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Background of the invention

Natural products are a relevant source of lead compounds of pharmacological importance, particularly in the anti-infective and anticancer areas, with actinomycetes representing one of the most prolific microbial groups.

The natural compound Ge2270 is produced by the rare actinomycete Planobispora rosea ATCC53733 and is defined as a thiazolylpeptide or thiopeptide, a class of antibiotics having a chemical structure characterized by a macrocyclic peptide rich in thiazoles, oxazoles, dehydroaminoacids and with a pyridine/piperidine core. These antibiotics inhibit bacterial protein synthesis by affecting either one of two targets: the loops defined by 23 S rRNA and the LI 1 protein, as for example thiostrepton (Porse BT et al. J.Biol.Mol. 1998, 276, 391) or the elongation factor Tu (EF-Tu), as for Ge2270 (Landini P. et al. Biochem.J. 1992, 283, 649.).

Ge2270 is a 14-aa thiazolyl peptide with a triazole-substituted pyridine core at the junction of the macrocycle and the side chain, three additional thiazole and one oxazoline heterocycles. Diversely from other thiopeptides, Ge2270 possesses unique modifications such as β-hydroxylation at the Phe residue, an N-methylation at the Asn residue, and C- and O-methylations at some thiazole rings, resulting in a complex of closely related different congeners.

According to the present invention, with the term "congener" it is intended a biosynthetic variants of a common chemical structure. Among the congeners one is usually preferentially produced while the others are present in smaller amounts. Known "congeners" of Ge2270 are characterized by having R ₂ = NH ₂ , R = OH and X = Gly while showing chemical variations at positions Rj, R4 and R5 and more precisely known congeners of Ge2270 are those reported in table 1 in which their original nomenclature is indicated. Table 1 :

Ge2270 exhibits a potent activity against Gram-positive bacteria, including resistant pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus sp. (VRE). However its poor solubility and poor pharmacokinetics have limited clinical progress. Increasing knowledge on thiopeptide biosynthesis might provide the basis for generation of new derivatives with improved pharmacological applications.

As related thiopeptides, Ge2270 is ribosomally synthesized: the mature antibiotic arises from a precursor peptide that contains an N-terminal leader peptide and a C-terminal region, the final thiopeptide framework, that undergoes a series of posttranslational modifications catalyzed by lantibiotic-like dehydratases, cyclodehydratases and dehydrogenases, and other enzymes involved in thiazoles/oxazoles and pyridine/piperidine ring formation. In all thiopeptides characterized so far, these enzymes are encoded by a set of 6 genes localized in the flanking regions of the structural gene, highly conserved in the biosynthetic pathways, showing homologs in other tailored ribosomal peptides biosynthetic pathways such as lantibiotics and cyanobactins. Ge2270 is also produced by Nonomuraea strain WU8817. The genes and their products, which are involved in the synthesis, post translational modifications, regulation and export of the precursor peptide from the Nonomuraea strain, have been described in International Patent Application PCT /WO 2009/019289.

Description of the invention

The present invention relates to novel antibiotic compounds, having general formula (I), a process for their preparation, and pharmaceutical compositions containing them, their pharmaceutical acceptable salts and their use as antibacterial agents.

The process for their preparation include the identification of the Ge2270 biosynthetic gene cluster in Planobispora rosea ATCC53733, its heterologous expression in a different microorganism, the genetic manipulation of the cluster by gene modification and/ or inactivation experiments, the recovery of the compounds of formula (I), according to the present invention, from the mycelium and/or from the fermentation broth and the isolation of the pure substances, for example by chromatographic means. The present invention also refers to a process for the preparation of antibiotic derivatives according to formula (I) comprising modifications through chemical reactions of the antibiotics directly obtained from culturing the heterologous host microorganism according to the above.

According to the present invention, compounds of formula (I) have the following general formula:

wherein X represents a natural amino acid

Ri represents H, CH ₂ OH, CH ₂ OMe

R.2 represents OH, NH ₂ , NRgR ₇ wherein one of R$ and R ₇ is selected as:

■ a cycloalkyl of 5 to 7 carbon atom optionally substituted by one or two substituents independently selected from lower alkyl of 1 to 4 carbon atoms, phenyl, phenyl-lower alkyl of 1 to 4 carbon atoms, acyl-phenyl, phenoxy, phenoxy-lower alkyl of 1 to 4 carbon atoms, wherein said phenyl and the phenyl portion of said phenyl lower-alkyl, acyl-phenyl, phenoxy and phenoxy-lower alkyl group is optionally substituted by one or two substituents selected from halo, cyano, CF3, lower alkyl of 1 to 4 carbon atoms, and lower alkoxy of 1 to 4 carbon atoms.

■ a 5-7 member cycloalkyl containing one or two N atoms optionally substituted by one or two substituents independently selected from lower alkyl of 1 to 4 carbon atoms, phenyl, phenyl-lower alkyl of 1 to 4 carbon atoms, acyl-phenyl phenoxy, phenoxy-lower alkyl of 1 to 4 carbon atoms, wherein said phenyl and the phenyl portion of said phenyl lower-alkyl, acyl-phenyl, phenoxy and phenoxy-lower alkyl group is optionally substituted by one or two substituents selected from halo, cyano, CF3, lower alkyl of 1 to 4 carbon atoms, and lower alkoxy of 1 to 4 carbon atoms.

^■ a phenyl optionally substituted by one or two substituents independently selected from lower alkyl of 1 to 4 carbon atoms, phenyl, phenyl-lower alkyl of 1 to 4 carbon atoms, acyl-phenyl phenoxy, phenoxy-lower alkyl of 1 to 4 carbon atoms, wherein said phenyl and the phenyl portion of said phenyl lower-alkyl, acyl-phenyl, phenoxy and phenoxy-lower alkyl group is optionally substituted by one or two substituents selected from halo, cyano, CF3, lower alkyl of 1 to 4 carbon atoms, and lower alkoxy of 1 to 4 carbon atoms.

■ A 5 or 6 member heteroaromatic cycle containing 1 to 3 nitrogen atoms optionally substituted by one or two substituents independently selected from lower alkyl of 1 to 4 carbon atoms, phenyl, phenyl-lower alkyl of 1 to 4 carbon atoms, acyl-phenyl phenoxy, phenoxy-lower alkyl of 1 to 4 carbon atoms, wherein said phenyl and the phenyl portion of said phenyl lower-alkyl, acyl-phenyl, phenoxy and phenoxy-lower alkyl group is optionally substituted by one or two substituents selected from halo, cyano, CF3, lower alkyl of 1 to 4 carbon atoms, and lower alkoxy of 1 to 4 carbon atoms

while the other of and R ₇ is selected as H, C 1-C6 alkyl, Ci-C(, alkylamino, Ci-C ₆ alkylamino-(Ci-C4)alkylene

R.3 represents H, OH

R4 represents H, CH3

R ₅ represent H, CH ₃

provided that: - when R ₂ is selected as NH ₂ or OH, R ₃ is selected as OH, X is selected as Gly and Ri is selected as CH ₂ OMe, then both R4 and R5 must be selected as H

and that

- when R is selected as NH ₂ or OH, R ₃ is selected as OH, X is selected as Gly and Ri is selected as CH ₂ OH or H, then R4 must be H.

In particular, according to the present invention and according to the above formula (I), compounds here indicated as Na (N being a progressive integer number, i.e. l a, 2a, 3a ...) are characterized by the fact that X is a natural amino acid different from Gly, compounds indicated as Nb in the present application (N being a progressive integer number, for example lb, 2b, 3b ...) are characterized by the fact that R ₃ is selected as H, compounds here indicated as Nc (N being a progressive integer number for example lc, 2c, 3c ...) are characterized by the fact that R ₂ is selected as OH.

Moreover compounds indicated as defined above as Nc, can be further submitted to chemical amidation of the carboxylic acid, thus generating compounds indicated as Ncn in the contest of the present application, (for example lcl, lc2, ect.) wherein n is a progressive integer number in agreement with Table 2, as below indicated. These compounds indicated as Ncn, belong to general formula (I) where R ₂ is selected as NRfiR ₇ .

Always according to the present invention, the following compounds are preferred: la (named "Na" as above defined, where N is selected as 1 ) (example 5): wherein, with reference to general formula (I) according to the present invention, X = Ala; Ri = CH ₂ OMe; R ₂ = NH ₂ ; R ₃ = OH; R4 = CH ₃ ; R ₅ = CH ₃ .

lb (named "Nb" as above indicated, where N is selected as 1 ,) (example 2): wherein, with reference to general formula (I) according to the present invention, X = Gly; Ri = CH ₂ OMe; R ₂ = NH ₂ ; R ₃ = H; R4 = CH ₃ ; R ₅ = CH ₃ .

Ic2 (named "Ncn" as above indicated, where N is selected as 1 and n is selected as 2,) (example 4): wherein, with reference to general formula (I) according to the present invention, X = Gly; Ri = CH OMe; R ₂ = NR _$ R ₇ wherein R _$ is selected as H and R ₇ is selected as 6 member cycloalkyl containing one nitrogen atom, substituted by one substituent selected as phenyl-lower alkyl where said lower alkyl is a CH ₂ group and said phenyl is substituted by CF ₃ ; R ₃ = OH; R4 = CH ₃ ; R ₅ = CH ₃ .

2b (named " b" as above indicated, where N is selected as 2,) (example 10): wherein, with reference to general formula (I) according to the present invention, X = Gly; Ri = CH ₂ OH; R ₂ = NH ₂ ; R ₃ = H; R4 = CH ₃ ; R ₅ = CH ₃

5a (named "Na" as above indicated, where N is selected as 5,) (example 11): wherein, with reference to general formula (I) according to the present invention, X = Ala; Ri = H; R ₂ = NH ₂ ; R ₃ = OH; R4 = CH ₃ ; R ₅ = CH ₃ .

The above indicated genetic manipulation leads to the modification of complex composition in which the main congener Ge2270A is no longer produced while other congeners became prevailing.

In particular, gene inactivation of pbtl 7 in cosmid 2F7 leads to a compound which, according to the present invention and with reference to general formula (I), is defined as follows: X = Gly; R, = CH ₂ OH; R ₂ = NH ₂ ; R ₃ = OH; R4 = CH ₃ ; R ₅ = CH ₃ .

Gene inactivation oi pbt6 in cosmid 2F7 leads to compounds defined as follows: X = Gly; Ri = CH ₂ OCH ₃ ; R ₂ = NH ₂ ; R ₃ = OH; R4 = CH ₃ ; R ₅ = H and X = Gly; Ri = H; R ₂ = NH ₂ ; R ₃ = OH; R4 = CH ₃ ; R ₅ = H.

Gene inactivation of pbtl6 in cosmid 2F7 leads to compound defined as follows,: X = Gly; Ri = H; R ₂ = NH ₂ ; R ₃ = OH; R4 = CH ₃ ; R ₅ = CH ₃ .

Gene inactivation of pbt7 in cosmid 2F7 leads to compounds defined as follows: X = Gly; Ri = CH ₂ OCH ₃ ; R ₂ = NH ₂ ; R ₃ = OH; R4 = H; R ₅ = CH ₃ and X = Gly; Ri = CH ₂ OCH ₃ ; R ₂ = NH ₂ ; R ₃ = OH; R4 = H; R ₅ = H.

Heterologous expression of Ge2270A

A draft genome sequence for the Planobispora rosea ATCC53733 strain, producer of the thiopeptide antibiotic Ge2270, was obtained by the use of the 454 sequencing technology (454 Life Sciences, Roche Diagnostics, http://www.454.com). By Blast analysis (http:/ blast.ncbi.nlm.nih.gov/Blast.cgi), the predicted Ge2270 precursor peptide was used to identify a 167-bp open reading frame (ORF) named pbt8. The pbt8 product consisted of the 39-aa N-terminal leader peptide followed by the 16-aa core peptide. The C-terminal Ser and Ala are missing in the mature Ge2270 molecule. Sequence analysis of the genomic region surrounding pbt8 revealed 17 ORFs, designated pbtl to pbtl 7, assigned to the Ge2270 cluster, spanning 21.4 Kb region (figure 1, SEQ ED No. 1). The overall organization of the pbt cluster is fully syntenic to the Ge2270 cluster from Nonomuraea WU8817 and shows good similarity to other thiopeptide clusters. By cluster comparison, putative roles for most genes could inferred. A high quality and high coverage cosmid library of the P. rosea genome was generated in the conjugative SuperCos3 vector (M. Sosio, unpublished results) using the Gigapack® III XL Packaging Extract (Stratagene). From the screening of the cosmid library using specific PCR primers for pbtl (Table 3), and pbt 16 (Table 3), a positive cosmid was identified and named 2F7. End-sequencing of 2F7 confirmed an insert of 44-Kb containing the complete Ge2270 gene cluster with 5.5-kb upstream the pbtl and 17.1 -kb downstream the pbtl 7 (figure 1 ).

Table 3 shows the oligonucleotide sequence of several primers used for the screening of cosmid library containing the pbt cluster and for the gene manipulation and/or inactivation experiments. Thus, the primers shown in table 3 are used for the screening of the cosmid library 2F7 (and/or mutant generation thereof) and they are able to recognize oligonucleotides of SEQ ED No. 1. In each primer the term "F" or "R" is reported, which respectively refers to primer "forward" or "reverse". F and/or R also contain a progressive integer number for example Fl , Rl, R2 etc.. when different primers are constructed for different portion of oligonucleotide sequence belonging to the same ORF.

Table 3

The term "cosmid 2F7" herein refers to the cosmid which contains the complete Ge2270 gene cluster arbitrarily named "pbt".

The term "2F7-A pbt... " herein refers to cosmid 2F7 wherein at least one pbt ORF was deleted. For example 2F7-Apbt4 represents the cosmid 2F7 wherein the ORF pbt4 was deleted.

In the present description the terms "pbtl ", "pbt2 ", "pbt3 ", etc.. up to "pbt 17 "are referred to ORFs 1 -17 belonging to pbt gene cluster encoding to Ge2270.

The cosmid 2F7 was first transferred in E.coli ET12567/pUB307 and then by conjugation to different actinomycete hosts which do not naturally produce Ge2270. At first, Streptomyces lividans 1326 and Streptomyces lividans TK24 were successfully conjugated. However, Ge2270 could never be detected in any of the production media used. When cosmid 2F7 was used to site-specifically integrate the pbt cluster into the Nonomuraea sp. ATCC39727 chromosome following conjugation, the resulting ex-conjugants produced Ge2270A, as confirmed by bioassay, LC-MS and Ή-NMR. As expected, no peak or MS corresponding to Ge2270 was observed in the control sample {Nonomuraea sp. ATCC39727 containing the empty vector). Notably Nonomuraea sp. ATCC39727 is rarely used as heterologous hosts and it is the first time that is used for the production of a thiopeptide.

NEW Ge2270 analogs (Na, Nb, Ncn with N=l)

Modifications of the Ge2270 were done chemically on the purified compound and since the molecule presents a limited number of reactive groups only a limited number of modifications could be done. The present invention offers the possibility of genetically modify the Ge2270 molecule by engineering the gene cluster in E.coli and then transferring the modified cluster in a suitable host for expression and production. Genetic engineering according to the present invention, allowed the introduction of new reactive groups as hydroxyl groups, etc. in the molecule, which may be suitable for further chemical modifications.

Mutagenesis of the Ge2270 gene cluster was performed in E.coli DH10B carrying the cosmid 2F7 using the procedure based on the λ Red recombination system (Datsenko A and Wanner BL, P oc Natl Acad Sci U S A. 2000, 97:6640-5). In short, a DNA fragment composed of the desired "mutagenic cassette" (a selectable marker as chloramphemcol, cat, or kanamycin, kan, resistance genes plus any other element to be inserted in the target site of the cosmid) flanked by short sequences homologous to the target site was electrotransferred to an E. coli strain containing cosmid 2F7 and expressing λ Red genes. The transformants obtained by homologous recombination between the target sites and the homologous flanking regions of the mutant allele are the candidate mutants. The antibiotic resistance genes are flanked by directly repeated FRT sites, which allow the excision of the resistance cassette by FLP-mediated site-specific recombination. Upon excision of the antibiotic resistance genes a scar, in the range of 80-85 nt, substitutes the disrupted gene.

The engineered cosmids were then introduced first in E. coli ET12567/pUB307 and then by conjugation in the recipient strain Nonomuraea ATCC39727.

Gene modification (i.e. substitution) of the residue Glycine at position 7, in the 16-aa core peptide of pbt$ product, in the residue Alanine (G7A) in cosmid 2F7 leads to compound la, as described below.

Gene inactivation of pbt4 in cosmid 2F7 leads to compound lb, as described below. Gene inactivation of pbtl5 in cosmid 2F7 leads to compound lc that can be further subjected to chemical amidation of the carboxylic acid thus generating compounds lcn, as described below.

Genetic manipulation to obtain further compounds according to general formula (I) (N=l):

Following the procedures described before, always according to the present invention, the following compounds were obtained:

gene inactivation of pbt4 in cosmid 2F7 -pbt8G7A can lead to a compound wherein, with reference to general formula (I): X = Ala, Ri = CH ₂ OMe, R ₂ = NH ₂ , R ₃ = H, R4 = CH ₃ , R ₅ =CH ₃ .

Gene inactivation of pbtl5 in cosmid 2F7-Apbt4 can lead to a compound wherein, with reference to general formula (I): X = Gly, Ri = CH ₂ O e, R ₂ = OH, R ₃ = H, R4 = CH ₃ , R ₅ =CH ₃ . Gene inactivation of pbtl5 in cosmid 2F7-pbt8G7A can lead to a compound wherein, with reference to general formula (I): X = Ala, Ri = CH ₂ OMe, R ₂ = OH, R ₃ = OH, R4 = CH ₃ , R ₅ =CH ₃ .

Gene inactivation of pbt4 and pbtl5 in cosmid 2F7-pbt8G7A can lead to a compound wherein, with reference to general formula (I): X = Ala, Ri = CH ₂ OMe, R ₂ = OH, R ₃ = H, R4 = CH ₃ , R ₅ =CH ₃ .

Some of the above described compounds can be further subjected to chemical reaction (amidation of the carboxylic acid) thus generating additional compounds according to the invention.

Genetic manipulation to produce additional compounds according to the invention

Using a similar procedure, gene inactivation of pbtl 7 can be performed in the cosmids 2F7-pbt8G7A, 2F7-Apbt4, and 2F7-Apbtl5 so that, once conjugated in Nonomuraea ATCC39727, the compounds 2a, 2b, and 2c can be produced.

Using a similar procedure, gene inactivation of pbt6 can be performed in the cosmids 2F7-pbt8G7A, 2F7-Apbt4, and 2F7-Apbtl5 so that, once conjugated in Nonomuraea ATCC39727, the compounds 3a, 3b, 3c, 4a, 4b, and 4c can be produced.

Using a similar procedure, gene inactivation of pbtl6 can be performed in cosmid 2F7-pbt8G7A, 2F7-Apbt4, and 2F7-Apbtl5 so that, once conjugated in Nonomuraea ATCC39727, the compounds 5a, 5b, and 5c can be produced.

Using a similar procedure, gene inactivation of pbt7 can be performed in cosmid 2F7-pbt8G7A, 2¥7-Apbt4, and 2F7-Apbtl5 so that, once conjugated in Nonomuraea ATCC39727 the compounds 6a, 6b, 6c, 7a, 7b, and 7c can be produced.

According to the present invention, new compounds Nc as above defined, can be further subjected to chemical reaction (amidation of the carboxylic acid) thus generating additional compounds in agreement with data reported in Table 2.

2

3

4

5

6

7

8

9

10

11

12

FERMENTATION

The production of compounds of formula (I) is achieved by cultivating the suitable strain and isolating the resulting compounds of formula (I) from the whole culture broth and/or from the separated mycelium and/or from the filtered fermentation broth, and purifying the isolated compound by chromatographic means. According to one preferred embodiment the production of compounds of formula (I) is carried out under aerobic conditions in an aqueous nutrient medium containing easy digestible or usable sources of carbon, nitrogen, and inorganic salts. Many of the nutrient media usually employed in fermentation field can be used, however preferred carbon sources are starch, dextrin, glucose, maltose, glycerol, and the like. Preferred nitrogen sources are soybean meal, peptone, meat extract, hydrolyzed casein, tryptone, corn steep liquor, cottonseed meal, yeast extract, and the like.

Soluble salts capable of yielding sodium, potassium, iron, zinc, cobalt, magnesium, calcium, ammonium, chloride, carbonate, sulphate, phosphate, nitrate, and the like ions can be incorporated in certain media.

Preferably, the strain producing compounds of formula (I) is cultured in a fermentation tube or in a shake flask, then the culture is used to inoculate jar reactors for fermentation for the production of substantial quantities of substances. The medium used for the pre-culture can be the same as that employed for larger fermentations, but other media can also be employed.

The temperature for growing strains producing compounds of formula (I) is 26-35 °C, preferably 28-32°C. During the fermentation, production of compounds of formula (I) can be monitored by HPLC analyses with a maximum production that generally occurs after 72 hours and before 192 hours of fermentation.

EXTRACTION AND PURIFICATION OF COMPOUNDS OF FORMULA (I)

Compounds of formula (I) can be found both in the mycelium and in the filtered fraction of the fermentation broth. The culture may be processed to separate the mycelium from the cleared broth and the mycelium may be extracted with a water- miscible solvent to obtain a solution containing the compounds of formula (I), after removal of the spent mycelium. This mycelium extract may then be processed separately or in pool with the supernatant according to the procedures reported hereafter for the supernatant fraction. The term "water-miscible solvent" as used in this application, is intended to have the meaning currently given in the art of this term and refers to solvents that, at the conditions of use, are miscible with water in a reasonably wide concentration range. Examples of water-miscible organic solvents that can be used in the extraction of the compounds of the invention are: lower alkanols, e.g. (C1 -C4) alkanols such as methanol, ethanol, and propanol, butanol; lower ketones, e.g. (C3-C4) ketones such as acetone and ethyl methyl ketone; cyclic ethers such as dioxane and tetrahydrofuran; lower amides such as dimethylformamide and diethylformamide; acetic acid dimethylsulfoxide and acetonitrile.

The recovery of the compounds of formula (I) from the supernatant of the fermentation broth of the producing microorganism is conducted according to known per se techniques which include extraction with solvents, precipitation by adding non-solvents or by changing the pH of the solution, by direct or reverse phase chromatography, ion exchange chromatography, matrix adsorption and the like or a combination of two or more of said techniques.

In any case, whatever may be the procedure adopted for recovering the crude compounds of formula (I), the successive purification step is usually carried out on the mixture of the crude materials resulting from the combination of the products originating from the separate extraction stages. Purification of the crude compounds of formula (I), can be accomplished by any of the known techniques but is preferably conducted by means of chromatographic procedures including chromatography on stationary phases such as silica gel, alumina, activated magnesium silicate and the like or reverse phase chromatography on silanized silica gel having various functional derivatization, and eluting with suitable organic solvents or aqueous mixture of water-miscible solvents of the kind mentioned above.

Accordingly, dried preparations of purified compounds of formula (I) are obtained as a white powder. As usual in this art, the production as well as the recovery and purification steps may be monitored by a variety of analytical procedures including HPLC or HPLC coupled with mass spectrometry.

AMIDATION

The present invention also concerns a process for the preparation of compounds of formula (I) characterized in that it comprises at least one additional step of a condensation reaction between at least a starting compound of formula (I) wherein R.2 is OH, and at least a selected amine of general formula HNR R.7, wherein Rf, and R.7 are defined as above, in the presence of a condensing agent. The reaction is carried out in the presence of a condensing agent, in the presence of a solvent. Preferred inert organic aprotic solvents useful for the condensation reaction are those solvents which do not interfere with the reaction course and are capable of at least partially solubilizing the starting material. For example compounds chosen among those previously indicated in Formula (I). Solvents can be chosen among organic amides, ethers of glycols and polyols, phosphoramide derivatives, sulfoxides. Preferably, solvents are chosen among: dimethyl formamide, dimethylsulphoxide, dioxane, dichloromethane and mixtures thereof. Preferably, dimethylformamide (DMF) is employed. The condensing agent according to the present invention is one suitable for forming amide bonds in organic compounds and, in particular, in peptides. Representative examples of condensing agents are diisopropylcarbodiimide (DIC), dicyclohexylcarbodiimide (DCC) with or without or hydroxybenzotriazole (HOBT), Ν,Ν,Ν',Ν'- tetramethyl-0-(benzotriazol-l-yl)uroniumtetrafluoroborate (TBTU), N,N,N',N'-tetramethyl-0-(7oxabenzotriazol-l-yl)uranium hexafluorophosphate (HATU), benzotriazolyl-oxy-tris-

(dimethylamino)phosphoniumhexafluorophosphate (HBTU), benzotriazolyloxy- tris- (pynOlidino)phosphoniumhexafluorophosphate (PyBOP) and (C1-C4) alkyl, phenyl or heterocyclic phosphorazidates such as diphenylphosphorazidate, dimorpholyl- phosphorazidate. The preferred condensing agent is PyBOP. The condensing agent is generally employed in a slight molar excess, such as from 1.2 to 5; preferably the molar excess of condensing agent is about 2.5 times the molar amount of starting compounds of formula (I). According to the present method, the amine is normally used in slight molar excess with respect to the compound of formula (I). In general, a 1.2 to 40-fold molar excess of the selected amine is used, while a 5-30 fold molar excess is preferred. When the amine HNRsR is used as a corresponding salt, for example the hydrochloride salt, it is necessary to add a suitable base in at least a molar proportion to obtain the free base of the amine H R6R which reacts with compounds of formula (1). In this case, an excess of the base is generally preferred. It is convenient to add a salt-forming base to the reaction mixture in an at least equimolar amount, and preferably in about 1.2 fold molar excess with respect to the amine HNR^Ry. Examples of said salt-forming bases are tertiary organic aliphatic or alicyclic amines such as trimethylamine, triethylamine (TEA), N-methylpyrrolidine or heterocyclic bases such as picoline and the like, alkali metals (e.g. sodium and potassium) hydrogen carbonates and carbonates. The reaction temperature will vary considerably depending on the specific starting materials and reaction conditions. In general, it is preferred to conduct the amidation reaction at temperature from 0°C to 50°C preferably at room temperature. Also the reaction time varies considerably, depending on the other reaction parameters; in general the condensation is completed in about l-4h. When the amine HNI^R _? contains a further primary amino group it might be protected, if necessary, as known in the art, in order to get the desired product. Any typical protecting group of the amino rest, which is resistant to the conditions applied during the process of this invention and may be readily removed under conditions which do not affect the stability of the compounds of formula (I) core portion can be utilized here. Suitable protecting groups of the amino function can be selected, for instance, from the groups described in: T. W. Greene, "Protective Groups in Organic Synthesis", J. Wiley, N. Y., 1981. Generally, the reaction course is monitored by HPLC according to methods known in the art. On the basis of the results of this assay it will be possible to evaluate the reaction course and decide when to stop the reaction and start working up the reaction mass according to per se known techniques which include, for instance, precipitation by addition of non- solvents, extraction with solvents, in conjunction with further common separation operations and purification, e.g. by column chromatography. According to the methodologies of the present invention as well as according to the above examples, a series of compounds can be prepared, always according to the present invention. Always according to the present invention, compounds belonging to general formula (I) or its pharmaceutically acceptable addition salts, are used as medicaments and, particularly, for use in the treatment of bacterial infections, particularly deriving from Gram positive bacteria.

BRIEF DESCRIPTIONS OF FIGURES

Fig. l shows DNA gene cluster obtained from the analysis of Planobispora rosea ATCC53733 genome. Thick line indicates the cosmid 2F7 (44.0 kb) containing the pbt cluster (21.4 kb), the genetic organization thereof is represented by arrows wherein each arrow indicates an ORF (ORFs 1-17).

Fig.2 shows LC-MS analysis of compound lb

Fig.3 shows 'H-NMR of compound lb

Fig.4 shows LC-MS analysis of compound lc2

Fig.5 shows LC-MS analysis of compound l a

Fig.6 shows LC-MS analysis of compound 7

Fig.7 shows LC-MS analysis of compound 2b

Fig.8 shows LC-MS analysis of compound 5a

EXAMPLES

The following examples are given in a non-limitative manner just as exemplification of the content of the present invention.

EXAMPLE 1 (COMPOUND 1) (X = GLY, Ri = CH ₂ OMe, R ₂ = NH ₂ , R ₃ = OH, R4 = CH ₃ , R _S =CH ₃ )

The cosmid 2F7 (carrying apramycin resistance, aacIV), which contains the complete Ge2270 gene cluster, pbt, was introduced in E.coli ET12567/pUB307 (carrying chloramphenicol, tetracyclin and kanamycin resistance, cat, tet and kan respectively) by transformation and selection of the positive clones. Two positive clones were analyzed for the presence of the cosmid by PCR amplification with primers specific for pbtl (Table 3) and pbtl6 (Table 3) of the Ge2270 gene cluster. Then, E.coli ET12567/pUB307 carrying the cosmid 2F7 was conjugated with Nonomuraea ATCC39727 as described in Stinchi et al. FEMS Microbiol Lett., 2003, 225:53-57 with minor modifications. A 3-day-old 10-ml Nonomuraea culture in RARE3 (dextrose lOg/L; malt extract lOg/L; glycerol 5g/L; yeast extract 4g L; bacto-peptone 2g/L; MgCl ₂ 2g/L; pH7.2) medium was diluted with 90-ml of fresh broth and grown for an additional 24 h at 28°C. Then, a 50-ml aliquot was centrifuged, resuspended in 10 ml of fresh medium, homogenized and fragmented by sonication (using 5 cycles of 30s each using Pabisch sonicator set at 20 kHz). After further growth for 2-3 h at 28°C, the culture was centrifuged, resuspended in 2.5-5 ml fresh RARE3 medium (dextrose lOg L; malt extract lOg/L; glycerol 5g/L; yeast extract 4g/L; bacto-peptone 2g/L; MgCl ₂ 2g L; pH7.2). At this stage, the Nonomuraea titer ranged from 5xl0 ⁷ to 5xl0 ⁸ CFU ml ^"1 .

The donor strain E. coli ET12567/pUB307, carrying the desired conjugative plasmid, was prepared by diluting an overnight culture in Luria Bertani medium (NaCl lOg/L; tryptone lOg/L; yeast extract 5g/L) with antibiotic selection (dilution 1 : 10) and growing the culture to OD6oo=0.7-0.8. The cells were washed twice with an equal volume of LB medium (NaCl lOg/L; tryptone l Og/L; yeast extract 5g/L) without antibiotics and resuspended in 1/10 initial volume of LB medium (NaCl l Og/L; tryptone 10g/L; yeast extract 5g L). The E.coli titer ranged from l lO ⁸ to 5 l0 ⁸ CFU ml ^"1 .

During each mating experiment, donor and recipient cells were mixed in 1 : 1 ratio (200μ1:200μ1) and plated on MV0.1X agar (soluble starch 2.4g/L; dextrose 0.1 g/L; 0.3 beef extract 0.3g/L; yeast extract 0.5g/L; tryptose 0.5g/L; agar 15g/L; pH7.2) in the presence of lOmM MgCh. After a 20-h incubation at 28°C, each plate was overlaid with 3 ml soft agar containing 200 μg nalidixic acid and 250 μg apramycin and further incubated at 28°C.

Nonomuraea colonies usually appeared after 7-10 days; they were then streaked on BTT agar (soluble starch 2.4g L; dextrose 0.1 g/L; 0.3 beef extract 0.3g/L; yeast extract 0.5g/L; triptose 0.5g/L; agar 15g/L; pH7.2) + 25 μ§/πι1 nalidixic acid + 20 μg/ml apramycin and BTT agar + 25 μg/ml nalidixic acid only, as control.

Two independent clones were then grown in liquid using RARE3 medium (dextrose lOg/L; malt extract lOg/L; glycerol 5g/L; yeast extract 4g/L; bacto-peptone 2g/L; MgCl ₂ 2g/L; pH7.2) with 20μg/ml apramycin. Genomic DNA was extracted using the Invisorb Genomic DNA Kit III (Stratec, Invitek, Germany) and analyzed by PCR amplification for the presence of pbt. Both clones gave the expected PCR product using the primers specific for pbtl (Table 3) and pbt 16 (Table 3). After 3 days of growth in RARE3 (dextrose l Og/L; malt extract lOg/L; glycerol 5g/L; yeast extract 4g/L; bacto-peptone 2g/L; MgCl ₂ 2g L; pH7.2) without selection of Nonomuraea containing the cosmid 2F7, methanolic extraction was performed by adding 2 volumes of methanol to 450 μΐ of broth and incubating lh at 40°C. After centrifugation, 20 μΐ of the supernatant was analyzed by HPLC on a Shimadzu instrument (HPLC analysis conditions: LC 2010A-HT liquid chromatograph, Shimadzu Corporation, Japan) equipped with a column LiChrosphere CI 8 5um, 4.6 100mm (Merck, Darmstadt, Germany) eluted at 1 ml/min flow rate and at 50°C temperature. Elution was with a linear gradient from 10% to 90% of phase B in 30 min. Phase A was 0.1% TFA (v/v) in water and phase B was acetonitrile. UV detection was set at 270 nm and 310 nm.

Under these HPLC conditions, Ge2270A (standard) showed a retention time of 17.28 min. Methanolic extract of the broth exhibited a peak in the UV310 nm trace, which corresponded to Ge2270A purified standard.

The same sample was analysed by LC-MS. HPLC-MS analysis conditions: Agilent 1 100 series liquid chromatograph equipped with a column Ascentis express Supelco RP18, 2.7μ (50 x 4.6 mm) eluted at 1 ml/min flow rate and at 40°C temperature. Elution was with a multistep program: time=0 (5% phase B); time=6 min (95% Phase B); time=7 min (100 % phase B); time=7.2 min (5% phase B); time=10 min (5% phase B). Phase A and phase B were 0.05% TFA (v/v) in water and acetonitrile, respectively. UV detection was at 220 nm. The effluent from the column was split in a 1 :1 ratio, with one part diverted to photodiode array detector and the remaining part diverted to the ESI interface of a Bruker Esquire3000 plus ion trap mass spectrometer. The mass spectrometric analysis was performed under the following conditions:

sample inlet conditions: sheat gas ( ₂ ) 50 psi; dry gas 10 1/min; capillary heater 365°C;

sample inlet voltage settings: positive polarity; capillary voltage -4000V; end plate offset -500V;

scan conditions: maximum ion time 200 ms; ion time 5 ms; full micro scan 3;

segment: duration 10 min, scan events positive (100-2400 m/z).

Under these analytical HPLC-MS conditions Ge2270A (standard) showed retention time of 4.6 min and a positive charged peak at m/z of 1312 amu, corresponding to its sodium adduct. Mass of the compound produced by Nonomuraea containing the cosmid 2F7 (compound 1) matched the mass of Ge2270A from the wild-type producer. Fermentation optimization and scale-up was necessary to obtain an amount of compound 1 for NMR analysis. We selected medium M8-20 (dextrose 20g/L; soluble starch 20g/L; casein hydrolizated 4g/L; yeast extract 2g L; meat extract 2g/L; CaC0 ₃ 3g L; pH8.0) as best for compound 1 production and 1 L fermentation was done.

lL-culture was centrifuged at 3000 rpm for 10 min and the supernatant was discarded. The mycelium was extracted with 240 mL of methanol (c.a. 3 fold the volume of the mycelium) on a rotary shaker, overnight at room temperature. The mixture was centrifuged at 3000 rpm for 10 min, the exhausted mycelium was discarded and the methanolic extract was concentrated under vacuum (20 mL) and transferred in a separating funnel. An equal volume of butanol was added along with few mL of water to facilitate the phase separation. The butanolic phase was evaporated to dryness, dissolved in 2 mL dichloromethane and purified by medium pressure chromatography on 12g of normal phase silica Flash RediSep RF column by using a CombiFlash RF Teledyne Isco Medium Pressure Chromatography System. The silica was previously conditioned for 2 min at 30 mL/min with pure dichloromethane, then brought to 50% of methanol in 13 min. The fractions containing compound 1 were pooled and concentrated under vacuum. HPLC analysis showed compound 1 with a retention time of 17.28 min. HPLC-MS analysis showed compound 1 with a retention time of 4.6 min and a positive charged peak at m/z of 1312 amu, corresponding to its sodium adduct. NMR analyses conditions: Ή- and ^{l 3} C- I D and 2D NMR experiments were recorded in DMSO-i/ ₆ at 25°C on a Bruker AMX 400 spectrometer. As internal standard the residual signal of DMSO at 2.54 ppm is considered. NMR spectra of the purified compound 1 produced by Nonomuraea containing the cosmid 2F7 were indistinguishable from Ge2270A produced by the wild-type host.

EXAMPLE 2 (compound lb) (X = Gly, R, = CH ₂ OMe, R ₂ = NH ₂ , Rj = H, R4 = CH ₃ , Rs =CH ₃ )

Gene inactivation of pbt4 in cosmid 2F7 with a cat cassette (chloramphenicol resistance gene) flanked by FRT sites (1.1 -kb) was performed using the procedure based on the λ Red recombination system described by Datsenko KA and Wanner BL, Proc Natl Acad Sci U S A. 2000, 97:6640-5. p D3 was used as template for the cat cassette and primers PBT4-Fl and PBT4R1 (Table 3) were used for the PCR amplification. The primers were 70-nt and 72-nt long, respectively, including 20-nt priming sequence for p D3 and 50-nt upstream the starting codon and 52 nt nucleotide centered on the stop codon of the pbt4 gene as homologous regions. E.coli carrying the Red helper plasmid (tetR) were first transformed with the 2F7 cosmid, carrying apramycin resistance gene aacIV, and clones were selected at 30°C. Then the obtained strain was made electrocompetent and transformed with the amplified DNA cassette by electroporation using a BioRad Cell-Porator system with the PCR amplified DNA. Shocked cells were added to 1 ml LB (NaCl lOg L; tryptone lOg/L; yeast extract 5g/L) and incubated lh at 37°C. Cells were spread on LB (NaCl lOg/L; tryptone lOg/L; yeast extract 5g/L) to select apramycin and chloramphenicol resistance transformants. The positive clones were colony-purified on the same selection medium at 37°C and then confirmed by PCR. Two PCR reactions were done by using flanking locus-specific primers PBT4-F2 and PBT4-R2 (Table 3) with the respective common test primers, cl and c2 (Table 3), for cat in order to check for both new junction fragments. A third PCR reaction was carried out with the flanking locus-specific primers and the amplified fragment was sequenced to verify simultaneous loss of the parental fragment and gain of the new mutant. The modified cosmid was named 2F7-Apbt4::cat.

Excision of the cat cassette was then also performed. To do this, DH10B cells carrying the cosmid 2F7-Apbt4::cat (aacIV and cat) were transformed with the plasmid 707-FLPe tetR (GeneBridges, http://www.genebridges.com/) and selection was carried out at 30°C. Several independent colonies were picked and grown in 1ml LB medium (NaCl lOg/L; tryptone lOg/L; yeast extract 5g/L) at 30°C for 2-3 hour then increasing to 37°C for overnight incubation. The suspension was diluted to obtain single cells and plated at 37°C on LB agar (NaCl lOg/L; tryptone lOg/L; yeast extract 5g L; agar 20g/L) containing apramycin. Several clones were streaked in parallel on LB agar (NaCl l Og/L; tryptone lOg/L; yeast extract 5g/L; agar 20g L) with apramycin and apramycin+chloramphenicol to identify the colonies with successful removal of the FRT-flanked fragment. PCR amplification of the colonies using flanking locus primers PBT4-F2 and PBT4R2(Table 3) confirmed the loss of the cat cassette. The cosmid 2F7 -Apbt4 was transformed in E.coli ET12567/pUB307 and the new strain was used to conjugate the actinomycete Nonomuraea ATCC39727 following the same procedure described previously. Two ex-conjugants have been tested for the presence of the cosmid by genomic DNA preparation and PCR amplification. Fermentations in RARE3 medium (dextrose lOg/L; malt extract lOg L; glycerol 5g L; yeast extract 4g L; bacto-peptone 2g/L; MgCl ₂ 2g/L; pH7.2) have been carried out. Methanolic extracts of the broth have been analysed in HPLC and LC-MS as described previously highlighting a peak presenting mass and fragmentation compatible with compound lb.

To perform NMR experiments on this peak, a 1 L fermentation scale in medium Medium CI (soluble starch 35g/L; dextrose l Og/L; hydrolyzed casein 5g/L; meat extract 3.5g/L; yeast extract 20g/L; soybean meal l Og/L; CaC0 ₃ 2g/L; pH7.2) was done. The culture was centrifuged at 4000 rpm for 10 min and the supernatant was discarded. The mycelium was extracted overnight with 500 mL methanol, at room temperature on a rotary shaker. The mixture was centrifuged and the exhausted mycelium was discarded. The methanolic extract was evaporated to reduced volume (50 mL), splitted into 5xl0-mL portions, and roughly purified using a 5g Flash CI 8 prepacked column (Isolute, Biotage) placed on a Vac Master system (Stepbio). Each loaded solution was washed with acetonitrile:water 25:75 (10 mL) and eluted with acetonitrile: water 75:25 (15 mL). The eluted fractions were pooled, evaporated to dryness and dissolved in lmL acetonitrile:water 75:25. This solution was purified by HPLC using a Shimadzu Series 10 system (Kyoto, Japan), equipped with a reversed- phase column, LiChrosphere CI 8 5μιη, 4.6 100mm (Merck, Darmstadt, Germany) and a diode array detector (190-800 nm). The purification was obtained using a 22- min linear gradient of eluent A (H ₂ 0) and eluent B (acetonitrile) from 10% to 80% of eluent B. This procedure allowed the isolation compound lb showing a retention time of 18.47. HPLC-MS analysis of the compound showed a retention time of 4.8 min and a positive charged peak at m/z of 1274 amu, corresponding to its proton adduct (figure 2). 1H-NMR and 2D experiments were recorded in DMSO-d ₆ at 25°C on a Bruker AMX 600 spectrometer. ¹ H-NMR analysis of compound lb showed the disappearance of the signal at 5.01 ppm relative to the β-CH of the hydroxy-phenylalanine present on Ge2270A (figure 3).

EXAMPLE 3 (compound lc) (X = Gly, Ri = CH ₂ OMe, R ₂ = OH, R ₃ = OH, R, = CH ₃ , R ₅ =CH ₃ )

Gene inactivation of pbtl5 in cosmid 2F7 with a cat cassette (chloramphenicol resistance gene) flanked by FRT sites (l . l -kb) was performed using the procedure based on the λ Red recombination system (Datsenko KA and Wanner BL, Proc Natl Acad Sci U S A. 2000, 97:6640-5) described in example 2. p D3 was used ad template for the cat cassette and primers PBT15-F1 and PBT15-R1 (Table 3) were used for the PCR amplification. The positive clones were tested as described in example 2 using two nearby locus-specific primers PBT15-F2 and PBT15-R2 (Table 3) and the common test primers, cl and c2 (Table 3), for cat. The modified cosmid was named 2F7-Apbtl5::cat. Excision of the cat cassette was performed as described in example 2 generating the cosmid 2F1-Apbtl5.

The cosmid 2FT-Apbtl5 was transformed in E.coli ET12567/pUB307 and the new strain was used in conjugation with the actinomycete Nonomuraea ATCC39727 following the same procedure described previously. Two ex-conjugants have been tested for the presence of the cosmid by genomic DNA preparation and PCR amplification. Fermentations in RARE3 medium (dextrose l Og/L; malt extract lOg L; glycerol 5g/L; yeast extract 4g/L; bacto-peptone 2g/L; MgCl ₂ 2g/L; pH7.2) have been carried out. Methanolic extracts of the broth have been analysed in HPLC and LC-MS as described previously showing a peak with a m/z 1291 amu, compatible with the protonated mass of compound lc.

To perform further analysis of the produced metabolite and to have available starting material for semi-synthetic derivatives, a fermentation of 1 L scale in medium C2 (soluble starch 35g/L; dextrose lOg L; hydrolyzed casein 5g/L; meat extract 3.5g L; yeast extract 14g L; soybean meal 7g/L; CaCOj 2g/L; pH7.2) was done. l L-culture was centriruged at 4000 rpm for lOmin and the supernatant was discarded. 400 mL methanol was added to the mycelium and the mixture was shaken for 2h at room temperature. The exhausted mycelium was discarded by centrifugation and the methanolic extract was concentrated under vacuum to 7 mL. This solution was purified by medium pressure chromatography on 30g of reversed phase Biotage Snap Cartridge P-C18-HS by using a CombiFlash RF Teledyne Isco Medium Pressure Chromatography System. The column was previously conditioned for 1 min at 20 mL/min with pure water, then brought to 40% of acetonitrile in 5 min, successively brought to 60% of acetonitrile in 13 min and washed with 95% of acetonitrile for 2 min. The fractions containing compound lc were pooled and concentrated under vacuum. HPLC analysis of the purified compound 1 c showed a peak with a retention time of 17.83 min. HPLC-MS analysis of the purified compound lc showed a retention time of 4.4 min and a positive charged peak at m/z of 1291 amu, corresponding to its proton adduct.

LC-HRMS analyses of the purified compound lc were run to have additional confirmations of its structure. LC-HRMS analyses were performed on a HPLC system Surveyor Accela (Thermo Fisher Scientific, San Jose.USA) connected to the benchtop mass spectrometer Exactive™ (Thermo Fisher Scientific, Bremen, Germany), equipped with a NSI-ESI ion source. Samples were injected on a C8 reversed phase column (BioBasic C8, 100 x 0.18mm, 5μηι, 300A, Thermo Fisher Scientific, San Jose, Ca, USA) and were eluted through an acetonitrile gradient (eluent A, 0.1 % formic acid in water; eluent B, 0.1% formic acid in ACN): the gradient profile was 5% eluent B for 3 minutes, 5 to 65 % B in 17 minutes, 65 to 95% B in 5 minutes; the flow rate was 100 μί/πιίηυίε split in order to achieve a final flux of 2 μ τϊά νΛΐ. The observed peak was at m/z 1291.2557 [M+H] ⁺ (calcd for CseHssN OnSe, 1291.2493).

EXAMPLE 4: Synthesis of compound lc2:

To a stirred solution of 2.5 mg (c.a. 2 μπιοΐ) of compound lc, purified as described above in 0.5 mL DMF, 0.6 mg of l-(4-(trifluoromethyl)benzyl)piperidin-4-amine and 4 mg of PyBOP were added and the reaction mixture was kept under stirring at room temperature for 2 hours. The reaction was quenched by addition of 0.5 mL of water. HPLC-MS analysis of the obtained compound l c2 showed a retention time of 5.1 min and a positive charged peak at m/z of 1553 amu, corresponding to its sodium adduct (figure 4).

EXAMPLE 5 (compound la) (X = Ala, Ri = CH ₂ OMe, R ₂ = NH ₂ , R ₃ = OH, Rj = CH ₃ , R ₅ =CH ₃ )

Generation of Ge2270 analogs was performed using the λ Red recombination system (Datsenko A and Wanner BL, Proc Natl Acad Sci U S A. 2000, 97:6640-5) properly modified. The cat cassette (chloramphenicol resistance gene) flanked by FRT sites (1.1-kb) was amplified with primers PBT8GA-F1 and PBT8-R1 (Table 3) using p D3 plasmid DNA as substrate. The forward primer was designed with 20-nt priming sequence for p D3 and 70-nt upstream the stop codon (included) of the structural gene pbt8, carrying a nucleotide substitution , the GGC codon coding for Gly was mutated in GCC coding for Ala. The reverse primer included 20-nt priming sequence for p D3 and 50-nt starting from the stop codon of pbt8.

E.coli carrying the Red helper plasmid (tetR) and the cosmid 2F7 (aacIV) was electroporated with the amplified DNA fragment and selection was performed for apramycin and chloramphenicol resistant colonies at 37°C. The positive clones were confirmed by two independent PCR reactions: the first reaction using a nearby locus- specific primer PBT8-F2 (Table 3) with the test primer cl (Table 3) for cat; the second reaction with the flanking locus-specific primers (PBT8F2 and PBT8R2, Table 3). The amplified fragments were sequenced to verify the presence of the nucleotide substitution. The modified cosmid was named 2F7 -pbt8G7A::cat.

Excision of the cat cassette was performed as described in example 2 generating the cosmid 2Vl-pbt8G7A. The cosmid l¥l-pbt8G7A was transformed in E.coli ET12567/pUB307 that subsequently was used for conjugation to Nonomuraea ATCC39727, following the same procedure described previously. Two ex- conjugants have been tested to confirm the presence of the cosmid by PCR amplification. Fermentations in RARE3 medium (dextrose lOg/L; malt extract lOg L; glycerol 5g/L; yeast extract 4g/L; bacto-peptone 2g/L; MgCl ₂ 2g L; pH7.2) have been carried out. Methanolic extracts of the broth have been analyzed in HPLC and LC-MS, as described previously, showing a peak with a m/z 1326 amu, compatible with the sodiated mass of compound la. The recovery of compound l a was obtained using the method described in the example 2, starting from a culture of Nonomuraea-2Fl-pbt8G7A prepared as described above. HPLC analysis of the purified compound la showed a peak with a retention time of 18.28 min. HPLC-MS analysis of the purified compound la showed a retention time of 4.5 min and a positive charged peak at m/z of 1326 amu, corresponding to its sodium adduct (figure 5).

EXAMPLE 6 (compounds 2) (X = Gly, R, = CH ₂ OH, R ₂ = NH ₂ , R ₃ = OH, R4 = CH ₃ , R _S =CH ₃ )

Gene inactivation of pbtl l in cosmid 2F7 with a kan cassette (kanamycin resistance gene) flanked by FRT sites (1.4-kb) was performed using the procedure based on the λ Red recombination system described in Datsenko KA and Wanner BL, Proc Natl Acad Sci U S A. 2000, 97:6640-5). pKD13 was used ad template for the kan cassette and primers PBT17-F1 and PBT17-R1 (Table 3) were used for the PCR amplification. E.coli carrying the Red helper plasmid (tetR) and the cosmid 2F7 (aacIV) was electroporated with the amplified DNA fragment and selection was performed on LB agar (NaCl lOg/L; tryptone lOg/L; yeast extract 5g/L; agar 20g/L) with apramycin and kanamycin at 37°C. The positive clones were confirmed by PCR reactions: two PCR experiments were conducted using flanking locus-specific primers PBT17-F2 and PBT17-R2, (Table 3) with the respective common test primers, kl and k2 (Table 3), for kan; one PCR experiment was conducted using the flanking locus-specific primers PBT17-F2 and PBT17-R2, (Table 3). The amplified fragment was sequenced to verify the inactivation of pbtl 7. The modified cosmid was named 2F7 -Apbtl 7::kan.

The cosmid 2F7 -Apbtl 7:: kan was transformed in E.coli ET12567/pUB307, then used to conjugate the actinomycete Nonomuraea ATCC39727, following the same procedure described previously. Two ex-conjugants have been tested for the presence of the cosmid by PCR amplification. 15 mL fermentations in RARE3 medium (dextrose lOg/L; malt extract lOg/L; glycerol 5g/L; yeast extract 4g L; bacto-peptone 2g/L; MgCl ₂ 2g/L; pH7.2) have been carried out. Methanolic extracts of the broth have been analysed in HPLC and LC-MS as described previously. HPLC analysis showed a main peak with a retention time of 15.07 min. HPLC-MS analysis showed a main with a retention time of 4.1 min and a positive charged peak at m/z of 1298 amu, [M+Na] ⁺ . 1 L Fermentation was done in medium M8-20 (dextrose 20g/L; soluble starch 20g/L; casein hydrolizated 4g/L; yeast extract 2g L; meat extract 2g/L; CaC0 ₃ 3g L; pH8.0) and the recovery of compound 2 was obtained following the same procedure described in example 1. Ή-NMR ID- and 2D experiments were recorded in acetone-i/^ at 25°C on a Bruker AMX 600 spectrometer. As internal standard the residual signal of DMSO at 2.05 ppm is considered. Ή-NMR analysis demonstrated that the obtained compound is Ge2270 D2 congener.

EXAMPLE 7 (compounds 3) (X = Gly, R, = CH ₂ OMe, R ₂ = NH ₂ , R ₃ = OH, | = CH ₃ , R _s =H) and (compound 4) (X = Gly, Ri = H, R ₂ = NH ₂ , R ₃ = OH, R = CH ₃ , Rs =H)

Gene inactivation of pbt6 in cosmid 2F7 with a kan cassette (kanamycin resistance gene) flanked by FRT sites (1.4-kb) was performed as described in example 6. p D13 was used ad template for the kan cassette and primers PBT6-F1 and PBT6- R I (Table 3) were used for the PCR amplification. The positive clones were tested by PCR reaction using flanking locus-specific primers PBT6-F2 and PBT6-R2, (Table 3) and the common test primers, kl and k2 (Table 3), for kan as described in example 6. The modified cosmid was named 2F7-Apbt6::kan.

The cosmid 2 l-!spbt6::kan was transformed in E.coli ET12567/pUB307 then used to conjugate the actinomycete Nonomuraea ATCC39727 following the same procedure described previously. Three ex-conjugants have been tested for the presence of the cosmid by PCR amplification. 15 mL culture in RARJE3 medium (dextrose lOg/L; malt extract lOg L; glycerol 5g/L; yeast extract 4g/L; bacto-peptone 2g/L; MgCl ₂ 2g/L; pH7.2) have been carried out and methanolic extracts of the broth, analysed in HPLC and LC-MS as described previously, showed two main peaks at 16.56 min of 15.22 min corresponding to compound 3 and 4 respectively. HPLC-MS analysis: compound 3 at of 4.4 min and at m/z of 1298 amu [M+Na] ⁺ , compound 4 at 4.2 min and m/z of 1254 amu, [M+Na] ⁺ .

1 L Fermentation was done in medium Medium CI (soluble starch 35g/L; dextrose 10g L; hydrolyzed casein 5g/L; meat extract 3.5g/L; yeast extract 20g/L; soybean meal 10g L; CaC0 ₃ 2g/L; pH7.2) and the recovery of compound 3 and 4 were obtained using the method described in the example 1 NMR analysis demonstrated that compound 3 is Ge2270Bl congener and compound 4 is congener Ge2270Dl . EXAMPLE 8 (compounds 5) (X = Gly, Ri = H, R ₂ = NH ₂ , R ₃ = OH, R4 = CH ₃ , R ₅ =CH ₃ )

Gene inactivation of pbtl6 in cosmid 2F7 with a cat cassette (chloramphenicol resistance gene) flanked by FRT sites (1.1 -kb) was performed using the procedure based on the λ Red recombination system described in example 2. p D3 was used ad template for the cat cassette and primers PBT16-F2 and PBT16-R2 (Table 3) were used for the PCR amplification. The positive clones were tested by PCR reactions using the flanking locus-specific primers PBT16-F3 and PBT16-R3 (Table 3) and the test primers, cl and c2 (Table 3), for cat as described in example 2.The modified cosmid was named 2F7-Apbtl6::cat.

The cosmid 2Fl-&pbtl6::cat was transformed in E.coli ET12567/pUB307 and the new strain was used in conjugation with the actinomycete Nonomuraea ATCC39727 following the same procedure described previously. Two ex-conjugants have been tested for the presence of the cosmid by genomic DNA preparation and by PCR amplification. Fermentations in RARE3 medium (dextrose lOg/L; malt extract lOg/L; glycerol 5g/L; yeast extract 4g/L; bacto-peptone 2g/L; MgCl ₂ 2g L; pH7.2) have been carried out. Methanolic extracts of the broth have been analysed in HPLC and LC-MS as described previously. HPLC analysis showed a peak with a retention time of 15.82 min. HPLC-MS analysis showed a retention time of 4.2 min and a positive charged peak at m/z of 1268 amu, compatible with sodium adduct of compound 5.

1 L Fermentation was done in medium Cl (soluble starch 35g L; dextrose lOg/L; hydrolyzed casein 5g/L; meat extract 3.5g/L; yeast extract 20g/L; soybean meal lOg/L; CaC0 ₃ 2g/L; pH7.2) and the recovery of compound 5 was obtained using the method described in the example 1. NMR analysis demonstrated that compound 3 is Ge2270 Cl congener.

EXAMPLE 9 (compounds 6, (X = Gly, Ri = CH ₂ OMe, R ₂ = NH ₂ , R ₃ = OH, R4 = H, R ₅ =CH ₃ ) and (compound 7) (X = Gly, Ri = CH ₂ OMe, R ₂ = NH ₂ , R ₃ = OH, R, = H, R ₅ =H) Gene inactivation of pbt7 in cosmid 2F7 with a cat cassette (chloramphenicol resistance gene) flanked by FRT sites (1.1 -kb) was performed using the procedure based on the λ Red recombination system described in example 2. pKD3 was used ad template for the cat cassette and primers PBT7-F1 and PBT7-R1 (Table 3) were used for the PCR amplification. The positive clones were tested by PCR reactions using the flanking locus-specific primers (PBT7-F2 and PBT7-R2, Table 3) and the test primers, cl and c2 (Table 3), for cat as described in example 2. The modified cosmid was named 2F7-Apbt7::cat.

The cosmid 2F1-Apbt7::cat was transformed in E.coli ET12567/pUB307 and the new strain was used in conjugation with the actinomycete Nonom raea ATCC39727 following the same procedure described previously. Two ex-conjugants have been tested for the presence of the cosmid by PCR amplification. Nonomuraea-2F7- Apbt7::cat was cultured in RARE3 medium(dextrose lOg/L; malt extract lOg/L; glycerol 5g/L; yeast extract 4g/L; bacto-peptone 2g/L; MgCl ₂ 2g L; pH7.2) . Methanolic extracts of the broth have been analysed in HPLC and LC-MS as described previously. HPLC analysis showed two peaks: peak a with retention time of 15.57 min and peak b with a retention time of 16.20 min. HPLC-MS analysis of peak b showed a retention time of 4.1 min and a positive charged peak at m/z of 1298 amu, compatible with sodium adduct of compound 6. HPLC-MS analysis of peak a showed a retention time of 4.0 min and a positive charged peak at m/z of 1284 amu, compatible with sodium adduct of compound 7.

1L fermentation was performed in medium C l (soluble starch 35g/L; dextrose 10g L; hydrolyzed casein 5g/L; meat extract 3.5 g/L; yeast extract 20g L; soybean meal lOg/L; CaCC«3 2g/L; pH7.2) and the recovery of compound 6 and 7 was obtained using the method described in the example 1.

NMR recorded as above demonstrated that compound 6 is Ge2270 C2b congener while compound 7 is a new congener of the Ge2270 family (figure 6).

EXAMPLE 10 (compound 2b) (X = Gly, Ri = CH ₂ OH, R ₂ = NH ₂ , R ₃ = H, R, = CH3, Rs =CH3)

Gene inactivation of pbtl 7 in cosmid 2FT-Apbt4 with a kan cassette (kanamycin resistance gene) flanked by FRT sites (1.4-kb) was performed using the procedure based on the λ Red recombination system described in example 6. pKD13 was used ad template for the kan cassette and primers PBT17-F1 and PBT17-R1 (Table 3) were used for the PCR amplification. The positive clones were tested as described in example 6 using two nearby locus-specific primers (PBT17-F2 and PBT17-R2, Table 3) and the common test primers, kl and k2 (Table 3), for kan. The modified cosmid was named 2¥1-Apbt4 -Apbtl 7::kan. The cosmid 2 1-Apbt4 -Apbtl 7::kan was transformed in E.coli ET12567/pUB307, then used in conjugation with the actinomycete Nonomurae ATCC39727 following the same procedure described previously. Two ex-conjugants have been tested for the presence of the cosmid by PCR amplification. The two exconjugants Nonomuraea-2F7-Ap6r4 -Apbtl 7::kan were cultured in RARE3 medium (dextrose lOg/L; malt extract lOg/L; glycerol 5g/L; yeast extract 4g L; bacto-peptone 2g/L; MgCl ₂ 2g/L; pH7.2). Methanolic extracts of the broth have been analysed in HPLC (retention time 16.45 min) and LC-MS (retention time 4.4 min) as described in example 1 showing a peak with a m/z 1260 amu, corresponding to the protonated mass of compound 2b (figure 7).

EXAMPLE 11 (compound 5a) (X = Ala, R, = H, R ₂ = NH ₂ , R ₃ = OH, R, = CH ₃ , Rs =CH ₃ )

Gene inactivation of pbtl 6 in cosmid 2?l-pbt8G7A with a cat cassette (chloramphenicol resistance gene) flanked by FRT sites (1.1-kb) was performed as described in example 2. pKD3 was used as template for the cat cassette and primers PBT16-F2 and PBT16-R2 (Table 3) were used for the PCR amplification. The positive clones were tested by PCR reaction using flanking locus-specific primers (PBT16-F3 and PBT16-R3, Table 3) and the common test primers, cl and c2 (Table 3), for cat as described in example 2. The modified cosmid was named 2F7-pbt8G7A -Apbtl6::cat.

The cosmid 2Yl-pbt8G7A -Apbtl6::cat was transformed in E.coli ET12567/pUB307 and the E. coli recombinant strain was used in conjugation with the actinomycete Nonomuraea ATCC39727 following the same procedure described previously. Two Nonomuraea 2F7-pbt8G7A -Apbtl6::cat ex-conjugants have been tested for the presence of the cosmid by PCR amplification. 15 mL cultures in RARE3 medium (dextrose lOg/L; malt extract l Og/L; glycerol 5g/L; yeast extract 4g/L; bacto-peptone 2g/L; MgCl ₂ 2g/L; pH7.2) have been carried out. Methanolic extracts of the broth have been analysed in LC-MS (retention time 4.4 min) as described previously showing a peak with a m/z 1282 amu, corresponding to the sodiated mass of compound 5a (figure 8).

EXAMPLE 12: in vitro antibacterial activity of compounds belonging to Table The compounds of formula (I) of the present invention can be effectively employed as active ingredients of antimicrobial preparation used in human or animal medicine for the prevention and treatment of infectious diseases caused by pathogenic bacteria which are susceptible to said active ingredients, in particular for the treatment of infections caused by streptococci, enterococci and staphylococci. Minimal inhibitory concentrations (MICs) for aerobic and anaerobic bacteria have been determined by broth microdilution methodology, according to Clinical and Laboratory Standards Institute guidelines (CLSI documents M100-S16 and M27-A, NCCLS, Wayne, PA) using inocula of 1 -5x10 ⁵ CFU/mL for Gram positive and negative bacteria

Staphylococcus aureus, Enter ococcus faecalis, and Escherichia coli were grown in Cation Adjusted Mueller Hinton (CAMHB) broth, Streptococcus pyogenes in Todd Hewitt broth. All media were from Difco Laboratories, Detroit, MI, USA. MICs for anaerobic bacteria are determined by the broth dilution method in Brucella broth (BB) supplemented with hemin (5 μg/mL), vitamin Kl (1 g/mL), lysed horse blood (5%) and Oxyase (1 :25 v/v) (CLSI documents Ml 1 -A6, NCCLS, Wayne, PA). Test results were scored after 24 hours of incubation at 37°C for all tested strains.

All strains used are clinical isolates or strains from American Type Culture Collection (ATCC). The results of the tests are reported in Table 4.

Compounds l a, lb, lc, lc2 and 2b (prepared as described under Example 5, Example 2, Example 3, Example 4 and Example 10 respectively) and teicoplanin and clindamycin are dissolved in 10% DMSO to obtain a 10 mg/mL stock solution, and subsequently diluted in the test media to obtain working solutions. Microplates are always pre-coated with 0.02% bovine serum albumin to prevent non-specific adhesion of compounds. Table 4 Antimicrobial activity of compounds l a, lb, lc, lc2 and 2b against aerobic bacteria.

SEQ ID No 1

CATAATGACTATCCGTATCCGGTCGTGCAACGCGCTGCGGTCGCGGCGGG AAAGCGCGTG

ACGAGCTCGCGCAGTAATGCCGAACGAGTGGTCCGAATCATCGACGCAT CTGCGGAGCTT

TTACTCCAGAGGGGATATCGACGGGTGACCGTCGAGGAGGTCGCCAGCC ATGCCGGTGTC TCCAAGAGC AGCGTCTACCTGCACTGGAAC ACG AAGGACGACATCTTCT ACG AC GCTCTC

GACCGCGAGTGCGCCGCGCTCGTGTGCGAGGCCGTCGACCGCGTCAGAC GCAACCCGGCC

GAAATCCTGGCTCACCGGATGGCGGCCAATCTGCTCCGGATCATCCTGGA CAGGCCGCTG

CTGCGGGCGCTGCTGATGGGTGACCAGGCGATTCTGGGATCGCTCCGCCA TGCGAAGTCG TCCGCTCTCCGATCCCGGACGGCGGCAATTGACGAGCTGATTCACCGATA TCTCTCAGCG CTGCAGAAAAACCAACTCATCTGTCCCGACATCGATCTGCGCATCACCCG GAAAGCGGTG

TGGGAAATGCTGCGCGGCATGATTTTCTCGGCGGGGACGGAGCCGTTGG GCAAGCCGCGC

TCCGCCGAGCTGGCGCAGGTCATGACGGTTACCGTGCGGCGGGCGTTCG AGCCCAGGGGC

GTACCGGGGATCGACCGGATCACCGCCGCCGCGGCCGAGGTCTTCGAGG CGTTCGACGAA

CTCATTCCGACCGCGGAGAACCTGGACTTCGAGCGCCCGATGGTGCTATG ACCGTGGCGC CGGCATATCTTTCTCCGGTTTCGAGGACGCTTGTCGGAAACGGTGTGCTC GCCGACCGGC

TCTCGGCGGTGCCGGCCGGGCTCGATCCGGATCTGGTGCTGGCCGTATCG GACGCGCCCG

ATCCCGCGGTGCTCGCCGCGGCGAGAGCGACGGGCCGCCCGGTGCTGCC GGTGATCGCCG

AGATGGGGCACGTCCGCATCGGACCGCTGGAACGGCCGGGCGAGCCGGG GTGCTCCGACT

GCCTGAGCATCCGCAGACATCGTGCCGCCACCCGGTCGGCCGAACGCGA GGCGGTGTGGC TGAG AC ACGGCG AG AGCATGGCCGGTGCGATGTCGCCGCTGCTCGTGCC GGCGGCACTGG

ACCTGGTGGCCGCGCTCGTCGCCGGAGCGGCGGCGGGCCGCGGCGGCAT TCTGCTGGTCG

ACCTCGCCGACCTCGCCGTGACGCCGCACTCGTTCCTGCCCGATCCGCTC TGCGGCCGGT

GCGGGGAACTGCCGGACGACAGCCCGGAGCGTGCGCGCGTCACGTTGGT GCCGCGGCCGA AGCCCGCTCTGGACCAGTACCGGGTGTGGGACGCCGAACACGAACTGGA CCGCCTGATCC GGACCTACGTCGACGACCACACCGGTCTCGTGAACTCGCTCACCCCGGCG GCGCTCGGTT

CGCTCGCCGTCGCGGGAGCGGCGATCCGGCTACGCGGGACGACGGTGTT CGAACCGGGCT

TCGGCCGGTCCCGCAGCTACCGCCGCAGCTCGGCCATCGCGTTGCTCGAA GCGCTGGAGC

GGTACGGCGCCATCGGCCCCGGCGGCAGGCGCGGAACCGTACGCGCGAG CTACGCGTCCC

TGGGCGACCGGGCGGTGGATCCACGGTCGCTGGGCCTGCACCCGCCCAC GCACTACGCGC TGCCGGACTTCCCGTACCGGCCGTTCACGGTCGACGCGGTGTGCCGTTGG GTGTGGGGGC

ACTCGTTCGCGGCCGGTGGTCCCGTGCTGGTGCCGGAGCGCAACGTCTAC TACGGGCGCT

CCGACGACCAGCCGTTCTGCTACGAGCTCGCCAACGGCTGCGCCCTGGGA TCGTGCCTGG

AGGAGGCGATCTTCCACGGCATCCTGGAGGTGCTGGAACGCGACGCGTT CCTGCTGACCT

GGTACACCCGTGCGCGGGCCCCGCGCATCGATCTCGGCACGGCCCGCGA TCCGGGGATCC CGCTGGTCGCCGCGGCGATCACCGCCGAGACCGGTTACCTCGTCGAGTGC TACGACATCA

CGCCCGACCACGGAGTGCCGTGCGTGTGGGCGCTGGCCCGTACGCTGTCC GGTGAGCCCG

CCACGATCAGCGCCGCCGCCGCCGGGACCAGCCTGGAACACGCCGCCGC GGGGGCGCTCG

CCGAACTCGGCCCGATGGTGCCGACCGTGCGCGAGCACTTCCCCCCGAAC GCCGACCGAG CACGGACGCTGGCCGCCGACGGCAGCCGGGTACGGTCGATGATCGACCA TTACCTGGTGT ACGGAGTGCGGTCGGCGGCGGAGCGGTTGTCGTTCCTCACCGAGGGGAC GACGCGGGTGC

CGTTCCCACCGCCTCCCGAAGGGTTCCGGCACCAGGATCTGACCCTCGAC CTGGAGTTTC

TGATCGACCGGCTCGCGGACGTCGGGCTCGACGTGATCGTGGTCGATCTG ACGACACCGG

AGCATCGCGCGGGCGGCCTGCGCTGCGTGAAGGTGCTCGTGCCCGGTGC GGTCCCGATGA

CGTTCGGGGAGCAGAACCGCCGCACCTGGGGGCTGCCGCGACTGCTGGA CCCGGCCGCGG TGCGCGGACGGGGGATGCCGGTGCGTGTCCACGCCGACCTGAACCCCGA CCCCCACCCGT

TCCCGTGAACCGCGCGGTCGCCAGGGTCGGCGGACTGCCGCTGTCCGCTC TGGACGCTCT

GGCCTGCCCTGACGCCACGGCTCTGGCCGCCTGTGTCGTCTCGCTCACCG ACGAGTTGTC

CCGGCGGGCCGCGGTGCTGTCCGACCTCCTGTACGAGGTGATCGGCGCCG CGGAGGAGCA

CAAGCCCGTCCTGGTGGCGATCCGGCGGGACCTGCACGGGCTGCGTCGG CCCAAGCGGAT CGAGGTGCTGCCCGCGGCGCTGCTGGATCGGGTCC ATGAGTGGATCTCCC TCTGGGAACA

GCGCGCGCGGGCCCGCGCCGCGTTGCCCGAGGTGCTCGCGGGTGAGGCC CGGACGGCCTG

GGCGTCGCTGCGGGAGCTGGCCGCCGCTCCGGCGGTACGGCACGGGCTC GCGCACGCCAG

CCCGGACCTGTCCGCGGAACTGGAGAAGTGGCTCGCCGACCCGGGGCGA CGGCCCCGGGC GGGGACACTGGCGAGCCTGCTGCGCTACGTCACCCGGGTGGCGGCCAAG ACGAGTCCTTT CAGCACGTTCACGAGCGTTCACCGCGTGCACTGGGAGATGGAGGGGACG GGCTGGGAGAT

ACCCGACTCACCGCCGACCGTGGTGGTCGAGGCCGATGTGGGACTCCGG CTGCTGGTGGA

GTTCATGGTGCCACGATGGCCAGGACCGGCCGCGGCGCGTACGGTCCGG CTGTCGCCGAC

CGCGTACATGTCCGGGCGGAAGCTGCTCTTCCTCGGCCCGGACGGGCGG ACGCGCGCACT

CGAACGGACGGCGGCGCTGGATGCCCTCGTCGAGTTGCTGCGTGCCGAG CACGGGGCCCG GTGGGACGTGGTGGCCGGCAAGCTCGCCGCAGAAGACCGCGAGCAGGGG GAGGAGACCCT

GGCCCGGCTGGTGCGCGGCGGGCTGGTGGAGGCGGTGGTGCCGGTGCCC GGCCAGGCGGC

GAGGCCGTTCGCGGCCCTCGCGGACTGGGCCCGGGCCGCCGAGGTCGCC CATCCGCTGAA

CCGCATCCAGCAGGCACTGGACCTCACCGGACCCCTCGGCACCGGTGAC CCGGCGACATC

GGCCTGCACCGAGGCGGCGCGTCGCGTCACCGCCGAACTCCCCGCGCTC AGCCTTCCCGC CATGCCGCTCCCCGAGCTGCGCCGCCGCGTTCTGCGCGAGTCGACCGTCG GCGCGCCCGT

CACCTGCGCGCTCCCGGAGTGGCGACCGGCCCTGGCCGACCTGGAACGG GTCCGCCGCTG

GCTGGCGGTCCACGATCCGATGTTGCCGCTCCGCCTTTCCCTGGCGGACC ACGTCCGCGA

CTGGTTCGGCCCGGACTCCAGCCCGCCGCTGCTGGAGGTGTACCTCCGGG TCGAGGCGGC TCAGCCGGGGTCGCCGCTCCACCCCGACTTCCTGGAACGCCCCGACCCCC TGGCGGATGT CGCCGACCCGCGCCTGCGCGGACTGCGCGACCTGCGCGTCCGGACGATC GAGGCGCTTCA

CGGAGGGCGGGCCGAGGAGATGCTCGGCGAACTGCCGGGGTGGATCGGC GACCCGGGACC

GGTCACCTGCTACGTCCAGCCCTTCCATGAGGAGGGCGAGCTCAGGCTGG TCCTCAACAC

CGCGCACGGCGGCCACGGCCGGGGCATCACCCGCTGGAGCAGACTTCTG GGCGGCAGTCC

GGTGCGGGTCTCCCCCGGCTACCTGGCCGCCGAGCTGCCGGGGGTCTTCG GGCACAGCTT GAACCTGCGCGCTCCCGGCACGGAATGGGAACTCGACTACCCGGGCGCG GTAGGCCAGGC

ACCGCCGGATCGCCGCATTCCGCTCACCGAGTTGCAGGTCCGGCACGACC AGGCCAGGGG

TCTCGTCACCCTGTGGTGGCCGCGTGCCGGCCGGCGGGTCGTCCCGGTGC ACGCGGGCAT

GATGTCGGAGACGCTCCTGCCGCCGCTGGCGCGGCTGCTCGTCGAGGCGT TCGGTACCAC

CTACCTGACCCACCCCACCCTCCCGGCGATCCCGCGGGCGAGCGGCCCTC GGATCGATCT CGGCCGTGTCACCCTCGCCAGAGCCCAGTGGACGGTCGGCCAGGACGCC GTTCCCCGGCG

GGAAGACGACGACGCCGATCACTTCGTCGCCGTCCAGCGGTGGCTGCGC CGCACCGGGAT

TCCGCGCCGTTGCTTCGTGCGCGTCCGGGAACGCCAGGTGAGCAGGGAC CGGATCGCGTT

CGACAAACGGCACAAGCCGGTGTTCATCGACTTCGGCAGCTGGCCGTCG GTGCTGGAGTT CGACCGGATGGTCGAGCGGACGACCGGCGAGCTGGAAGTGGCGGAGGC GCTGCCCGACGG CGATCGGGCCGTGGAGTTCGCGATCGAGGTGGGGGAACCGTGACGCAGT ATCCACTGAGC

CGTCCGGAGCCGCTCGGGGTCCACCCCGACTACCGGCGGCTCCGTGAGA CATGCCCGGTG

GCGCGGGTCGGCTCGCCCTACGGCCCGGCCTGGCTGGTCACCCGCTACGC GGACGTGGCG

GCGGTGCTGACCGACGCGCGGTTCAGCCGTGCGGCGGCGCCGGAGGACG ACGGTGGCATC

CTGCTCAACACGGACCCGCCGGAGCACGACAGGCTGCGCAAGCTCATCG TGGCGCACACC GGAACGGCGCGGGTCGAGCGGCTCCGGCCGAGGGCCGAGGAGATCGCG GTCGCGCTGGCA

CGGCGGATCCCGGGTGAGGGTGAGTTCATCAGCGCGTTCGCCGAGCCGTT CTCGCATCGG

GTGCTCAGCCTGTTCGTCGGGCACCTGGTCGGCCTGCCCGCCCAGGACCT GGGTCCGCTG

GCCACCGTCGTGACGCTCGCGCCGGTGCCCGACCGGGAGCGGGGAGCCG CCTTCGCCGAG

CTGTGCCGGCGGCTCGGGCGCCAGGTGGACCGGGAAACGCTCGCGGTGG TGCTCAACGTC GTCTTCGGCGGGCACGCGGCGGTGGTGGCCGCGCTGGGCTACTGCCTGCT GGCCGCGCTG

GACGCACCGCTGCCGAGGCTGGCGGGCGATCCCGAAGGCATCGCCGAGC TGGTCGAGGAG

ACGCTCCGCCTCGCGCCACCGGGCGACCGGACTCTGCTGCGCCGTACCAC GGAGCCGGTG

GAGCTGGGCGGGCGGACACTCCCCGCGGGCGCGTTGGTGATCCCGTCGA TAGCGGCGGCG AACCGCGACCCGGACCGGCCCGTCGGCCGGCGCATGCCCCGGCATCTGG CGTTCGGCCGA GGCGCGCACGCGTGCCTCGGGATGGCGCTCGCCAGGATGGAGCTGCAGG CGGCGCTCAAG

GCGCTGGCCGAGCACGCCCCCGACGTGCGGCTTCCGGCCGGGACGGGCG CCCTCGTGCGA

ACACATGAAGAATTGTCGGTGAGCCCTCTTGCGGGAATCCCGATTCAGAG GTAAGAGCGA

AAAGTCGAGAATACGGTTTTGGTCAGCGGGTTCGGATTCCTTGCGATGTC CGTAACGCGA

CAATAGCCTCGAATCAGCGAGCAGAAAGACTAGCGCTGTAGGGATTCGA GGGAGAACCGT GTCGGCAGAACACCCTTACCACGACAGGCTGCGCGCCTTGTTCGCGTATC TTCGGAAAGT

CGACAGCGACCCGGCCGTGGCGTCCGAGCTCCAGGAGGACCCGGAGAAA GCCCTGCGTGC

AGCAGGTGTGGATCAGGCATTCGACCGTCCGGAAGCCTTTCAGGCTTTCG TCGGGAAACT

GTCGGCGCTGAGCGGTGAAGCGTGGCTCGCGACTGTTCACTCGATGATTG AATTATGTGA

GAACGGCTCCGACGTGCAGCCGCCGACGGGGCCGAATATCTCATTCCGG CTCTCCGGGGA CGGCAGTGTCACCGCTATCGCCAACCGCGGCGAAGTCGCCAAGAAGGTG CAGCCCAATCC

GTTCTACACGAGTGGGAAGAGCGCGTCGGCGGGCAGCCGGCTGCGCATT TACCCCGAGTA

CGCGACCAGCGAGCTGTCGGCGCGGCTGAGCGAGCGTTATCTCTCGACGT TCTACCAGCG

CACCCTGCTCAAGCGCGTCGTTCTCGACCCGGCCACCGTCGTCGAGGACG CGGACGCCGG TGAGGGCGTCACCGTGAGCCGGTCGCACTACAAGGGCGTCGAGTTCGAC CTGCACACCAG GGCCGAGGGCGCGGACCGCGAGATCATCGCCGCGTTCGTGCGCTGATCA TCGGCACAACG

ACCACGACATCACATCCACGGAGGCTCGGGATGCTCAGCAGCGCCCTGG AAGTAGACATC

GACGAGGCGGCCGTCGCCGCCGACCTCAGAGAACTGGCCGCGGCACTCG ACCGCAGCGGC

TACGGAGAGATCCTGACGTGCTTCCTGCCGCAGAAAGCGCAGGCCCACA TCTGGGCGCAG

ACCGCGGCGAAGATCGATGGTCCGCTGCGGACCCTGATGGAGCTCTTCCT GCTCGGCCGC GCGGTGCCGCAGGACGACCTCCCGCCCCGGATCGCCGCGGTGATCCCCG GCCTCGTCTCC

GCGGGGCTGGTCAAGACGGGGCAGGGCGCCGTGTGGCTGCCCAACCTCA TCCTGCTCCGG

CCGATGGGCCAGTGGCTGTGGTGCCAGCGCCCGCACCCCTCGCCCACCAT GTACTTCGGC

GACGACTCGCTCGCGCTGGTGCACCGCATGGTGACCTACCGCGGCGGCC GCGCCCTCGAC

CTGTGCGCGGGGCCGGGCGTGCAGGCGCTGACGGCGGCGCTGCGCAGCG AGCACGTCACG GCGGTCGAGATCAACCCGGTGGCGGCGGCGCTGTGCCGTACCAACATCG CCATGAACGGA

CTCTCGGACCGGATGGAGGTCCGCCTCGGCAGCCTCTACGACGTCGTGCG GGGCGAGGTC

TTCGACGACATCGTGTCGAACCCGCCGCTGCTGCCGGTCCCCGAGGACGT GCAGTTCGCG

TTCGTCGGCGACGGCGGCCGTGACGGCTTCGACATCTCCTGGACCATCCT CGACGGCCTG CCCGAGCATCTGTCCGACCGCGGAGCATGCCGCATCGTCGGCTGCGTGCT GAGCGACGGC TACGTGCCCGTCGTGATGGAAGGCCTCGGCGAATGGGCGGCGAAGCACG ACTTCGACGTG

CTGCTGACCGTCACCGCCCATGTCGAGGCGCACAAGGACTCCTCCTTCCT GCGCAGCATG

TCGCTGATGTCGAGCGCGATCTCCGGCAGGCCCGCCGAGGAACTGCAGG AGCGCTACGCC

GCGGACTACGCCGAGCTCGGCGGCTCCCACGTGGCGTTCTACGAGCTGTG CGCGCGGCGC

GGCGGCGGCTCGGCCCGGCTCGCGGACGTCTCCGCCACCAAGCGCTCCG CCGAGGTCTGG TTCGTCTGACCTGCCACCGACCGGACGGGATCGCTATGTCGACGACACTT CGCGGAACAG

GCGGCGCAGTCGCCGAGCAGCCCTTGCTGATCTACGTGAACGTTCCGTTC TGCAACTCCA

AATGCCACTTCTGCGACTGGGTCACCGAGGTGCCCCTTGCGGATCTGCGG CTCACGCCGG

ACTCCTCGCCGCGGCAGCGTTACATCGCCGCGCTGGTGCAGCAGATCGAG ACCCACGCCC

CGGTCCTGACCGGCTTCGGCTACCGGCCGGAGATCATGTACTGGGGTGGC GGCACGGCCA GCATCCTCTCGATCGACGAGATCGAGGCGGTGGCCGGCGCGCTGTCGTCG CGGTTCGACA

TGAGCGGACTCACCGAGGCCACCATCGAGGGCAGTCCGGAATCCCTGGA CCCGGACAAAC

TGAAGCTGTTCCGCGCGGCCGGGTTCAACCGGATCAGCATCGGCGTCCAG GCGTTCGATG

ACGCCCGCCTGCGTCGCATCGGCCGCGTCCACTCCGCGGAACAGGCGGT GCGCGCGGTCG AGATGGCCGCCGAGGCGGGGTTCGACAACATCAACATCGACCTGATCGT CGGATTCCCCG GCCAGGAGGTCGACGAGGTGTCCCACATGATCCAGCGGGCCGTCACCCT GCCGGTCAACC

ACTTCTCCGTGTACCCGTACCGTCCGACGAACGGGACGGTGATGCGCAAA CAGGTACGGC

GCGGCAACAGCGAGATCGATGTGGACGAGCAGCTGCGGTCCTACGCCTA CGCCCGTGACC

TGCTCGCCGAACACGGATTCGACGAGTACGCCACCGCCTACTTCGGCGGC CCCCGGTGCG

AGTCCGACGAGGTCTACTACAAGCTCACCATGGACTGGATCGGATTCGGC TCCGGTGCGA ACTCCCTGATCGGGACGCGGTTCCTGCTGAACGAGCGGGGGGCCCTCCAC CGGTTCAGCG

CGGCGCCGCAGCGGTTCGACTCCGACATCCCGGCGTCCTCACCCCACCTG ACACGCCATT

TCCTGGCGCAGGCGCTGACCACCGTGGACGGCATGGACGCGCGGACGTT CCAGCAGCGCA

CCGGCCGTTCGCTGCGGGCGGCGTGCGAGGAGCCCGCCGTACGGCGGAT GCTCGAGCAGA

TCAACCGGCGTGGCCGGCTGATCATCGACTCGCGCGGCATCCGTCTGCAC CGCGACGACA TGGCCTCGACCTACATCACGATGAACAGCGTCGACCTGTACGCCGCGACC GAGCAGATCG

GCGGATGACCACGGGTCCTCCCGCCGATCCCGCTCCACAGTCTTCGCGAT CCCGTGTGTC

CGTCCCATGGGGGGACGGGCGTGCCGGTCGACCGCCATGGGGTCGTGGA GGAACGACAAG

ACAAGGAAAGGAGATGAGCGAGATGGAGTTGAACCTTAACGACCTGCCC ATGGACGTCTT CGAGATGGCCGACAGCGGCATGGAAGTCGAGTCACTCACCGCGGGACAC GGGATGCCCGA GGTCGGTGCCTCGTGCAACTGCGTGTGCGGCTTCTGCTGTTCCTGCAGCC CGTCCGCGTG

ACGATCAAGGAGTGCCGCCGGCCTCGGCCGGCGGCACTCCGCCTGCGAG GGGGAGACGAT

GCGTGTGGAGAAAGGCCACGAGATCGTGGTCCGGGTCGCGGGGGTGCCC GCGGCCGTTCT

CGGCGGACTGCGCCTGCCGCACTCCGCCGCACTCGTCACGCATCTCATCG CGGAGGACCA

CCGGCTGACGGCCGAGGCCGCCGTGCTCTCCGACGACCTCTTCGAGCTGA TCGGCGACGC CGGACCCGCGCGCGCCGCGCTGGTCGGCCTGCGACGCGCGCTCGCCCCC GGACGTCGCCG

CCCCTCCGCGCGACTGATCGAACGGTGCCCCCTGCCGGAGCCCCTGGCCG GCCGGATCAC

CGCGTGGGCGCGGGCACGGGCGGAATGGGACGGACGGCGCCGCGAACTC GACGACCTGCT

GACGAAGGAACGCGCCGACGTGCTCGGCCACGTACGCGCGGCCTGCGCC GACCCCGCGTT

CCGGCGGGGCCTGTTCCTGTCCGGCGCCGAACTGTCCGGCACGCTCGACC GCTGGCTGGC CGACCCGGACCGCCCGCCCCGGCCCGGAAAGGTGCTCAGACTCGTC AAA TACCTCGCGCG

CGCCTCGGCCAAGACCAGCCCGTTCGGCTCCTTCATGGTGAGCGCGCTCA CCGGCTGGAG

CGACGACGGCATGGGCGCACCGGAGTCCGTCGAGGTGACCGAGCCGTCC GGCGCGTTCCT

CGACGCCGTCCGTGACGCGTTGCTCGCCGACCCGCGGCTGGCTGACCGGG TGCCGTTGCG TCCCAACCCGAGCCTGACCGAGGCCGGCGACGCGCTGATGTTCGTGTCCG GGCAGACCGG ¹ GGAGCGGATCGCCACGGTCGGGCGGGTGCCCGCGGTCGAGCTCTGCCTG CGGCACGCCGA

GTCCCGGCCCACCGCGCCCCGGCTGGCCGGGCTGCTGACCGAGGCGGGT GCCGACCCCGG

CGAGGCCGCCCGGTTCGTCTCCCGGATGGTGACGGCGCAGCTGCTGGTGC CGTGCCCGCC

GGTCACCGACGACGATCACGACCCGTTCGGCGCCTGGGCCCGCTGGGCG GACCTTCCCGA

ACTGCGCGAGCTGTCGGCCGCGTTGCGTCCCGTACGGCTCGGACGGCATG GCCGCTCCGG ACCCTCCGGACCGTCCGGCCCGGACGCCCACCGGCGGCGCCGGGAGAGG ACCGCCGCCGC

GCTCACCGCCGTGGCGGAACGTCTCGGCATCGACCCGCCCGCCGAACCC GCGCACGAGGT

CGAGGTCGGCGTCGGCAGACCGGCGCCGCCGGCGCTGCCCGACGACGTG CTCGCCGATCT

GGACGCCGTCCGCCGATGGCTGTCGGTGTTCGACTGGAAGATCCCCGTCC GGGTGAGCGT

GGGCGCGTACTGCCGGGAGCACTTCGGCCCCGGTTCCCGGACACCGTTCC TGCTGGTGTG CCGGCAGGCCACGGCGGCCTTGCCCCACTTGTTCGGACCGGCCGCGATGC CGTGGTTCTC

CGACCTGACCGGCAGCCCACGCCTGCGCGAGCTGGACCGCCTCCGGGAG CGGGCCCGCGG

GCTGGCCCGCTCGGGCGCTCTCGACCGCCGGGAGGTCCTGGACGACACC GCCGACTGGCC

CGCGTGGCTGACCTCACCCGCCTCCGTCGGCTTCTACCTGCAGCTCCTTCC CGGAGAGCC CGGGAAGGTCGTGCTGAACGCCGTCCACGCCGGACACGGCCGGGCGTCC GGACGCCTCCA CCACCTCCTCGGCCGGGCGGGCGCCGCACCGGAGCGCCCCGCGCGGCCG GGCCCGCCCCT

GGCCGAGATCGGCGGGAGGTTCGGTTCGGCGCTCAACACCCGCACGCCG AGCACGCTCCT

GGAGATCGACCATCCCGGCGCGGCCTCCGGCCGCGACCCCGGCCATCGG ATCCGTCTCGG

CGAGCTCATGGTCGTCCACGATCCGGAGACCGATCTGGCCTTCCTGCACA GCGAGCGGTT

CGGGCGGATCGAACCGGTGCACCTCGGCATGATGGGCGAGCTGGCGCTG CCCGCCGTGGC CGGGTTCCTCGAACGCGCCTTCGCGCCCACCTACCTGTTCCACCCGAGCG TCCCCCCGCT

GATCTCCCTGCGAGACCTCGCCGGAACCGCCGCGACCCGGCGGTTCCCCC GCGTGTCCGT

GGGCGGCGTCATCGTGCAGCGTGCCCGCTGGACCGTGCCCGCCGACCGG GTGCCCGCCCG

TTCGGGACCGGACGCGGACCATTCGGGATCGGACGGAGACCATCTGCTC GCCCTCGCCCG

GTGGCGGCACGCCGAAGGCATCCCCGAACGGTGCTTCGTCCGCGGCTGG AGCCCCGGCGC CGCGTTCGGCAAGGCCCGCAAACCCCTGTACGTCGATTTCGCCTCCTGGC ACCTGACGAC

CCTGTTCGAGCGGGAGGCACGCTCGAACGCCGCCGTCGTCATCGACGAG GCCCTGCCGGA

CCCCCTGGCCGAAGGCGCTCCGGCCCACGTGACCGAGTACCACATCGAG ATCGACACCGG

AGGGACGGCTGATGCCTGACCAGCCGACCTGGCTCGGAGCGCACCTGCA CTACCGTGGCG ACCTCGACATGATGTTGCGGGACGCGGTGGCGCCGCTGGTGCGCGCGCTC GGCACCGACT TCTTCTTCCTGCGCTACTGGGACGGAGGCAGCCACGTGCGGCTGCGGCTG CGCGGAGCCG

ACGAGGCCGTCGTCGCCGACCACATGAACGCGTACTTCGCCGCGCATCCC GCACCGGAGA

CCATGACCCAGCAGGAGTACGCCCGGGTCGCCGCGGTGCTGGCCGACCG CGAGGGCATGA

CGCACCACCTCACATCGCTCCGGCCGAACAACTCCGTGGAGTTCGTGCCC TACCGGCCCG

AGACCGGCAAATACGGCACCGGGGAGACGTTACGCGCCGTCGAGCGGCA CTTCGTCGAAT CCAGCCGGTCCGCGCTGGACATCATCGGCCGCTCACCGACCGGCAACCA GCGGGAACTCG

CCGTGCTGGGCATCCTGCTGCTGGCCTGGTACGCCGCCGGGGTGCCGGAG GAACGGCTGC

CCGGCGCGGCCGAGACGTTGTGCCGGGGCTGGCGCGGTGGTCACGACCT GCCCGAGGACC

GGGTGGAACGAGAGTTCGCCGGCGTGCGGGAGCGGGTCGTGCGACTGGC CGGCTCGCTGC

GCGGCCTCGAACCGAGGCCGGATCATCCCGGCACCAGCCTGCACGCGTG GGCGGCGACGT TCGCGCGGCTCGGCGCCGCGCTGGCCGGACCGGACCGGCTGCGGGTGCT CGACAACTGCG

CTCACCTGGCGGCCAACCGGCTGGGCGTGTCCATGGCGGCCGAGGTGCG CCTGCGGCTGC

TCGCCGTACGGGCGTTGCGTGAGACGGCTTCCCCGAGACCGTTTCGGTGC GGAGGTGACG

GTGACCTGGCGACGCTTTGACGTGGCCTACCACGACCCGGATCTCGACCG GCTGATCCTG GCGGCGCGGCCGCTGCTCTCGGAGAGCCCCGGCCGCGGCTGGTTCCAGC GGCACTGGGTA CGCGGCCCGCATCTGGAGCTCTGGTTCGACCACCCGGAACCCTCGTGGGA GCGGGTGCGG

GAGGTGCTGGGCACCCACCTGCGCGCCCACCCGTCCCGCACCGGGATCG ACCCGGACCGG

CTGCTGCCCCAGCACCGCCGCCTGGCCCTCGCCGAGCAGATCGACGAGCC GCTGCTGCCC

TTCTACGACGACAACACGCTGCACCGGGCCGTGCCGCGGTCCCGCGTGCA CGTGCTGGGC

AGCGCGGCGGCCGAGGATCTGTTCCACGACTTCCACGCCGCCGCCAGCA CCGCGGCGTTC GACCAGCTCGACGCGGTGGTGGCGGGGGAGTCCCGGCTGGGGCTGGCGT TCGAGCTGATG

ATCGCGGCGGCGCACGCCCACGCCGAGGGCGGCATCACCGGCGGGTTCG TGTCGTTCCGC

TCACACGCCGAGGCGTTCCTGGCCGGCGCCGCCGGCCTGCGCGAGCGCT GGGAGGCCGAG

TACCGCACCCGCGCGGAGGCGTTGCGCGCGCAGGTCGCCGCCGTGGTCA CCGGGACCCCG

CGCGGCCGGGCCTGGACCGGGTTGCTcGACGGGTTCGCCGGCCGCGGCGA CGAGTTGaTC GCGTCCGGCgCCCTGACgGTCGAgCCCGCCTCACCGACCgCGGCCGCCGAG cCGGACACC

GAGTTCCACCGGGCGTTACGCGCCAACCGGACCTgGCACGACGAGGTGCT GCGGTCACCG

TCGTTCCGCCGGTACCGGCTGCTGCTCAACCTGACGTACCTGCAGATGTC CCGGCTGGGG

GTGACCGCCGTGCAGCGGTCGCTGCTGTGCCACTTCGCCGCCTCGGCGGT CGAGGAGGAG TACGGGGTGTCCGCCATCGAGATCGCCGTAGGAGGCATGTGATGTCAGT ACAGGCTGATT CGGTCGTGGCGCACCGGTGGGCGCTGCGGTCCGGCGTCTACCGCGCCACC GCGGCCAACg

GCGACCTCATGCTCGCGGCCTGGCCGCACACCGCGATGCTCGGGCATGCG

TcCCCGCAGC

TGCTGGCGTTGCTGGACGCGCTGGCCGAAGGaCCGGTGCCGGTCGACGAG CCCGGCATGT

CGGcCACGCTGGACCgGTTGCGCGCGGGCGGCTGGCTCTcCAgGACGGTCT CCTGCGCGG

GACGCGACCTCTACACGGTCAcTcCGCTCGCCGCGCCCACCGAGGCTCCG GCGCCCGCgG GGGAGCTGAgGcTGTCCCGctTCgCgGTgCtgCgGAACAccCCgGAggGGCTgGT GCTGG

AgATGcCCGGCTCGTgGTgCGAcAtCCGCGTGcACGACCCCGCGGTGGCCGC GCTGCTCG

CCGACCCGTCCGGTgACGCGGGGCTGcccGCCGACGCCGCCgcggcGGTgCG CGCCGATC

TggTCGCCGCCGGgATGCTGgTGgcGGAGGAGgAGGAGCGGgAGCCGTTCG AACGgcGGC

AGTGgAGCACgcACGAaCtGTGGTtCCACGAACGCAGCAGgCtCGGCAACCG CGgcTGGT TCGgCGGCGCccAcTTCGGCGGcAccTTCTGGGcGCGcGGCGTGCACGagCCC CCGCCcG

CgCGGCCGTCGCCCTACCCCGGCGAGGCcGTCCCCcTCGCACGGCcGgATC TCGCGACGC

TCCGCCGCACCGAtCCGACGCTCACGACCGTGCTGGAGGACCGGGAGAGC GTCCGCGACC

ACGACGACGACGCCCCGATCACCGCCGAGCAGCTGGGTGAGTTCCTCTA CCGCTGCGCAC GGGTGCGCCTGCTCCGCACCATCGAGGGGTTCGAGTACTCCAGCAAGCCC TATCCCGGCG GCGGGTCCGCGTACGAGCTGGAGGTGTACCCGATCGTCCGGCTGGCCGC GGACCTGACGG

CGGGCATGTACCACTACGACGCCCATGACCACCTGCTCcGCCCCGTCCAG CCGCTGGGCC

ACCCGTCGGTGCGCAGGTTGCTGAAGGTCGCCACCGAGTCGTCCGTGACG AAGGCGCCGC

CCCAGGTGCTGCTCGTGATCAGCGCACGGGTCGGCCGCATCATGTGGAA GTACGAGGCCA

TGGGCTACGCCCTGATGCTCAAGCACGTCGGCGTGCTGCAGCAGACGAT GTACGCGGTCG CGACCGCCATGGGCCTGGCCCCCTGTGCGCTGGGCAGCGGCGACGACCT GGCGTTCACCG

GCGCCACCGACCGGGACCGGCTCACCGAGTGCGCGGTCGGGgAATTCAT GATCGGCAGCC

GTAGGAAGGAGCTCGCGACATGGCAGCTCTGAACGTCCTGCTGcGCCCCG ACGCGTACTA

CGCCGAGGTCGACGGCGGCGTCTACTTCATCAGCCACCAGGGCGAGACG TTCATCGCCGG

TCCTACGGTGCACCAGTGGCTCGACCGCcTCGCGCCGCTGCTCGACGGCA CCCGCACCCT CGACCGGCTCACcGCGGGCCTGCCcGCCGACCGGGCCGCGTtCGTCACCAA ACTCGTCGG

CGTCCTCGCcGAGCGCGgCCTGgTGcGCATGGTCGgCCCcGGcACGCCGGAC ACGCTCAC

CGACGCCGAACGCCACGAgTACCGCGGACTGCTGTCCTATCTCGGCTACT TCAGCGACTC

ACCCGGCcACGTCCTCGAAAACGTCCTCGACACCCCGACCGTGATCATCG GATCGGACCc GCTGGCGGCCGAGCTGAGCCGCGCGTGCGCCGCCGCGGGACTGCGGAAG GTCGAGATGGC CGACgAGGTGGGCACCGCCCAGGTCGTGGTACACGTGGCCGACCGCGCC GAACCGGAGCG

AGCCGTACGGCTGGAGCGGCTCTGCGCCGCGTCCGGAGCCCTGCTGGCG CAGGTCATGCC

CGTGGCGGACGGCATCTGGTGGCAGCCcGCGGCCcGGGgCGGTGGCTTGG CGAGCGCCTG

GCGCAGGCACCGGGCACTGGCAGgTCCGGTCCCGGCGGCCGGCCCTCCG GACCCGGTGgC

GGCCAAGGTGGTGgCGAACCAGGTGGCGCACGACGTGTTCcGgCTCCTGA CcGGTCTGCG CGAGGAGACcGCGCCccGGCTGGTGGTGTTCGACCCGCGGACCATGGCGA GcACCACGCA

TCCGATCATTCCGCACCcGCTCGACCTGCCCGCCGAACCGCTGGACGAGG CCGCGTTCCT

CGACCGGATCACCTCGCTGCGCACCGCCCCCGCGGTGAGCGAGGAGGAG TTCTCCCGGCG

GGCCAAGGGGCTGATGGACGGGAGAACCGGACTGTTCGCCGAGATCGAC GAGGGCGATCT

CGCGCAGTTGCCGCTGCACGTCACGGCGACCACGGTGTCCGACCcGTGCG GGAGACTGGG CGACGCGCCCAGGCCGGTGGTCACCGgCGCCGGTTTCACCTTCGAGGAGG CCCGCTACCG

GGCGGCGCTCGCGGCCcTGGCGCGCGCCGGCACGCTGACGCTCGACGAA CGGCGgCTgAT

CGACGGCCACGTGCACGCCTACGACCTGGTGgACCACACGGCACGCCTGG TGCCGGCGGA

GACCGTCTTCGGCGCGTCGCGTGGTGCCGCGGCGGCGTACTcATGGGACG AGGCGGTCGC CGCGGGCCTGACCGCCCACGCGGCCGCGCTCACCCTCGACGGGATCGCG CACGTCACCGA GCCGTTCGGGCGGGTGGACCTGACGGAGGCGCCGGAGTACTGCCTGGCG ATGATCCGCGC

GCTCGGCGAGAAGCCGGCCGTGTACGACGTCACCGGACcGCTGGGTgCGC CcACGGTGGT

CGGgACGCTCTCCGGCGGGGCCACCGCGTGCGGTGCGGCCCTGACGATCG AGGCCGCCGT

GGCCGCATGCCTCCGCGACCTGCTGCTGgTCCGCCAGGCCGAGATCAACG ATCAGCCGGT

CTACGCCCCGGCGCCGTGCGCGCCCCCGTCCccGGCGCTTcAGGGGGATCA CCTGGCGCC CCGGCGgCcGGgCACCGATGTGCCCACGCTCGTGGCGAGAcTGGCCGAGC AGGgCCGCCG

TCcGCTGGCGgTGCCGCTCGACCACGACCCGGCcGTGCACGCGgCCCTgCCc TtCGTCGT

CcAGGTGGTgTGCTGATGATCcGTGTGCCGGACAGCGGCGAACTGACCGT CGCGCTGGAG

AAAGGCCCGAGGCTCCCGGTCCGGACGGAACTGGGCCGGATCACCGTCG GCCcGCTGGAA

CGgCCGGGCGTGCCGGGGTGCCGGGAATGCGTGCGCGTCCGGGAGAGGC GCGTCCAGCCC GACGCCCGCAAGGCCGAAGCCGTGCGCGCCCTGCACACCCCGGCAGCGA GTCAGTGGCTC

ACCCCGCTCGCCACCGACCTGGTCCACACCCTGGTCGCGAGCGAGGCCGC CGCGCTGGCG

GCCGACGCGGAGCCCCGGACCGTGAACGCCGTGCTGGAGATCGACCTGA TGACCCTGGAG

ATCACCCGGCACCGGTTCCTGCCGGATCCGCTGTGCGCGCACTGCGGGGA CCTGCCGCCG GACGCCCCGGCGGAGCTCACCCTCCGGTCCCGGCGCAAGCTCGGCGGCA GCCCGCGGACC AGGGAGATCGAGCTCGACGCGCTCTTGGAGACCTACGTGGACGGCCGGA CCGGCATGATC

CGCCCGCTGAAGACCGGCGTGCAGGGCGGTCTCACGGTGGCGAGCGCGA TGCTGCCGATC

CGCGCGGGCAACGGCCTCGAACCGGGCGTCGGCCGCACCCGGAACTACC GCGCCAGCAAG

CTCACCGCCGTGCTGGAGGCGCTGGAACGCTACGGCGGCGTGAGTCCCG GGGGCCGCCGC

ACGGTGATCAGGTCCGCCTACCGCGACATCGCCGGTGACGCCGTCCACCC CGATGGGTTC GGCACGCATCCGGAGGAGAACTACGACCGTCCCGACTTCGAGTTCCGCC GGTTCACCGAG

GACACGGTGTGCCGATGGGTGTGGGGCTACTCGTTCGCGCAGTCGCGGCC CGTCCTGGTG

CCGGAGAATCAGGTCTACTACTACGCCCGGCACATGCCCGACGGCGAGA AGCCGTTCGTC

TTCGAGGTCTCCAACGGATGCGCGCTCGGCTCGTGTCTGGAGGAGGCGAT CCTCCACGGC

CTGCTGGAGGTCGTCGAACGCGACGCGTTCCTGCTCACCTGGCATGCCCG GCGGCAGGTC CcGGTGCTGGACCCCGCGCTGGCGGCCGACCCGGTGCTGCCGATGCAGGT CGCCGCGATC

ACTGCTGAGACGGGACATCGGGTGCTGTGCTTCGACACCACGGCGGAAC ACGGCATCCCG

AGCGTGTGGGTCATGGCGGTGGACGCCGAACGCCGCCCGgACCAACCCG CCACCGCCCAC

GCCGCCGGCTCGgCGCTCACGCTGGAGAAGGCCACGATGAACGCCCTCA GCGAACTGGGA CCGCTGCTGGCGGATGTGATCCGCCGCTACCCGCAGCAGCGGGAGCGGG CGCAGGCCATG GTCCGCGACCCGGAGGAGGTCGTCACCATGCACGACCACTCGATCCTCTA CGCCGCGCcG

GAGGCGGCGGACCGGCTGGACTTCCTCATCGGCCGCGCCGACGGCCCGA AGGCGGGgTTC

GGCACGGACCGGTTCACCGGTGACGACCTCACCGCGgACCTGCGGGCCAT GATCGATGCG

GTGCTGgCGGCCGGAATGgACGTGGTGGTGGTCGACcAGACcACCCCCGA GCACCTGGCG

GGCGGATTCCGCTGCGTGAAGGTGCTGGTGCCCGGCGCGTTGCCGATGAC GTTCGgCCAT CGGCACCGGCGGCTGGGAAACCTGCCGCGGCTGGAGACCGTACGGACCA CCGATCCGCAC

CCGTTCCCGTGAGCAGGAAGGATCCGCCATGTACGTGGTGATCGTCGCGT TCGACCTGAA

GGACTCCGCCATCGACTTCGCCGAACTGCGCGCCTGGGTGCACGACCGG GCGGCGGACGA

TTACTCCAGGCTGCCGGGGATGCGCTTCAAGGCGTGGTTCTCCGACGAGA GGAAACGGCT

GTGGGGCGCGGTCTACCTGGTGGAGTCGATGTCGTCGTTCGACCGGGACA AGATCCCGCT GCTGCCCGACGGAGCCACCGgACCGGTCGGCACCCGGCCGACCTCGATCA TGTTCCTGGA

ACTTGAGGCGTTCGTCGCCGGACCCGACGGCATCGCCGGGATCGAAGCG CTCACCCGGCA

GGGGCTGAGCATGGTGGGAGGCAGTCATGACCACTGAAGCGATCACAAA CGCACTTCCGC

TGACCGGGCCGAAGACGGACGAGCCGTTGCTGCTCTACGTCAACGTCCC GTTCTGCAACT CCAAATGCCACTTCTGCGACTGGGTGGTGGACGTCCcGGTCTCCGACCTG CGGCTCACTC CCGTGTCAGCGGGCCGGATCGACTACCTGGAGGCGCTGCGCACCCAGAT CCGCATCCACG

CCCCCGCCcTGCGGGAGGCCGgTTACcGCAGCGAGGTCATGTACTGGGGC GGGGGCACGG

CGACCGTtCTCACCGAACGGGAGATCGAGCAGACCTACGCGTGCCTGGCG GCGGAGCTCG

ACCTGTCCTCCCTGGCCGAGGCGACGATCGAAGGCAGTCCCGAGTCGGT GGACCTCGCCA

AGCTGACGTTCATGCGCGACCTCGGCTTCGACCGGGTGAGCCTCGGCGTG CAGTCGTTCG ACGAGACGAGGCTGCGCCGCATCGGCCGCGCGCACTCcGCGGgCCAGGCC GTGCAGAGCG

TCGAGGCCGCGCACGCGGCGGGGTTCGACAACATCAACATCGATCTGAT CGTCGGTTTCC

CGGACCAGTCACTGGAGGAGGTCGAGGAGATGATCCGGCGGGCGCTCGA CCTGCCGGTGA

ACCACTTCTCGGTCTACTCGTACCGGGCGACCGAGGGCACGGTGATGCGC AAGCAGATCG

AGCGCAGCGGTACCGAGATCCTGCTGGAGCATCAGCTGCGGTCGTACCG GCTCGCCGCGG ACATGCTGGCCGCGGCGGGgCACCCcGAGTACGCGGTCTCGTACTTCGGC GTGCCGAGGT

GCCTCGCAGACGAGGCGTACTACCGGCTCAGCATGGACTGGATCGGCTTC GGGACGGGCG

CCAACTCGCTGATCAATCAGCGCTACCTGCTGAACGGCCGCGGCCGGATG CGGGACTTCA

CCAGCAAGCCGGGGGAGTTCGAGGTGAACCTGCCGGCCGCCACCGGATC GCTCACCGTGC AATGGCTGCCCAGGgCGCTGGGCACCTCGGAGGGGATCGACGCGgTCACC TTCCAGCGCC GCACCGGCATGTCGTTACGCGCGGCGTGCGAGGAGCCCGACCTGAACGC GTtCCTGAGGC

AGGTCAACAGGTTCGGCGATCTGGTGGTGGACCGCACCGGCATCCGCCTC GCGGACGACG

ACCGGTCGAGCGTGCTGTCCCGTACGTTCGCCGCGATGGGCTGGGTGTCC TGAGTGGAGG

CGATGCGCACGGTGCTCGCCGCCGCGGACCTGGCGACGCCCATGGCCGT CCGGGTCGCGG

CGACGCTGAGGCTGGCCGACCGCATCGCGGCCGGAACGGATTCCGCGGC CGCACTCGCCG CGGCCGCGGGGGCCGATGAGGCGGCACTGACGCGGTTGCTGCGCTACCT GGTCGCGCGGG

ACATCTTCCGCGAACCGTCACCGGGGCGTTtCGCCCTGAGCCCGGCCGCG GAGCTGCTGC

GCGGCGACCGCCCGGAGCGGTTGCGCGACTGGCTGGACCTCACCGGCCcG ATCGGCCGCG

CGGACCTGGCGTTCGGgTCGCTGCTGGACGTCGTGCTCACCGGCAGGCCC GGATACCCGA

TGATCCATGGACGGGGCTTCTGGGACGACCTCGCCGGCCGGCCCGAGCTC GCCGCcGCGT ACGACGCCCTGATGGGCGGCAAGCGCGACTGGgCGGCGACCACCCTCGC CGCGCTCGACT

GGAGCCGTTCCCGGCATGTGGTGGACGTCGGCGGCGGCAACGGCACCCT GCTGTCGTGTG

TCCTGgCCGCGCATCGGCATCTGCGCGGCACTGTCGTGgACCGgCCCtCGT CCGCCGAGG

CcGCCGGGGCGGTGCTGGCGTCGGCGGgCGTTGCGGACCGCTGCGAGTTC CGGgCGGGGg ACTTCTTcGAACCGTTGCCGGTGAAGGGTGCCGACACCTATCTGCTGAGT TCGATCGTGC ACGACTGGGACGACTCCTCGgCGGTCGCGATCCTGCGCcGCTGCGCGGAG GCCGCCGGGC

CGGGCGGCCGGATACTGCTGTgTGAGCTGATGGCCGGAGGCGGgCGGGAC CAGCGGGCGG

TGACACAGATGGACCTGTGCATGCTCGTGTACTTCGGCGGgCGGGAACGC ACCGGGgACG

AGTTCGCCGCGCTGGCCGAGCGTGCCGGACTGGAACTGCGGACGGTGAC GCCGCTGCCGC

CGCATGACTGGGGCAACTCGCTCATCGAGTGCGTGGTCACCGGGGGCTG AGGCCTGCCCG GCGGCTCCTTCCGGGGTTCGCGACGGGGGGTCGGCGGGCCcGGGGACGTC TCGGGGGATA

TACTTCTCGTCGAGCCCGATGCGGACGGCGTCACCCCCTCGAAGGGGGCG TCTCTCTCCG