CN101287746B - 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

Differentially protected orthogonal lanthionine technology

The cross reference of related application

The application requires the rights and interests of U.S.'s sequence number 60/708,086 of applying on August 12nd, 2005 and U.S.'s sequence number 60/808,907 of applying on May 26th, 2006, and these two document integral body are hereby incorporated by.

Background of invention

In twentieth century second half, development of antibiotics makes medical practice generation dramatic change.In this period since the mortality ratio that causes of communicable disease significantly descend.Armstrong etc., (1999) PAMA.281,61-66.Yet since nineteen eighty-two, dead soaring steadily because of what communicable disease caused, the antibiotics resistance pathogenic agent is on the increase simultaneously.The various medically important bacteriums microbiotic commonly used in the treatment of anti-further clinical infection that just becoming.At nearest 20 years, the report and the books of thousands of these phenomenons of record appearred in document.Armstrong etc., (1999) PAMA.281,61-66; Dessen etc., (2001) Curr.Drug Targets Infect.Disord.1,11-16; Rapp (2000) Surg Infect (Larchmt) .1,39-47; Benin &Dowell (2001) Antibiotic resistance and implications for the appropriateuse of antimicrobial agents, Humana Press, Totowa, NJ.

Although need to lecture antibiotic usage preferably, the more important thing is needs new antibiotic.Vancomyein is considered to the last line of defense of anti-many severe bacterial infections.The discovery of vancomyein resistance pathogenic strains is troubling; But indicating, it use the irremediable multi-drug resistance pathogenic agent of current medicament to increase.Worry that part is, only if just develop new antibiotic soon, otherwise in fact we will get back to the preceding epoch of microbiotic.

One type of microbiotic little, new texture is arranged, is called lantibiotics (I bacterioid plain), its can based on they chemical structure and biosynthesizing difference and be divided into 5 subclass: A (I) class, A (II) class, category-B, two-pack class and unknown structure class.Know such microbiotic existing decades, but still do not have the potential operability of its treatment communicable disease of extensive testing, even if known many lantibioticss not only effectively but also have an especially broad spectrum of activity of resisting gram-positive kind.The major cause of this situation is generally to be difficult to effectively to measure and obtain these molecules with enough, cost, so that they can be tested and commercialization.

Nisin A (Nisin A) (Fig. 1) provides the good example of lantibiotics, and the quantity of the chemical complexity relevant with lantibiotics and the good example of type are provided.Lantibiotics be rich in sulfur-containing amino acid-L-lanthionine (Lan, ala-S-ala), often be 3-methyl-L-lanthionine (MeLan, abu-S-ala).Lan is made up of alanine residue, and these alanine residues connect through the thioether bridging, to set up the ring structure crucial to biological activity.Typically, 3-5 such ring is arranged on lantibiotics, these rings have and manyly often overlap each other.Lan and MeLan are considered to always have the meso stereochemical structure.Except Lan with the MeLan residue; The amino acid (Fig. 2) that in lantibiotics, also can have other posttranslational modification; For example 2; 3-two dehydroalanines (Dha), 2,3-two dehydrogenation butyrines (didehydrobutyrine) (Dhb), unsaturated L-lanthionine verivate (for example S-amido vinyl-D-halfcystine (AviCys) and S-amino-D-methyl halfcystine) and D-L-Ala, 2-oxo propionyl group, 2-oxobutanoyl and hydroxy propionyl group residue.As under the situation of nisin A, the ring structure of being made up of Lan and MeLan can be eclipsed (for example encircling D and E), and this has further increased the complexity of molecule.

Gram positive bacterium is responsible for the known lantibiotics of biosynthesizing.They use a series of orderly enzymatic steps that act on rrna synthetic pre-pro-peptide to make ripe molecule.Common bunch of collection of gene of being responsible for the coding modifying enzymes is on the dna fragmentation of 8-10Kb, and this dna fragmentation can be positioned on the karyomit(e), on the plasmid or as the integral part of transposon.In A (I) type of lantibiotics; All dewatered by the enzyme of lanB genes encoding by all Serines in the rrna synthetic propetide of lanA genes encoding and threonine residues, the amino acid of these dehydrations is participated in and is more formed thioether bond near the localized contiguous cysteine residues of the carboxyl terminal of molecule.This reaction is by the albumen catalysis of lanC genetic expression.For some lantibiotics such as epidermin (epidermin) and variation rhzomorph 1140 (mutacin 1140), C-holds halfcystine by the enzyme decarboxylation of lanD genetic expression, and changes S-amido vinyl-D-halfcystine into.After being transported cell by the product of lanT gene, then the leader sequence of the pre-pro-peptide of modified is produced ripe microbiotic by the extracellular protease excision of lanP coding.Ra etc., (1996) Microbiology-Uk.142,1281-1288; Kupke & Gotz (1996) Antonie Van LeeuwenhoekInternational Journal of General and Molecular Microbiology.69,139-150; Kuipers etc., (1996) Antonie Van Leeuwenhoek InternationalJournal of General and Molecular Microbiology.69,161-169.

The lantibiotics that is difficult to obtain q.s or enough purity has hindered the research of the potentially useful property that its treatment is used to be attempted.At about 40 kinds lantibiotics (Chatterjee etc. that characterize up to now; (2005) Chemical Reviews.105; 633 683) in; Have only the nisin A of A (I) type lantibiotics-produce by streptococcus uberis (Streptococcus lactis) to find its widespread use at nearest 50 years as food antiseptics with the technical scale manufacturing.Use nisin A widely for a long time and remarkable resistance (DelvesBroughton etc. do not take place; (1996) Antonie Van Leeuwenhoek InternationalJournal of General and Molecular Microbiology.69 193-202) provides powerful impellent for developing other lantibiotics that is used for various uses.

The zymotechnique that the use of large-scale production of nisin A has been proficient in for many years carries out.The purification process of nisin A has been applied for USP (USPA 2004/0072333) recently.This method is used after the expensive proteinase mixture succeeded by column chromatography.But, nisin A purification process that do not announced, viable commercial.This has confirmed the value that current searching is produced the proper method of pure nisin A and treated other lantibiotics of usefulness.

The multiple potential selection that is used for the scale operation lantibiotics has appearred.Stand on the position of raw materials cost, zymotechnique undoubtedly should be the best way.The current amount production that is used for the fermentation process of many lantibioticss with every liter of microgram, this is not enough to drug development.

Perhaps, developed the produced in vitro of using lantibiotics modification means with regard to A (I) type of lantibiotics.Kupke and Gotz (1996) Antonie Van LeeuwenhoekInternational Journal of General and Molecular Microbiology.69,39-150; Kuipers etc., (1996) Antonie Van Leeuwenhoek InternationalJournal of General and Molecular Microbiology.69,161-169.Enzyme non-activity in acellular lysate of being responsible for posttranslational modification lantibiotics pre-pro-peptide is by the entity of purifying perhaps, but LanD is an exception.Kupke and Gotz (1996) Antonie VanLeeuwenhoek International Journal of General and MolecularMicrobiology.69,139-150; 10; Kupke and Gotz (1997) Journal ofBiological Chemistry.272,4759-4762; Kupke etc., (1992) Journal ofBacteriology.174,5354-5361; Kupke etc., (1993) Fems MicrobiologyLetters.112,43-48; Kupke etc., (1995) Journal of Biological Chemistry.270,11282-11289; Kupke etc., (1994) Journal of Biological Chemistry.269,5653-5659.For A (II) type of lantibiotics, in Science, have report recently, external synthetic lacticin 481 is possible.The molecule that belongs to this group and category-B lantibiotics only uses single bull enzyme (multiheaded enzyme) LanM to accomplish the formation of Dha, Dhb, Lan and MeLan residue.Xie etc., (2004) Science.303,679-681.Lacticin 481 biosynthetic reports do not provide any details of relevant output and purity, but their work is carried out with the nanogram scale.The technology of in this report, describing demonstrates little but significant progress, and it is praised that widely ground acceptance has further shown exploitation lantibiotics pressing for as therapeutical agent.

It is unlikely that use is cloned into that suitable expression vector selects with the 3rd of industrial-scale production lantibiotics with the lan gene cluster among the non-sensibility host, and reason is the expression of the several genes that the complicacy of system is related with needing the difference adjusting.Lan gene cluster with gallidermin was cloned in the Bacillus subtilus (Baeillus subtilis) already, attempted to improve the condition of production of this specific lantibiotics.But this strategy does not have to produce the output that greatly increases, and is inappropriate for all lantibioticss, because the known regulatory site is variable between planting and planting.Method involving uses the artificial gene of being cloned into the variation rhzomorph 1140 in the intestinal bacteria (Escherichia coli).This artificial gene has replaced participation and the halfcystine codon forms the Serine of thioether bridge and the natural codon of threonine residues.The gene clone of this modified in pET32, and is expressed in intestinal bacteria Origami bacterial strain, so that the disulfide linkage maximization.Develop new chemical process,, change them into thioether thus to evict a sulphur atom from from disulfide group.In general, this method proves feasible, but yielding poorly of being obtained, reason is the multiple arrangement of disulfide linkage and is difficult to activity form and nonactive isomer separation are opened.

To the biological activity of nisin A and other lantibiotics crucial be frequent eclipsed ring structure, these eclipsed ring structures have caused the problem that is difficult to overcome on synthetic.Broad research be used for the synthetic external compound method that contains the biologically active peptides and the lantibiotics of various L-lanthionines.The antibiotic challenge of synthetic wool sulphur is arduous, does not does not also research and develop comprehensive synthesis strategy up to now.In document, reported the method for several kinds of synthetic wool methyllanthionines.These methods comprise uses alkalescence or nucleophilic condition based on the halfcystine unit in the preparatory assembled peptide of original position desulfurization.Galande etc., (2003) Biopolymers (Peptide Science) 71,543-551; Galande & Spatola (2001) Letters in Peptide Science.8,247-251.Sulfur method does not still demonstrate any commercial viability, and reason is not have cis-selectivity and yields poorly.Also used bionic method, wherein the Dha residue produces in preformed peptide, then carries out the Michael addition, to form the L-lanthionine ring.The pre-organized of peptide possibly cause cis-selectivity Michael addition.Burage etc., (2000) Chemistry A European Journal.6,1455-1466.Also used the peptide cyclisation on the oxime resin, the wherein synthetic linear peptides that contains the L-lanthionine of orthogonally protect, then cyclisation and cracking cyclic peptide product.Melacini etc., (1997), J.Med.Chem.40,2252-2258; Osapay etc., (1997) Journal of Medicinal Chemistry.40,2441-2251.These methods have prospect, but do not have to produce the ability of the lantibiotics with overlapping thioether ring structure.This considers most of known lantibiotics particularly important that becomes when containing overlapping ring people.

From conceiving, develop external compound method, comprise that improved solid-phase peptide synthesizes (SPPS) method, than biological and bionic method remarkable advantages is arranged.At first, molecular composition is not limited to other physiological amino acid of normal group; Might design amino acid analogue and use well-established solid-phase synthesis to mix them.Can also implement parallel synthesizing, sharply increase the quantity of candidate's substrate thus.Because this method is fully in external enforcement, so eliminated many misgivings that caused by synthesis of biologically active molecule in the body.For example, product degradation can not be the misgivings factor during the fermentation, and bioactive molecules can not be the misgivings factor to production microbial cell toxic effect yet.

In order to reach external synthetic purpose, used different methods to be designed for the orthogonal lanthionine of the potential suitable protection base of having of SPPS, for example the Michael addition reaction of halfcystine forms Dha in advance.Probert etc., (1996) Tetrahedron Letters.37,1101-1104.This method produces 1: 1 mixture of diastereomer, therefore demonstrates not have commercial value basically.Also reported with shielded halfcystine to make the open loop of Serine lactone, but this produces the mixture of L-lanthionine and thioesters.Studied the open loop method of Soluol XC 100, but shown that it produces regional isomer intermixture, reason is that Soluol XC 100 breaks off at α and β position.Dugave& Menez (1997) Tetrahedron-Asymmetry.8,1453-1465; Swali etc., (2002) Tetrahedron.58,9101-9109.Most of up-to-date report prompting make the halfcystine alkylation of due care can cause the synthetic wool methyllanthionine with shielded β-bromine L-Ala, but this method does not allow to make up the molecule with overlapping ring.Zhu(2003)European Journal ofOrganic Chemistry.20，4062-4072。

Because the commercially available analogue that can be used for the Fmoc/Boc protection of SPPS is not enough to solve the challenge of the synthetic wool sulphur microbiotic and the biologically active peptides of other conformation constraint; This area has needs synthetic peptide with intramolecularly bridge; Said intramolecularly bridge is set up the inner loop structure, comprises a plurality of rings and overlapping ring structure.Specifically, need the antibiotic in vitro method of extensive synthetic wool sulphur.

Summary of the invention

Therefore, the present invention provides the method for the polypeptide of bridging in the synthetic molecules, and said polypeptide comprises at least one intramolecularly bridge, and said method comprises:

A) with the free carboxy end of the differentially protected quadrature intramolecularly bridge of following formula

With solid support or with the free ammonia cardinal extremity coupling of amino acid that randomly is combined with solid support or polypeptide, wherein L ⁿBe covalently bound amino acid side chain; Wherein D, E and G are the protection bases; Said respectively protect base all under the differential responses condition being selected property remove, basic those conditions of the reaction conditions that wherein is used to slough the basic D of protection and the amino group of amino acids protection that is used to slough the polypeptied chain rest part are different;

B) slough the basic E of protection, form the free ammonia cardinal extremity;

C) with the shielded aminoacid addition of amino to the free ammonia cardinal extremity, make said amino acid deprotection then, obtain new free ammonia cardinal extremity;

D) randomly repeat c) one or many;

E) slough the basic G of protection, form the free carboxy end;

F) with e) free carboxy end and the coupling of free ammonia cardinal extremity;

G) slough the basic D of protection, form the free ammonia cardinal extremity; With

H) randomly with the shielded aminoacid addition of amino to the free ammonia cardinal extremity, make said amino acid deprotection then, obtain new free ammonia cardinal extremity; With

I) randomly repeat h) one or many.

The present invention also provides the method for bridging polypeptide in the synthetic molecules, and said polypeptide comprises two eclipsed intramolecularly bridges, and said method comprises:

A) with first of following formula by the free carboxy end of differentially protected quadrature intramolecularly bridge

With solid support or with the free ammonia cardinal extremity covalent attachment of amino acid that randomly is combined with solid support or polypeptide, wherein L ⁿBe covalently bound amino acid side chain; Wherein D, E and G are the protection bases; Said respectively protect base all under the differential responses condition being selected property remove, basic those conditions of the reaction conditions that wherein is used to slough the basic D of protection and the amino group of amino acids protection that is used to slough the polypeptied chain rest part are different;

B) slough the basic E of protection, form the free ammonia cardinal extremity;

C) with the shielded aminoacid addition of amino to the free ammonia cardinal extremity, make said amino acid deprotection then, obtain new free ammonia cardinal extremity;

D) randomly repeat c) one or many;

E) with second of following formula by the free carboxy end of differentially protected quadrature intramolecularly bridge

With free ammonia cardinal extremity covalent attachment, wherein L ⁿAs above definition; Wherein M, Q and T are the protection bases; Said respectively protect base all under the differential responses condition being selected property remove, wherein D and M just are removed under different condition, wherein G and T just are removed under different condition; Basic those conditions of the reaction conditions that wherein is used to slough the basic M of protection and the amino group of amino acids protection that is used to slough the polypeptied chain rest part are different, and wherein E and Q are with those conditions that will slough D with will slough under those condition various conditions of M and be removed;

F) slough the basic Q of protection, form the free ammonia cardinal extremity;

G) randomly with the shielded aminoacid addition of amino to the free ammonia cardinal extremity, make said amino acid deprotection then, obtain new free ammonia cardinal extremity;

H) randomly repeat g) one or many;

I) slough first by the protection base G of differentially protected quadrature intramolecularly bridge, form the free carboxy end;

J) with free carboxy end and the coupling of free ammonia cardinal extremity;

K) slough first by the protection base D of differentially protected quadrature intramolecularly bridge, form the free ammonia cardinal extremity;

L) randomly with the shielded aminoacid addition of amino to the free ammonia cardinal extremity, make said amino acid deprotection then, obtain new free ammonia cardinal extremity;

M) randomly repeat l) one or many;

N) slough second by the protection base T of differentially protected quadrature intramolecularly bridge, form the free carboxy end;

O) with free carboxy end and the coupling of free ammonia cardinal extremity;

P) slough second by the protection base M of differentially protected quadrature intramolecularly bridge, form the free ammonia cardinal extremity; With

Q) randomly with the shielded aminoacid addition of amino to the free ammonia cardinal extremity, make said amino acid deprotection then, obtain new free ammonia cardinal extremity; With

R) randomly repeat q) one or many.

In addition, the present invention provides the method for the interior bridging polypeptide of synthetic molecules, and said polypeptide comprises two intramolecularly bridges, and wherein said two intramolecularly bridges form two series connection rings or two the embedding rings like this paper definition.The present invention also provides the synthetic method that comprises the lantibiotics of nisin A.

On the other hand, the present invention provides through this paper disclosed method synthetic intramolecularly bridging polypeptide.

Another aspect, the present invention provides the differentially protected orthogonal lanthionine of following formula:

Wherein D and E are the different protection bases, for example are Fmoc, Alloc or IvDde, and G is the protection base, for example alkynes propyl ester and benzyl ester.

Description of drawings

Fig. 1 has shown the structure of nisin A [SEQ ID NO:1], comprises between residue 7 and 10 the intramolecularly bridge of setting up ring A between the intramolecularly bridge of setting up ring B between the intramolecularly bridge of setting up ring C between the intramolecularly bridge of setting up ring D between the intramolecularly bridge of setting up ring E, the residue 9 and 12, the residue 16 and 22, the residue 24 and 27 and residue 28 and 32.Ring A, B and the C series connection ring structure of having given an example, and ring D and the E overlapping ring of having given an example.Shown that also synthetic nisin category-A is like thing [SEQ ID NO:2].

Fig. 2 has shown the amino acid whose limiting examples of posttranslational modification.

Fig. 3 has shown the retrosynthesis strategy of the L-lanthionine that is used for the manufacturing variation protection.

Fig. 4 has shown the synthesis strategy of the halfcystine of Fmoc-protection.

Fig. 5 has shown the synthesis strategy of the L-lanthionine 1 that is used for orthogonally protect, comprises the synthetic of N (Alloc)-D-β-bromine L-Ala alkynes propyl ester.

Fig. 6 has shown the synthesis strategy of the L-lanthionine 2 that is used for orthogonally protect, comprises the synthetic of N (ivdDe)-D-β-bromine alanine benzyl ester.

Detailed Description Of The Invention

Herein disclosed is the differentially protected orthogonal lanthionine technology (DPOLT) that is used for solid phase synthesis of peptide.This technology relies on the mass production of multiple orthogonally protect peptide bridge, and the pendant carboxylic group of said peptide bridge and amino protecting group can be removed by difference.It is synthetic that the peptide bridge of orthogonally protect for example can be used for solid-phase peptide, and the biologically active peptides with the constraint of preparation conformation wherein contains the intramolecularly bridge that forms ring structure.DPOLT especially can be used for the synthetic polypeptide that contains more than one intramolecularly bridge and have overlapping ring structure.

Although be not limited to this, DPOLT can carry out the baroque lantibiotics produced in vitro of (comprising those lantibioticss with overlapping ring structure) with the mode of viable commercial.The for example conventional solid-phase peptide compound method of the synthetic use of lantibiotics peptide is carried out, and this method is incorporated into the L-lanthionine analogue in the peptide, but the pendant carboxylic group of said L-lanthionine analogue and the amino protection base orthogonally protect that removes with difference.This method can be provided for for example treating the new antibiotic of application continuously.

Abb.

The following abb. that this paper uses has following implication:

■ Alloc=allyloxy carbonyl

■ Boc=tert-butoxycarbonyl

■ DMAP=Dimethylamino pyridine

■ DMF=N

■ Fmoc=9-fluorenyl methoxy carbonyl

The relevant spectrum of ■ HMBC=heteronuclear multikey

The relevant spectrum of ■ HMQC=heteronuclear volume

■ HPLC=performance liquid chromatography

■ ivDde=1-(4,4-dimethyl--2,6-dioxo-cyclohexylidene)-3-methyl-butyl

■ LC-MS=liquid chromatography-mass spectrography

■ MS=mass spectrum

■ NMR=NMR spectrum

■ NOESY=nuclear Overhauser effect spectrum

■ TFA=trifluoroacetic acid

■ TLC=thin-layer chromatography

■ TOCSY=total correlation spectrum

Intramolecularly bridging polypeptide

This paper disclosed method can be used for bridging polypeptide in the synthetic molecules, includes but not limited to lantibiotics.The term " polypeptide " that this paper uses, " albumen " and " peptide " refer to the polymer be made up of the chain that amino acid monomer is connected through amido linkage.Polypeptide can form through condensation or the linked reaction between an amino acid whose alpha-carbon carboxyl and another amino group of amino acids.Therefore, have free amine group, and have free carboxy at the end amino acid of the other end (carboxyl terminal) of chain at the end amino acid of the end (aminoterminal) of chain.Intramolecularly bridging polypeptide of the present invention randomly available various (comprising on amino and/or the carboxyl terminal) functional group or protection base are modified or protection.

Term " intramolecularly bridging peptide " or " intramolecularly bridging polypeptide " that this paper uses are meant the peptide chain with at least one intramolecularly bridge.The structure that term " intramolecularly bridge ", " peptide bridge ", " the intramolecularly bridging part " or " bridge " that this paper uses forms when being meant two amino-acid residues comprising in the single peptide chain or preparation is used for mixing single peptide chain each other through their side chain covalent attachment.This key has produced the polypeptide of internal crosslinking.The crosslink part that the term " ring " that this paper uses or " ring structure " are meant intramolecularly bridging polypeptide promptly need be between two covalently bound amino-acid residues and the structure of the covalent linkage that forms together with the side chain by them of the polypeptied chain that comprises these two covalently bound amino-acid residues.

Intramolecularly bridging peptide of the present invention has following general formula:

Wherein A is H or aminoterminal protection base; Z is H or carboxyl terminal protection base; X ⁿIt is the peptide chain of covalent linkage, single amino acids or at least 2 amino acid longs; R ⁿIt is the amino-acid residue that forms the intramolecularly bridge through its side chain.Between the side chain in single " X " peptide chain or be arranged between the amino acid of difference " X " peptide chain and can have the intramolecularly bridge in addition.

The term that this paper uses " aminoterminal protection base " and " carboxyl terminal protection base " refer to any chemical part that can add to reaction site or randomly removed by reaction site (being respectively amino and carboxyl in the case), to allow at this reaction site site operating chemical entity in addition.

The amino acid of intramolecularly bridging polypeptide of the present invention can comprise 20 kinds of natural amino acids and alpha-non-natural amino acid, amino acid analogue and peptide mimics.Spatola, (1983) are stated from Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins, Weinstein edits, Marcel Dekker, New York, the 267th page.All amino acid that use among the present invention all can be D-or L-optical isomer.In preferred embodiments; Intramolecularly bridging polypeptide of the present invention comprises the one or more following residue of arbitrary combination: 2; 3-two dehydroalanines (Dha), (Z)-2,3-two dehydrogenation butyrines (Dhb), hydroxyl propionyl group, 2-oxobutanoyl and 2-oxo propionyl group (referring to Fig. 2).

One of ordinary skill in the art will appreciate that intramolecularly bridging peptide of the present invention can have more than one intramolecularly bridge, produce various possibility structures.For example, for the intramolecularly bridging polypeptide that comprises two intramolecularly bridges, the intramolecularly bridge can be placed in-line, embedding or eclipsed as follows.

At two intramolecularly bridges is under the eclipsed situation; Between two amino acid of first intramolecularly bridge, and another amino acid of second intramolecularly bridge is before or after two amino acid of first intramolecularly bridge in the one-level aminoacid sequence for an amino acid that means second intramolecularly bridge.At two intramolecularly bridges is under the placed in-line situation, means that two amino acid of second intramolecularly bridge are positioned in the one-level aminoacid sequence before or after two amino acid of first intramolecularly bridge.At two intramolecularly bridges is under the situation about embedding, two amino acid that mean second intramolecularly bridge in the one-level aminoacid sequence between two amino acid of first intramolecularly bridge.

Have under the situation of 3 above intramolecularly bridges at intramolecularly bridging peptide, can form more a large amount of possible structures.For example can there be a plurality of overlapping rings.In limiting examples, intramolecularly bridging polypeptide can have 5 intramolecularly bridges, wherein has 2 to form overlapping ring structure in 5 bridges, and 3 remaining bridges are one another in series or connect with overlapping ring.Lantibiotics nisin A presents such structure (referring to Fig. 1).

In preferred embodiments, intramolecularly bridging polypeptide of the present invention is the lantibiotics peptide.In a more preferred embodiment, intramolecularly bridging polypeptide of the present invention is nisin A and analogue thereof.

Differentially protected quadrature intramolecularly bridge

The intramolecularly bridge of orthogonally protect of the present invention has following general formula:

Wherein L represents covalently bound amino acid side chain, and D and E are hydrogen or aminoterminal protection base, and G and J are hydrogen or carboxyl terminal protection base.

The key that contains amino acid side chain can be but is not limited to thioether bond, disulfide linkage, amido linkage or ehter bond.In preferred embodiments, the intramolecularly bridge comprises thioether bond.

In polypeptide is synthetic, mix " differentially protected " or " orthogonally protect " intramolecularly bridge the selectively removing to their protection base is provided, the removing of the protection base on other part of this selectively removing and peptide chain (comprising other intramolecularly bridge) separated and had nothing to do.In other words, select the protection base of bridge in the specific molecular, make their cracking condition not damage other protection base or the stability of functional group on the polypeptide.Cross reactivity in the deprotection process of these groups is minimum, can be through the monitoring of standard mass-spectrometric technique.Title product can come with these impurity purifying through standard HPLC or other technology.The priority that cracking can be selected is arbitrarily implemented.

The protection base for example is described in " Protective Groupsin Organic Chemistry, " Plenum Press, London, N.Y.1973 with the mode that they are introduced into and remove; " Methodender organischen Chemie, " Houben-Weyl, the 4th edition, the 15/1st volume, Georg-Thieme-Verlag, Stuttgart 1974; And Theodora W.Greene, " Protective Groups in Organic Synthesis, " John Wiley & Sons, New York1981.Many protections base is characterised in that they for example can promptly not have undesirable side reaction to take place through solvolysis, reduction, photolysis, through using organo-metallic catalyst such as organic palladium and organic cobalt catalyst or under physiological condition, easily being removed.

Numerous protection bases are known in the art.Nonrestrictive illustrative protects basic list to comprise methyl, formyl radical, ethyl, ethanoyl, the tertiary butyl, anisyl, benzyl, trifluoroacetyl group, N-hydroxy-succinamide base, tert-butoxycarbonyl, benzoyl-, 4-methyl-benzyl, sulfo-anisyl (thioanizyl), sulfo-tolyl, benzyloxymethyl, 4-nitrophenyl, benzyloxycarbonyl, 2-nitro benzoyl, 2-nitrophenyl sulfinyl, 4-tosyl group, pentafluorophenyl group, diphenyl methyl, 2-chlorine benzyloxycarbonyl, 2; 4; 5-trichlorophenyl, 2-bromo-benzyloxy-carbonyl, 9-fluorenyl methoxy carbonyl, trityl group and 2; 2; 5; 7,8-pentamethyl--chroman-6-alkylsulfonyl.About the argumentation of the amino and the carboxyl-protecting group of number of different types, referring to for example No. the 5th, 221,736, USP (on June 22nd, 1993 granted patent); USP the 5th, 256, No. 549 (on October 26th, 1993 granted patent); USP the 5th, 049, No. 656 (on September 17th, 1991 granted patent); With USP the 5th, 521, No. 184 (on May 28th, 1996 granted patent).

Said protection base can use the protection base of any combination, as long as can optionally be removed in the bridging polypeptide building-up process in target molecule.In a preferred embodiment, aminoterminal protection base is selected from Fmoc, Alloc and IvDde.In a further preferred embodiment, carboxyl terminal protection base is selected from alkynes propyl ester and benzyl ester.

In preferred embodiments, the intramolecularly bridge of orthogonally protect is the L-lanthionine or the L-lanthionine verivate of orthogonally protect.In a more preferred embodiment, the intramolecularly bridge of orthogonally protect is L-lanthionine (Lan), Beta-methyl L-lanthionine (MeLan), S-[(Z)-2-amido vinyl]-D-halfcystine (AviCys) or S-[(Z)-2-the amido vinyl]-2-methyl D-halfcystine (referring to Fig. 2) of aminoterminal and/or carboxyl terminal protection.The intramolecularly bridge of these orthogonally protects can be synthetic through methods known in the art.

In a more preferred embodiment, the intramolecularly bridge is a L-lanthionine.Protected L-lanthionine can be as using ordinary method synthetic shown in the retrosynthesis among Fig. 3.The stereochemical structure of L-lanthionine product can begin through the correct steric isomer with suitable amino acid (for example halfcystine and Serine) and guaranteed in this stage.

In a more preferred embodiment, the intramolecularly bridge is L-lanthionine 1 or L-lanthionine 2:

L-lanthionine 1 L-lanthionine 2

It can respectively as summarize synthetic in Fig. 5 and 6.In brief, with reference to Fig. 5, for L-lanthionine 1, the D-Serine is changed into the protected Alloc verivate of its aminoterminal, changes the protected alkynes propyl ester of carboxyl terminal subsequently into.N (Alloc)-D-Serine alkynes propyl ester is changed into its corresponding β-bromine alanine derivatives.Change and for example can realize as follows: N (Alloc)-D-Serine alkynes propyl ester is dissolved in the methylene dichloride, and handles this solution with 1 normal carbon tetrabromide and triphenyl phosphine.These reactions be non-normal temperature and, be used for routinely changing hydroxyl into bromide.Zhu(2003)European Journal of Organic Chemistry.20，4062-4072。Perhaps, use the phosphorus tribromide in solvent such as toluene or methylene dichloride to realize synthesizing, then carry out gentle alkaline aftertreatment, to obtain the D-β-bromine L-Ala of expectation.Olah etc., (1980) Journal of Organic Chemistry.45,1638-1639.Also can use other method.At last, β under suitable alkylation conditions-bromine alanine derivatives and Fmoc-L-Cys reaction generate L-lanthionine 1.L-lanthionine 2 can be synthetic similarly like general introduction in Fig. 6.

Synthesizing of intramolecularly bridging polypeptide

Intramolecularly bridging polypeptide of the present invention can be synthetic through any means that provides for the intramolecularly bridge of using and mix orthogonally protect, and said method includes but not limited to that solid-phase peptide is synthesized (SPPS), solution phase peptide is synthetic, native chemical connects, the albumen of intein mediation connects with chemistry and is connected or its combination.In preferred embodiments, it is synthetic that intramolecularly bridging polypeptide of the present invention uses the standard SPPS of improved form.Intramolecularly bridging polypeptide of the present invention can be through artificial SPPS or through using the commercially available robotization SPPS synthesizer that gets synthetic.

SPPS has been exactly known (Merrifield, R.B., J.Am.Chem.Soc., 85:2149-2154,1963) in this area since the sixties in 20th century is early stage, and is widely used.Universal method has several known variations.(referring to for example " Peptide Synthesis; Structures; andApplications " 1995 by Academic Press; The 3rd Zhanghe White (2003) FmocSolid Phase Peptide Synthesis; A practical Approach, Oxford UniversityPress, Oxford).Say very simply, in solid-phase peptide is synthetic, the C-terminal amino acid residue and the solid support coupling of expectation.Follow-up amino acid to peptide chain to be added is protected by Boc, Fmoc or other suitable protection base on its aminoterminal, and its carboxyl terminal is with the activation of standard coupling reagent.Make the reaction of amino acid whose free ammonia cardinal extremity of support bonded and follow-up amino acid, again two amino acid are coupled at together.The aminoterminal of growthing peptide chain is repeated this process by deprotection, until the polypeptide of accomplishing expectation.

According to method of the present invention, intramolecularly bridging peptide can synthesize through differentially protected quadrature intramolecularly bridge is incorporated among the standard SPPS.The polypeptied chain part that is not the integral part of intramolecularly bridge can be synthetic through standard SPPS technology known in the art.In preferred embodiments, use by the amino acid of Fmoc-or Boc-protection, in a more preferred embodiment, is used the SPPS based on Fmoc at aminoterminal.The protection base of sloughing its active amino and carboxyl through selectivity is incorporated into differentially protected quadrature intramolecularly bridge in the polypeptied chain.

Method of the present invention can be used for synthetic intramolecularly bridging polypeptide with bridge in the individual molecule shown in general formula III:

Wherein A, X ⁿAnd R ⁿAs before formula I being defined.This polypeptide uses the interior bridge preparation of the individual molecule of general formula I V:

Wherein L represents covalently bound amino acid side chain, and D and E are aminoterminal protection bases, and G is a carboxyl terminal protection base.

Say that simply the intramolecularly bridge is through its free carboxy end and the peptide chain coupling that is connected with solid support, or direct and solid support coupling.Other amino acid is in intramolecularly bridge deprotection (sloughing E) back and its free ammonia cardinal extremity coupling.Slough the protection base (G) on all the other carboxyls of intramolecularly bridge, with of the free ammonia cardinal extremity coupling of this carboxyl with the polypeptied chain that so forms.Other amino acid randomly can be added on the remaining amino subsequently.

In polypeptide of the present invention is synthetic, on the polypeptied chain one " free ammonia cardinal extremity " will be only arranged all and treat and one of free ammonia cardinal extremity link coupled " free carboxy end " increasing at any one time.When adding amino acid again with its deprotection, initiate amino acid all will seal the free ammonia cardinal extremity, if initiate amino acid subsequently by deprotection, then will form new free ammonia cardinal extremity.Those skilled in the art will recognize that a free ammonia cardinal extremity is only arranged in these cases.

More particularly, in having individual molecule, in the intramolecularly bridging polypeptide of bridge synthetic, select D, make the reaction conditions that is used to slough the basic D of protection can not cause sloughing of E or G, and/or the sloughing of the amino group of amino acids of polypeptied chain rest part protection base.Opposite situation also is suitable for.In other words, as limiting examples,, then select D, under the condition of not sloughing E, G and/or Fmoc so that it can being selected property remove if use synthetic polypeptide based on the SPPS of Fmoc.Similarly, select D and G, make the condition of sloughing Fmoc can not cause the cracking of D or G.In preferred embodiments, amino protecting group E is equal in the polypeptied chain amino group of amino acids protection base of the integral part that is not the intramolecularly bridge.Therefore, for example under the situation of use based on the SPPS of Fmoc, E is preferably Fmoc.

The synthetic coupling with C-terminal amino acid and solid support of intramolecularly bridging polypeptide begins.Term " solid support " refers to any solid phase material of synthetic polypeptide above that.Solid support comprises term like " resin ", " solid phase " and " support ".Solid support can be made up of organic polymer such as PS, Vilaterm, Vestolen PP 7052, PVF, T 46155 and SEPIGEL 305 and multipolymer thereof and graftomer.Solid support also can be inorganic, for example has glass, silica, controlled pore glass (CPG) or the reverse phase silica gel of suitable group, and amino acid can be connected to said suitable group and be removed in an easy manner.The configuration of solid support can be the form on pearl, ball, particle, particulate or surface.The surface can be plane, general plane or on-plane surface.Solid support can be foraminous or atresia, and can have swelling or non-swollen characteristic.Solid support can be the form of hole, pit or other container.Most solid supports can transmit the addressable array format of reagent by robot, perhaps can be according to the detection method setting, and said detection method comprises with laser radiation scanning and copolymerization Jiao or deflection optically focused.Many solid supports are commercial.The coupling of first amino acid and solid support can be through mensuration monitoring performance known in the art.

In preferred embodiments, Fmoc amino acid is used for synthetic polypeptied chain.Fmoc amino acid is commercial, perhaps can be synthetic through methods known in the art.Other amino acid can use standard SPPS method to be added on the polypeptied chain.For example, using under the amino acid whose situation of Fmoc, the Fmoc amino protecting group of C terminal amino acid in case with the resin coupling, just can be sloughed through the DMF solution that for example contacts 20% piperidines.Standard coupling chemistry be can adopt, next Fmoc amino acid and polypeptied chain coupling made.Amino acid with active side chain can protect with suitable protection base, so be still shielded in the middle of whole the synthesizing of their side chain bridging polypeptide in target molecule.Coupling and deprotection steps can use suitable amino acid to repeat as required.This has accomplished the X of general formula III ³Synthetic.

Through standard coupling chemistry with the intramolecularly bridge with increase the polypeptied chain coupling.Perhaps, if the intramolecularly bridge drops on the C-terminal of intramolecularly bridging polypeptide, then the intramolecularly bridge can the direct and resin coupling through its free carboxy.Selectivity is sloughed the basic E of protection under suitable condition then, for example uses the DMF solution of 20% piperidines, and wherein E is Fmoc.With reference to general formula III, R ²Now and the polypeptied chain coupling.One or more amino acid can be added into the polypeptied chain (X of general formula III through sequential coupling and deprotection subsequently ²) on.

Then, selectivity is sloughed the basic G of protection under suitable condition.In preferred embodiments, G is the dichloromethane solution cracked propargyl that can use cobalt octacarbonyl, or can use method for hydrogenation cracked benzyl ester, and said method for hydrogenation uses the dichloromethane solution of palladium carbon and cyclohexadiene.This has accomplished the R of general formula III ¹Interpolation, be incorporated in the polypeptide the intramolecularly bridge is complete thus, form ring structure.

Can under suitable condition, optionally slough the basic D of protection then.In preferred embodiments, D can use 20mol%Pd (PPh ₃) ₄With 20-25 equivalent PhSiH ₃Dichloromethane solution cracked Alloc, or can be by the DMF solution cracked ivDde of 2-10% hydrazine.Intramolecularly bridging polypeptide can prolong (the X in the general formula III with its deprotection through the other amino acid of sequential coupling subsequently again ¹).

Can use synthetic similarly the intramolecularly bridging polypeptide of single differentially protected intramolecularly bridge, promptly have the intramolecularly bridging polypeptide of more than one intramolecularly bridge with a plurality of series connection rings.Randomly, each other difference only is that the differentially protected intramolecularly bridge more than of their protection base can be used for synthetic polypeptide with a plurality of rings.The variable a plurality of differentially protected intramolecularly bridge of their side-chain structure (for example Lan and MeLan) also can be used for mixing different intramolecularly bridging parts.Protection base on these follow-up bridges can be identical or different with the protection base on first intramolecularly bridge that is incorporated in the polypeptied chain.Intramolecularly bridging polypeptide with a plurality of series connection rings is synthetic as follows: be incorporated in the polypeptied chain first intramolecularly bridge is complete; Form first ring structure, slough terminal amino group protection base, randomly again its deprotection is prolonged polypeptied chain through the other amino acid of sequential coupling; Intactly mix second intramolecularly bridge (identical or different) through its carboxyl terminal with first intramolecularly bridge; Randomly prolong polypeptide, and repeat these steps as required, with bridging polypeptide in the synthetic target molecule.

A plurality of overlapping or embed the intramolecularly bridging polypeptide of ring for having, must use the intramolecularly bridge of an above orthogonally protect.Although the side-chain structure of the intramolecularly bridge of a plurality of orthogonally protects can be identical or different, the protection base must be by difference ground orthogonally protect, to allow optionally to make their amino and carboxyl deprotections separately.The quantity of these bridges depends on overlapping or embeds the quantity of encircling.For example, two rings of intramolecularly bridging polypeptide overlap each other or situation about being embedded in another under, use two different differentially protected quadrature intramolecularly bridges; For example 3 rings overlap each other or situation about embedding each other under, use 3 different differentially protected quadrature intramolecularly bridges, like that.

In limiting examples, the bridging polypeptide comprises under the situation of two overlapping rings in target molecule, uses two differentially protected quadrature intramolecularly bridges of general formula V and VI:

L wherein ¹And L ²Be covalently bound amino acid side chain (L ¹Can with L ²Identical or different), D, M, E and Q are aminoterminal protection bases, and G and T are carboxyl terminal protection bases; Wherein D and M only can be by cracking under different condition; Wherein E and Q can be under the same conditions by cracking; Wherein E and Q under those conditions that are different from cracking D and cracking M by cracking; Wherein G and T under different condition just by cracking.In preferred embodiments, amino protecting group E and Q are equal in the polypeptied chain amino group of amino acids protection base of the integral part that is not the intramolecularly bridge.Therefore, for example under the situation of use based on the SPPS of Fmoc, E and Q are preferably Fmoc, but are not limited to this.In this case, E and Q also can be for example Boc.

According to method of the present invention, the intramolecularly bridging polypeptide that comprises two overlapping rings can be through at first synthesizing C-terminal amino acid and solid support coupling.Randomly, can use standard SPPS method with other aminoacid addition to polypeptied chain.In preferred embodiments, Fmoc amino acid is used for synthetic polypeptied chain.Amino acid with active side chain can be used suitable protection base protection, and their side chain is still protected in bridging polypeptide synthetic in whole target molecule like this.The step of coupling and deprotection can use suitable amino acid to repeat as required.Intramolecularly bridge with general formula V passes through its free carboxy and growthing peptide chain coupling then, sloughs E subsequently.D and G remain unaffected.In preferred embodiments, E is Fmoc.Randomly carry out cyclisation then through coupling and the deprotection steps of secundum legem SPPS, can with one or more amino acid sequentially with the free ammonia cardinal extremity coupling of polypeptide.Then, the intramolecularly bridge of general formula VI through its free carboxy and growthing peptide chain coupling, is sloughed Q subsequently.D, G, M and T remain unaffected.In preferred embodiments, Q is Fmoc.Moreover, then randomly can with one or more amino acid sequentially with the free ammonia cardinal extremity coupling of polypeptide.For forming first ring, in being to use suitable deprotection chemical cracking G, with the free carboxy that produces and the free ammonia cardinal extremity coupling of polypeptied chain.Protect basic D, M and T to remain unaffected.Subsequently, under suitable condition, slough the basic D of protection, expose free amine group.Basic M of protection and T remain unaffected in sloughing the process of D.Then randomly can be with other amino acid and the free amine group coupling that is positioned at polypeptide N-end.For forming second ring, and form overlapping ring thus, cracking T under suitable condition is with the free ammonia cardinal extremity coupling of free carboxy that is obtained and polypeptied chain.Can under suitable condition, slough the basic M of protection then, and further prolong polypeptied chain through the other amino acid of sequential coupling.

According to method of the present invention, comprise two intramolecularly bridging polypeptide that embed ring and can use two differentially protected quadrature intramolecularly bridges of general formula V and VI synthetic similarly.Comprise two synthesizing of intramolecularly bridging polypeptide and synthesizing quite of the intramolecularly bridging polypeptide that comprises two overlapping rings that embed ring, difference only is the order of the intramolecularly bridge of deprotection and coupling formula V and VI.Specifically, with the free ammonia cardinal extremity coupling of intramolecularly bridge and the peptide chain of formula V, peptide chain is connected with solid support through its carboxyl terminal, perhaps with the intramolecularly bridge of formula V directly and the solid support coupling.Excise E subsequently, then randomly coupling through secundum legem SPPS and deprotection steps can with one or more amino acid sequentially with the free ammonia cardinal extremity coupling of polypeptide.Then, the intramolecularly bridge of general formula VI through its free carboxy and growthing peptide chain coupling, is sloughed Q subsequently.Moreover, then randomly can with one or more amino acid sequentially with the free ammonia cardinal extremity coupling of polypeptide.For forming first ring, in being to use suitable deprotection chemical cracking T, with the free ammonia cardinal extremity coupling of free carboxy that is obtained and polypeptied chain.Subsequently, under suitable condition, slough the basic M of protection, expose free amine group.Then randomly can be with other amino acid and the free amine group coupling that is positioned at polypeptide N-end.For forming second ring, and form thus and embed ring, under suitable condition, excise G, the free ammonia cardinal extremity coupling of free carboxy that is obtained and polypeptied chain.Can under suitable condition, slough the basic D of protection then, and further prolong polypeptied chain through the other amino acid of sequential coupling.

Those of skill in the art will recognize that and to prepare more complicated molecule through the variation of above method similarly.For example, having method that the polypeptide of two overlapping rings and other 3 series connection rings can be through will disclosedly being used for the synthetic intramolecularly bridging polypeptide that contains overlapping ring is used for synthetic method with intramolecularly bridging polypeptide of the ring of connect and makes up synthesizing with disclosed.

In the middle of intramolecularly bridging polypeptide synthetic, the synthetic progress randomly can be monitored through multiple technologies known in the art with precision, include but not limited to Maldi and LC-MS.When synthetic the completion, intramolecularly bridging polypeptide under appropriate condition from the solid support under the cracking.Contain at the synthetic polypeptide under the situation of sulphur of significant quantity (for example for the polypeptide that contains L-lanthionine), can use the mixture of TFA/ thioanisole/water/phenol/dithioglycol (82.5/5/5/5/2.5).The progress of scission reaction can be passed through LC-MS or other suitable technique periodic monitoring.According to selected Side chain protective group, can the process of cracking polypeptide from resin or with independent step, realize their cracking.End product for example can pass through by the cold diethyl ether precipitate and separate, and includes but not limited to the reversed-phase HPLC purifying through currently known methods.

Can analyze intramolecularly bridging polypeptide of the present invention and analyze its biochemical function from structure through known technology.Structural analysis can realize through the technology that includes but not limited to two-dimentional NMR and X-radiocrystallography.Used two-dimentional NMRTOCSY (the Braunschweiler & Ernst (1983) that obtains with the 60ms mixing time; Journal of Magnetic Resonance53,521-528) and the NOESY that obtains with 200ms, 400ms, 450ms successfully analyzed intramolecularly bridging polypeptide from structure.Kumar etc., (1980), Biochem.Biophys.Res.Commun.95,1-6.Smith，J.L(2002)Dissertation，University of Florida，Gainesville。Smith etc., (2000), European Journal of Biochemistry 267,6810-6816.

In preferred embodiments, method of the present invention is used for the synthetic intramolecularly bridging polypeptide that comprises one or more L-lanthionines or L-lanthionine verivate.In a more preferred embodiment, the inventive method is used for synthetic wool sulphur microbiotic.In a more preferred embodiment, the inventive method is used for synthetic nisin A and analogue thereof.

Can use currently known methods to measure biological activity (Hillman etc., (1984), Infection and Immunity 44, the 141-144 of nisin A and analogue thereof; Hillman etc., (1998), Infection and Immunity 66,2743-2749).Can through with the three-dimensional structure (1991 of the nisin A that produces with biological process that had before confirmed through NMR by Van De Yen etc.; European Journal of Biochemistry 202; 1181-1188) contrast, auxiliary structural analysis through the inventive method synthetic nisin A and analogue thereof.Morning, covalent structure was confirmed the amino acid arrangement that work is made according to this, possible fast characterizing covalent linkage, and differentiate that all relevant length apart from NOE, are used for through the structure of the inventive method synthetic nisin A and analogue thereof definite.

The DPOLT The Application of Technology

DPOLT is a kind of platform technology that is produced by multidisciplinary method.There are several advantages that this technology is so catered to the need.At first and most important, its candidate's lantibiotics that can synthesize remarkable quantity fast is with other biologically active peptides and screen their potential application in the treatment field, and needn't devote considerable time and expense designs fermentation and purification process for analyzing them.The lantibiotics that contains overlapping thioether bridge near 50 kinds is arranged, and more come to light every year in addition, and they all can be synthetic through this paper disclosed method.These lantibioticss comprise A (I) type of lantibiotics nisin A, nisin Z, subtilyne, Quercetin S (Ericin S), Quercetin A, streptin, epidermin, [Vall-Leu6]-epidermin, Gallidermin, variation rhzomorph 1140, variation rhzomorph B-Ny266, variation rhzomorph III, variation rhzomorph I, Pep5, Epilancin K7 and Epicidin 280; A (II) class lantibiotics lacticin 481, Variacin, variation rhzomorph II, streptostacin (Streptococcin) A-FF22, SalivaricinA, [Lys2-Phe7]-salivaricin A, the plain C of plant lactobacillus, Sublancin 168, Butyrivibriocin OR79A; Category-B lantibiotics cinnamycin, Moli 1901, Moli 1901 B, Moli 1901 C, curamycin C, ancovenin, Mersacidin, actagardin (Actagardine), Ala (0)-actagardin and Subtilocin A; Two-pack lantibiotics lacticin 3147A1, lacticin 3147A2, staphylococcin C55 α, staphylococcin C55 β, the plain W α of plant lactobacillus, the plain W β of plant lactobacillus, cytolysin L _LWith cytolysin L _SWith other lantibiotics, for example Ruminococcin A, Carnocin UI 49, Macedocin, Bovicin HJ50, NukacinISK-1 and SapB morphogen.(referring to for example Chatterjee etc., 2005.Chem.Rev.105,633-83).

According to the experience in past, as if might be that many fermentations and the purification process that is used for multiple lantibiotics can not obtain fast.The nisin A that found before more than 50 year is still the target of further investigation, so that find quick and suitable purification process, is used for its exploitation as therapeutical agent.Up-to-date U.S. Patent application (U.S. Patent application 2004/0072333) is attempted reaching this target, but has used proteolytic enzyme and a plurality of purification step of multiple costliness.The SPPS method that DPOLT uses is very likely far to realize re-set target as the cost efficient manner.At present, surpassing 35 kinds, to use SPPS method synthetic bioactive moleculess be commercial, for example pitocin, kind peaceful (sandostatin) and Fu Zeang (fuzeon), and demand can increase undoubtedly as time passes.The use of DPOLT allows locus specificity replacement amino acid and analogue thereof, even if in the combinatorial library method, this method provides discovery to be used for the best approach of the therapeutical agent new and improvement of its re-set target.Thus, DPOLT is used for synthetic unique prior art with molecule of overlapping ring, has except that lantibiotics, also to prepare the multiple potentiality that are used for the bioactive molecules of various application.DPOLT can the for example baroque lantibiotics of produced in vitro (comprising those lantibioticss with overlapping ring structure), makes with the mode of viable commercial to use conventional solid-phase peptide compound method.

DPOLT provides two kinds of significant advantages of the new lantibiotics of screening and exploitation commercial applications: stand in obviously preferred fermentation method on the position of production material cost, but possibly make us hanging back in the time and efforts that the starting stage of drug discovery is optimized these methods and needs.In addition, resemble with regard to nisin A, possibly be not easy to realize the purifying of high yield fermented product.The purifying of end product is not prominent question usually in SPPS.DPOLT has a large amount of potentially useful compounds are screened in permission with the immediate mode that is used for Clinical Laboratory advantage.For seeming promising compound, DPOLT provides the shortcut that leads to market, has also indicated the molecule that those time and efforts that possibly be fit to provide essential are developed fermentation method.For the compound of the essential characteristic that is not used in further exploitation, for example have those compounds of bad activity profile, defective pharmacokinetics, toxicity problem etc., DPOLT allows fast and eliminates effectively the misgivings to these.At last, because DPOLT relies on solid-phase peptide synthetic, will be simple so screening and exploitation have the analogue of improved characteristics (overcoming the characteristic of bacterial resistance like those).Therefore, this method can be applicable to other lantibiotics and target peptide, and differentiate have expect on the function with the lantibiotics and the target peptide of favourable characteristic economically.

DPOLT and be medical science and the veterinary science treatment of infectation of bacteria through the most important purposes of the inventive method synthetic lantibiotics.Also have several kinds of other potential application.Lantibiotics is fully confirming and attractive substituting other antiseptic-germicide that uses in food antiseptic and the makeup.DelvesBroughton etc., (1996) Antonie Van LeeuwenhoekInternational Journal of General and Molecular Microbiology.69,193-202; Rollema etc., (1995) Applied and Environmental Microbiology.61,2873-2878; Liu & Hansen, (1990) Applied and EnvironmentalMicrobiology.56,2551-2558; Huot etc., (1996) Letters in AppliedMicrobiology.22,76-79; Delvesbroughton, (1990) Food Technology.44,100; Delvesbroughton (1990) Journal of the Society of Dairy Technology.43,73-76; Delvesbroughton etc., (1992) Letters in Applied Microbiology.15,133-136; Thomas & Wimpenny (1996) Applied and EnvironmentalMicrobiology.62,2006-2012; Sahl & Bierbaum (1998) Annual Review ofMicrobiology.52,41-79.In addition, to lantibiotics as partly sterilised's agent, especially study as the collutory that promotes oral Health, obtain certain achievement.Howell etc., (1993) Journal of Clinical Periodontology.20,335-339.

Lantibiotics class medicine has very big potentiality, is most possibly fully accepted by medical circle.As long as then the market of application of antibiotics is just still very high and will keep so for disease although be infectious, most of antibiotic whole life cycle is of short duration, and reason is to suddenly change and bacterial resistance.The benefit of lantibiotics class antibiotic medicine is that they have the validation record that relative antibacterium adapts to, and has come to light numerous anti-other antibiotic bacterial pathogens are had and effectively kill bacterial activity.

All patents that this paper anywhere mentions, patented claim and other science or technological document all integral body are incorporated herein by reference.The method and composition that this paper describes as present representational preferred embodiment is exemplary, the intention of the unrestricted scope of the invention.Variation wherein it will be apparent to those skilled in the art that with other purposes, and is included in the spirit of the present invention.The present invention that this paper illustrative is described can implement under the situation that does not have this paper concrete not disclosed any one or a plurality of key element, one or more restrictions aptly.Therefore, for example, under every kind of situation of this paper, term " comprises ", " basically by ... form " with " by ... form " in any all available all the other two term in any replacement, and do not change its common implication.Term and the statement used are used as descriptive term; Unrestricted effect; And be not intended to use these terms and statement get rid of shown in any equivalents thereto of institute's characteristic of describing or its part, but what will recognize is that multiple modification possibly fall in the scope of the invention that requirement protects.Therefore, should be understood that although the present invention is open particularly by embodiment and optional feature, the modification of the disclosed design of this paper and change are considered to fall in the specification sheets and the scope of the invention that claims limited of enclosing.

In addition, in the time can selecting key element to describe characteristic of the present invention or aspect, those skilled artisans will appreciate that each member or each member group in Ma Kushi key element or other key element is similarly integral part of the present invention according to Ma Kushi key element or other.

Can understand the present invention better according to following examples, these embodiment only are used for illustration purpose, shall not be construed as and limit scope of the present invention by any way.

Embodiment

Embodiment 1: differentially protected orthogonal lanthionine synthetic

A.Fmoc-Cys's is synthetic

The halfcystine (Fig. 3, structure B) of Fmoc-protection is synthetic by the L-Gelucystine with two steps order like general introduction in Fig. 4.(4.6g, 43.6mmol) (5.0g 20.8mmol) is dissolved in the water (200mL) with the L-Gelucystine with yellow soda ash.The solution that is obtained is cooled to 10 ℃.(11.85g 45.8mmol) is dissolved in the diox (80mL), the drips of solution that is obtained is added to the aqueous solution of L-Gelucystine with FmocCl.Stirred these solution 2 hours in 10 ℃, and make it be warming up to room temperature gradually.Obtain the thickness white precipitate, it is filled on the sintered glass funnel.Product grinds with ether (50mL), and vacuum-drying 2 days.Obtain N, N '-two (Fmoc)-L-Gelucystine (14.0g, yield 98%) for white powder.

With N, (12.0g 17.5mmol) is dissolved in the methyl alcohol (300mL) N '-two (Fmoc)-L-Gelucystine.(12.0g) adds this solution with mossy zinc, the mixture that uses the magnetic stirring apparatus vigorous stirring to be obtained.(75mL 1mol) is added dropwise in the reaction mixture, in 12 hours time period of stirring at room with trifluoroacetic acid in 2 hour time period.Through C-18 anti-phase HPLC (HPLC) and thin-layer chromatography (TLC, chloroform/methanol/acetate=30: 1: 0.1, volume) monitoring reaction.At N, when N '-two (Fmoc)-L-Gelucystine disappears, filter reaction mixture, and on rotatory evaporator, concentrate, volume is reduced to about 100mL.Add methylene dichloride (400mL).With 2N aqueous hydrochloric acid purging compound.Water layer is used dichloromethane extraction, and the organic layer of merging is through dried over mgso.Enriching soln obtains N-(the Fmoc)-L-halfcystine (8.8g 73%) (Fig. 3 and 4, structure B) into white powder.

Synthesizing of B.N-(Alloc)-D-Serine alkynes propyl ester

Synthetic carry out as follows (referring to Fig. 5) of N-(Alloc)-D-Serine alkynes propyl ester (Fig. 3, structure A).With the D-Serine (10.5g, 100mmol) and yellow soda ash (11.1g 105mmol) is dissolved in the water (100mL).(50mL) joins in this solution with acetonitrile, and mixture is cooled to 5 ℃ in ice bath.In 30 minutes time period, drip allyl chlorocarbonate (11.7mL, 13.3g, 110mmol).Reaction mixture is warming up to room temperature gradually, and stirred 12 hours.Vacuum concentrated mixture to remove acetonitrile, is cooled to 0-5 ℃ with resistates to about 100mL.Through adding the pH regulator to 2.0 of concentrated hydrochloric acid aqueous solution (about 10mL) with solution.(5 * 40mL) extraction products, extract is through anhydrous magnesium sulfate drying with ETHYLE ACETATE.Solvent removed in vacuo on rotatory evaporator obtains being revealed as N-(the Alloc)-D-Serine (16.9g, 89%) of light yellow oil.

(16g 85mmol) is dissolved among the DMF (70mL) with N-(Alloc)-D-Serine.(7.9g 94mmol) adds gained solution with sodium hydrogencarbonate.In 20 minutes time period in room temperature dripping bromine propine (80% toluene solution, 10.5mL, 94mmol).Reaction mixture was in stirring at room 2 days.Vacuum concentration reaction mixture on rotatory evaporator is dissolved in resistates in the ETHYLE ACETATE (100mL).With sodium bicarbonate aqueous solution (2 * 50mL) and water (2 * 50mL) washing solns, and through dried over mgso.Solvent removed in vacuo on rotatory evaporator obtains N-(Alloc)-D-Serine alkynes propyl ester (18g, yield 93%).

Synthesizing of C.N-(ivDde)-D-Serine (benzyl) ester

Prepare N-(ivDde)-D-Serine (Fig. 3 by D-Serine and ivDde-OH; Structure C), through in the presence of pyridine, methone O-acidylate being synthesized, then use previous reported method to make formed 3 Methylbutanoic acid 5 with aluminum chloride with isoveryl chloride; 5-dimethyl--3-oxo hexamethylene-1-alkenyl esters is reset (Akhrem; A.A. etc., Synthesis 1978,925).Specifically, in 15 minutes time period with isoveryl chloride (13.5mL, 13.3g, methylene dichloride 110mmol) (50mL) drips of solution add to stirring methone (14g, 100mmol) and pyridine (9.7mL, 9.5g, methylene dichloride 120mmol) (150mL) solution.Stirred reaction mixture 1.5 hours, and with the 2N aqueous hydrochloric acid (2 * 50mL), the washing of water and saturated sodium bicarbonate aqueous solution (50mL), then through dried over mgso.Through the rotatory evaporator solvent removed in vacuo, obtain being revealed as the 3 Methylbutanoic acid 5 of faint yellow oily thing, 5-dimethyl--3-oxo hexamethylene-1-alkenyl esters (22.4g, yield 100%).In 30 minutes time period, (16.0g in methylene dichloride 120mmol) (100mL) stirred suspension, drips 3 Methylbutanoic acid 5,5-dimethyl--3-oxo hexamethylene-1-alkenyl esters (11.2g, 50mmol) solution to refrigerative aluminum chloride on ice bath.Make reaction mixture be warming up to room temperature, and stirred 1 hour.Then reaction mixture is poured in the mixture of 37% aqueous hydrochloric acid (50mL) and ice (150g) lentamente, simultaneously in cooled on ice, temperature is not above 5 ℃ like this.Salt solution (200mL) is added mixture, and product is with methylene dichloride (6 * 50mL, the thoroughness of extraction is through the TLC inspection) extraction.Extract with salt solution (2 * 50mL) washing, through dried over mgso, and on rotatory evaporator vacuum concentration.Crude product is through using hexane to ETHYLE ACETATE: the gradient of hexane (1: 10) obtains being revealed as the ivDde-OH (10.5g, 94%) of light yellow oil through the silica gel column chromatography purifying.

Synthetic as follows then N-(ivDde)-D-Serine: to ivDde-OH (1.1g, 5mmol) with the D-Serine (0.6g, 5.75mmol) with the mixture of methyl alcohol (50mL) add the N-diisopropylethylamine (3.4mL, 2.6g, 20mmol).Stirred reaction mixture spends the night under refluxing.TLC check (ethyl acetate/hexane 1: 4) shows does not have free ivDde-OH.Reaction mixture is cooled to room temperature, through the rotatory evaporator solvent removed in vacuo.Resistates is dissolved in the water (40mL), and is cooled to 5-10 ℃, be acidified to pH 2 through dripping the 2N aqueous hydrochloric acid.Stirred the mixture 30 minutes, filtering-depositing, water cleans, and vacuum-drying obtains N-(the ivDde)-D-Serine (1.5g, 96%) into white micro-crystals.

Be prepared as follows N-(ivDde)-D-Serine benzyl ester: to N-(ivDde)-D-Serine (0.93g, 3mmol) and sodium hydrogencarbonate (0.34g, 4mmol) with the mixture of DMF (20mL) in; Add bromotoluene (0.43mL; 0.62g, 3.6mmol), in stirring at room mixture 24 hours.Vacuum concentrated mixture on rotatory evaporator is dissolved in resistates in the ETHYLE ACETATE (40mL).The water cleaning solution, water layer is with ETHYLE ACETATE (2 * 30mL) extractions.The organic layer that merges with saturated sodium bicarbonate aqueous solution (2 * 40mL) and water (40mL) wash.Organic layer is dry through magnesiumcarbonate, and solvent removed in vacuo on rotatory evaporator obtains N-(the ivDde)-D-Serine benzyl ester (1.03g, 86%) into the white needles thing.

Synthesizing of D.N-(Alloc)-D-β-bromine L-Ala alkynes propyl ester and N-(ivDde)-D-β-bromine alanine benzyl ester

Corresponding β-bromine the alanine derivatives of N (alloc)-D-Serine (propargyl) ester and N (ivDde)-D-Serine (benzyl) ester is synthetic as follows: the ester that 1 equivalent is suitable is dissolved in the methylene dichloride (or similar aprotic solvent), and handles this solution with 1 equivalent carbon tetrabromide and triphenyl phosphine.In the stirring at room reactant, accomplish until observing, through purification by flash chromatography target beta-bromine alanine derivatives according to TLC.Perhaps, it is synthetic to use the solution of phosphorus tribromide in solvent such as toluene or methylene dichloride to realize, then carries out gentle alkaline aftertreatment, the D-that obtains expecting β-bromine L-Ala.Except bromination, can in following alkylation step, use tosylation or other leavings group, produce final shielded L-lanthionine.

E. L- lanthionine 1 and 2 synthetic

L-lanthionine 1 synthesizes (Fig. 5) through make N (alloc)-D-β-bromine L-Ala alkynes propyl ester alkylation with (Fmoc)-L-halfcystine.L-lanthionine 2 synthesizes (Fig. 6) through make N (ivdDe)-D-β-bromine alanine benzyl ester alkylation with (Fmoc)-L-halfcystine.

Following β-bromine L-Ala the alkylation that makes correspondence with (Fmoc)-L-halfcystine: 1 equivalent β-bromine L-Ala is dissolved in the methylene dichloride (or similar aprotic solvent), and under phase-transfer catalyst such as Tetrabutyl amonium bromide, tetrabutylammonium iodide or Aliquat336, handles with (Fmoc) halfcystine.The catalytic amount that needs is 5-50mol%, and can be optimised, to obtain the product of good speed of reaction and respective pure form.Temperature of reaction also can be optimised in 10-50 ℃ scope.

Thus obtained product is through the flash column chromatography purifying; Degree of purity of production and structure are confirmed through NMR, HPLC, mass spectrum and/or TLC.L- lanthionine 1 and 2 synthetic route are direct relatively, and the expection product is stable, make can easily realize amplifying with extensive synthetic (＞10g).

Embodiment 2: use L- lanthionine 1 and 2 synthetic wool sulphur microbiotic nisin category-As seemingly Thing

A. the nisin category-A is synthetic like the solid-phase peptide of thing

Following general introduction is synthesized the nisin category-A like thing [SEQ ID NO:2] according to the present invention.The L-Ala that this analogue contains 33 dehydrogenation butyrine (dehydrobutarine) and 30 and 2 s' dehydroalanine replaces.Considerable evidence shows, does not significantly act on (Kuipers etc., (1996) with respect to this activity profile to product of natural nisin A with rendeing a service; Devos etc., (1995), Molecular Microbiology 17,427-437; Sahl etc., (1995), European Journal of Biochemistry 230,827-853; Bierbaum etc., (1996), Applied and Environmental Microbiology 62,385-392).

Except as otherwise noted, otherwise all schemes all are the standard FmocSPPS methods of in document, reporting.White(2003)Fmoc Solid Phase Peptide Synthesis，Apractical Approach，Oxford University Press，Oxford。Nisin A begins to synthesize (referring to Fig. 1) with the substep mode by its carboxyl terminal.

1. with N ^α-Fmoc-Lys-N ^εThe carboxyl of-tertbutyloxycarbonyl-L-Methionin (residue 1) connects CLEAR-Acid Resin ^TM(Peptide International).Check resin with triketohydrindene hydrate, to confirm the reaction performance.

2. the DMF solution that uses 20% piperidines realizes being positioned at the deprotection of the Fmoc group on the Methionin acid amides in room temperature.

3. use Fmoc L-amino acid (commercially available getting) separately repeats the coupling and the deprotection of above step (1-2), to connect L-Ala, Xie Ansuan, Histidine, Isoleucine and Serine (residue 2-6) in order.Amino acid such as Histidine, Methionin and Serine have the tertiary butyl that is connected with its active side chain, so that these radical protections are got up.

4. after the DMF solution that uses 20% piperidines is sloughed the Fmoc group on the orthogonal lanthionine 1, use orthogonal lanthionine 1 to carry out next coupling.

5. coupling Fmoc Histidine (residue 8).

6. the DMF solution with 20% piperidines makes Fmoc Histidine deprotection, with Histidine and orthogonal lanthionine 2 couplings.

7. use the propargyl on the dichloromethane solution cracking orthogonal lanthionine 1 of cobalt octacarbonyl.Use the Fmoc aminoterminal of the DMF solution exposure orthogonal lanthionine 2 of 20% piperidines.The N-end coupling of the C-end of the orthogonal lanthionine 1 that exposes and the orthogonal lanthionine 2 that exposes.Accomplish the synthetic of ring E in this step.

8. pass through with 20mol%Pd (PPh ₃) ₄With 20-25 equivalent PhSiH ₃Dichloromethane solution handle peptide-based resin and reach 15-20 minute for twice, slough N (Alloc) group of L-lanthionine 1.

9. with the N-end and Fmoc L-Ala (residue 11) coupling that expose.Use the DMF solution of 20% piperidines to make the Fmoc group deprotection on the L-Ala.

10. use the transfer hydrogenation scheme to make all the other C-end deprotections of L-lanthionine 2 with the dichloromethane solution of palladium on carbon and cyclohexadiene.

11. the N-end coupling of the C-of the L-lanthionine 2 that exposes end and L-Ala (residue 11).Synthesizing in this step of overlapping ring E and D accomplished.In order to check synthetic correct product, take out low amounts of resin, use cleavage mixture to make peptide cracking get off (vide infra).The peptide that is obtained is analyzed through Maldi and LC-MS.

12. use the DMF solution of 2-10% hydrazine to slough the ivDde on the L-lanthionine 2, the free ammonia cardinal extremity that is obtained prolongs with Methionin, methionine(Met) and the l-asparagine ( residue 13,14 and 15) of Fmoc protection sequentially.

13. L-lanthionine 1 is connected with the N-end of the deprotection of l-asparagine.(still, any in L-lanthionine 1 or the L-lanthionine 2 all can be used for accomplishing the synthetic of ring C, B and A).

14. the Fmoc group of L-lanthionine 1 is by deprotection, and sequential and Fmoc glycocoll, methionine(Met), L-Ala, leucine and glycocoll (residue 17-21) coupling, forms ring C.

15. the propargyl of the C-of L-lanthionine 1 end uses 1 normal cobalt octacarbonyl to slough, and with the N-end coupling of glycocoll (residue 21), accomplish ring C.

16. the Alloc group on the N-end of L-lanthionine 1 is sloughed according to the method for describing in the step 8, and with Fmoc Methionin (residue 23) coupling.

17. the N-of Methionin end is by deprotection, with the N-end coupling of L-lanthionine 1 with Methionin.

18. the Fmoc group of L-lanthionine 1 is by deprotection, and sequential and Fmoc glycocoll and Fmoc proline(Pro) (residue 25 and 26) coupling.

19. the propargyl of the C-of L-lanthionine 1 end uses 1 equivalent cobalt octacarbonyl to slough, and with the N-end coupling of the deprotection of proline(Pro), form ring B thus.

20. the Alloc group on the N-end of L-lanthionine 1 is sloughed according to the method described above, and with L-lanthionine 1 coupling.

21. the Fmoc group of L-lanthionine 1 is by deprotection, and sequential and Fmoc leucine, L-Ala and Isoleucine (residue 29-31) coupling.

22. the propargyl of the C-of L-lanthionine 1 end uses 1 equivalent cobalt octacarbonyl to slough, and with the N-end coupling of the deprotection of Isoleucine, form ring A thus.

23. the Alloc group on the N-end of L-lanthionine 1 is sloughed according to the method described above, and ground, sequential ground and Fmoc L-Ala and Isoleucine (residue 33 and 34) coupling.This has accomplished nisin category-A synthesizing like thing.

B. the cracking from the resin of synthetic peptide is got off

Because the synthetic peptide comprises a large amount of sulphur, so use the mixture contain TFA/ thioanisole/water/phenol/dithioglycol (82.5/5/5/5/2.5) to make peptide cracking from the resin get off (White2003).Clean resin up hill and dale with methylene dichloride,, and use above mixture process with DMF and other remaining organism of removing trace.The optimization of cracking time point realizes as follows: on the 15-20mg resin, react, follow the tracks of reaction with interval hourly through LC-MS and reach 18 hours.Optimized conditions is used to amplify cracking.The peptide that little by little cracking is got off is poured in the cold diethyl ether, thus precipitation of peptides.Sedimentary peptide cleans with cold diethyl ether, and dry.

C. the purifying of the peptide that gets off of cracking

Said peptide is purifying through reconstruct in containing the water of 1%TFA.Solution uses acetonitrile gradient on the C-18 reversed-phase column: the Biorad HPLC that the water gradient is carried out HPLC and adopted the quadtech detector.Collect each peak, and through the time-of-flight analysis of ground substance assistant laser desorption ionization, to confirm the structure of product.Collection contains flow point and the freeze-drying of expecting peptide, to obtain purified product.Use HPLC, MS and NMR to confirm purity.

Embodiment 3: the nisin category-A of purifying is like the structure and the biological analysis of thing

A. the nisin category-A is like the biological assay of thing

With so carrying out five equilibrium and freeze-drying at synthetic lantibiotics described in embodiment 1 and 3 with purifying.The product of weighing and being obtained calculates ultimate yield.Confirm the biological activity of nisin category-A through extension antagonism assay method known in the art like thing; Said extension antagonism measure allow to confirm the nisin category-A like the minimum inhibition concentration of thing with kill bacterial concentration (Hillman etc.; (1984); Infection and Immunity 44,141-144; Hillman etc., (1998), Infection and Immunity 66,2743-2749).Can confirm accordingly than living with natural nisin A contrast.Biological assay is carried out as follows:

In 96 hole microtiter plates, use acetonitrile: water (80: 20), will test 2 times of active grade of sample (20 μ l) serial dilutions that divides of nisin A.Concentration is in the scope of 20-0.08 μ g/mL.With pancreas casein soybean broth (Difco) with 1: 1000 (about 10 ⁶Cfu/mL) dilute micrococcus luteus (Micrococcus luteus) the strains A TCC272LS overnight culture of (spontaneously resisting 100 μ g/mL Streptomycin sulphates), and grow to OD in 37 ℃ ₆₀₀=0.2.600 μ l cells are added to the pancreas casein soybean broth top-layer agar (0.75% agar) that 15mL has been cooled to 45 ℃; And be poured on and cover the big petridish surface contain pancreas casein soy agar (containing 100 μ g/mL Streptomycin sulphates) (Streptomycin sulphate prevents the pollutent growth that possibly exist, but does not influence the ability of measuring the nisin A live vol that exists).After top-layer agar solidifies, get 5 μ L samples of 2 times of serial dilutions that level to be tested divides, point sample and makes it air-dry to planar surface.

With flat board in 37 ℃ of incubations 24 hours, and the growth inhibition zone of check indicator strain.The inverse of the high dilution that the tiring of sample is regarded as that micrococcus luteus (M.luteus) indicator strain produces that visible growth suppresses.As contrast, dilute real nisin A, and point sample as stated.Concentration is in the scope of 20-0.08 μ g/mL.The result make it possible to as based on as the percentage of the purity level of these compounds of formerly confirming in the step confirm the biological activity of synthetic analogues with respect to natural nisin A.

Use synthetic and natural nisin A to more than at least ten kinds of Gram-positive kinds; Comprise multi-drug resistance streptococcus aureus (Staphylococcus aureus), enterococcus faecalis (Enterococcus faecalis) and monocyte hyperplasia listeria spp (Listeriamonocytogenes), carry out above biological assay.Other microbiotic of the target species that one or more are suitable for being tested is parallel running also, is used for contrast.

B. the nisin category-A is like the structural analysis of thing

Confirm the three-dimensional structure of nisin category-A through using TOSCY and NOESY NMR to contrast natural nisin A like thing.With TV 700 μ L at H ₂O/D ₂Prepare sample (3-5mM) synthetic and natural nisin A in O/3-(trimethyl silyl)-propionic acid-D4, the sodium salt (TSP) (90.0: 9.9: 0.1%).Gather NMR data in 25 ℃ with 600MHz with freezing Bruker Avance spectrograph, and be the center with the water resonance with carrier frequency, water resonance was suppressed by presaturation in the middle of the relaxation delay at 1.5 seconds.TOCSY experiment with the 60ms mixing time use the MLEV-17 sequence obtain (Bax & Davis (1985), Journal of MagneticResonance 65,355-360).The NOESY experiment obtains with 200ms, 400ms and 450ms mixing time.With producing or be adjusted to respectively time of lag of refocused anti-phase coherency 3.5ms (140Hz coupling) and 60ms (8.5Hz coupling) in HMQC and the HMBC experiment.

All 2D data all with 2048 complex point collections, are collected with the complex point between 256-512 for indirect dimension in obtaining dimension.The phase place susceptibility indirect detection of all experiments all use the method for States-TPPI accomplish (Marion etc., (1989), Journal of MagneticResonance 85,393-399). ¹The H chemical shift is with reference to TSP.With NMRpipe processing data (Delaglio etc.; (1995); Journal of Biomolecular NMR 6; 277-293): at first eliminate the residual water signal through deconvolution, the cosine square function that the data in two dimensions multiply by cosine square function or have 60 ° of displacements is (for HMBC's ¹The H dimension), a zero clearing, Fourier's (Fourier) conversion and baseline calibration.With interactive computer program NMR View analytical data (Johnson & Blevins (1994), Journal of Biomolecular Nmr 4,603-614).According to standard method (W ü thrich, K. (1986) NMR of Proteins andNucleic Acids., Wiley; New York) uses TOCSY (Braunschweiler & Ernst (1983); Journal of Magnetic Resonance 53,521-528) and NOESY (Kumar etc., (1980); Biochem.Biophys.Res.Commun.95,1-6) experiment is confirmed ¹H resonance.Use HMQC (Bax etc., (1983), Journal of Magnetic Resonance 55,301-315; Muller (1979); Journal of the American Chemical Society 101; 4481-4484) and HMBC (Bax & Summers (1986); Journal of the AmericanChemical Society 108,2093-2094) some the equivocal zone in distinct TOCSY of experiment and the NOESY spectrum.

Methionin, Isoleucine, leucine, glycocoll and asparagine residue have completely different and easy sign ¹The H spin mode that resonates, this confirms their easily in 2D TOCSY and NOESY experiment.These residues are differentiated out earlier.The thioether bond pattern is examined through long distance beta proton N OE connectivity pattern.Length between 3 and 7,8 and 11,13 and 19,23 and 26 and 25 and 28 s' residue estimates it is identifiable apart from NOE.To as at Smith etc.; 2002 (Structural and Functional Characterization of theLantibiotic Mutacin 1140; University of Florida, the three-dimensional modeling described in Gainesville) use long apart from NOE (＞i ^{+ 2}).

Detect NOE intersection peak intensity with NMR View.Use relational expression r _Ab ⁶=r _Cal ⁶(V _Cal/ V _Ab) computed range, wherein r _AbBe the distance between atom a and the b, V _AbBe NOESY a-b intersection peak volume, r _CalBe known distance, V _CalIt is the corresponding volume at NOESY calibration intersection peak.The distance that is used to calibrate is the β proton of Isoleucine.Only the intersection of the NOE between residue peak is used as distance restraint in calculating.Potential well is used the upper and lower force constant definition of 1kcal/mol/

.

InsightII software is all used in all conformation modelings, and (Accerlys, San Diego CA) carries out.Molecular dynamics simulation use in a vacuum have cross term, Morse electromotive force and 40

carries out with 500K, specific inductivity 4.0 by the cvff field of force of distance.Peptide uses the person of foundation (builder) function among the InsightII to make up.At first, linear peptides is minimized, make unconfined molecular dynamics operation 10ps then.After this, only increase i+2 or bigger distance restraint.When i+2 in forming the residue of each thioether ring or bigger distance restraint are satisfactory, regularly stop molecular dynamics simulation.Form ring A earlier, form ring B and ring C afterwards, ring D and E then tangle.In case formed the thioether ring, just the i+1 distance restraint is increased to i+2 or bigger distance restraint, use the cvff field of force to make molecular dynamics simulation operation 5ns with 500K, specific inductivity 4.0 with cross term and Morse electromotive force.Make the molecular dynamics simulation 20ns that reruns with all constraints then.

Every 10ps writes dynamic (dynamical) history file.Adopt all NMR constraints; Use 2000 step steepests to meet conjugate gradient and Newton-Raphson after descending; Make from 200 structural energies that begin also every 100ps history file at interval with 1ns to minimize, until rootmean-square (RMS) gradient that reaches 0.01kcal/mol/

energy.Use NMR constraint violation situation (Laskowski, R.A., the Rullmann of 200 energy minimization structures of PROCHECK-NMR software inspection; J.A.C., MacArthur, M.W.; Kaptein, R.& Thornton, J.M. (1996) AQUA and PROCHECK-NMR:Programs for checking the qualityof protein structures solved by NMR; Journal of Biomolecular Nmr.8,477-486).Use the XCluster program that the textural classification of energy minimization is the (Shenkin of family; P.S.& McDonald; D.Q. (1994) Cluster-Analysis ofMolecular-Conformations, Journal of Computational Chemistry.15,899-916).With conformation with by VanDeVen etc., 1991 (European Journal ofBiochemistry 202, the 1181-1188) natural structure of definite nisin A contrasts.

Sequence table

<110>Hillman，Jeffrey D

Orugunty，Ravi S

Smith，James

< 120>differentially protected orthogonal lanthionine technology

<130>05-654-C

<150>US 60/708,086

<151>2005-08-12

<150>US 60/808,907

<151>2005-05-26

<160>2

<170>PatentIn version 3.3

<210>1

<211>34

<212>PRT

< 213>artificial sequence

<220>

< 223>synthetic peptide

<220>

< 221>other characteristics

<222>(2)..(2)

< 223>Xaa is Dhb

<220>

< 221>other characteristics

<222>(5)..(5)

< 223>Xaa is Dha

<220>

<221>MOD_RES

<222>(8)..(8)

<223>Abu

<220>

<221>MOD_RES

<222>(13)..(13)

<223>Abu

<220>

<221>MOD_RES

<222>(23)..(23)

<223>Abu

<220>

<221>MOD_RES

<222>(25)..(25)

<223>Abu

<220>

< 221>other characteristics

<222>(33)..(33)

< 223>Xaa is Dha

<400>1

Ile Xaa Ala Ile Xaa Leu Ala Xaa Pro Gly Ala Lys Xaa Gly Ala Leu

1 5 10 15

Met Gly Ala Asn Met Lys Xaa Ala Xaa Ala His Ala Ser Ile His Val

20 25 30

Xaa Lys

<210>2

<211>34

<212>PRT

< 213>artificial sequence

<220>

< 223>synthetic peptide

<400>2

Ile Ala Ala Ile Ala Leu Ala Ala Pro Gly Ala Lys Ala Gly Ala Leu

1 5 10 15

Met Gly Ala Asn Met Lys Ala Ala Ala Ala His Ala Ser Ile His Val

20 25 30

Ala Lys

Claims (13)

1. the method for bridging polypeptide in the synthetic molecules, said polypeptide comprises two eclipsed intramolecularly bridges, and said method comprises:

A) with first of following formula by the free carboxy end of differentially protected quadrature intramolecularly bridge

With solid support or with the free ammonia cardinal extremity covalent attachment of amino acid that randomly is combined with solid support or polypeptide, wherein L ⁿBe covalently bound amino acid side chain; Wherein D, E and G are the protection bases; Said respectively protect base all under the differential responses condition being selected property remove, basic those conditions of the reaction conditions that wherein is used to slough the basic D of protection and the amino group of amino acids protection that is used to slough the polypeptied chain rest part are different;

B) slough the basic E of protection, form the free ammonia cardinal extremity;

C) with the shielded aminoacid addition of amino to the free ammonia cardinal extremity, make said amino acid deprotection then, obtain new free ammonia cardinal extremity;

D) randomly repeat c) one or many;

E) with second of following formula by the free carboxy end of differentially protected quadrature intramolecularly bridge

With free ammonia cardinal extremity covalent attachment, wherein L ⁿAs above definition; Wherein M, Q and T are the protection bases; Said respectively protect base all under the differential responses condition being selected property remove, wherein D and M just are removed under different condition, wherein G and T just are removed under different condition; The reaction conditions that wherein is used to slough the basic M of protection is different with those conditions of the amino group of amino acids protection base that is used to slough the polypeptied chain rest part, and wherein E and Q are with those conditions that will slough D with will slough under those condition various conditions of M quilt and sloughed;

F) slough the basic Q of protection, form the free ammonia cardinal extremity;

G) randomly with the shielded aminoacid addition of amino to the free ammonia cardinal extremity, make said amino acid deprotection then, obtain new free ammonia cardinal extremity;

H) randomly repeat g) one or many;

I) slough first by the protection base G of differentially protected quadrature intramolecularly bridge, form the free carboxy end;

J) with free carboxy end and the coupling of free ammonia cardinal extremity;

K) slough first by the protection base D of differentially protected quadrature intramolecularly bridge, form the free ammonia cardinal extremity;

L) randomly with the shielded aminoacid addition of amino to the free ammonia cardinal extremity, make said amino acid deprotection then, obtain new free ammonia cardinal extremity;

M) randomly repeat l) one or many;

N) slough second by the protection base T of differentially protected quadrature intramolecularly bridge, form the free carboxy end;

O) with free carboxy end and the coupling of free ammonia cardinal extremity;

P) slough second by the protection base M of differentially protected quadrature intramolecularly bridge, form the free ammonia cardinal extremity; With

Q) randomly with the shielded aminoacid addition of amino to the free ammonia cardinal extremity, make said amino acid deprotection then, obtain new free ammonia cardinal extremity; With

R) randomly repeat q) one or many.

2. the method for claim 1, said method also comprises:

A) with the differentially protected quadrature intramolecularly bridge of following formula

With the coupling of free ammonia cardinal extremity, wherein L ⁿBe covalently bound amino acid side chain; Wherein D, E and G are the protection bases; Said respectively protect base all under the differential responses condition being selected property remove, basic those conditions of the reaction conditions that wherein is used to slough the basic D of protection and the amino group of amino acids protection that is used to slough the polypeptied chain rest part are different;

B) slough the basic E of protection, form the free ammonia cardinal extremity;

C) with the shielded aminoacid addition of amino to the free ammonia cardinal extremity, make said amino acid deprotection then, obtain new free ammonia cardinal extremity;

D) randomly repeat c) one or many;

E) slough the basic G of protection, form the free carboxy end;

F) with e) free carboxy end and the coupling of free ammonia cardinal extremity;

G) slough the basic D of protection, form the free ammonia cardinal extremity;

H) randomly with the shielded aminoacid addition of amino to the free ammonia cardinal extremity, make said amino acid deprotection then, obtain new free ammonia cardinal extremity;

I) randomly repeat h) one or many; With

J) randomly repeating step is a)-i).

3. the process of claim 1 wherein that said polypeptide comprises two intramolecularly bridges.

4. the method for claim 3, wherein said two intramolecularly bridges form two placed in-line rings.

5. the method for claim 3, wherein said two intramolecularly bridges form two rings, and one of them ring is embedded in another ring.

6. the process of claim 1 wherein that said differentially protected quadrature intramolecularly bridge is a L-lanthionine.

7. the process of claim 1 wherein that said intramolecularly bridging polypeptide is a lantibiotics.

8. the process of claim 1 wherein that D, E, M and Q are selected from Fmoc, Alloc and IvDde.

9. the process of claim 1 wherein that G and T are selected from alkynes propyl ester and benzyl ester.

10. the method for claim 6, wherein said intramolecularly bridge are selected from Beta-methyl L-lanthionine (MeLan), S-[(Z)-2-amido vinyl]-D-halfcystine (AviCys) and S-[(Z)-2-amido vinyl]-2-methyl D-halfcystine.

11. the method for claim 7, wherein said lantibiotics are selected from nisin A, nisin Z, subtilyne, Quercetin S, Quercetin A, streptin, epidermin, [Vall-Leu6]-epidermin, Gallidermin, variation rhzomorph 1140, variation rhzomorph B-Ny266, variation rhzomorph III, variation rhzomorph I, Pep5, Epilancin K7, Epicidin 280, lacticin 481, Variacin, variation rhzomorph II, streptostacin A-FF22, SalivaricinA, [Lys2-Phe7]-salivaricin A, the plain C of plant lactobacillus, Sublancin 168, Butyrivibriocin OR79A, cinnamycin, Moli 1901, Moli 1901 B, Moli 1901 C, curamycin C, ancovenin, Mersacidin, actagardin, Ala (0)-actagardin, Subtilocin A, lacticin 3147A1, lacticin 3147A2, staphylococcin C55 α, staphylococcin C55 β, the plain W α of plant lactobacillus, the plain W β of plant lactobacillus, cytolysin L _LWith cytolysin L _S

12. the method for claim 11, wherein said lantibiotics are nisin A or its analogue.

13. the process of claim 1 wherein L ⁿComprise the chemical bond that is selected from thioether bond, disulfide linkage, amido linkage or ehter bond.

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