120 CGTCACCGAA 2130 CTGGCCGGGC 2140 AACTGCTCGA 2150 CGCCGTCGAC 2160 GGCGACACG7
2170 TCGACCTCGT 2180 CGCCGACTTC 2190 GCCTACCCGC 2200 TGCCCGTGAT 2210 CGTC-ATCC-CC 2220 C-AACTCCTCG
2230 GCGTGCCGCC 2240 CGCCGACCGC 2250 ACCCTGTTCC 2260 GCTCCTGGTC 2270 CGACCGC-ATG 2280 CTGCAGATGC
2290 AGGTCGCCGA 2300 CCCGGCGGAC 2310 ATGCAGTTCG 2320 GCGACGACGC 2330 CGACGAGGAC 2340 TACCAACGCC
2350 TCGTCAAAGA 2360 ACCCATGCGC 2370 GCCATGCACG 2380 CCTACCTCCA 2390 CGACCACGTC 2400 ACCGACCGCC
2410 GCGCCCGCCC 2420 CGCGAACGAC 2430 CTGATCTCCG 2440 CACTCGTCGC 2450 CGCCCGCGTG 2460 GAGGGCGAAC
2470 GACTCACCGA 2480 CGAGCAGATC 2490 GTCGAATTCG 2500 GGGCGCTGCT 2510 GCTGATGGCC 2S20 GGCCACGTCT
2530 CCACCTCCAT ' 2S40 GCTGCTCGGC 2550 AACACCGTGC 2560 TGTGCCTGAA 2570 GGACCACCCC 2580 CGGGCCGAGG
2590 CCGCCGCCCG 2600 CGCCGACCGG 2610 TCCCTGATCC 2620 CCGCCCTGAT 2630 CGAAGAAGTA 2640 CTGCGGCTGC
2650 GGCCGCCGAT 2660 CACCGTCATG 2670 GCCCGCGTCA 2680 CCACCAAGGA 2690 CACCGTCCTC 2700 GCCGGCACCA
2710 CCATCCCCGC 2720 CGGACGCATG 2730 GTCGTGCCCT 2740 CCCTGCTGTC 2750 CGCCAACCAC 2760 GACGAACAGG
2770 TCTTCACCGA 2780 CCCCGACCAC 2790 CTCGACCTCG 2800 CCCGCGAAGG 2810 CCGCCAGATC 2820 GCCTTCGGCC
2830 ACGGCATCCA 2840 CTACTGCCTG 2850 GGCGCCCCGC 2860 TCGCCCGCCT 2870 GGAGGGCCGC 2880 ATCGCCCTGG
2890 AAGCCCTCTT 2900 CGACCGATTC 2910 CCCGACTTCT 2920 CGCCCACCGA 2930 CGGCGCAAAA 2940 CTGCGCTACC
2950 ACCGCGACGG 2960 ACTGTTCGGC 2970 GTCAAGAACC 2980 TGCCGCTGAC 2990 CGTACGGCGC 3000 GGCTGACACA
3010 GACAAGGGGG 3020 CCACCTGGTG 3030 CGCACCGTGC 3040 GAACCCTGCT 3050 GATCGACAAC 3060 TACGACTCGT
ΑΡ/Ρ/ 9 5 / 0 0 7 5 2
136
3070 TCACCTACAA 3080 CCTCTTCCAG 3090 ATGCTGGCCG 3100 AGGTGAACGG 3110 CGCCGCTCCG 3120 CTCGTCGTCC
3130 3140 3150 3160 3170 3180
GCAACGACGA CACCCGCACC TGGCAGGCCC TGGCGCCGGG CGACTTCGAC AACGTCGTCG
3190 3200 3210 3220 3230 3240
TCTCACCCGG CCCCGGCCAC CCCGCCACCG ACACCGACCT GGGCCTCAGC CGCCGGGTGA
3250 3260 3270 3280 3290 3300
TCACCGAATG GGACCTGCCG CTGCTCGGGG TGTGCCTGGG CCACCAGGCC CTGTGCCTGC
3310 3320 3330 3340 3350 3360
TCGCCGGCGC CGCCGTCGTC CACGCACCCG AACCCTTTCA CGGCCGCACC AGCGACATCC
3370 3380 3390 3400 3410 3420
GCCACGACGG GCAGGGCCTG TTCC-CGAACA TCCCCTCCCC GCTGACCGTG GTCCGCTACC
3430 3440 3450 3460 3470 3480
ACTCGCTGAC CGTCCGGCAA CTGCCCGCCG ACCTGCC-CGC CACCGCCCAC ACCGCCGACG
3490 3500 3510 3520 3530 3540
GGCAGCTGAT GGCCGTCGCC CACCGCCACC TGCCCCGCTT CGGCGTGCAC- TTCCACCCCG
3550 3560 3570 3580 3590 3600
AATCGATCAG CAGCGAACAC GGCCACCGGA TGCTCGCCAA CTTCCGCGAC CTGTCCCTGC
3610 3620 3630 3640 36S0 3660
GCGCGGCCGG CCACCGCCCC CCC-CACACCG AACGCATACC CGCACCCGCA CCCGCCCCCG
3670 3680 3690 3700 3710 3720
CCCCCGCCCC CGCACCGGCA CCGCCCGCGT CCGCGCCGGT GGGGGAGTAC CGGCTGCATG
3730 3740 3750 3760 3770 3780
TGCGCGAGGT CGCCTGCGTG CCCGACGCGG ACGCCGCGTT CACCGCCCTG TTCGCCGACG
3790 3800 3810 3820 3830 3840
CCCCGGCCCG GTTCTGGCTC GACAGCAGCC GCGTCGAGCC GGGCCTCGCC CGCTTCACCT
3850 3860 3870 3880 3890 3900
TCCTCGGCGC CCCCGCCGGC CCGCTCGGCG AACAGATCAC CTACGACGTC GCCGACCGGG
3910 3920 3930 3940 3950 3960
CCGTGCGCGT CAAGGACGGT TCAGGCGGCG AGACCCGCCG GCCCGGCACC CTCTTCGACC
3970 3980 3990 4000 4010 4020
ACCTGGAACA CGAACTGGCC GCCCGCGCCC TGCCCC-CCAC CGGCCTGCCC TTCGAGTTCA
4030 4040 4050 4060 4070 4080
ACCTCGGCTA CGTCGGCTAC CTCGGCTACG AGACCAAGGC CGACAGCGGC GGCGAGGACG
4090 4100 4110 4120 4130 4140
CCCACCGCGG CGAACTGCCC GACGGCGCCT TCATGTTCGC CGACCGGATG CTCGCCCTCG
4150 4160 4170 4180 4190 4200
ACCACGAACA GGGGCGGGCC TGGCTCCTGG CACTGAGCAG CACCCGACGG CCCGCCACCG
4210 4220 4230 4240 4250 4260
CACCCGCCGC CGAACGCTGG CTCACCGACG CCGCCCGGAC CCTCGCCACC ACCGCCCCCC
4270 4280 4290 4300 4310 4320
GCCCGCCCTT CACCCTGCTG CCCGACGACC AACTGCCCGC CCTGGACGTC CACTACCGCC
4330 4340 4350 4360 4370 4380
ACAGCCTGCC CCGCTACCGG GAACTGGTCG AGGAATGCCG CCGCCTGATC ACCGACGGCG
4390 4400 44 10 4420 4430 4440
AP/P/ 9 5 / 0 0 7 5 2 . 0 0 5 6 2
- 137 AGACCTACGA GGTGTGCCTG ACGAACATGC TCCGGGTGCC CGGCCGGATC GACCCGCTCA
4450 4460 4470 4480 4490
CCGCCTACCG CGCCCTGCGC ACCGTCAGCC CCGCCCCCTA CGCCGCCTAC CTGCAG (6) INFORMATION FOR SEQ ID NO: 5 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1065 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double
(D) TOPOLOGY : linear
(ii) MOLECULE TYPE : CDNA
(iii) HYPOTHETICAL: no CJ
ir>
(iii) ANTISENSE : no r-
o
(vi) ORIGINAL SOURCE: o
(A) ORGANISM : Streptomyces —
pristinaespiralis
ATG GAG ACC TGG GTC CTG GGC CGG CC-C GAC GTC GCC GAG GTG GTG GCC GCC GTC 54 O>
Met Glu Thr Trp Val Leu Gly Arg Arg Asp Val Ala Glu Val Val Ala Ala Val 18 Ol
GGC CGC GAC GAA CTC ATG CGC CGC ATC ATC GAC CGC •CTC ACC GGC GGA CTG GCC 108 0··
Gly Arg Asp Glu Leu Met Arg Arg lie He Asp Arg Leu Thr Gly Gly Leu Ala 36 <
GAG ATC GGC CGC GGC GAG CGG CAC CTG TCC CCG CTG CGC GGC GGA CTG GAA CGC 162
Glu lie Gly Arg Gly Glu Arg His Leu Ser Pro Leu Arg Gly Gly Leu Glu Arg 54
AGC GAA CCC GTG CCC GGC ATC TGG GAA TGG ATG CCG CAC CGC GAA CCC GGC GAC 216
Ser Glu Pro Val Pro Gly lie Trp Glu Trp Met Pro His Arg Glu Pro Gly Asp 72
CAG ATC ACC CTC AAG ACC GTC GGC TAC AGC CCC GCC AAC CCC GGC CGC TTC GGC 270
His lie Thr Leu Lys Thr Val Gly Tyr Ser Pro Ala Asn Pro Gly Arg Phe Gly 90
CTG CCG ACC ATC CTG GGC ACC GTC GCC CGC TAC GAC GAC ACC ACC GGC GCC CTG 324
Leu Pro Thr He Leu Gly Thr Val Ala Arg Tyr Asp Asp Thr Thr Gly Ala Leu 106
ACC GCC CTG ATG GAC GGC GTG CTG CTC ACC GCC CTG CGC ACC GGC GCC GCC TCC 378
Thr Ala Leu Met Asp Gly Val Leu Leu Thr Ala Leu Arg Thr Gly Ala Ala Ser 126
GCC GTC GCC TCC CGC CTG CTG GCC CGC CCC GAC AGC CAC ACC CTG GGA CTG ATC 432
Ala Val Ala Ser Arg Leu Leu Ala Arg Pro Asp Ser His Thr Leu Gly Leu He 144
GGC ACC GGC GCC CAG GCC GTC ACC CAA CTG CAC GCC CTG TCC CTG GTA CTG CCC 486
Gly Thr Gly Ala Gin Ala Val Thr Gin Leu His Ala Leu Ser Leu Val Leu Pro 162
138
CTG CAA CGG GCC CTG GTG TGG GAC ACC GAC CCC GCC CAC CGG GAA AGC TTC GCC 540
Leu Gin Arg Ala Leu Val Trp Asp Thr Asp Pro Ala His Arg Glu Ser Phe Ala 180
CGG CGC GCC GCG TTC ACC GGC GTC AGC GTC GAG ATC GCC GAG CCC GCC CGG ATC S94
Arg Arg Ala Ala Phe Thr Gly val Ser Val Glu lie Ala Glu Pro Ala Arg He 198
GCC GCC GAG GCC GAC GTC ATC TCC ACC GCC ACC TCG GTA GCC GTC GGC CAG GGC 648
Ala Ala Glu Ala Asp Val lie Ser Thr Ala Thr Ser Val Ala Val Gly Gin Gly 216
CCG GTC CTG CCC GAC ACC GGC GTC CGC GAG CAC CTG CAC ATC AAC GCC GTC GGC 702
Pro Val Leu Pro Asp Thr Gly Val A.rg C-lu His Leu His lie Asn Ala Val Gly 234
C-CG GAC CTC GTC GGC MG ACC- GAA. CTG CCG CTC GGC CTG CTC GAG CGG GCG TTC 756
Ala Asp Leu Val Gly Lys Thr C-lu Leu Pro Leu Gly Leu Leu Glu Arg Ala Phe 252
GTC ACC GCC GAC CAC CCC GAG CAC- C-CC- CTG CC-C GAG GGC GAG TGC CAG CAA CTC 810
Val Thr Ala Asp His Pre C-lu C-lr. Ala Leu Arc C-lu Gly Glu Cys Gin Gin Leu 270
TCC GCC GAC CGG CTC GGC CCG CAC- CTC- GCC CAC CTG TGC GCC GAC CCG GCG GCC 864
Ser Ala Asp Arg Leu Gly Pro Gln Leu A.la His Leu Cys Ala Asp Pro Ala Ala 288
GCC GCC GGC CGG CAG GAC ACC CTG AGC C-TC TTC GAC TCC ACC GGC TTC GCC TTC 918
Ala Ala Gly Arg Gin Asp Thr Leu Ser Val Phe Asp Ser Thr Gly Phe Ala Phe 306
GAC- GAC GCC CTG GCG ATG GAA GTG TTC CTC GAG GCC GCC GCC GAA CGG GAC CTG 972
Glu Asp Ala Leu Ala Met Glu Val Phe Leu Glu Ala Ala Ala Glu Arg Asp Leu 324
GGC ATC CGG GTG GGC ATC GAA CAC CAC CCC GGC GAC GCC CTG GAC CCC TAC GCC 1026
Gly lie Arg Val Gly lie Glu His His Pro Gly Asp Ala Leu Asp Pro Tyr Ala 342
CTC CAG CCC CTG CCC CTG CCC CTG GCC GCC CCC GCC CAC 1065
Leu Gin Pro Leu Pro Leu Pro Leu Ala Ala Pro Ala His 355
AP/P/ 9 5 / 0 0 7 5 2 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1194 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: no (iii) ANTISENSE: no (vi) ORIGINAL SOURCE:
(A) ORGANISM: Streptomyces pristinaespiralis
AP. Ο Ο 5 6 2
- 139 -
(xi) DESCRIPTION DE LA SEQUENCE: SEQ id : NO: 6
ATG CCC CCC ACC CCC CGG CCC ACC ACC GAC GAC GGC GGC CGT GAA CTG CTC GCC 54
Met Pro Pro Thr Pro Arg Pro Thr Thr Asp Asp Gly Gly Arg Glu Leu Leu Ala 18
TGG CTG CGC GAG ATG CGC CAC CAC CAC CCC GTC CAC GAG GAC GAA TAC GGT GCC 108
Trp Leu Arg Glu Met Arg His His His Pro Val His Glu Asp Glu Tyr Gly Ala 36
TTC CAC GTC TTC CGG CAC GCC GAC GTC CTC ACC GTC GCC TCC GAC CCC GGC GTC 162
Phe His Val Phe Arg His Ala Asp Val Leu Thr Val Ale Ser Asp Pro Gly Val 54
TAC TCC TCC CAG CTC AGC CGG CTA CGG CCC GGC TCC CAG C-CG TTG AGC GAA CAG 216
Tyr Ser Ser Gin Leu Ser Arg Leu Arg Pro Gly Ser Gin Ala Leu Ser Glu Gin 72
ATC CTG TCG GTC ATC GAC CCG CCC- ATG CAC CGC ACC CTC- CGC CGC CTG GTC AGC 270
He Leu Ser Val lie Asp Pro Pro Met His Arg Thr Leu Arg Arg Leu Val Ser SO
CAC- GCC TTC ACC CCC CGC ACC GTC GCC C-AC CTC GAA CCA CGC GTC ACC GAA CTG 324
Gln Ala Phe Thr Pro Arg Thr Val Ala Asp Leu Glu Pre Arg Val Thr Glu Leu 108
GCC GGG CAA CTG CTC GAC GCC GTC GAC GGC GAC ACG TTC GAC CTC GTC GCC GAC 378
Ala Gly Gin Leu Leu Asp Ala Val Asp Gly Asp Thr Phe Asp Leu Val Ala Asp 126
TTC GCC TAC CCG CTG CCC GTG ATC GTG ATC GCC GAA CTC CTC GGC GTG CCG CCC 432
Phe Ala Tyr Pro Leu Pro Val lie Val He Ala Glu Leu Leu Gly Val Pro Pro 144
GCC GAC CGC ACC CTG TTC CGC TCC TGG TCC GAC CGG ATG CTG CAG ATG CAG GTC 486
Ala Asp Arg Thr Leu Phe Arg Ser Trp Ser Asp Arg Met Leu Gin Met Gln Val 162
GCC GAC CCG GCG GAC ATG CAG TTC GGC GAC GAC GCC GAC GAG GAC TAC CAA CGC 540
Ala Asp Pro Ala Asp Met Gin Phe Gly Asp Asp Ala Asp Glu Asp Tyr Gin Arg 180
CTC GTC AAA GAA CCC ATG CGC GCC ATG CAC GCC TAC CTC CAC GAC CAC GTC ACC 594
Leu Val Lys Glu Pro Met Arg Ala Met His Ala Tyr Leu His Asp His Val Thr 198
GAC CGC CGC GCC CGC CCC GCG AAC GAC CTG ATC TCC GCA CTC GTC GCC GCC CGC 648
Asp Arg Arg Ala Arg Pro Ala Asn Asp Leu lie Ser Ala Leu Val Ala Ala Arg 216
GTG GAG GGC GAA CGA CTC ACC GAC GAG CAG ATC GTC GAA TTC GGG GCG CTG CTG 702
Val Glu Gly Glu Arg Leu Thr Asp Glu Gin He Val Glu Phe Gly Ala Leu Leu 234
CTG ATG GCC GGC CAC GTC TCC ACC TCC ATG CTG CTC GGC AAC ACC GTG CTG TGC 756
Leu Met Ala Gly His Val Ser Thr Ser Met Leu Leu Gly Asn Thr Val Leu Cys 252
CTG AAG GAC CAC CCC CGG GCC GAG GCC GCC GCC CGC GCC GAC CGG TCC CTG ATC 810
Leu Lys Asp His Pro Arg Ala Glu Ala Ala Ala Arg Ala Asp Arg Ser Leu He 270
CCC GCC CTG ATC GAA GAA GTA CTG CGG CTG CGG CCG CCG ATC ACC GTC ATG GCC 864
Pro Ala Leu lie Glu Glu Val Leu Arg Leu Arg Pro Pro He Thr Val Met Ala 288
CM
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DL
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140
CGC GTC ACC ACC AAG GAC ACC GTC CTC GCC GGC ACC ACC ATC CCC GCC GGA CGC
Arg Val Thr Thr Lys Asp Thr Val Leu Ala Gly Thr Thr lie Pro Ala Gly Arg
ATG GTC GTG CCC TCC CTG CTG TCC GCC AAC CAC GAC GAA CAG GTC TTC ACC GAC
Met Val Val Pro Ser Leu Leu Ser Ala Asn His Asp Glu Gin Val Phe Thr Asp
CCC GAC CAC CTC GAC CTC GCC CGC GAA GGC CGC CAG ATC GCC TTC GGC CAC GGC
Jro Asp His Leu Asp Leu Ala Arg Glu Gly Arg Gin He Ala Phe Gly His Gly
ATC CAC TAC TGC CTG GGC GCC CCG CTC GCC CGC CTG GAG GGC CGC ATC GCC CTG
lie His Tyr cys Leu Gly Ala Pro Leu Ala Arg Leu C-lu Gly Arg lie Ala Leu
GAA GCC CTC TTC GAC CGA TTC CCC GAC TTC TCG CCC ACC GAC GGC GCA AAA CTG
Glu Al a Leu Phe Asp Arg Phe Pro Asp Phe Ser Pro Thr Asp Gly Al a Lys Leu
CGC TAC CAC CGC GAC GGA CTG TTC GGC GTC AAG AAC CTG CCC- CTG ACC GTA CGG
Arg Tyr His Arg Asp Gly Leu Phe Gly Val Lys Asn Leu Pro Leu Thr Val Arg
918
306
972
324
1026
342
1080
360
1134
378
1188
396
CGC GGC 119*
Arg Gly 300
(8) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1561 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: no (iii) ANTISENSE: no (vi) ORIGINAL SOURCE:
(A) ORGANISM: Streptomyces pristinaespiralis
AP/P/ 9 5 / 0 0 7 5 2 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7
10 20 30 40 50 60
AAGCTTCCCG ACCGGGTGGA GGTCGTCGAC GCGTTCCCGC TGACCGGCCT CAACAAGGTC
70 80 90 100 110 120
GACAAGAAGG CCCTGGCGGC CGACATCGCC GCCAAGACCG CCCCCACCCG CCCCACCACC
130 140 150 160 170 180
GCCGGCCACG GCCCGACCAC GGACGGCGAT ACGGCCGGTG GGGGTGGGTC CGCGGGCGGG
190 200 210 220 230 240
GTGACGGCCG CCGGTGGCGG GCGGGAGGAG GCGGCGTGAG CGGGCCCGGG CCCGAGGGCG
250 2 60 270 280 2 90 300
GCTACCGGGT GCCGTTCGCG CGACGCGGTT CGGI'GGTGGG CGZ.GCCGGZkC CTGGCGGCGC
AP . Ο Ο 5 6 2
- 141 -
310 TGGGCGAACT 320 GGTCCGCTCG 330 GGCCGGTCGC 340 TGACGTCGGG 350 GGTGTGGCGG 360 GAGCGGTTCG
370 AGGAACAGTT 380 CGCCCGCCTG 390 ACCGGCGCCC 400 GGCACGCGCT 410 CAGTGTCACC 420 AGCGGCACCG
430 TCGCGCTGGA 440 ACTGGCGGTG 450 CGGATGCTGG 4 60 ACCTGGCGCC 470 GGGCGACGAG 480 GTGATCGCCA
490 CCCCGCAGAC 500 GTTCCAGGCG 510 ACGGTGCAGC 520 CGCTGCTCGA 530 CCACGACGTG 540 CGGCTGCGGT
550 TCTGCGACAT 5 60 CGACCCGGAC 570 ACCCTCAACC 580 TCGACCCGGC 590 GGTGCTGGAG 600 ACGCTGATCA
610 CCGACCC-CAC 620 CCGGGCGATC 630 CTGCTCGTCC 640 ACTACGGCGG 650 CAACCCGGCC 660 GACATGGACC
670 GCATCATGGC 680 CCTGGCCCGC 650 AAGCGCGGCA 700 TCA.TCC-TCGT 710 CGAGGACAGC 720 GCGCACGCGC
730 TGGGCGCCGT 740 GTACCGGGGG 750 CGGCGGCCGG 760 GGGCACTGGC 770 GGACATCGGC 780 TGCTTCACTT
790 TCCACTCCAC 800 GAAGAACATC 810 ACCACCCTCG 820 GCGAGGGCGG 830 CATGATCACC 840 CTGTCGCGTG
850 ACGAGTGGGC 860 CCAGCGGGTG 870 GGACGTATCC 880 GCGACAACGA 890 GGCCGACGGC 900 GTGTACGCGG
910 CGCTGCCGGA 920 CTCCGCGCGG 930 GCGGGTGCTC 940 CGGCGCTGCT 950 GCCGTGGATG 960 AAGTTCGCGG
970 AGGGTGTGTA 980 CGGTCACCGG 990 GCGGTCGGGG 1000 TCCGCGGGGC 1010 GGGCACGAAC 1020 GCGACGATGT
1030 CGGAGGCGGC 1040 GGCGGCGGTG 1050 GGCGTGGTGC 1060 AACTGGCGTC 1070 GCTGGAGCGG 1080 TTCGTGGCCC
1090 GGCGCCGGAG 1100 CATCGCGCAG 1110 CGGCTGGACG 1120 AGGCCGTGGC 1130 CTCGGTGGCC 1140 GGCACCCGGC
1150 TGCACCGGGC 1160 GGCGGCGGAC 1170 AGTCTGCACG 1180 CCTACCACCT 1190 GTACACGTTC 1200 TTCCTCACCG
1210 GCGGCCGGCA 1220 GGTGCGGGAG 1230 CGGTTCGTGC 1240 GCGCCCTGGA 1250 CCGGCTGGGT 1260 GTGGAGGTCC
1270 AGTTGCGGTA 1280 CTTCCCGCTC 1290 CATCTGTCGC 1300 CCGAGTGGCG 1310 GCTGCGCGGC 1320 CACGGGCCGG
1330 GCGAGTGTCC 1340 GACGGCCGAA 1350 CGGGTCTGGT 1360 TCGAGGAGCA 1370 CATGAACCTG 1380 CCGTGCCATC
1390 CCGGTCTGAG 1400 TGACGGCCAG 1410 GTCGACTACA 1420 TGGTCGAGGC 1430 GGTCACCCGC 1440 GCCCTGCACG
1450 AGGCCCACGG 1460 CACGGGGACG 1470 CGGGTGGCGG 1480 CCGGGCACCT 1490 GTGACACCGT 1500 CCGCATCCGG
1510 CCGGTGGTTT 1520 TCCAAGACCG 1S30 AGGGAGAGGC 1540 AGGCGTATGC 1550 CGTTCATCGA 1560 AGTGAAGATC
ΑΡ/Ρ/ 95/00752 τ
142 (9) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1233 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: no (iii) ANTISENSE: no (vi) ORIGINAL SOURCE:
(A) ORGANISM: Streptomyces pristinaespiralis
CXJ in (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8
GTG CCG TTC GCG CGA CGC GGT TCG GTG GTG GGC GAG GCG GAC CTG GCG GCG CTC- S4
Val Pro Phe Ala Arg Arg Gly Ser Val Val Gly Glu Ala Asp Leu Ala Ala Leu ιε
GGC GAA CTG GTC CGC TCG GGC CGG TCG CTG ACG TCG GGG GTG TGG CC-G GAC- CGG 108
Gly Glu Leu Val Arg Ser Gly Arg Ser Leu Thr Ser Gly Val Trp Arg Glu Arg 36
TTC GAG GAA CAG TTC GCC CGC CTG ACC GGC GCC CGG CAC GCG CTC AC-T GTC ACC 162
Phe Glu Glu Gin Phe Ala Arg Leu Thr Gly Ala Arg His Al a Leu Ser Val Thr 54
AGC GGC ACC- GTC GCG CTG GAA CTG GCG GTG CGG ATG CTG GAC CTG GCG CCG GGC 216
Ser Gly Thr Val Ala Leu Glu Leu Ala Val Arg Met Leu Asp Leu Ala Pro Gly 72
GAC GAG GTG ATC GCC ACC CCG CAG ACG TTC CAG GCG ACG GTG CAG CCG CTG CTC 270
Asp Glu Val He Ala Thr Pro Gin Thr Phe Gin Ala Thr Val Gin Pro Leu Leu 90
GAC CAC GAC GTG CGG CTG CGG TTC TGC GAC ATC GAC CCG GAC ACC CTC AAC CTC 324
Asp His Asp Val Arg Leu Arg Phe Cys Asp He Asp Pro Asp Thr Leu Asn Leu 108
GAC CCG GCG GTG CTG GAG ACG CTG ATC ACC GAC CGC ACC CGG GCG ATC CTG CTC 378
Asp Pro Ala Val Leu Glu Thr Leu He Thr Asp Arg Thr Arg Ala He Leu Leu 126
GTC CAC TAC GGC GGC AAC CCG GCC GAC ATG GAC CGC ATC ATG GCC CTG GCC CGC 432
Val His Tyr Gly Gly Asn Pro Ala Asp Met Asp Arg He Met Ala Leu Ala Arg 144
AAG CGC GGC ATC ATC GTC GTC GAG GAC AGC GCG CAC GCG CTG GGC GCC GTG TAC 486
Lys Arg Gly lie He Val Val Glu Asp Ser Ala His Ala Leu Gly Ala Val Tyr 162
CGG GGG CGG CGG CCG GGG GCA CTG GCG GAC ATC GGC TGC TTC ACT TTC CAC TCC S40
Arg Gl y Arg Arg Pro Gly Ala Leu Ala Asp lie Gl y Cys Phe Thr Phe His Ser 180
AP/P/ 9 5 / 0 0 7
AP . Ο Ο 5 6 2
- 143 -
ACG AAG AAC ATC ACC ACC CTC GGC GAG GGC GGC ATG ATC ACC CTG TCG CGT GAC 594
Thr Lys Asn lie Thr Thr Leu C-ly Glu Gly Gly Met He Thr Leu Ser Arg Asp 196
GAG TGG GCC CAG CGG GTG GGA CC-T ATC CGC GAC AAC GAG GCC GAC GGC GTG TAC 64 6
Glu Trp Ala Gin Arg Val Gly Arc lie Arg Asp Asn Glu Ala Asp Gly Val Tyr 216
GCG GCG CTG CCG GAC TCC GCG CC-C- GCG GGT GCT CCG GCG CTG CTG CCG TGG ATG 702
Ala Ala Leu Pro Asp Ser Ala Arg Ala Gly Ala Pro Ala Leu Leu Pro Trp Met 234
AAG TTC GCG GAG GGT GTG TAC C-GT CAC CGG GCG GTC GGG GTC CGC GGG GCG GGC 756
Lys Phe Ala Glu Gly Val Tyr Gly His Arg Ala Val Gly Val Arg Gly Ala Gly 252
ACG AAC GCG ACG ATG TCG GAC- GCC- GCG GCG GCG GTG GGC C-TG GTG CAA CTG GCG eio
Thr Asn Ala Thr Met Ser Glu A.la Ala Ala Ala Val Gly Val Val Gin Leu Ala 270
TCG CTG GAG CGG TTC GTG GCC CC-G CGC CGG AC-C ATC GCG CAG CGG CTG GAC GAG 864
Ser Leu Glu Arg Phe Val Ala Arc A.rc Arg Ser lie Ala Gin Arg Leu Asp Glu 288
GCC GTG GCC TCG GTG GCC GGC ACC CGG CTG CAC CGG GCG GCG GCG GAC AGT CTG 916
Ala Val Ala Ser Val Ala Gly Thr Arg Leu His Arg Ala Ala Ala Asp Ser Leu 306
CAC GCC TAC CAC CTG TAC ACG TTC TTC CTC ACC GC-C GGC CGG CAG GTG CGG GAG 972
His Ala Tyr His Leu Tyr Thr Phe Phe Leu Thr Gly Gly Arg Gin Val Arc Glu 324
CGG TTC GTG CGC GCC CTG GAC CGG CTG GGT GTG GAG GTC CAG TTG CGG TAC TTC 1026
Arg Phe Val Arg Ala Leu Asp Arc Leu Gly Val Glu Val Gin Leu Arg Tyr Phe 342
CCG CTC CAT CTG TCG CCC GAG TGG CGG CTG CGC GGC CAC GGG CCG GGC GAG TGT 1080
Pro Leu His Leu Ser Pro Glu Trp Arg Leu Arg Gly His Gly Pro Gly Glu cys 360
CCG ACG GCC GAA CGG GTC TGG TTC GAG GAG CAC ATG AAC CTG CCG TGC CAT CCC 1134
Pro Thr Ala Glu Arg Val Trp Phe Glu Glu His Met Asn Leu Pro Cys His Pro 378
GGT CTG AGT GAC GGC CAG GTC GAC TAC ATG GTC GAG GCG GTC ACC CGC GCC CTG 1189
Gly Leu Ser Asp Gly Gin Val Asp Tyr Met Val Glu Ala Val Thr Arg Ala Leu 396
CAC GAG GCC CAC GGC ACG GGG ACG CGG GTG GCG GCC GGG CAC CTG 1233
His Glu, Ala His Gly Thr Gly Thr Arg Val Ala Ala Gly His Leu 411
c\

Claims (29)

1. A compound of formula I wherein
- Rj and R4 are independently hydrogen or a methyl group;
- R3 is hydrogen or a hydroxyl group;
- X is a CO, CHOH or CH2 group; and
AP/P/ 9 5 / 0 0 7 5 2 wherein, when
10 A, C, D and E are hydrogen, then B is - a halogen atom,
AH . Ο Ο 5 6 2
- 145 -
- a monoalkylamino or dialkylamino group,
- an ether group,
- a thioether group,
- a Cx to C3 alkyl group, or .5 - a trihalogenomethyl group; and when A, B, D and E are hydrogen, then C is
- a halogen atom,
- an NR5R6 group with R5 and R6 independently being
10 - hydrogen,
- a straight-chain or branched C3 to C4 alkyl group, with the proviso that when one of the substituents Rs or R6 is a methyl group, then the other is an ethyl group,
15 - an alkyl cycloalkylmethyl group in which the cycloalkyl group contains 3 or 4 carbon atoms,
- an optionally substituted C3 or C4 cycloalkyl group,
- a straight-chain or branched C3 or C4 20 alkenyl group, with the proviso that when one of the substituents R5 or R6 is alkyl, the other is not methyl or C3 to C6 cycloalkyl ;
- a substituted or unsubstituted N-pyrrolidinyl group,
25 - an ether group,
- a thioether group,
- an acyl or alkoxycarbonyl group,
- a Cj to C6 alkyl group which is straight-chain or branched,
30 - a alkylthio methyl group,
- an aryl group, or
- a trihalogenomethyl group; and when A, D and E are hydrogen then B is
- a halogen atom,
35 - a monoalkylamino or dialkylamino group
- an ether group,
AP/P/ 9 5 / 0 0 7 5 2
- 146 -5
- a thioether group, or
- a Cj to C3 alkyl group, and
C is
- a halogen atom,
- an amino, monoalkylamino or dialkylamino group with the proviso that B is not a bromine or chlorine atom, or a substituted or unsubstituted allyl group,
- an ether group,
- a thioether group,
- a C3 to C6 alkyl group, or
- a trihalogenomethyl group; and when Β, E and D are hydrogen then A and C are methyl groups .
2. A compound according to claim 1 wherein A,
C, D and E are hydrogen or wherein A, D and E are hydrogen, and B is fluorine; or wherein A, D and E are hydrogen and C is fluorine
3. A compound according to claim 1 wherein A,
C, D, and E are hydrogen or wherein A, D and E are hydrogen; and B is an alkylamino group in which the alkyl group is methyl or ethyl or a dialkylamino group in which one or both of the alkyl group or groups is methyl or ethyl.
4. A compound according to claim 1 wherein A,
C, D, and E are hydrogen or wherein A, D and E are hydrogen; and B is a trifluoromethyl group.
5. A compound according to claim 1 wherein A,
B, D and E are hydrogen and C is methyl, isopropyl or tertbutyl.
6. A compound according to claim 1 wherein A,
B, D and E are hydrogen and C is phenyl.
7. A compound according to claim 1 wherein A,
B, D and E are hydrogen and C is trifluoromethyl.
8. A compound according to claim 1, selected from
AP/P/ 95/00752
4 ζ-methylthio-de (4 f-dimethylamino) pristinamycin
AP. Ο Ο 5 6 2
- 147 -
I».
4/-methylthio-de(4/-dimethylamino) pristinamycin
Ifi t
57-hydroxy-4/-methylthio-de (4/5 dimethylamino)pristinamycin IH,
4/-methyl-de (4/-dimethylamino) pristinamycin IA,
4/-methyl-de(4/-dimethylamino)pristinamycin IH,
4 /-methoxy-de (4 /-dimethylamino) pristinamycin IA, 4/-methoxycarbonyl-de(4/10 dimethylamino)pristinamycin IA,
4/-chloro-de (4/-dimethylamino) pristinamycin IA,
4/-bromo-de(4/-dimethylamino)pristinamycin IA,
4/-bromo-de(4/-dimethylamino)pristinamycin IH,
4/-iodo-de(4/-dimethylamino)pristinamycin IA,
15 4/-iodo-de(4/-dimethylamino)pristinamycin IH,
4/-trifluoromethyl-de(4/-dimethylamino)pristinamycin IA,
4/-trifluoromethyl-de(4/-dimethylamino)pristinamycin IH,
20 4/-tert-butyl-de (4/-dimethylamino) -pristinamycin
Ia,
4/-isopropyl-de (4/-dimethylamino) -pristinamycin
Ia,
4/-isopropyl-de(4/-dimethylamino) -pristinamycin
25 IE,
4 e -methylamino-de (4 /-dimethylamino) -pristinamycin Ia, . 4 e-methoxy-de (4/-dimethylamino) pristinamycin IA,
4c-methoxy-de(4/-dimethylamino)pristinamycin IH,
30 4e-fluoro 4/-methyl-de(4/-dimethylamino)pristinamycin IA,
4/-amino-de(4/-dimethylamino)pristinamycin IA,
4/-ethylamino-de(4/-dimethylamino) -pristinamycin
Ia,
4/-diethylamino-de (4/-dimethylamino)pristinamycin IA,
AP/P/ 95/007 5 2
148
4 f-allylamino-de(4 f-dimethylamino)-pristinamycin
4 f-diallylamino-de(4 f-dimethylamino)pristinamycin IA, '5 4 ζ-allylethylamino-de(4 ζ-dimethylamino)pristinamycin IA,
4 ζ-ethylpropylamino-de(4 f-dimethylamino)pristinamycin IA,
4 ζ-ethylisopropylamino-de(4 ζ-dimethylamino)10 pristinamycin IA,
4 ξ-ethylcyclopropylmethylamino-de(4 ζdimethylamino)pristinamycin IA,
4 ζ-(1-pyrrolidinyl)-de(4 ζ-dimethylamino)pristinamycin IA,
15 4 f-trifluoromethoxy-de(4 ζ-dimethylamino)pristinamycin IA,
4 f-allyloxy-de(4 f-dimethylamino)pristinamycin IA, 4 f-ethoxy-de(4 f-dimethylamino)pristinamycin IA,
4 f-ethylthio-de(4 f-dimethylamino)-pristinamycin
20 IA,
4 f-methylthiomethyl-de(4 f-dimethylamino)pristinamycin IA,
4f-(2-chloroethoxy)-de(4f-dimethylamino)pristinamycin IA,
25 4ζ-acetyl-de(4ζ-dimethylamino)pristinamycin IA,
4 f-ethyl-de(4 ζ-dimethylamino)pristinamycin IA,
4 f-ethyl-de(4 f-dimethylamino)pristinamycin IH, 4e-dimethylamino-de(4ζ-dimethylamino)pristinamycin IA,
30 4e-methylthio-de(4f-dimethylamino)- pristinamycin
IA, and
4e-ethoxy-de(4f-dimethylaminolpristinamycin IA,
9. A process for preparing streptogramins, which process comprises culturing a mutant
35 streptogramin-producing microorganism strain having a genetic modification which affects the biosynthesis of a
AP/P/ 9 5 / 0 0 7 5 2
AP. Ο Ο 5 6 2
- 149 - precursor of a group B streptogramin, in an appropriate culture medium supplemented with a group B streptogramin precursor different from that whose biosynthesis is altered by the modification, and recovering the streptogramins '5 therefrom.
10. A process according to claim 9, wherein the mutant strain possesses genetic modification located within a gene involved in the biosynthesis of a group B streptogramin precursor.
10
11. A process according to claim 10, wherein the gene whose expression is altered is involved in the biosynthesis of L-2-aminobutyric acid, 4-dimethylamino-Lphenylalanine (DMPAPA), pipecolic acid, L-phenylglycine or 3-hydroxypicolinic acid.
15
12. A process according to claim 10 or 11, wherein the gene is papA, papM, papC (SEQ ID No. 2), papB (SEQ ID No. 3), pipA (SEQ ID No. 5), snbF (SEQ ID No. 6) or hpaA (SEQ ID No. 8).
13. A process according to one of claims 10 to
20 12, wherein the genetic modification renders a gene involved in the biosynthesis of group B streptogramin precursors partially or totally incapable of expressing the natural enzyme that the gene encodes.
14. A process according to one of claims 10 to
25 13, wherein the genetic modification comprises a disruption of a gene involved in the biosynthesis of group B streptogramin precursors.
15. A process according to one of claims 10 to 14, wherein the mutant strain is derived from
AP/P/ 9 5 / 0 0 7 5 2
30 S. pristinaespiralis.
16. A process according to claim 15 wherein the mutant strain is strain . SP212 or SP92. 17 . A process according to claim 16 wherein the strain is SP92 ::pVRC508. 35 18 . A process according to claim 16 wherein the
strain is SP92pipA::Dam*.
150
19. A process according to claim 16 wherein the strain is SP92hpA::PamR.
20. A process according to any one of claims 9 to 19, wherein the precursor introduced into the culture *5 medium is a derivative or analogue of an amino acid or of an alpha-ketocarboxylic acid.
21. A process according to any one of claims 9 to 20 wherein the precursor is related to the precursor whose biosynthesis is altered.
10
22. A process according to claim 20 or 21 wherein the precursor is a derivative of phenylalanine and the gene whose expression is altered is involved in the biosynthesis of DMPAPA.
23. A process according to any one of claims 9
15 to 22 wherein the streptogramin produced is pristinamycin
IB.
24. A nucleic acid sequence, selected from:
(a) all or part of one of the genes papC (SEQ ID No. 2), papB (SEQ ID No. 3), pipA (SEQ ID No. 5), snbF (SEQ
20 ID No. 6) and hpaA (SEQ ID No. 8), or a sequence complementary thereto;
(b) a nucleic acid sequence which hybridizes to all or part of a gene defined in (a) and (c) a nucleic acid sequence related to those
25 defined in (a) and/or (b) owing to the degeneracy of the genetic code.
25. A nucleic acid according to claim 24, selected from the papC (SEQ ID No. 2), papB (SEQ ID No. 3), pipA (SEQ ID No. 5), snbF (SEQ ID No. 6) and hpaA (SEQ ID
30 No. 8) genes.
26. A recombinant DNA comprising a gene as defined in claim 25.
27. A nucleic acid vector, comprising a nucleic acid sequence according to claim 24 or 25 or a recombinant
35 DNA according to claim 26.
28. Use of a sequence according to claim 24 or
AP/P/ 9 5 / 0 0 7 5 2
AP. Ο Ο 5 6 2
- 151 -
25 and/or of a vector according to claim 26 for preparing metabolites .
29. A polypeptide which results from the expression of a sequence according to claim 24 or 25.
*5 30. A mutant Streptomyces pristinaespiralis strain, which possesses genetic modification within one of its papC (SEQ ID No. 2), papB (SEQ ID No. 3), pipA (SEQ ID No. 5), snbF (SEQ ID No. 6) or hpaA (SEQ ID No. 8) genes.
31. A mutant strain according to claim 30, which
10 is strain SP92pipA::DamR.
32. A mutant strain according to claim 30, which is strain SP92hpaA::QamR.
33. A mutant S. pristinaespiralis strain, which comprises a papA gene disrupted by double homologous
15 recombination.
34. A strain according to claim 33 which is derived from SP212.
35. A precursor compound for the biosynthesis of a group B streptogramin selected from
20 4-trifluoromethoxyphenylalanine,
3- methylaminophenylalanine, 3-methylthiophenylalanine,
3 -fluoro-4-methylphenylalanine, 4 -me thylaminophenylpyruvic acid, 3-ethoxyphenylalanine, 4-allylaminophenylalanine,
4- diallylaminophenylalanine,
25 4-allylethylaminophenylalanine,
4-ethylpropylaminophenylalanine,
4 -ethylisopropylaminophenylalanine,
4-ethylcyclopropylmethylaminophenylalanine,
4 - (1-pyrrolidinyl) phenylalanine,
30 4-ethylthiomethylphenylalanine,
4-0-(2-chloroethyl)tyrosine, 3-dimethylaminophenylalanine and 3-ethylaminophenylalanine
36. A pharmaceutical composition comprising a compound according to any one of claims 1 to 8 and a
35 pharmaceutically acceptable carrier.
37. A pharmaceutical composition according to
AP/P/ 9 5 / 0 0 7 5 2
152 claim 29 further comprising a group A streptogramin.
APAP/P/1995/000752A 1994-07-08 1995-07-06 Novel streptogramins and a process for preparing streptogramins by mutasynthesis. AP562A (en)

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