MX2008001798A - Differentially protected orthogonal lanthionine technology - Google Patents

Abstract

The present invention provides a method of synthesizing an intramolecularly bridged polypeptide comprising at least one intramolecular bridge. The present invention further provides a method of synthesizing an intramolecularly bridged polypeptide comprising two intramolecular bridges, wherein the two intramolecular bridges form two overlapping ring, two rings in series, or two embedded rings. The present invention also provides methods for synthesizing lantibiotics, including Nisin A. Additionally, the invention provides intramolecularly bridged polypeptides synthesized by the methods disclosed herein and differentially protected orthogonal lanthionines.

Description

ORTHOGONAL LANTIONIN TECHNOLOGY DIFEKENTIALLY PROTECTED BACKGROUND OF THE INVENTION The development of antibiotics developed the practice of medicine in the second half of the 20th century. Mortality due to infectious diseases decreased markedly during this period. Armstrong et al., (1999) PAMA. 281, 61-66. Since 1982, however, deaths from infectious diseases have increased continuously in parallel with the increase in antibiotic-resistant pathogens commonly used in the treatment of clinical infections. Thousands of reports and books have appeared in literature during the past 20 years documenting this phenomenon. Armstrong et al., (1999) PAMA. 281, 61-66; Dessen et al., (2001) Curr. Drug Targets Infect. Disord. 1, 11-16; Rapp (2000) Surg Infect (Larchmt). 1, 39 -47; Bening & Dowell (2001) Atibiotic resistance. and implications for the appropriate use of antimicrobial agents, Humana Press, Totowa, NJ. While there is a need to teach more appropriate uses of antibiotics, a need for new antibiotics is more important. Vancomycin is considered to be the last line of defense against many serious bacterial infections. The discovery of vancomycin-resistant strains of a pathogenic bacterium is No. Ref. : 189731 alarming; announces the increase of pathogens resistant to multiple drugs that would be intractable with currently available drugs. The concern is that, in effect, it would return to the pre-antibiotic era unless new antibiotics are developed soon. There is a small, structurally new class of antibiotics called lantibiotics (Class I bacteriocins) which can be divided into 5 subclasses based on the differences in their chemical compositions and biosynthesis: Type A (I), Type A (II), Type B , Two-Components and those of unknown structures. This class of antibiotics has been known for decades but has not been extensively tested for its potential utility in the treatment of infectious diseases even though many lantibiotics are known to be both potent and have a broad spectrum of activity, notably against Gram-positive species. The main reason for this is the general difficulty of obtaining these molecules in sufficient quantities, of cost effective to allow their evaluations and commercialization. Nisin A (Figure 1) provides a good example of a lantibiotic, and the number and types of chemical complexity associated with lantibiotics. Lantibiotics are rich in sulfur-containing amino acids, lanthionine (Lan, ala-S-ala) and, frequently, 3-methyl-lanthionine (MeLan, abu-S-ala). Lan consists of alanine residues that are connected by means of thioether bridges to create ring structures that are critical for bioactivity. Typically there are 3-5 such rings in a lantibiotic, and frequently many of the rings overlap one another. The Lan and MeLan are considered to invariably have meso-stereochemistry. In addition to the Lan and MeLan residues, there may be other post-translationally modified amino acids (Figure 2) found in lantibiotics, such as 2, 3-didehydroalanine (Dha), 2,3 didehydrobutyrin (Dhb) derivatives, unsaturated lanthionine derivatives such as S-vinylamino-D-cysteine (AviCys) and S-amino-D-methylcysteine, as well as D-alanine, 2-oxopropionyl, 2-oxobutyryl, and hydroxypropionyl residues. As in the case of Nisin A, the ring structures made of Lan and MeLan can overlap (for example, rings D and E), an additional aggregation to the complexity of the molecule. Gram-positive bacteria are responsible for the biosynthesis of known lantibiotics. They make the molecule mature using a series of sequential enzymatic steps that act on a ribosomally synthesized prepropeptide. The genes responsible for coding the modification enzymes are typically grouped into an 8-10 Kb DNA fragment that can reside on the chromosome, a plasmid, or as a part of transposon. In type A (I) antibiotics, all serine and threonine residues in the synthesized pre-peptide ribosornamente encoded by the lanA gene involved in the formation of thioether bonds with a nearby cysteine residue that is located more towards the carboxyl terminus of the molecule. This reaction is catalyzed by the protein expressed by the lanC gene. In the case of certain lantibiotics, such as epidermin and mutacin 1140, the C-terminal cysteine is decarboxylated by the enzyme expressed by the lanD gene and converted to an A-vinylamino-D-cysteine. After being transported out of the cell by the lanT gene product, the targeting sequence of the modified prepropeptide is then divided by an extracellular protease encoded by lanP to produce a mature antibiotic. Ra et al., (1996) Microbiology-Uk. 142, 1281-1288; Kupke & Gotz (1996) Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology. 69, 139-150; Kuipers et al, (1996) Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology. 69, 161-169. Attempts to study lantibiotics for their potential utilities in therapeutic applications have been impeded by the difficulty of obtaining them in sufficient quantities or with sufficient purity. Of the 40 or lantibiotics so characterized to date (Chatterjee et al., (2005) Chemical Reviews 105, 633 683) only Type A (I) lantibiotic, Nisin A, produced by Streptococcus lactis, has been produced in commercial quantities, and has found a wide application as a food preservative for the past 50 years. The extended and long-term use of Nisin A without the development of significant resistance (Delves Broughton et al, (1996) Antonie Van Leeuenhoek International Journal of General and Molecular Microbiology 69, 193-202) has provided strong impetus for the development of Latibiotics additional for several applications. The large-scale production of Nisina A is carried out with the use of a fermentation process that has been refined over the years. A purification protocol for Nisin A has recently been presented according to a Patent. North American (USPA 2004/0072333). The protocol used a cocktail of expensive proteases followed by column chromatography. However, a commercially viable procedure for the purification of Nisin A has not been published. This demonstrates the current interest in finding a suitable method for producing Nisin A · pure and other lantibiotics for therapeutic applications. Several potential options are present by themselves for the large-scale production of lantibiotics. From the point of view of the cost of materials, the fermentation process would undoubtedly be the best method. The current fermentation methods for many lantibiotics produce quantities of micrograms per liter, which is not sufficient for the development of the drug. Alternatively, in vitro production using lantibiotic modification machinery has been explored in Type A (I) lantibiotics. Kupke & Gotz (1996) Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology. 69, 39-150; Kuipers et al, (1996) Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology. 69, 161-169. Responsible enzymes for post-translational modification of the lantibiotic propeptide are not active in lysates of free cells or as purified entities, with the exception of LanD. Kupke & Gotz (1996) Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology. 69, 139-150; 10; Kupke & Gotz (1997) Journal of Biological Chemistry. 272, 4759-4762; Kupke et al, (1992) Journal of Bacteriology. 174, 5354-5361; Kupke et al, (1993) Fems Microbiology Letters. 112, 43-48; Kupke et al, (1995) Journal of Biological Chemistry. 270 ,. 11282-11289; Kupke et al, (1994) Journal of Biological Chemistry. 269, 5653-5659. In the case of Type A (II) lantibiotics, it has recently been reported in Science that in vitro synthesis of lacticin 481 is possible. Molecules belonging to this group and Type B lantibiotics use only a single multifactor enzyme, LanM, to achieve the formation of the Dha, Dhb, Lan, and MeLan residues. Xie et al., (2004) Science. 303, 679-681. The report of the biosynthesis of lacticin 481 did not provide any detailed information regarding the production or purity, if not that his work was performed on the nanogram scale. The progress described in this report represents a small but significant step forward, and its widely acclaimed reception further emphasizes the pressing need to develop lantibiotics as therapeutic agents. A third option for commercial scale production of lantibiotics with the use of the gene pool cloned into an appropriate expression vector (s) and a non-sensitive host is unlikely due to the complexity of the system and the likely need to differentially regulate the expression of several genes involved. The gene group for galidermin has been cloned into Bacilus subtilis in an attempt to improve the production of this particular lantibiotic type. However, this strategy does not result in greatly increased yields and is appropriate for all lantibiotics since regulatory sites of genes are known to vary from species to species. A related approach made use of an artificial gene for mutacin 1140 cloned in Escherichia Coli. This artificial gene replaced the natural codons for the serine and threonine residues involved in thioether bridge formation with cysteine codons. This modified gene was cloned in pET32 and expressed in the Origami strain of E. coli to maximize the disulfide bonds. New chemical methods were developed to extrude a single sulfur atom from the disulfide groups thereby converting them to thioethers. In general, this method proved feasibility, but the yields obtained were low due to the multiple permutations of the disulfide bonds and the difficulty in separating the active form from the non-active isomers. The frequent overlapping ring structures are critical for the bioactivity of Nisin A and other lantibiotics, creating a problem that is difficult to overcome synthetically. Synthetic methods in vi have been extensively investigated for the synthesis of various lanthionines containing bioactive peptides as well as latibiotics. The challenge of synthesizing lantibiotics is arduous and, until now, exhaustive synthetic strategies have been developed. Several methods to synthesize lanthionines have been reported in the literature. These include desulphurisation methods of cystine units in pre-assembled peptides with the use of basic or nucleophilic conditions. Galande et al, (2003) Biopolymers (Peptide Science) 71, 543-551; Galande & Spatola (2001) Letters in Peptide Science. 8, 247-251. The desulfurization methods have not yet shown any commercial viability due to the lack of diastereoselectivity and poor yields. Biomimetic approaches have also been used where D a residues are generated in a preformed peptide followed by a Michael addition to form the lanthionine ring. The organization of the peptide presumably leads to a Michael diastereoselective addition. Burage et al, (2000) Chemistry A European Journal. 6, 1455-1466. Oxime resin cycle formation has also been employed wherein a linear peptide containing an orthogonally protected lanthionine is synthesized followed by cyclization and cleavage of the cyclic peptide product. Melacini et al, (1997), J. Med. Chem. 40, 2252-2258; Osapay et al, (1997) Journal of Medicinal Chemistry. 40, 2441-2251. These methods are promising but lack the ability to produce lantibiotics with thioether rings that overlap. This becomes particularly important when one takes into account that most known lantibiotics contain ring overlaps. Conceptually, there are clear advantages in developing synthetic approaches in vitro, including modifications of solid phase peptide synthesis methods (SPPS), in comparison with biological and biomimetic approaches. First, the composition of the molecules is not limited to the normal configuration of physiological amino acids; it is possible to design analogue amino acids and incorporate them with the use of sufficiently well known solid phase synthesis methods. A parallel synthesis can also be taken to port, thereby dramatically increasing the number of substrate candidates. Because the approach is performed in vitro, many of the problems that arise from in vitro synthesis of bioactive molecules are eliminated. For example, the degradation of products during fermentation would not be a problem, nor would the cytotoxic effects of the bioactive molecule on the producing microorganism be a problem. In order to achieve the goal of an in vitro synthesis, orthogonal lanthionines with potentially appropriate protective groups have been designed for SPPS with the use of different approaches, such as addition of cisha ichael to preformed Dha. Probert et ah, (1996) Tetrahedron Letters. 37, 1101-1104. This method leads to a 1: 1 mixture of diastereomers and, therefore, was shown to have little commercial value. The opening of serine lactone ring with protected cysteines has also been reported but this leads to a mixture of lanthionines and thioesters. The ring opening of aziridines has been investigated but was shown to produce regioisomeric mixtures due to the opening of aziridine in the OI and ß position. Dugave & Menez (1997) Tetrahedron-Asymmetry. 8, 1453-1465; Swali et al., (2002) Tetrahedron. 58, 9101-9109. More recent reports suggest that altering an appropriately protected cyst with a protected β-bromoalanin may result in the synthesis of lanthionines, but this method does not allow the construction of molecules with overlapping rings. Zhu (2 0 0 3) European Journal of Organic Chemistry. 2 0, 4 0 62 - 4 07 2. Because the protected Fmoc / Boc analogs commercially available for SPPS are not sufficient to solve the challenge of synthesizing lantibiotics and other bioactive peptides restricted in form, there is a need in the prior art for peptide synthesis with intramolecular bridges that they create internal ring structures, including multiple ring and overlapping ring structures. In particular, there is a need for methods to synthesize lantibiotics in vitro on a large scale. SUMMARY OF THE INVENTION Accordingly, the present invention provides a method for synthesizing an intramolecular bridged polypeptide comprising at least one intramolecular bridge: a) coupling the free carboxy terminus of a differentially protected orthogonal intramolecular bridge of formula to a solid support or to the free amino terminus of an amino acid or polypeptide optionally linked to a solid support and wherein Ln represents side chains of covalently linked amino acids, wherein D, E, and G are protecting groups, each of which is selectively extracted under different conditions of reaction, and wherein the reaction conditions for the extraction of the protecting group D are different from those of the extraction of the amino protecting group from the amino acids of the remainder of the polypeptide chain; b) extract the protecting group E to form a free amino terminus; c) adding an amino-protected amino acid to the free amino terminus and then deprotecting the amino acid to produce a new free amino terminus; d) optionally repeating c) one or more times; e) extract protecting group G to form a free carboxy terminus; f) coupling the free carboxy terminus of e) with the free amino terminus; g) extract protecting group D to form a free amino terminus; and h) optionally adding an amino-protected amino acid to the free amino terminus and then deprotecting the amino acid to produce a free amino terminus; and i) optionally repeating h) one or more times. The present invention further provides a method for synthesizing an intramolecular bridged polypeptide comprising two overlapping intramolecular bridges comprising: a) covalently binding the free carboxy terminus of a differentially protected first orthogonal intramolecular bridge of formula to a solid support or to the free amino terminus of an amino acid or polypeptide optionally linked to a solid support and wherein Ln represents side chains of covalently linked amino acids, wherein D, E, and G are protecting groups, each of which is selectively extracted under different reaction conditions, and wherein the reaction conditions for the extraction of the protecting group D are different from those of the extraction of the amino protecting group from the amino acids of the remainder of the polypeptide chain; b) extract protecting group E to form a free amino terminus; c) add an amino-protected amino acid to the free amino terminus and then deprotect the amino acid to produce 0 a new free amino terminus; d) optionally repeating c) one or more times; e) covalently linking the free carboxy terminus of a second differentially protected orthogonal intramolecular bridge of formula to the free amino terminus, where Ln is as defined above, where M, Q, and T are protective groups, each of which is selectively extracted under different reaction conditions, and where D and M are extracted only under different conditions, wherein G and T are extracted only under different conditions, wherein the reaction conditions for the extraction of the protective group M are different from those of the extraction of the amino protective group of the amino acids of the rest of the chain of polypeptide; and where E and Q are extracted under conditions different from those with D, and those with which M will be extracted; f) extracting the protecting group Q to form a free amino terminus; g) optionally adding an amino-protected amino acid to the free amino terminus and then deprotecting the amino acid to produce a new amino terminus 1 ibre; h) optionally repeating g) one or more times; i) extracting the protecting group G of the first differentially protected orthogonal intramolecular bridge to form a free carboxy terminus; j) attaching the free carboxy terminus to the free amino terminus; K) extract protecting group D from the first differentially protected orthogonal intramolecular bridge to form a free amino terminus; and 1) optionally adding an amino-protected amino acid to the free amino terminus and then deprotecting the amino acid to produce a new free amino terminus; m) optionally repeating 1) one or more times; n) Extract the protective group T of the second differentially protected orthogonal intramolecular bridge forming a free carboxy terminus; o) coupling the free carboxy terminus to the free amino terminus; p) extract protecting group M from the second differentially protected orthogonal intramolecular bridge to form a free amino terminus; and q) optionally adding an amino-protected amino acid to the free amino terminus and then deprotecting the amino acid to produce a new free amino terminus; and r) optionally repeating q) one or more times. Additionally, the present invention provides methods for synthesizing polypeptides with intramolecular bridges comprising two intramolecular bridges, wherein the two intramolecular bridges form two rings in series or two rings incorporated as defined herein. The present invention further provides methods for synthesizing ibérico ions, including Nisin A. In another aspect, the invention provides polypeptides with intramolecular bridges synthesized by the methods described herein. In a further aspect, the invention provides differentially protected orthogonal lanthionines of formula: wherein D and E are different protecting groups and are, for example, Fmoc, Alloc, or IvDde, and G is a protecting group, for example propargyl ester or benzyl ester. BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows the structure of Nisin A [ID of SEC. No.:l], which includes intramolecular bridges between residues 7 and 10, creating ring E, between residues 9 and 12, creating ring D, between residues 16 and 22, creating ring C, between residues 24 and 27, creating ring B, and between residues 28 and 32, creating ring A. Rings A, B, and C exemplify ring structures in series, and rings D and E exemplify overlapping rings. A synthetic analogue Nisin A is also shown [SEQ. ID. NO.:2]. Figure 2 shows non-limiting examples of post-translationally modified amino acids. Figure 3 shows a retrosynthetic strategy to elaborate differentially protected lanthionines. Figure 4 shows the synthetic strategy for Fmoc-protected cistern. Figure 5 shows the synthetic strategy for a Orthogonally protected lanthionine 1, including the synthesis of propargyl ester of (Alloc) -D- -Bromoalanine. Figure 6 shows the synthetic strategy for an orthogonally protected lanthionine 2, including the synthesis of Benzyl ester of N (ivdDe) -D-p-Bromoalanin.

DETAILED DESCRIPTION OF THE INVENTION The Technology of Differentially Protected Orthogonal Lanthionine (DPOLT) for solid phase synthesis of peptides is described herein. The technology depends on the volumetric manufacture of several orthogonally protected peptide bridges whose protective carboxyl and amino active groups can be differentially extracted. The orthogonally protected peptide bridges can be used in, for example, solid phase peptide synthesis, to prepare conformation-restricted bioactive peptides containing intramolecular bridges that form ring structures. In particular, DPOLT can be used to synthesize polypeptides that contain more than one intramolecular bridge and that have ring structures that overlap. While not limited, DPOLT allows the in vitro production of structurally complex lantibiotics (including those with overlapping ring structures) to be processed in a commercially viable manner. The synthesis of lantibiotic peptides is developed with the use of, for example, routine solid phase peptide synthesis methods which incorporate in the peptide lanthionine analogues whose active carboxyl and amino groups are protected orthogonally with protective groups that can be differentially extracted. This method can provide a constant flow of antibiotics for, for example, therapeutic applications. Abbreviations As used herein, the following abbreviations have the following meanings: - Alloc = allyloxycarbonyl - Boc = t-butoxycarbonyl - DMAP = dimethylaminopyridine - DMF = dimethylformamide - Fmoc = 9-fluorenylmethoxy carbonyl - HMBC = Heteronuclear Correlation of Multiple Links - HMQC = Multiple Quantum Heteronuclear Correlation - HPLC = high performance liquid chromatography ivDde = 1- (4,4-dimethyl-2,6-dioxo-cyclohexylidene) -3-methyl-butyl-LC-MS = liquid chromatography-spectrometry mass - MS = mass spectrometry - NMR = nuclear magnetic resonance spectrometry - NOESY = nuclear overhauser effect spectrometry - TFA = trifluoroacetic acid - TLC = thin layer chromatography - TOCSY = total correlation spectroscopy Polypeptides with Intramolecular Bridges The methods here described can be used to synthesize polypeptides with intramolecular bridges that include, but not limited to, lantibiotics. As used herein, the terms "polypeptide", "protein" and "peptide" refer to polymers composed of chains of amino acid monomers linked by amide bonds. The polypeptides can be formed by a condensation or coupling reaction between the carboxyl group α-carbon of one amino acid and the amino group of another amino acid. The terminal amino acid at one end of the chain (amino terminal) therefore has a free amino group, while the terminal amino acid at the other end of the chain (carboxy terminal) has a free carboxyl group. The intramolecular bridge polypeptides of the invention can be optionally modified or protected with a variety of functional groups or protecting groups, including at the amino and / or carboxy terminus. As used herein, the terms "peptide with intramolecular bridges" or "polypeptide with intramolecular bridges" refer to a peptide chain having at least one intramolecular bridge. The terms "intramolecular bridge", "peptide bridge", "fraction with intramolecular bridge" or "bridge", as used here ", refers to the structure formed when the amino acid residues, contained within a single peptide chain, or prepared to be incorporated into a single peptide chain, are covalently linked to one another by their side chains. Such bond creates an internally crosslinked polypeptide. According to as used herein, the term "añilo" or "ring structure" refers to the cross-linked portion of the polypeptide with intramolecular bridges, in this case the structure that forms the polypeptide chain between, and that includes the two residues of covalently linked amino acids, together with the covalent bond formed by their side chains. The polypeptides with intramolecular bridges of the invention have the general formula: Formula I wherein A is any H or an amino end protecting group; Z is either H or a carboxy-terminus protecting group; Xn is a covalent bond, a single amino acid, or a peptide chain in at least 2 amino acids in length; and Rn is an amino acid residue that forms an intramolecular bridge in its side chain. There may additionally be intramolecular bridges between side chains within a single "X" peptide chain or between amino acids located on different "X" peptide chains. As used herein, the terms "amino end protecting group" and "carboxy terminus protecting group" refer to any chemical moiety with the ability to aggregate to, and optionally be extracted from, a reactive site (an amino group or a carboxy group, respectively, in this case) to allow the manipulation of a chemical entity at different sites of the reactive site. The amino acids of the intramolecular bridging polypeptides of the invention can include the 20 naturally occurring amino acids as well as non-natural amino acids, amino acid analogs, and peptidomimetics. Spatola, (1983) in Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins, Weinstein, ed. , Marcel Dekker, New York, p. 267 All the amino acids used in the present invention can be any of the optical isomers-D or -L. In a preferred embodiment, the intramolecular bridging polypeptides of the invention contain one or more of the following residues, in any combination: 2, 3 -dydhydroalanine (Dha), (Z) -2,3-didehydrobutyrine (Dhb), hydroxypropionyl, 2-oxobutyryl, and 2-oxopropionyl (see Figure 2). It will be appreciated by one of ordinary skill in the art that the intramolecular bridged peptides of the invention may have more than one intramolecular bridge, creating a wide range of possible structures. For example, for a polypeptide with intramolecular bridges containing two intramolecular bridges, the intramolecular bridges may be incorporated in series, or overlapped as shown below.

-N- -X1 R1 X2 R; 3 X3 2 X4 R4 Xs Q - O H Overlapped series AN X1 RI X2 Ri3 X3 R14 X4 R2 X5 / ~ -OZH Incorporated Where two intramolecular bridges are overlapped, it means that one amino acid of the second intramolecular bridge is between, in the main amino acid sequence, the two amino acids of the first bridge intramolecular and the other amino acid of the second intramolecular bridge is either before or both both amino acids of the first intramolecular bridge. Where two intramolecular bridges are in series, it means that both amino acids of the second intramolecular bridge are, in the main amino acid sequence, before both or both amino acids of the first intramolecular bridges. Where the two intramolecular bridges are incorporated, it means that both amino acids of the second intramolecular bridge are found between, in the main amino acid sequence, the two amino acids of the first intramolecular bridge. Where the peptide with intramolecular bridges has three or more intramolecular bridges, a greater number of possible structures can be formed. There may be multiple rings that overlap, for example. In a non-limiting example, a polypeptide with intramolecular bridges can have 5 intramolecular bridges, wherein 2 of the 5 bridges form overlapping ring structures and the remaining 3 bridges are in series with each other and with the rings overlapping. The Nisin A Lantibiotic represents such a structure (see Figure 1). In a preferred embodiment, the intramolecular bridged polypeptides of the invention are lantibiotic peptides. In a more preferred embodiment, the intramolecular bridged polypeptides of the invention are Nisin A and analogs thereof. Differentially Protected Orthogonal Intramolecular Bridges The orthogonally protected intramolecular bridges according to the invention have the following general formula: Formula II wherein L represents covalently bonded amino acid side chains, D and E are hydrogen or an amino-terminal protecting group, and G and J are hydrogen or a carboxy-terminus protecting group. The linkage comprising the side chains of amino acids can be, but is not limited to, a thioether, a disulfide, an amide, or an ether. In a preferred embodiment, the intramolecular bridge comprises a thioether linkage. The incorporation of intramolecular bridges "differentially protected" or "orthogonally protected" in the synthesis of polypeptides provides for the selective extraction of their protecting groups to separate and remove the protecting groups in other portions of the peptide chain, including other intramolecular bridges. In other words, the protective groups of a particular intramolecular bridge are selected so that their segmentation conditions do not compromise the stability of other protective or functional groups in the polypeptide. Cross-reactivity during deprotection of these groups is minimal and can be monitored by standard mass spectrometry techniques. The desired product can be purified without these impurities by standard HPLC or other techniques. Segmentations can be affected in any order of priority selected. Protective groups, and the manner in which they are introduced and extracted are described, for example, in "Protective Groups in Organic Chemistry," Plenum Press, London, N.Y. 1973; and in "Methoden der organischen Chemie," Houben-Weyl, 4th edition, Vol. 15/1, Georg-Thieme-Verlag, Stuttgart 1974; and in Theodora W. Greene, "Protective Groups in Organic Synthesis," John Wiley & Sons, New York 1981. A characteristic of many protecting groups is that they can be easily extracted, in this case, without the existence of undesirable side reactions, for example by solvolysis, reduction, photolysis, by the use of organometallic catalysis such as sodium catalyst. organpalladium and organocobalt, or alternatively under physiological conditions. Numerous protecting groups are known in the prior art. An illustrative, non-limiting list of protecting groups includes methyl, formyl, ethyl, acetyl, t-butyl, anisyl, benzyl, trifluoroacetyl, N-hydroxysuccinimide, t-butoxycarbonyl, benzoyl, 4-methylbenzyl, thioanizyl, thiocresyl, benzyloxymethyl, -nitrophenyl, benzyloxycarbonyl, 2-nitrobenzoyl, 2-nitrophenylsulfenyl, 4-toluenesulfonyl, pentafluorophenyl, diphenylmethyl, 2-chlorobenzyloxycarbonyl, 2,4,5-trichlorophenyl, 2-bromobenzyloxycarbonyl, 9-fluorenylmethyloxycarbonyl, triphenylmethyl, and 2, 2, 5, 7, 8-pentamethyl-chroman-6-sulfonyl. For discussions of several different types of de-amino and -carboxy protecting groups, see, for example, U.S. Patent No. 5, 221, 736 (filed June 22, 1993); U.S. Patent No. 5, 256, 549 (filed October 26, 1993); U.S. Patent No. 5, 049, 656 (filed September 17, 1991); and U.S. Patent No. 5, 521, 184 (filed May 28, 1996). Any combination of protective groups can be used, provided that the protective groups can be selectively extracted during the synthesis of the polypeptides with objective intramolecular bridges. In a preferred embodiment, the amino terminal protecting groups are selected from the group consisting of Fmoc, Alloc, and IvDde. In another preferred embodiment. The terminal carboxy protecting groups are selected from the group consisting of propargyl ester and benzyl ester. In a preferred embodiment, the orthogonally protected intramolecular bridge is an orthogonally protected lanthionine or lanthionine derivative. In a more preferred embodiment, the orthogonally protected intramolecular bridge is amino-terminally and / or carboxy-terminally protected lanthionine (Lan), β-methyllanthionine (MeLan), S - [(Z) -2-Aminovinyl] -D-cysteine (AviCis), or S- [(Z) -2-Aminovinyl] -2-methyl-D-cysteine (see Figure 2). Such orthogonally protected intramolecular bridges can be synthesized by methods known in the art. In a more preferred embodiment, the intramolecular bridge is lanthionine. Protected lanthionines can be synthesized according to the retrosynthetic arrangement shown in Figure 3, using a routine methodology. The stereochemistry of the lanthionine products can be ensured at this stage by starting with the correct stereoisomers of the appropriate amino acids, for example cysteine and serine. In a more preferred embodiment, the intramolecular bridge is either Lanthionine 1 or Lanthionine 2: Lanthionine 1 Lanthionine 2 which can be synthesized, for example, as indicated in Figures 5 and 6, respectively.

In summary, with reference to Figure 5, for lanthionine 1, D-serine is converted to its amino-terminally protected Alloc derivative and subsequently converted to the carboxy-protected terminal propargyl ester. The propargyl ester N (Alloc) -D-Serine is converted to its corresponding β-bromoalanine derivative. The conversion can be carried out, for example, by dissolving propargyl ester N (Alloc) -D-Serine in dichloromethane and treating the solution with one equivalent of carbon tetrabromide and triphenylphosphine. These reactions are quite moderate and have been routinely used to convert hydroxyls to bromides. Zhu (2003) European Journal of Organic Chemistry. 20, 4062-4072. Alternatively, the syntheses are performed with the use of phosphorus trobromide in a solvent such as toluene or dichloromethane followed by a moderate basic analysis to provide the D-bromoalanines. Olah et. to the. (1980) Journal of Organic Chemistry. 45, 1638-1639. Other methods can also be used. Finally, the β-bromoalanine derivative is reacted with Fmoc-L-Cys under appropriate alkylation conditions to form Lanthionine 1. Lanthionine 2 can be synthesized in a similar manner as indicated in Figure 6. Synthesis of Polypeptides with Intramolecular Bridges The polypeptides with intramolecular bridges of the invention can be synthesized by any means maintaining the use and incorporation of orthogonally protected intramolecular bridges, including, but not limited to, solid phase peptide synthesis (SPPS), synthesis of peptide in solution phase, natural chemical ligation, intein-mediated protein ligation, and chemical ligation, or a combination thereof. In a preferred embodiment, the intramolecular bridge polypeptides of the invention are synthesized using a modified version of standard SPPS. Polypeptides with intramolecular bridges can be synthesized by any manual SPPS or with the use of commercially available automated SPPS synthesizers. SPPS has been known in the prior art since the begig of the 1960s (Merrifield, R.B., J. Am. Chem. Soc, 85: 2149-2154, 1963), and is widely employed. There are several known variations in the general approach. (See, for example, "Peptide Synthesis, Structures, and Applications" © 1995 by Academic Press, Chapter 3 and White (2003) Fmoc Solid Phase Peptide Synthesis, A practical Approach, Oxford University Press, Oxford). Very briefly, in the synthesis of peptides in solid phase, the desired C-terminal amino acid residue is coupled to a solid support. The subsequent amino acid to be added to the peptide chain is protected at its amino terminus with Boc, Fmoc, or other appropriate protecting group, and its carboxy terminus is activated with a standard coupling reagent. The free amino end of the aniino acid bound to the support is allowed to react with the subsequent amino acid, coupling the two amino acids. The amino terminus of the growing peptide chain is deprotected, and the process is repeated until the desired polypeptide is complete. In accordance with the methods of the invention, polypeptides with intramolecular bridges can be synthesized by incorporating differentially protected orthogonal intramolecular bridges in standard SPPS. Portions of the polypeptide chain that are not part of the intramolecular bridge can be synthesized with standard SPPS techniques known in the prior art. In a preferred embodiment, protected amino acids-Fmoc p -Boc terminally amino are used. In a more preferred embodiment, SPPS based on Fmoc is used. The differentially protected orthogonal intramolecular bridges are incorporated into the polypeptide chain through selective deprotection of their amino and carboxy groups. The methods of the invention can be used to synthesize a polypeptide with intramolecular bridges having a single intramolecular bridge according to that shown in Formula III: Formula III wherein A, Xn, and Rn are according to what is defined by Formula I. The polypeptide is prepared with the use of a single intramolecular bridge of general formula IV: Formula IV wherein L represents covalently linked amino acid side chains, D and E are amino end protecting groups, and G is carboxy-terminal protecting group. By abbreviating, the intramolecular bridge is coupled via its carboxy terminus with a peptide chain attached to a solid support, or directly to the solid support. Additional amino acids are coupled to the free amino end of the intramolecular bridge followed by their deprotection (extraction of E). The protecting group (G) in the remaining carboxy group of the intramolecular bridge is extracted and the carboxy group is coupled to the free amino terminus of the polypeptide chain thus formed. Additional amino acids can optionally be added subsequently to the remaining amino group. During the synthesis of the polypeptides of the invention, at any time there will be a single "free amino terminus" in the growing polypeptide chain and a single "free carboxy terminus" to be coupled to the free amino terminus. Each time an amino acid is added and unprotected the free amino ends will be blocked by the added amino acid, if the recently added amino acid is subsequently deprotected, a new free amino acid end will be formed. A person skilled in the art will understand that under such circumstances there is only a single free amino terminus. More specifically, in the synthesis of a polypeptide with intramolecular bridges having a single intramolecular bridge. D is selected so that the reaction conditions for removing the protecting group D do not result in the extraction of E or G and / or the amino protecting group from the amino acids of the remainder of the polypeptide chain. The conversion also applies. In other words, according to a non-limiting example, if the polypeptide is synthesized with the use of SPMO based on Fmoc, D is selected so that it can be selectively segmented under conditions that do not extract E, G, and / or Fmoc. Similarly, D and G are selected so that the conditions for the extraction of Fmoc do not result in the cleavage of D or G. In a preferred embodiment, the amino protecting group E is equivalent to the amino protecting group of the amino acids of the chain of polypeptide that are not part of the intramolecular bridge. Therefore, where, for example, SPPS based on Fmoc is used, E is preferably Fmoc. The synthesis of the polypeptide with intramolecular bridges begins with the coupling of the C-terminal amino acid to a solid support. The term "solid support" refers to any solid phase material on which a polypeptide is synthesized. Solid support includes terms such as "resin", "solid phase", and "support". A solid support can be composed of organic polymers such as polystyrene, polyethylene, polypropylene, polyfluoroethylene, polyethyleneoxy, and polyacrylamide, as well as copolymers and grafts thereof. A solid support can also be inorganic, such as glass, silica, controlled pore glass (CPG), or a reverse phase silica with appropriate groups on which the amino acids can be coupled and segmented in a manner easy. The configuration of a solid support can be in the form of beads, spheres, particles, granules or a surface. The surfaces can be flat, substantially flat, or non-flat. The solid supports may be porous or non-porous, and may have characteristics of inflammation or non-inflammation. A solid support can be configured in the form of a well, depression or other container. A plurality of solid supports can be configured in an array, treated for a robotic supply of the reagents, or by means of detection that includes scanning by laser illumination and scattering of confocal or deflected light. The coupling of the first amino acid to the solid support can be monitored for completion by assays known in the prior art. In a preferred embodiment, Fmoc amino acids are used in the synthesis of the polypeptide chain. The amino acids are commercially available or can be synthesized by methods known in the prior art. Additional amino acids can be added to the polypeptide chain using a standard SPPS methodology. Where, for example, Fmoc amino acids are used, the amino protective group Fmoc of the C-terminal amino acid, once coupled to the resin, can be extracted by, for example, exposure to 20% piperidine in DMF. The next amino acid Fmoc can be coupled to the peptide chain using standard chemical coupling techniques. Amino acids having reactive side chains can be protected with appropriate protecting groups so that their side chains remain protected throughout the synthesis of the polypeptide with intramolecular bridges of interest. The coupling and deprotection steps may be repeated as desired with the use of the appropriate amino acids. This completes the synthesis of X3 of general formula III. The intramolecular bridge is coupled to the polypeptide chain that grows by standard chemical coupling techniques. Alternatively, if the intramolecular bridge falls at the C-terminal end of the polypeptide with intramolecular bridges, the intramolecular bridge can be coupled directly to the resin via its free carboxy group. The protecting group E is then selectively extracted under appropriate conditions, for example using 20% piperidine in DMF where E is Fmoc. With reference to general formula III, R2 is now coupled to the polypeptide chain. One or more amino acids can be added subsequently to the polypeptide chain through sequential coupling and deprotection (X2 of general formula III). Subsequently, the protecting group G is selectively extracted under appropriate conditions. In a preferred embodiment, G is either a propargyl group, which can be cleaved with the use of dicobalto-octacarbonyl in diclomethane, or benzyl ester, which can be cleaved with the use of a hydrogenation protocol using palladium or carbon and cyclohexadiene in dichloromethane. This completes the addition of R1 of the general formula III thereby the intramolecular bridge is completely incorporated in the polypeptide, forming the ring structure. The protective group D can then be selectively deprotected under appropriate conditions. In a preferred embodiment, D is either Alloc, which can be cleaved with the use of 20 mol% of Pd (PPh3) 4 and 20-25 equivalents of PhSiH3 in dichloromethane, or ivDde, which can be cleaved by hydrazine at 2-10% in DMF. The polypeptide with intramolecular bridges can be subsequently enlarged by sequential coupling and deprotection of additional amino acids (X1 in general formula III). Polypeptides with intramolecular bridges with multiple rings in series, in this case having more than one intramolecular bridge, can similarly be synthesized with the use of. a single differentially protected intramolecular bridge. Optionally, more than one differentially protected intramolecular bridge, which differs from one another only by its protecting groups, can be used to synthesize a polypeptide having multiple rings. Multiple differentially protected intramolecular bridges, which vary in their side chain structures (eg, Lan and MeLan), can also be used to incorporate different fractions with intramolecular bridges. The protecting groups in such subsequent bridges may be the same or different from the protectants in the first intramolecular bridge incorporated in the polypeptide chain. The polypeptide with intramolecular bridges with multiple rings in series is synthesized by fully incorporating a first intramolecular bridge in the polypeptide chain, forming the first ring structure, extracting the amino terminal protecting group, optionally extending the polypeptide chain by means of a coupling and sequential deprotection of additional amino acids, fully incorporating a second intramolecular bridge (same or different than the first intramolecular bridge) by medium of its carboxy terminus, optionally extending the polypeptide, and repeating these steps as desired to synthesize the polypeptide with objective intramolecular bridges. For polypeptides with intramolecular bridges with multiple rings that either overlap or are incorporated, more than one orthogonally protected intramolecular bridge can be used. While the side chain structures of the multiple orthogonally protected intramolecular bridges may be the same or different, the protecting groups must be differentially protected orthogonally to allow selective deprotection of their respective amino and carboxy groups. The number of such bridges depends on the number of overlapped or incorporated rings. Where, for example, two polypeptide rings with intramolecular bridges overlap one another, or one is incorporated in the other, two differentially protected orthogonal intramolecular bridges are used.; where, for example, 3 rings overlap one another, or are incorporated within one another, three differentially protected orthogonal intramolecular bridges are used, etc. In a non-limiting example, wherein the polypeptide with intramolecular bridges of interest contains two overlapping rings, two orthogonal intramolecular bridges differentially protected from the general formulas V and VI are used: Formula V Formula VI wherein L1 and L2 represent covalent linkage amino acid side chains (L1 may be the same or different from L2), D, M, E, and Q without amino end protecting groups, and G and T are groups carboxy end protectors; where D and M can be segmented only under different conditions, where E and Q can be segmented under the same conditions; where E and Q are segmented under the same conditions different from those that will segregate to D and those that will segment M; and where G and T can be segmented only under different conditions. In a preferred embodiment the amino protecting groups E and Q are equivalent to the amino protecting group of the amino acids of the polypeptide chain that are not part of the intramolecular bridge. Therefore, where, for example, SPPS based on Fmoc is used, E and Q are preferably Fmoc, but they are not limited as such. In such a situation, E and Q are also, for example, Boc. In accordance with the methods of the invention, a polypeptide with intramolecular bridges containing two overlapping rings can be synthesized by first coupling the C-terminal amino acid to a solid support. Additional amino acids can be optionally added to the polypeptide chain with the use of a standard SPPS methodology. In a preferred embodiment, the Fmoc amino acids are used in the synthesis of the polypeptide chain. Amino acids having reactive side chains can be protected with appropriate protecting groups so that their side chains remain protected throughout the synthesis of the polypeptide with intramolecular bridges of interest. The steps of coupling and unprotecting can be repeated as desired with the use of appropriate amino acids. The intramolecular bridge of the general formula V is then coupled to the polypeptide chain that grows through its free carboxy group, and E is subsequently cleaved. D and G remain unaffected. In a preferred embodiment, E is Fmoc. One or more amino acids can then optionally be sequentially coupled to the free amino terminus of the polypeptide by setting a cycle by means of the coupling and unprotecting steps in accordance with standard SPPS. Subsequently, the intramolecular bridge of formula VI is coupled to the polypeptide chain that grows through its free carboxy group, and Q is subsequently cleaved. D, G, M, and T remain unaffected. In a preferred embodiment, Q is Fmoc. Again, one or more amino acids may optionally be sequentially coupled to the free amino terminus of the polypeptide. To form the first ring, G is then cleaved using appropriate deprotection chemistries and the resulting free carboxy group is coupled to the free amino terminus of the polypeptide chain. The protective groups D, M, and T remain unaffected. Subsequently, the protecting group D is extracted under appropriate conditions, exposing a free amino group. Protective groups M and T remain unaffected during segmentation of D. Additional amino acids can then optionally be coupled to the free amino group at the N-terminus of the polypeptide. To form the second ring, and the rings that overlap, T is cleaved under appropriate conditions and the resulting free carboxy group is coupled to the free amino terminus of the polypeptide chain. The protecting group M can then be cleaved under appropriate conditions, and the polypeptide chain is further extended through the sequential coupling of additional amino acids. In accordance with the methods of the invention, a polypeptide with intramolecular bridges containing two incorporated rings can be similarly synthesized with the use of two intramolecular bridges differentially protected from the general formulas V and VI. The synthesis of the polypeptide with intramolecular bridges containing two incorporated rings is comparable with the synthesis of a polypeptide with intramolecular bridges containing two overlapping rings, differ only in the order of the deprotection and coupling of intramolecular bridges of formulas V and VI. Specifically, the intramolecular bridge of formula V is coupled to the free amino terminus of a peptide chain connected through its carboxy terminus to a solid support, or the intramolecular bridge of formula V is coupled directly to the solid support. E is subsequently cleaved, and one or more amino acids can then optionally be sequentially coupled to the free amino terminus of the peptide chain by cycling through the coupling and unprotecting steps in accordance with standard SPPS. Subsequently, the intramolecular bridge of general formula VI is coupled to the peptide chain that grows through its free carboxy group, and Q is subsequently cleaved. Again one or more amino acids can then optionally be sequentially coupled to the free amino terminus of the polypeptide. To form the first ring, then T is cleaved with the use of appropriate deprotection chemistries and the resulting free carboxy group is coupled to the free amino terminus of the polypeptide chain. Subsequently, the protecting group M is extracted under appropriate conditions, exposing a free amino group. Additional amino acids then optionally can be coupled to the free amino group at the N-terminus of the polypeptide. To form the second ring and thus the incorporated rings, G is cleaved under appropriate conditions and the resulting free carboxy group is coupled to the free amino terminus of the polypeptide chain. The protecting group D can then be cleaved under appropriate conditions, and the polypeptide chain is further extended by sequential coupling of the additional amino acids. A person skilled in the art will appreciate that more complex molecules can be prepared in a similar way by means of variations of the above methods. For example, a polypeptide having two overlapping rings, and 3 additional rings in series can be synthesized by combining the methods described for the synthesis of polypeptides with intramolecular bridges that contain rings that overlap with the methods described for the synthesis of polypeptides. with intramolecular bridges that have rings in series. During the synthesis of a polypeptide with intramolecular bridges, the progress and precision of the synthesis can optionally be monitored by various techniques known in the prior art, including but not limited to the Maldi technique and LC-MS. Upon completion of the synthesis, the polypeptide with intramolecular bridges is cleaved from the solid support under appropriate conditions. Where the synthesized polypeptide contains significant amounts of sulfide (for example, for lanthionine containing polypeptides), a cocktail of TFA / thioanisole / water / phenol / ethanedithiol (82.5 / 5/5/5 / 2.5). The progress of the segmentation reaction can be monitored periodically by LC-MS or other appropriate technique. Depending on the selected side chain protecting groups, their cleavage may be affected during cleavage of the polypeptide from the resin, or alternatively in a separate step. The final product can be isolated by, for example, cold ether precipitation, and purified by known methods including, but not limited to, reverse phase HPLC.

The polypeptides with intramolecular bridges of the invention can be analyzed structurally and in terms of biochemical function by known techniques. Structural analysis can be achieved by techniques including, but not limited to, 3-dimensional NMR and X-ray crystallography. Polypeptides with intramolecular bridges have been successfully analyzed structurally with the use of 2-dimensional NMR TOCSY acquired in a 60 ms mix time (Braunschweiler &Ernst (1983), Journal of Magnetic Resonance 53, 521-528) and NOESY acquired in 200 ms, 400 ms, 450 ms. Kumar et. to the. (1980), Biochem. Biophys. Res. Commun. 95, 1-6. Smith, J.L. (2002) Dissertation, University of Florida, Gainesville. Smith et. to the. (2000), European Journal of Biochemistry 267, 6810-6816. In a preferred embodiment, the methods of the invention are used to synthesize polypeptides with intramolecular bridges containing one or more lanthionines or lanthionine derivative (s). In a more preferred embodiment, the methods of the invention are used to synthesize lantibiotics. In a more preferred embodiment, the methods of the invention are used to synthesize Nisin A and analogues thereof. Nisin A and analogs thereof can be evaluated for biological activity with the use of known methods. (Hillman et al. (1984), Infection and Immunity 44, 141-144; Hillman et al. (1998), Infection and Immunity 66, 2743-2749). The structural analysis of Nisin A and analogs thereof synthesized with the methods of the invention can be assisted with a comparison with the three-dimensional structure of the biologically produced Nisin A, previously determined by Van De Yen et al. by NMR (1991, European Journal of Biochemistry 202, 1181-1188). From the assignments of amino acids made from this work of determination of initial covalent structure, it is possible to quickly characterize the covalent bonds and identify all the long-range NOEs relevant for the structural determination of Nisin A and analogs thereof synthesized with the methods of the invention. Applications of the DPOLT Technology DPOLT is a technological platform that emerges from a multidisciplinary approach. There are several advantages that make this technology desirable. First and most relevant, it will allow a rapid synthesis and screening of a substantial number of candidate lantibiotics and other bioactive peptides for their application potential in the domain of therapies without having to devote large amounts of time and costs to conceive methods of fermentation and purification for your analyzes. There are approximately 50 lantibiotics that contain thioether bridges that overlap, with others being discovered each year, which can be synthesized with the methods described here. These lantibiotics include lantibiotics Nisin A Type A (I), Nisin Z, Subtilin, Ericin S, Ericin A, Streptin, Epidermin, [Vall-Leu6] -epidermin, Galidermin, Mutacin 1140, Mutacin B-Ny266, Mutacin III, Mutacin I , Pep5, Epilanin K7, and Epicidin 280; lantibiotics Type A (II) Lacticin 481, Variacin, Mutacin II, Streptococcin A-FF22, Salivaricin A, [Lys2-Phe7] -salivaricin A, Plantaricin C, Sublancin 168, and Butirivibriocin OR79A; Type B lantibiotics Cinnamicin, Duramycin, Duramycin B, Duramycin C, Curamycin C, Ancovenin, Mersacidin, Actagardine, Ala (O) -actagardine, and Subtilocin A; Two Component Lantibiotics Lacticin 3147A1, Lacticin 3147A2, Staphylococcin C55a, Staphylococcin C55p, Plantaricin Wa, Plantaricin WB, Cytolysin LL, Cytolysin Ls; and other lantibiotics such as Ruminococcin A, Carnocin UI 49, Macedocin, Bovicina HJ50, Nukacin ISK-I, and morphogen SapB. (See, for example, Chatterjee et al., 2005. Chem. Rev. 105, 633-83.) From previous experience, it seems likely that many methods of fermentation and purification for many lantibiotics will not be achieved quickly. Nisin A, which was discovered more than 50 years ago, remains the object of intense studies with the purpose of finding a method of rapid and appropriate purification for its development as a therapeutic agent. A recent North American Patent Application (US Application 2004/0072333) attempts to achieve this purpose, but uses a variety of costly proteases and multiple purification steps. It is extremely likely that the SPPS methods used by DPOLT will achieve the desired end in a much more cost efficient manner. Currently, more than 35 bioactive molecules are sold commercially that are synthesized with the use of SPPS methods, such as oxytocin, sandostatin and fuzeon, and, over time, the demand will certainly increase. The use of DPOLT allows site-specific substitution of amino acids and their analogues, even in a combinatorial library approach, which provides an optimal method for finding new and improved therapeutic agents for their intended purposes. "With respect to this, DPOLT is the only existing technology for the synthesis of molecules with overlapping rings, and has the potential to perform a variety of bioactive molecules, in addition to the latibióticos, to be used in several applications. DPOLT allows the in vitro production, for example, of structurally complex lantibiotics in a commercially viable manner with the use of solid phase peptide synthesis methods.

DPOLT provides two significant advantages in the screening and development of new lantibiotics for commercial applications: the fermentation approaches are clearly preferable from the point of view of cost of materials for production, but the time and effort required to optimize such methods can be prohibitive during the initial stages of drug discovery. Additionally, as in the case of Nisin A, the purification of high yield fermentations may not be easily obtained. The purification of the final product, typically, is not a significant problem in SPPS. DPOLT has the advantage of allowing a large number of potentially useful compounds to be screened in a rapid manner for clinical evaluation. For compounds that look promising, DPOLT provides a rapid route of commercialization, and also indicates which molecules could be functional by providing the necessary time and effort to develop fermentation methods. For compounds lacking the characteristics necessary for further development, such as those with a poor spectrum of activity, defective pharmacokinetics, toxicity problems, etc., DPOLT will allow rapid and efficient elimination of these based on considerations. Finally, since DPOLT depends on the synthesis of peptides in the solid phase, it will be simple to sift and develop analogues with improved characteristics, such as they 'overcome bacterial resistance. Thus, the method can be applied to other lantibiotics and peptides of interest and to identify those that have desirable and economically favorable functionality characteristics. The most obvious use for DPOLT and lantibiotics synthesized with the methods of the invention are the medical and veterinary treatment of bacterial infections. There are also several other potential applications. Lantibiotics are a well established or attractive alternative to other bactericidal agents for use in food preservation and cosmetics DelvesBroughton et al., (1996) Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology. 69, 193-202; Rollema et al., (1995) Applied and Environmental Microbiology. 61, 2873-2878; Liu & Hansen, (1990) Applied and Environmental Microbiology. 56, 2551-2558; Huot et al, (1996) Letters in Applied Microbiology. 22, 76-79; Delvesbroughton, (1990) Food Technology. 44, 100; Delvesbroughton (1990) Journal of the Society of Dairy Technology. 43.73-76; Delvesbroughton et al, (1992) Letters in Applied Microbiology. 15, 133-136; Thomas & Wimpenny (1996) Applied and Environmental Microbiology. 62, 2006-2012; Sahl & Bierbaum (1998) Annual Review of Microbiology. 52, 41-79. Additionally, lantibiotics have been studied with some success as topical disinfectants, particularly as mouth rinses to promote oral health. Howell et al, (1993) Journal of Clinical Periodontology. 20, 335-339. The lantibiotic drugs have enormous potential, and most likely will be well received by the medical community. Even when the antibiotic market remains high and you remain there whenever there are infectious diseases, the total life span of most antibiotics is short, due to mutation and baceterian resistance. The benefits of the class of antibiotic antibiotics are that they have a proven track record that they are relatively resistant to bacterial adaptation and have been found to have a bactericidal activity against a number of bacterial pathogens resistant to other antibiotics. All patents, patent applications, and other scientific and technical documents referred to here on either side are incorporated in their entirety as a reference. The methods and compositions described herein as currently representative of preferred embodiments are exemplary and are not intended to limit the scope of the invention. Changes in them and other uses will be evident for people experienced in the technique, and are considered to be within the spirit of the invention. The invention described illustratively herein may appropriately be practiced in the absence of any element or elements, limitation or limitations that are not described herein. Thus, for the examples, in each case here the term "comprising", "consisting essentially of", and "consisting of" can be replaced with any of the other two terms, without changing their usual meanings. The terms and expressions that have been used are used as terms of description, not limitation, and there is no intention in the use of such terms and expressions to exclude any of the equivalences of the characteristics shown and described or portions thereof, but that it is recognized that several modifications are within the scope of the claimed invention. Thus, it should be understood that although the present invention has been specifically described by the modalities and optional features, the modification and variation of the concepts described herein are considered to be within the scope of this invention as defined by the description and the appended claims. Additionally, where the features or aspects of the invention are described in terms of Markush groups or other groupings of alternatives, those skilled in the art will recognize that the invention is also why it is described in terms of any individual member or subgroup of members. from the Markush group or another group. The present invention may be better understood under the light of the following examples, which are for purposes of illustration only, and should not be construed as limiting the scope of the invention at all. EXAMPLES Example 1: Synthesis of Orthogonal Lanthionines Differentially Protected A. Synthesis of Fmoc-Cys The Fmoc-protected cysteine (Figure 3, structure B) was synthesized in a two-step sequence starting from L-cystine as indicated in Figure 4. Sodium carbonate (4.6 g, 43.6 mmol) and L-cystine (5.0 g, 20.8 mmol) were dissolved in water (200 mL). The resulting solution was cooled to 10 ° C and allowed to gradually warm to room temperature. FmocCl (11 85 g, 45.8 mmol) was dissolved in dioxane (80 mL), and the resulting solution was added dropwise to the aqueous solution of L-cystine. The solution was stirred for 2 h at 10 ° C and allowed to cool to room temperature. A thick white precipitate was obtained which was filtered on a sintered glass funnel. The product was precipitated with diethyl ether (50 mL) and dried in vacuum for 2 days. N, N '-Bis (Fmoc) -Z-cystine (14.0 g, 98% yield) was obtained as a white powder. N, iV'-Bis (Fmoc) -L-cystine (12.0 g, 17.5 mmol) was dissolved in methanol (300 mL). Granular zinc (12.0 g) was added to this solution. and the resulting mixture was vigorously stirred with the use of a magnetic stirrer. Trifluoroacetic acid (75 ml, 1 mol) was added dropwise into the reaction mixture over a period of 2 h stirred at room temperature for a period of 12 h. The reaction was monitored by reverse phase high pressure liquid chromatography C-18 (HPLC) and thin layer chromatography (TLC, chloroform / methanol / acetic acid = 30: 1: 0.1, v / v). During the disappearance of N, N'-bis (Fmoc) -L-cystine, the reaction mixture was filtered and concentrated on a rotary evaporator to reduce the volume to about 100 mL. Dichloromethane (400 mL) was added and the mixture was rinsed with 2N aqueous hydrochloric acid. The aqueous layer was extracted with dichloromethane and the combined organic layers were dried with magnesium sulfate. The concentration of the solution gave N- (Fmoc) -L-cysteine, 8.8 g, 73%) (Figure 3 and 4, structure B) as a white powder. B. Synthesis of Propargyl Ester of N- (Alloc) -Serine The propargyl ester synthesis of N- (Alloc) -D-serine (Figure 3, structure A) was developed as follows (see Figure 5). D-Serine (10.5 g, 100 mmol) and sodium carbonate (11.1 g, 105 mmol) were dissolved in water (100 mL). Acetonitrile (50 mL) was added to this solution and the mixture was cooled in an ice bath to 5 ° C. The allyl chloroformate (11.7 mL, 13.3 g, 110 mmol) was added dropwise over a period of 30 min. The reaction mixture was allowed to warm gradually to room temperature and was stirred for 12 h. The mixture was concentrated under vacuum to approximately 10 mL to extract the acetonitrile and the residue was cooled to 0-5 ° C. The pH of the solution was adjusted to 2.0 by adding concentrated aqueous HCL (approximately 10 mL). The product was extracted with ethyl acetate (5x40 mL), and the extract was dried with anhydrous magnesium sulfate. The solvent was removed on a rotary evaporator under vacuum to yield N- (Alloc) -D-serine (16.9 g, 89%) which was prepared as a light yellow oil. N- (Alloc) -D-serine (16 g, 85 mmol) was dissolved in DMF (70 mL). Sodium bicarbonate (7.9 g, 94 mmol) was added to the resulting solution. Propargyl bromide (80% in toluene, 10.5 mL, 94 mmol) was added dropwise over a period of 20 min at room temperature. The reaction mixture was stirred at room temperature for 2 d. The reaction mixture was concentrated under vacuum in a rotary evaporator and the residue was dissolved in ethyl acetate (100 mL). The solution was rinsed with aqueous sodium bicarbonate (2x50 mL) and water (2x50 mL), and dried with magnesium sulfate. The solvent was removed on a rotary evaporator under vacuum to provide propargyl ester of N- (Alloc) -D-serine (18 g, 93% yield). C. Synthesis of (Benzyl) N- (ivDde) -D-Serine N- (ivDde) -D-serine ester (Figure 3, structure C) was prepared from -serine and ivDde-OH which was synthesized by dimedone acylation-0 with isovaleryl chloride in the presence of pyridine followed by the rearrangement of 5,5-dimethyl-3-oxocyclohex-1- enyl 3-methylbutanoate with aluminum chloride using a previously reported method (Akhrem, AA, et al., Synthesis 1978, 925). In particular, a solution of isovaleryl chloride (13.5 mL, 13.3 g, 110 mmol) in dichloromethane (50 mL) was added dropwise over a period of 15 minutes to a solution of dimedone (14 g, 100 mmol) and pyridine ( 9.7 mL, 9.5 g, 120 mmol) in dichloromethane (150 mL). The reaction mixture was stirred for 1.5 h, and rinsed with 2N aqueous hydrochloric acid (2x50 mL), water, and saturated aqueous sodium bicarbonate (50 mL), and then dried with magnesium sulfate. The solvent was removed with a rotary evaporator under vacuum to provide 5, 5-dimethyl-3-oxocyclohex-l-enyl 3-methylbutanoate (22.4 g, 100% yield) which appeared as a light yellow oil. To a stirred suspension of aluminum chloride (16.0 g, 120 mmol) in dichloromethane (100 mL) cooled in an ice bath was added dropwise a solution of 5, 5-dimethyl-3-oxocyclohex-l-enyl 3-methylbutanoate (11.2 g, 50 mmol) for a period of 30 minutes. The reaction mixture was allowed to warm to room temperature and stirred for 1 h. Then the reaction mixture was poured slowly into a mixture of 37% aqueous hydrochloric acid (50 mL) and ice (150 g) with ice cooling so that the temperature did not exceed 5 ° C. Brine (200 mL) was added to the mixture and the product was extracted with dichloromethane (6x50 mL, the conclusion of the extraction was verified by TLC). The extract was rinsed with brine (2x50 mL), dried with magnesium sulfate, and concentrated on a rotary evaporator under vacuum. The crude product was purified by column chromatography on silica gel using a gradient of hexanes to ethyl acetate: hexanes (1:10) to give iVDde-OH (10.5 g, 94) which appeared as a yellow oil Clear. The N- (ivDde) - £ >; -serine was then synthesized as follows: To a mixture of ivDde-OH (1.1 g, 5 mmol) and L > Serine (0.6 g, 5.75 mmol) in methanol (50 mL) was added N-ethyldiisopropylamine (3.4 mL, 2.6 g, 20 mmol). The reaction mixture was stirred under reflux overnight. The TLC test (ethyl acetate / hexanes 1: 4) showed no free ivDde-OH. The reaction mixture was cooled to room temperature and the solvent was removed by rotary evaporation under vacuum. The residue was dissolved in water (40 mL), cooled to 5-10 ° C, and acidified to pH 2 by the dropwise addition of 2N aqueous hydrochloric acid. The mixture was stirred for 30 minutes and the aforesaid was filtered, rinsed with water and dried in vacuo to give N- (ivDde) -serine (1.5 g, 96%), as white microcrystals. Benzyl ester of N- (ivDde) -D-serine was prepared as follows: To a mixture of N- (ivDde) -D-serine (0.93 g, 3 mmol) and sodium bicarbonate (0.34 g, 4 mmol) in DMF (20 mL) was added benzyl bromide (0.43 mL, 0.62 g, 3.6 mmol) and the mixture was stirred at room temperature for 24 h. The mixture was concentrated under vacuum in a rotary evaporator, and the residue was dissolved in ethyl acetate (40 mL). The solution was rinsed with water and the aqueous layer was extracted with ethyl acetate (2x30 mL). The combined organic layer was rinsed with saturated aqueous sodium bicarbonate (2x40 mL), and water (40 mL). The organic layer was dried with magnesium carbonate, and the solvent was removed under vacuum on a rotary evaporator to provide Benzyl ester of N- (ivDde) -D-serine (1.03 g, 86%), as white needles. D. Synthesis of Propargyl Ester of N- (Alloc) -D-β-Bromoalanine and Benzyl Ester of N- (ivDde) -D-jS-Bromoalanin The corresponding β-bromoalanin derivatives of (propargyl) N ester (alloc) -D-serine and (benzyl) ester of N (ivDde) -D-serine are synthesized by dissolving an appropriate ester equivalent in dichloromethane (or a similar aprotic solvent) and treating the solution with one equivalent of carbon tetrabromide and triphenylphosphine. The reaction is stirred at room temperature until its conclusion was observed by TLC, and the β-bromoalanine derivative is purified by flash chromatography. Alternatively, the syntheses are achieved with the use of phosphorous tribromide in a solvent such as toluene or dichloromethane followed by a moderate basic analysis to produce the desired D-bromoalanines. In addition, bromination, tosylation or other leaving groups can be used in the alkylation step described below to produce the final protected lanthionine. E. Synthesis of Lanthionines 1 and 2 Lanthionine 1 is synthesized through the alkylation of propargyl ester with N (alloc) -D-bromoalanine with (Fmoc) -L-cysteine (Figure 5). Lanthionine 2 is synthesized through the alkylation of benzyl ester of N (ivdDe) -D - ^ - bromoalanine with (Fmoc) -L-cysteine (Figure 6). The respective β-bromoalanin is alkylated with (Fmoc) -L-cysteine as follows: an equivalent of β-bromoalanine is dissolved in dichloromethane (or a similar aprotic solvent) and treated with (Fmoc) cysteine under a phase transfer catalyst such such as tetrabutylammonium bromide, tetrabutyl ammonium iodide, or Aliquat 336. The amount of the catalyst required is 5-50 mol% and can be optimized to obtain a good reaction rate and clean product formation. The reaction temperature can also be optimized within a range of 10-50 ° C. The product thus obtained is purified by flash column chromatography; and the purity and identity of the product is determined by NMR, CLAR, mass spectrometry and / or TLC. The synthetic routes to lanthionines 1 and 2 are relatively straight, and the products are expected to be stable so that an increased and volumetric synthesis (> 10 g) can be easily achieved. Example 2: Synthesis of Lantibiotic Analog Nisin A With the Use of Lanthionines 1 and 2 A. Solid Phase Peptide Synthesis of the Analog Nisin A A Nisin A Analog [SEQ. ID. NO: 2] is synthesized according to the invention as indicated below. The analog contains alanine substitutions for dehydrobutarine at position 33 and dehydroalanines at position 30 and 2. Considerable evidence indicates that this will not have a significant effect on the spectrum of activity and potency of the product in relation to natural Nisin A (Kuipers et al, (1996), Devos et al. (1995), Molecular Microbiology 17, 427-437 Sahl et al (1995), European Journal of Biochemistry 230, 827-853; Bierbaum et al. (1996), Applied and Environmental Microbiology 62, 385-392). Unless otherwise indicated, all protocols are the standard SPPS Fmoc methodology reported in the literature. White (2003) Fmoc Solid Phase Peptide Synthesis, A practical Approach, Oxford University Press, Oxford. Nisin A is synthesized from its carboxy terminus in a gradual manner (see Figure 1). 1. The carboxyl of Na-Fmoc-Lys-NE-t-butyloxycarbonyl-L-lysine (Residue 1) is coupled to the CLEAR-Acid Resin ™ (Peptide International). The resin is verified with ninhydrin to verify the conclusion of the reaction. 2 . Deprotection of the Fmoc group located in the amide of lysine is achieved using 20% piperidine in DMF at room temperature. 3 . The above steps (1-2) of coupling and deprotection are repeated to join, in order, alanine, valine, histidine, isoleucine and serine (residues 2 by means of 6) using the respective amino acids-Fmoc L (commercially available). Amino acids such as histidine, lysine and serine have t-butyl groups coupled to their reactive side chains to protect these groups. Four . The next coupling is performed using orthogonal lanthionine 1 after which the Fmoc group in orthogonal lanthionine 1 is extracted with the use of 20% piperidine in DMF. 5 . The Fmoc histidine (residue 8) is coupled. 6 Fmoc histidine is deprotected with 20% piperidine in DMF and histidine is coupled with orthogonal lanthionine 2. 7 The propargyl group in orthogonal lanthionine 1 is cleaved with the use of dicobalt-octacarbonyl in dichloromethane. The amino terminus Fmoc of orthogonal lanthionine 2 is deprotected using 20% piperidine in DMF. The deprotected C-terminus of orthogonal lanthionine 1 and the deprotected N-terminus of orthogonal lanthionine 2 are coupled. The synthesis of ring E is completed in this step. 8. The N (Alloc) group of lanthionine 1 is extracted by treating the peptidyl resin twice with 20 mol% of Pd (PPh3) 4 and 20-25 equivalents of PhSiH3 in dichloromethane for 15-20 minutes. 9. The unprotected N-terminus is coupled with alanine Fmoc (residue 11). The Fmoc group in alanine is deprotected using 20% piperidine in DMF. 10 The remaining C-terminus of lanthionine 2 is deprotected using a transfer hydrogenation protocol using palladium on carbon and cyclohexadiene in dichloromethane. eleven . The deprotected C-end of lanthion 2 and the N-terminus of alanine (residue 11) is coupled. The synthesis of the rings that overlap E and D is completed in this step. In order to verify that the correct product is synthesized, a small amount of resin is taken and the peptide is segmented with the use of a segmentation cocktail (see below). The resulting peptide is analyzed by aldi and LC-MS. 12 The ivDde in lanthionine 2 is extracted using 2-10% hydrazine in DMF and the resulting free amino terminus is elongated sequentially with protected Fmoc lysine, methionine and asparagine (residues 1 3, 1 4 and 1 5). 13 Lanthionine 1 is linked to the unprotected N-terminus of asparagine. (Either lanthionine 1 or lanthionine 2, however, can be used to complete the synthesis of rings C, B and A.) 14. The Fmoc group of lanthionine 1 is deprotected and sequentially coupled with glycine Fmoc, methionine, alanine, leucine and glycine (residues 17 to 21) to form ring C. 15. The propargyl group at the C-terminus of lanthionine 1 is extracted using 1 equivalent of dicobaltoctacarbonyl and coupled to the N-terminus of glycine (residue 21), completing ring C. 16 The Alloc group at the N-terminus of lanthionine 1 is extracted according to the procedure described in step 8 and coupled to the Fmoc lysine (residue 23). 17 The N-terminus of lysine is deprotected, and lanthionine 1 is coupled to the N-terminus of lysine. 18 The Fmoc group of lanthionine 1 is deprotected and sequentially coupled with glycine Fmoc and Fmoc proline (residues 25 and 26). 19 The propargyl group at the C-terminus of lanthionine 1 is extracted using 1 equivalent of dicobaltoctacarbonyl and coupled to the deprotected N-terminus of the proline thus forming ring B. 20. The Alloc group at the N-terminus of lanthionine 1 is extracted according to the procedure described above and coupled to lanthionine 1. twenty-one . The Fmoc group of lanthionine 1 is deprotected and sequentially coupled with leucine Fmoc, alanine and isoleucine (residues 29 to 31.) 22. The propargyl group at the C-terminus of lanthionine 1 is extracted using 1 equivalent of dicobaltoctaccarbonyl and coupled to the unprotected N-terminus of isoleucine, thus forming ring A. 23. The Alloc group at the N-terminus of lanthionine 1 is extracted according to the procedure described above and sequentially coupled to alanine Fmoc and isoleucine (residues 33 and 34). This completes the synthesis of the analogue Nisin A. • B. Peptide Segmentation Synthesized from Resin Because the peptide synthesized contains significant amounts of sulfur, a cocktail containing TFA / thioanisole / water / phenol / ethanedithiol (82.5 / 5/5/5 / 2.5) is used to segment the peptide from the resin (White 2003). The resin is completely rinsed with dichloromethane to extract traces of DMF and other organic residues and treated with the previous cocktail. Optimization of the time given for segmentation is achieved by carrying out the reaction in 15-20 mg of the resin followed by LC-MS in hourly intervals up to 18 hours. Optimized conditions are used to increase segmentation. The segmented peptide is gradually poured into cold ether, thus precipitating the peptide. The precipitated peptide is rinsed with cold ether and dried. C. Purification of Segmented Peptide The peptide is purified by reconstituting it in water containing 1% TFA. The solution is subjected to CLAR with a QuadTech detector. The peaks are collected and analyzed by Maldi tof to confirm the identity of the product. The fractions containing the desired peptide are collected and lyophilized to obtain the purified product. The purity is determined using CLAR. MS and NMR. Example 3: Structural and Biological Analysis of Purified Nisin A Analog A. Bioassay of Nisin Analog A The lantibiotic thus is synthesized and purified according to that shown in Examples 1 and 3 are distributed in aliquots and lyophilized. The resulting product is weighed and the final productions calculated. The biological activity of the Nisin A analogue is determined by a deferred antagonism assay, known in the prior art, which allows the determination of the minimal inhibitory and bactericidal concentrations of the Nisin A analogue (Hiliman et al. (1984), Infection and Immunity 44, 141-144; Hillman et al. (1998), Infection and Immunity 66, 2743-2749). The comparison with the natural Nisin A allows the determination of the respective specific activities. The bioassay is conducted as follows: Samples (20 μ?) Of fractions to be evaluated for Nisin A activity are serially diluted 2-fold using acetonitrile: water (80:20) in 96 microtiter well plates. The concentrations are in the range from 20 to 0.08 pg / mL. An overnight culture of the Micrococcus luteus chain ATCC272LS (spontaneously resistant to 100 ug / mL streptomycin) is diluted 1: 1000 (ca. 106 cfu / mL) in a Tripticase soy broth (Difco) and allowed to grow at 37 ° C to OD600 = 0.2. Six hundred microliters of cells are added to 15 mL of Trypticase soy broth top agar (0.75% agar) that has been cooled to 45 ° C, and poured over the surface of a large Petri dish containing Trypticase soy agar containing 100 g / mL of streptomycin (streptomycin prevents the consequences of contaminants that may be present without affecting the ability to determine the amount of activity of Nisina S present). After the upper agar has been established, 5 μ ?? of samples of the dilutions in 2 serial occasions of the fractions to be tested are visible on the surface of the plates and are allowed to dry by air. The plates are incubated at 37 ° C for 24 hours and examined by areas of inhibition of growth of the indicator strain. The evaluation of the samples is taken as the reciprocal of the highest dilution that produces a visible inhibition of the growth of the M. luteus indicator strain. As a control, the authentic Nisin A is diluted and dotted as described. The concentrations were found in intervals of 20 to 0.08 ug / mL. The results allowed a determination of the bioactivity of the synthetic analogue in relation to natural Nisin A as a percentage based on the purity levels of these compounds according to that established in the previous step. The previous bioassay using synthetic and natural Nisin A is driven by at least a dozen Gram-positive species including Staphylococcus aureus, Enterococcus faecals, and Listeria monocytogenes resistant to multiple drugs. One or more other antibiotics appropriate for the target species that are evaluated are also run in parallel for a comparison. B. Structural Analysis of the Nisin A Analog The three-dimensional structure of the Nisin A analogue is determined by comparisons with natural Nisin A using TOSCY and NOESY NMR. Samples (3-5 mM) of the synthetic and natural Nisin A are prepared in H20 / D20 / 3- (trimethylsilyl) -propionic acid-D4, sodium salt (TSP) (90.0: 9.9: 0.1%) in one volume total of 700 pL. The NMR data are collected in a Bruker Avance spectrometer of cryoprobe at 25 ° C and the carrier frequency is centered on the water resonance, which is suppressed by presaturation during a lag delay of 1.5 seconds. The TOCSY experiments are performed with a mix time of 60 ms using the MLEV-17 sequence (Bax & amp;; Davis (1985), Journal of Magnetic Resonance 65, 355-360). The NOESY Experiments are carried out with mixing times of 200 ms, 400 ms, and 450 ms. The delay times to create or refocus a coherent amphase in the MC and NBC experiments are adjusted to 3.5 ms (140 Hz coupling) and 60 ms (8.5 Hz coupling), respectively. All 2D data are collected with 2048 complex points in the realized dimension and between 256 and 512 complex points for the indirect dimensions. Indirect phase-sensitive detection for all experiments is achieved with the use of the TPPI-State method (Marion et al. (1989), Journal of Magnetic Resonance 85, 393-399). The chemical changes of 1H are referenced to the TSP. The data are processed with MMRpipe (Delaglio et al. (1995), Journal of Biomolecular NMR 6, 277-293) by first extracting the residual water signal by deconvolution, multiply the data in both directions by means of a squared cosine function or a cosine-squared function with a change of 60 ° (for NBC's "H" dimension), once set to zero, a Fourier transform, and baseline correction, the data is analyzed with the interactive computer program NMRView (Johnson &Blevins (1994), Journal of Biomolecular Nmr 4, 603-614). The 1H resonances are assigned according to standard methods (Wüthrich, K. (1986) NMR of Proteins and Nucleic Acids., Wiley, New York) using TOCSY (Braunschweiler &Ernst (1983), Journal of Magnetic Resonance 53, 521-528) and NOESY experiments (Kumar et al. (1980), Biochem. Biophys.

Res. Commun. 95, 1-6). HMQC (Bax et al. (1983), Journal of Magnetic Resonance 55, 301-315; Muller (1979), Journal of the American Chemical Society 101, 4481-4484) and HMBC experiments (Bax &Summers (1986), Journal of the American Chemical Society 108, 2093-2094) are used to clarify some areas of ambiguity in the TOCSY and NOESY spectrum. The residues of lysine, isoleucine, glycine, and asparagine have distinct and easily characterized resonance spin patterns of '1H, which then makes it easy to assign in the 2D TOCSY and NOESY experiments. These residues are first identified. The thioether binding patterns are verified by means of proton NOE proton connectivity patterns of beta interval. The large interval NOEs presumably can be identified between residues at positions 3 and 7, 8 and 11, 13 and 19, 23 and 26, and 25 and 28. The NOEs of intervals. large (> i + 2) are used for three-dimensional models as described in Smith et. al, 2002 (Structural and Functional Characterization of the Lantibiotic Miration 1140, University of Florida, Gainesville). The peak-transverse intensities of NOE are measured in NMRView. The distances are calibrated with the use of the ratio rab6 = rcai6 (VCai / Vab), where rab is the distance between atoms a and b, Vab is the peak-transverse volume NOESY of aab, rcai is a known distance, and Vcai is the corresponding volume of the peak-transverse NOESY calibration. The distance used for the calibration is that of the proton beta of isoleucine. Only the transverse peaks of the NOE interresidue are used as a distance restriction in the calculations. The energy wells are defined with the use of a higher and lower force constant of 1 kcal / mol / Á2. All conformational modeling is developed with the use of the Insightll computer program ((Accerlys, San Diego, CA) Molecular dynamic simulations are run in a vacuum pump at 500K with a dielectric constant of 4.0 with the use of a force field cvff with crossed terminals, orse potentials, and 40 A cutoff distances The peptide is constituted with the μe of the builder function in Insightll Initially, the linear peptide is minimized, and then they are molecular dynamic runs without restriction pro 10 ps After this, only the distance restrictions of i + 2 or greater are added.Memory dynamics simulations are periodically stopped when the distance constraints i + 2 or greater are satisfied between the residues that constitute each ring The ring A is formed first followed by a ring B and ring C and then the rings D and E. The thioether molecules are formed, the distance constraints i + 1 are added to the distance constraints i + 2 or greater, and the simulation of the molecular dynamics is developed by 5 ns at 500K with a dielectric constant of 4.0 with the use of a cvff force field with crossed terminals and Morse potentials. The molecular dynamics simulations are then developed by another 20 ns with all the restrictions. Dynamics history files are written every 10 ps. Two hundred history file structures that start at 1 ns and spaced every 100 ps are minimized energy with all NMR constraints with the use of 2000 faster descent steps followed by conjugated gradients and Newton-Raphson until the gradient the root mean square (RMS, for its acronym in English of the energy of 0.01 kcal / mol / Á.) The 200 minimized energy structures are verified in terms of NMR restriction violations with the use of the computer program PROCHECK-NMR ( Laskowski, RA, Rullmann, JA C, MacArthur, MW, Kaptein, R. &Thornton, JM (1996) AQUA and PROCHECK-NMR: Programs to verify the quality of structures solved by NMR, Journal of Biomolecular Nmr. 8, 477-486) The minimized energy structures are grouped into families with the use of the XCluster program (Shenkin, PS &; McDonald, D. Q. (1994) Group Analysis of Molecular Conformations, Journal of Computational Chemistry. 15, 899-916). The conformations are compared with the natural structures of Nisin A determined by VanDeVen et. al., 1991 (European Journal of Biochemistry 202, 1181-1188). It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (17)

1. CLAIMS Having described the invention as above, the content of the following claims is claimed as property: 1. A method for synthesizing an intramolecular bridging polypeptide comprising at least one intramolecular bridge, characterized in that it comprises: a) coupling the free carboxy terminus of a differentially protected orthogonal intramolecular bridge of the formula to a solid support or to the free amino terminus of an amino acid or polypeptide optionally linked to a solid support and wherein Ln represents side chains of covalently linked amino acids, wherein D, E, and G are protecting groups, each of which is selectively extracted under different reaction conditions, and wherein the reaction conditions for the extraction of the protective group D are different from those of the extraction of the amino protecting group from the amino acids of the remainder of the polypeptide chain; b) extract protecting group E to form a free amino terminus; c) adding an amino-protected amino acid to the free amino terminus and then deprotecting the amino acid to produce a new free amino terminus; d) optionally repeating c) one or more times; e) extract protecting group G to form a free carboxy terminus; f) coupling the free carboxy terminus of e) to the free amino terminus; g) extract protecting group D to form a free amino terminus; and h) optionally adding an amino-protected amino acid to the free amino terminus and then deprotecting the amino acid to produce a free amino terminus; and i) optionally repeating h) one or more times.
2. 2. A method for synthesizing an intramolecular bridge polypeptide comprising two overlapping intramolecular bridges, characterized in that it comprises: a) covalently linking the free carboxy end of a first orthogonal intramolecular bridge "differentially protected from the formula to a solid support or to the free amino terminus of an amino acid or polypeptide optionally linked to a solid support and wherein Ln represents side chains of covalently linked amino acids, wherein D, E, and G are protecting groups, each of which is selectively extracted under different reaction conditions, and wherein the reaction conditions for the extraction of the protecting group D are different from those of the extraction of the amino protecting group from the amino acids of the remainder of the polypeptide chain; b) extract protecting group E to form a free amino terminus; c) adding an amino-protected amino acid to the free amino terminus and then deprotecting the amino acid to produce a new free amino terminus; d) optionally repeating c) one or more times; e) covalently linking the free carboxy terminus of a second differentially protected orthogonal intramolecular bridge of the formula to the free amino terminus, where Ln is as defined above, where M, Q, and T are protective groups, each of which is selectively extracted under different reaction conditions, and where D and M are extracted only under different conditions, wherein G and T are extracted only under different conditions, wherein the reaction conditions for the extraction of the protective group M are different from those of the extraction of the amino protective group of the amino acids of the rest of the chain of polypeptide; and where E and Q are extracted under conditions different from those with D and those with which M will be extracted; f) extracting the protecting group Q to form a free amino terminus; g) optionally adding an amino-protected amino acid to the free amino terminus and then deprotecting the amino acid to produce a new free amino terminus; h) optionally repeating g) one or more times; i) extracting the protecting group G of the first differentially protected orthogonal intramolecular bridge to form a free carboxy terminus; j) attach the free carboxy end to the free amino terminus; K) extract protecting group D from the first differentially protected orthogonal intramolecular bridge to form a free amino terminus; and 1) optionally adding an amino-protected amino acid to the free amino terminus and then deprotecting the amino acid to produce a new free amino terminus; m) optionally repeating 1) one or more times; n) extracting the protective group T from the second differentially protected orthogonal intramolecular bridge forming a free carboxy terminus; o) coupling the free carboxy terminus to the free amino terminus; p) extract protecting group M from the second differentially protected orthogonal intramolecular bridge to form a free amino terminus; and q) optionally adding an amino-protected amino acid to the free amino terminus and then deprotecting the amino acid to produce a new free amino terminus; and r) optionally repeating q) one or more times.
3. 3. The method according to claim 2, characterized in that it further comprises: a) extracting the amino terminal protecting group of the polypeptide bound to the solid support to form a free amino terminus b) linking a differentially protected orthogonal intramolecular bridge of the formula to the free amino terminus, where Ln represents side chains of covalently linked amino acids, wherein D, E, and G are protecting groups, each of which is selectively extracted under different reaction conditions, and wherein the reaction conditions for the extraction of the protective group D is different from that of the extraction of the amino protecting group from the amino acids of the rest of the polypeptide chain; c) extract protecting group E to form a free amino terminus; d) optionally adding an amino-protected amino acid to the free amino terminus and then deprotecting the amino acid to produce a new free amino terminus; e) optionally repeating d) one or more times; f) extract protecting group G to form a free carboxy terminus; g) attaching the free carboxy terminus of f) to the free amino terminus; h) extract protecting group D to form a free amino terminus; i) optionally adding an amino-protected amino acid to the free amino terminus and then deprotecting the amino acid to produce a new free amino terminus; j) optionally repeating i) one or more times; and k) optionally repeating steps b) -).
4. 4. The method according to claim 1, characterized in that the polypeptide comprises two intramolecular bridges.
5. 5. The method according to claim 4, characterized in that the two intramolecular bridges form two rings in series.
6. 6. The method according to claim 4, characterized in that the two intramolecular bridges form two rings in which one ring is incorporated with the other.
7. The method according to claim 2, characterized in that the differentially protected orthogonal intramolecular bridges are lanthionines.
8. 8. The method according to claim 2, characterized in that the polypeptide with intramolecular bridge is a lantibiotic.
9. 9. The method according to claim 2, characterized in that D, E, M, and Q are selected from Fmoc, Alloc, and IvDe.
10. The method according to claim 2, characterized in that G and T are selected from the group consisting of propargyl ester and benzyl ester.
11. The method according to claim 2, characterized in that the intramolecular bridges are selected from the group consisting of β-methyllanthionine (MeLan), S- [(Z) -2-Aminovinyl] -D-cysteine (AviCis), or S - [(Z) -2-Aminovinyl] -2-methyl-D-cysteine.
12. The method according to claim 8, characterized in that the lantibiotic is selected from the group consisting of Nisin A, Nisin Z, Subtilin, Ericin S, Ericin A, Streptin, Epidermin, [Val 1 -Leu 6] - epidermin, Galidermin , Mutacina 1140, Mutacina B-Ny266, Mutacina III, Mutacina I, Pep5, Epilancina K7, Epicidina 280; lant ibiotics Type A (II) Lacticin 481, Variacin, Mutacin II, Is treptococcin A-FF22, Salivaricin A, [Lys2-Phe7] -salivaricin A, Plantaricin C, Sublancin 168, But irivibriocin OR79A; Cinnamicine, Duramycin, Duramycin B, Duramycin C, Curamycin C, Ancovenin, Mersacidin, Actagardine, Ala (O) -actagardine, Subtilocin A; Lacticin 3147A1, Lacticin 3147A2, Staphylococcin C55, Is taf i lococcina 055ß, Plantaricina Wa, Plantaricina WB, Citolisina LL and Citolisina Ls.
13. 13. The method according to claim 12, characterized in that the lantibiotic is Nisin A or an analogue thereof.
14. 14. A polypeptide with intramolecular bridging, characterized in that it is formed by the method according to claim 1.
15. 15. An intramolecular bridging polypeptide, characterized in that it is formed by the method according to claim 2.
16. 16. A polypeptide with intramolecular bridging, characterized in that it is formed by the method according to claim 3.
17. 17. A differentially protected orthogonal lanthionine of the formula: characterized in that D and E are different protecting groups and are selected from the group consisting of Fmoc, Alloc, or IvDde, and G is a protective group selected from the group consisting of propargyl ester or benzyl ester.