CN1929860A - Site-specific chemical modification of HIV gp41-derived peptides - Google Patents

Site-specific chemical modification of HIV gp41-derived peptides Download PDF

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CN1929860A
CN1929860A CNA2005800073482A CN200580007348A CN1929860A CN 1929860 A CN1929860 A CN 1929860A CN A2005800073482 A CNA2005800073482 A CN A2005800073482A CN 200580007348 A CN200580007348 A CN 200580007348A CN 1929860 A CN1929860 A CN 1929860A
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布赖恩·布雷
张虎翼
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Abstract

Provided is a method for site-specific chemical modification of an HIV gp41 -derived peptide, wherein during synthesis one or more amine groups of the HIV gp41 -derived peptide are chosen to blocked by a chemical protecting agent, and one or more amine groups are selected to be unprotected, and remain free to be reacted with an amine reactive functionality. The resultant HIV gp41-derived peptide may be used to produce a substantially homogeneous conjugate comprised of HIV gp41-derived peptide and polymer by covalently coupling the polymer to the one or more free (unprotected) amine groups of the HIV gp41-derived peptide.

Description

The site-specific chemical modification of HIV gp41 derived peptide
Technical field
The present invention relates to the method for the site-specific chemical modification of HIV gp41 derived peptide; thereby between the synthesis stage of peptide; what add is one or more aminoacid; described aminoacid has the amido with the protection of chemoproection reagent chemical; one or more free aminos that selection stays synthetic peptide are unprotected, from but chemically reactive (" free ").The synthetic peptide of Chan Shenging can link with amine reactive polymer covalency then, form the conjugate that comprises HIV gp41 derived peptide of even matter basically, linked described polymer for described HIV gp41 derived peptide covalency in the selected and specific site of described synthetic peptide.
Background technology
Well-known at present, cell can be infected by HIV by the fusion process that takes place between cell membrane and viromembrane.The generally accepted model of this process is that the cell surface receptor on viral envelope glycoprotein complex (gp120/gp41) and the target cell membrane interacts.After gp120 is bonded to cell receptor (for example, CD4 and chemotactic factor coreceptor, for example CCR-5 or CXCR-4 in conjunction with), induce the conformation change in the gp120/gp41 complex and make gp41 insert the film of target cell and mediate film and merge.
The aminoacid sequence of gp41, and the variant in the middle of different HIV strains is well-known.Fig. 1 is the graphic extension (indicating the aminoacid sequence number can have slightly different according to the HIV strain) of generally accepted gp41 functional domain.Think that this fusogenic peptide (gene fusion (fusogenic) domain) relates to insertion and destroys target cell membrane.Comprise the membrane spaning domain of striding the film anchor series and be positioned at this proteinic C-end.Fusogenic peptide and to stride between the film grappling be the zone of two uniquenesses is called as seven peptides and repeats that (heptad repeat, HR) zone, each zone have a plurality of seven peptides.Comprise that the aminoacid sequence in HR1 zone and the aminoacid sequence that comprises the HR2 zone are the zones of high conservative separately in the HIV-1 envelope protein.Generally be described to comprise the amino acid residue of SEQ ID NO:1 or its polymorphic body (polymorphisms) (referring to, Fig. 2 for example) than the HR1 zone of more close this protein N-end in HR2 zone.The HR2 zone generally is described to comprise the amino acid residue of SEQ ID NO:2 or its polymorphic body (referring to, Fig. 3 for example).As among Fig. 1 further shown in, the HR zone has a plurality of 7 amino acid residues and extends or " seven peptides " (7 aminoacid in each seven peptide are called " a " and arrive " g "), it is advantageous that at first (" a ") and the 4th (" d ") position and be hydrophobic residue, in the 5th (" e ") and the 7th (" g ") position be charged residue often, and is the main determining factor that influences oligomeric state and chain direction at the aminoacid of " a " position and " d " position.
In external test and body in the clinical research, the peptide of all finding to derive from the native sequences of the HR1 zone (" HR1 peptide ") of HIV gp41 or HR2 zone (" HR2 peptide ") suppresses the propagation of HIV to host cell.For example, (also be called T20, enfuvirtide and Fuzeon  with DP178; SEQ ID NO:3), T651 (SEQ ID NO:4), T649 (SEQ ID NO:5) be the HR2 peptide of example, to be respectively 0.5ng/ml (at HIV-1 LAIEC50), 5ng/ml (IC50; HIV-1IIIB) and 2ng/ml (IC50; HIV-1IIIB) effectiveness blocking-up target cell infects.Carried out making great efforts to improve the biologic activity of HIV gp41 derived peptide, for example, by attempting stablizing the helical structure of this peptide.Also carried out the pharmacological property that various effort improve HIV gp41 derived peptide.
Used polymer to improve medicine widely, for example peptide, albumen and micromolecular pharmacokinetics and pharmacodynamics (improving pharmaceutical properties thus).The most widely used polymer that is used for medicinal application is Polyethylene Glycol (" PEG ")." PEGization " is a kind of process, by this process, medicine is chemically modified to produce one or more PEG molecules covalently bound (" binding ") (depending on that it is effective for reacting and close with the PEG yoke with PEG that what sites are arranged on this medicine) to this medicine.The pharmacology of the improvement relevant with the PEGization of medicine and biological property are known in drug world.For example, include but not limited to, thereby improve drug level by the degraded that reduces proteolytic enzyme; Thereby the size that increases its medicine that adheres to is improved the bio distribution of medicine; The protection epitope is to reduce the immunogenicity of expectation, and PEGization can improve treatment and render a service.Render a service by improving treatment, can reduce the frequency of administration and/or reach the required medication amount of therapeutic effect.
PEG has following general structure as the polyethers of linearity:
HO-(CH 2-CH 2O) n-CH 2CH 2-OH is n usually from about 10 to about 2000 wherein.
PEG has following general structure as ramose polyethers:
Figure A20058000734800071
Wherein T is joint or the molecule bridge that connects the PEG molecule, and Z is the functional group with chemical reactivity part.Many PEG revise, it forms PEG derivant (PEG and PEG derivant are called " PEG " in known technology), be at end group (" functional group "), add or change their chemical reactivity functional group, be used for the PEG molecule covalently bound to medicine.Various PEG derivants are well known in the art.For PEG is attached to medicine, the functional group of PEG molecule generally need be activated to become chemically reactive.The type of functional group and specificity are based on the selection to the chemical reaction gene that will link the PEG molecule on the medicine.For albumen with peptide is modal is, the chemical reaction group is present on the aminoacid of the group that is selected from following formation: with the internal amino acid of the side chain of free chemical reaction group (for example have, include but not limited to, lysine, cysteine, glutamic acid, serine, threonine, or the like),-terminal amino acid (has the N-terminal amido, or side chain amido, as free chemical reaction group), the C-end amino acid (has the terminal carboxylic acid of C-, or the side chain amido, as free chemical reaction group) and their combination.For with the site of the banded peptide of PEG, the most frequent selection is the N-terminal amido (" α amido ") of the-terminal amino acid of peptide, ε amido (" ε amido ") (lysine in the aminoacid sequence with lysine, it is not the-terminal amino acid or the C-end amino acid of peptide), or when lysine occurs as-terminal amino acid or as the C-end amino acid in peptide, be the ε amido of lysine.
Yet a difficult problem has appearred in this standard strategy of PEGization.Lysine is one of aminoacid the most general in the albumen.For HIV gp41, a plurality of lysine residues (referring to, Fig. 1-3 for example) are arranged in the aminoacid sequence in HR1 zone and HR2 zone.For example, two internal lysine residues are arranged in the peptide of these 36 amino acid residues for HIV gp41 derived peptide T20 (SEQ ID NO:1).Thereby, (thereby a large amount of pendant amine (ε amine) is arranged be reactive) and α amine because a large amount of lysine residues in the aminoacid sequence for the activated PEG that contains the reactive functional group of amine, exist a plurality of sites, on these sites, can link activated PEG by covalency with the reactive functional group of amine.The result of the standard P EGization of this peptide is the mixture of nonuniformity, is included in the colony of the banded PEG molecular amounts several conjugates different with the aspect, site that is connected.The heterogeneity of synthetic like this peptide-polymer conjugate usually is unwanted result.This be because, pharmacology and/or the biological property relevant with the PEGization of peptide will depend on some factors, for example (a) is connected to the quantity and (b) position in site of binding PEG on peptide of the PEG molecule of peptide.For example, the human growth hormone releasing factor's of PEGization extracorporeal biology activity depends on the site and the degree of PEGization.In addition, for standard P EGization,, be very difficult to the kind (having the PEG molecule number of expectation and the connection site of expectation) of using conventional isolation technics known in the art from the nonuniformity mixture, to isolate the peptide-polymer conjugate if fully if possible.This separation has attempted increasing required expense, time and the reagent of peptide-polymer conjugate of producing the expectation kind.A plurality of lysine residues in the aminoacid sequence of the peptide of wanting PEGization are considered to an a kind of like this difficult problem, thereby develop a kind of method of locus specificity PEGization, it comprises with being different from the aminoacid that lysine and shortage have the side chain of unhindered amina replaces lysine residue.
Thereby; in the formation of the conjugate that comprises HIV gp41 derived peptide (in its aminoacid sequence, containing one or more internal amino acid residues) and polymer with pendant amine; exist the demand of the site-specific sex modification of synthetic peptide; thereby what produce is a kind of synthetic peptide; it contains and one or morely has with the selected aminoacid of chemoproection reagent chemoprotectant pendant amine and have the aminoacid of one or more unprotected, available unhindered aminas.Therefore, selected according to synthesizing with banded people, polymer only covalency is attached to the specific site that synthesizes on the peptide, or several specific site.In addition, when wanting banded polymer to be branch, need be to the site-specific sex modification of synthetic peptide, thus only select a unhindered amina and polymer covalency to link, be attached to same a part of synthesizing peptide (with same a part of synthetic peptide crosslinked) with a plurality of branches of same a part of avoiding polymer.More specifically; containing above in the HIV gp41 derived peptide of a chemical reactivity (" dissociating ") amido; described chemical reactivity amido can link with the polymer with the reactive functional group of amine; what wish is chemically to protect one or more selected amidos, stays unprotected, free amido and polymer covalency and links.In addition, the useful HIV gp41 derived peptide that provides, it has the PEG that is attached to the one or more selected site (that is, in one or more selected amino acid positions) in the synthetic peptide in the locus specificity mode.
Summary of the invention
Summary of the invention
The invention provides a kind of between the synthesis stage of peptide the method for the site-specific chemical modification of HIV gp41 derived peptide, wherein synthetic peptide has one or more aminoacid with pendant amine.This method was included between synthesis stage, in described peptide or its fragment, mix at least one aminoacid, described aminoacid selected so that its pendant amine and chemoproection reagent generation chemical reaction, described chemoproection reagent protect described pendant amine to avoid subsequently and the reactive functional group generation of amine chemical reaction; With at least one aminoacid, its have unprotected and can with the amine of the reactive functional group reaction of amine, wherein unhindered amina is selected from by N-terminal amine, pendant amine and their group that constitutes.And, by this method generation is isolating HIV gp41 derived peptide, it has one or more aminoacid that contain pendant amine, wherein chemical reaction has taken place at least one amino acid whose pendant amine and chemoproection reagent, and described chemoproection reagent protects described pendant amine to avoid subsequently and the reactive functional group generation of amine chemical reaction; Have unprotected with at least one aminoacid of described synthetic peptide and can with the amine of the reactive functional group reaction of amine, wherein unhindered amina is selected from by N-terminal amine, pendant amine and their group that constitutes.
The present invention also provides a kind of production to comprise the method for the conjugate of even matter basically of HIV gp41 derived peptide and polymer; wherein said HIV gp41 derived peptide has the one or more aminoacid that are incorporated in its aminoacid sequence between synthesis stage; described aminoacid has the selected pendant amine to seal by the chemoproection group; (selected) of only allowing the expectation of described synthetic peptide do not seal free amino can with the polymer reaction that contains the amine reactive group, only will synthesize the peptide covalency and be attached on the polymer containing the specific site of unhindered amina (amino acid position of synthetic peptide).The invention still further relates to this method according to the present invention conjugate of even matter basically that produce, that comprise HIV gp41 derived peptide and polymer.
The present invention also provides a kind of method of locus specificity PEGization of HIV gp41 derived peptide, and wherein PEG is attached to HIV gp41 derived peptide with site-specific mode covalency.More specifically; described HIV gp41 derived peptide have between synthesis stage selected amino acid position be incorporated in its aminoacid sequence one or more amidos (for example; one or more α amine or ε amine); described amido has been sealed chemical reactivity with the reactive functional group of the amine of PEG by chemoproection reagent during PEGization, thereby the free amino that only stays at the selected amino acid position (by chemical modification) of described synthetic peptide can link with the PEG covalency.Use method of the present invention, provide the compositions of even matter basically of the HIV gp41 derived peptide that comprises PEGization, described HIV gp41 derived peptide contains and the bonded one or more amidos of PEG (as selected in carrying out the site-specific chemical modification method).
Method of the present invention may further include removes chemoproection reagent (" separating protection " step); so that the conjugate of even matter basically that comprises HIV gp41 derived peptide to be provided; described HIV gp41 derived peptide only combines with polymer at the specific site of synthetic peptide; described specific site (is for example selected when carrying out the inventive method; by using chemoproection), wherein said conjugate has kept suitable HIV (human immunodeficiency virus)-resistant activity (comparing with the HIV (human immunodeficiency virus)-resistant activity of the synthetic peptide that is not attached to polymer).It is (preferred that the present invention also provides treatment HIV to infect, the HIV-1 infection) method, comprise individual drug administration compositions to infected by HIV, described pharmaceutical composition comprises the conjugate of even matter basically, described conjugate comprises locus specificity and links (for example, in conjunction with) to the HIV gp41 of the polymer synthetic peptide of deriving.Preferably, described pharmaceutical composition is effectively to suppress the quantity that HIV propagates to target cell, and/or effectively suppress HIV the gp41 mediation be fused to the quantity of target cell.Also provide and suppress the method for HIV, be included in when having cell, make an amount of conjugate of even matter basically of described virus contact, effectively suppress the HIV infection cell according to of the present invention, polymer and synthetic peptide to cell propagation.In addition, provide and suppress the method for HIV, comprise to virus and cell and add an amount of conjugate of even matter basically, effectively suppress the HIV infection cell according to polymer of the present invention and synthetic peptide to cell propagation.The present invention also provides and (for example suppresses the HIV fusion, during the HIV target cell infection, the process that merges between HIV gp41 mediation viromembrane and the cell membrane) method, be included in when having cell, make an amount of conjugate of even matter basically of described virus contact, effectively suppress HIV and merge according to polymer of the present invention and synthetic peptide.These methods can be used for treating the individuality that HIV infects.
The present invention also provides the purposes of the conjugate of even matter basically that produce by the method according to this invention, polymer and synthetic peptide, be used for being manufactured on treatment HIV and (for example infect the medicine that uses, be used to suppress method, the method that inhibition HIV merges or the method for the treatment of the HIV infection that HIV propagates), as the described herein.Described medicine is the form of pharmaceutical composition preferably, comprises the conjugate and the pharmaceutically acceptable carrier of even matter basically according to polymer of the present invention and synthetic peptide.
Foregoing of the present invention, feature and advantage are presented in hereinafter the detailed Description Of The Invention, when read in conjunction with the accompanying drawings, will be conspicuous.
Description of drawings
Fig. 1 is the sketch map of HIV-1 gp41, and seven peptides that shown gp41 repeat 1 zone (HR1) and seven peptides repeat 2 zones (HR2) and other functional regions.For illustrative purposes, be HIV at gp160, strain IIIB, shown exemplary aminoacid sequence and amino acid position numbering corresponding to HR1 and HR2.
Fig. 2 has shown the comparison of sequence contained in the HR1 zone of HIV-1gp41, for illustrative purposes rather than the restriction, be according to measuring from each laboratory strains system and clinical isolates, wherein the explanation be in the aminoacid sequence some variations (for example, polymorphism), indicated by single-letter amino acid code.
Fig. 3 has shown the comparison of sequence contained in the HR2 zone of HIV-1 gp41, for illustrative purposes rather than the restriction, be according to measuring from each laboratory strains system and clinical isolates, wherein the explanation be in the aminoacid sequence some variations (for example, polymorphism), indicated by single-letter amino acid code.
Fig. 4 shows the sketch map that uses the synthetic HIV gp41 derived peptide of fragment condensation method, and wherein: the numbering representative is with respect to each amino acid position of synthetic HIV gp41 derived peptide; " K " representative lysine synthetic peptide sequence inside or that be used for the sheet intrasegmental part of synthetic described synthetic peptide; " Ac " represents the acetylation of N-end; " NH " represents the amidatioon of C-end.
Fig. 5 is the sketch map that shows the conjugate of the HIV gp41 derived peptide of the synthetic modification according to the present invention and polymer and synthetic peptide, and wherein: the numbering representative is with respect to each amino acid position of synthetic HIV gp41 derived peptide; " K " representative lysine synthetic peptide sequence inside or that be used for the sheet intrasegmental part of synthetic described synthetic peptide; " Ac " represents the acetylation of N-end; " NH " represents the amidatioon of C-end; " X " represents chemoproection reagent, and it links optionally to seal the further chemical reactivity of described amino acid side chain with selected amino acid whose ε amine;
Figure A20058000734800121
Representation polymer, it has and the banded specificity of free amino chemistry, combines with the amino acid whose unhindered amina that is not attached to chemoproection reagent in site-specific chemical modification.What produce is: have by the isolating HIV gp41 derived peptide of chemoprotectant at least one amido of the chemoproection reagent ε amine of the lysine residue of amino acid position 18 (for example); With the conjugate of the even basically matter that comprises HIV gp41 derived peptide and polymer, be attached to the described synthetic peptide ε amine place of the lysine residue of amino acid position 28 (for example) the polymer locus specificity.
Fig. 6 is the sketch map that shows the conjugate of the HIV gp41 derived peptide of the synthetic modification according to the present invention and polymer and synthetic peptide, and wherein: the numbering representative is with respect to each amino acid position of synthetic HIV gp41 derived peptide; " K " representative lysine synthetic peptide sequence inside or that be used for the sheet intrasegmental part of synthetic described synthetic peptide; " Ac " represents the acetylation of N-end; " NH " represents the amidatioon of C-end; " X " represents chemoproection reagent, and itself and ε amine link optionally to seal the further chemical reactivity of described amino acid side chain;
Figure A20058000734800122
Representation polymer, it combines with the amino acid whose unhindered amina that is not attached to chemoproection reagent in site-specific chemical modification.What produce is: have by the isolating HIV gp41 derived peptide of chemoprotectant at least one amido of the chemoproection reagent ε amine of the lysine residue of amino acid position 28 (for example); With the conjugate of the even basically matter that comprises HIV gp41 derived peptide and polymer, be attached to the described synthetic peptide ε amine place of the lysine residue of amino acid position 18 (for example) the polymer locus specificity.
The specific embodiment
Detailed description of the invention
Definition
When being used herein to description and claim, term " individuality " is meant mammal, preferably people.
When being used herein to description and claim, term " target cell " is meant the cell that can be infected by HIV.Preferably, this cell is people's cell or human cell; More preferably, the human cell can be by means of comprising that the process that film merges is infected by HIV.
When being used herein to description and claim, term " acceptable carrier on the materia medica " is meant not the mounting medium of the biological activity that significantly changes the active component that the adds conjugate of polymer of the present invention and synthetic peptide (for example, according to).Pharmaceutically acceptable carrier includes but not limited to, one or more of water, buffered water, saline, 0.3% glycine, aquiferous ethanol, isoosmotic aqueous solution; And can further comprise one or more materials, for example sodium, potassium, magnesium and ammonium of glycerol, oil, salt for example, phosphonate ester, carbonic ester, fatty acid, saccharide are (for example, mannitol), polysaccharide, excipient and antiseptic and/or stabilizing agent (for increase the shelf life or as required and be suitable for the manufacturing and the distribution of said composition).Preferably, pharmaceutically acceptable carrier is suitable for intravenous, intramuscular, subcutaneous or parenteral.
For this description and claim and relate to the synthetic peptide that uses among the present invention, term " aminoacid " is meant the molecule with at least one free amino and at least one free carboxy.Aminoacid can have more than a free amino, or more than a free carboxy, or can further comprise the one or more free chemical reaction base (for example, hydroxyl, sulfydryl etc.) except amine or carboxyl.This aminoacid can be aminoacid (for example D-aminoacid), synthetic aminoacid, the aminoacid of modification, amino acid derivativges, the amino acid precursor that naturally occurring aminoacid (for example, L-aminoacid), non-natural exist, and conservative substitution.Those skilled in the art is known, and the aminoacid of selecting to mix peptide will partly depend on and be the needed special physics of antiviral peptide, chemistry or biological characteristics.This characteristic ground is by 26S Proteasome Structure and Function (for example, antiviral activity; Mensuration as described in more detail in this) is determined.For example, those skilled in the art will can be made up of (D)-aminoacid that one or more naturally occurring (L)-aminoacid and non-natural exist from the aminoacid of distinguishing in this explanation the synthetic peptide.Preferred amino acids can be used for getting rid of the aminoacid except that preferred amino acid.
Aminoacid sequence about the synthetic peptide that uses among the present invention, " conservative replacement " is to be used for the term that description and claim are used hereinafter, be meant that one or more aminoacid are replaced in the sequence of synthetic peptide, and its biologic activity is constant basically (for example, if described peptide suppresses the fusion of HIVgp41 mediation under the concentration of nanomole scope before replacement, in the inhibition of under the nanomole scope, still observing after the replacement the fusion of HIV gp41 mediation).As known in the art, by aforesaid way definition " conservative replacement ", comprise having the amino acid whose replacement of going up identical electric charge, size, hydrophilic and/or armaticity with replaced aminoacid substantially.This replacement is that those of ordinary skills are known, includes but not limited to glycine-alanine-valine; Isoleucine-leucine; Tryptophan-tyrosine; Aspartic acid-glutamic acid; Arginine-lysine; Agedoite-glutamine; And serine-threonine.Relevant especially with the present invention is, the conservative replacement that this area suppresses also comprises with ornithine and replaces lysine, and free amino (for example, ε amine) is provided.For HIV gp41 derived peptide, this replacement can also comprise the polymorphism on each amino acid position in the relevant HR zone (HR1 or HR2) of gp41, it is to find in any one of HIV or a plurality of clade, laboratory strains system or clinical isolates, can easily obtain from public data base, be (example of property is also referring to Fig. 2 and Fig. 3 as an illustration) well known in the art.
When being used herein to description and claim, term " polymer " " be meant polymeric molecule; its: (a) when combining, in medicinal application, adopt to improve pharmacology and/or biological property (thereby being nontoxic basically and water miscible basically) with medicine; (b) have one or more functional groups, described functional group itself and/or activation become chemical reactivity is arranged after, can be used for linking with the unhindered amina covalency of medicine (for example, synthetic peptide), form the drug-polymer conjugate.For the latter, described polymer preferably is used for and the reactive functional group of the banded amine of synthetic peptide covalency.Polymer can include, but not limited to polylysine or poly-(D-L-alanine)-poly-(L-lysine), or polyhydric alcohol.Preferred polyhydric alcohols comprises water miscible poly-(oxyalkylene) polymer, and can have straight line or ramose chain.The preferably water miscible polyhydric alcohol of term " polyol ", it can include but not limited to, Polyethylene Glycol (" PEG "), polypropylene glycol (" PPG "), diethylene glycol, 2,2'-ethylenedioxybis(ethanol)., ethylene glycol, dipropylene glycol, comprise PPG copolymer (for example, ethylene glycol/PPG), comprise PEG copolymer (for example, PEG/PPG), mPEG (mono methoxy-poly-(ethylene) ethylene glycol), or the like.Polyhydric alcohol is contained homopolymer and copolymer, can further have the structure that comprises branched structure or linear structure as is known to persons skilled in the art.Preferably, when being used for the individual body planted agent time spent, described polymer is nontoxic basically.In preferred embodiment, described polymer has about 200 dalton to the molecular weight between about 40,000 dalton; In preferred embodiment, described polymer has about 400 dalton to the molecular weight between about 10,000 dalton.Be used for preferred polymer of the present invention and comprise Polyethylene Glycol (" PEG "), be used for preferred polymer of the present invention and comprise Polyethylene Glycol with certain molecular weight scope, wherein said molecular weight ranges is to be not less than about 400 dalton and not to be higher than about 20,000 dalton.As previously herein describing, exist various forms of PEG, its generally be used for the PEG molecule covalently bound to medicine end group or the chemical reactivity functional group aspect difference.Various PEG are well known in the art.Be used for having chemical reaction group (for example, " functional group ") according to the present invention with the banded preferred PEG of one or more unprotected amido of synthetic peptide, it can be used for the PEG covalency is attached to one or more unprotected amidos.PEG can include but not limited to, PEG-HFC-143a sulphonic acid ester (tresylate), isodigeranyl function PEG, the PEG dichlorotriazine, PEG succinimido carbonate, PEG 124 Triazole carbonate, PEG p-nitrobenzophenone carbonate, PEG trichlorophenyl carbonate, the PEG carbonylic imidazole, PEG succinimido succinate, mPEG succinyl phosphorons amino propyl acid salt, mPEG succinimido butyrate, the PEG butyraldehyde, the mPEG propionic aldehyde, PEG aldehyde, PEG acetaldehyde, PEG acetaldehyde diethyl acetal, the PEG carboxylic acid, mPEG phenyl ether succinimido carbonate, mPEG Benzoylamide succinimido carbonate, the PEG thioesters, linear PEG, PEG of branch and linear fork PEG.Preferred polymer can be applied to the present invention and get rid of preferred polymers polymer in addition.
Term " synthetic peptide " and " HIV gp41 derived peptide " are used with the free burial ground for the destitute at this, about the peptide that adopts among the present invention with concerning description and claim, be meant a kind of peptide, its (a) comprises length and is no less than about 15 aminoacid and no more than about 60 amino acid whose aminoacid sequences, and comprise at least a portion (preferred, at least 9 continuous amino acids) of aminoacid sequence contained in the HR1 zone of gp41 of HIV (being more preferably HIV-1) or the HR2 zone; (b) as measuring by evaluation antiviral activity in external and/or the body, can suppress HIV and propagate into target cell (preferably, by and the HR region composite of HIV-1 gp41 and suppress HIV-1 and target cell between fusion), will describe in more detail at this.Preferred, the synthetic peptide that adopts among the present invention can comprise length and be no less than 28 aminoacid and no more than about 51 amino acid whose sequences, and preferred again length is no less than about 36 aminoacid and no more than about 51 aminoacid.When synthetic peptide was used, term " isolating " was meant that it is substantially free of the composition of the integrally-built part that does not become peptide itself; For example, as when the process of using biology, biochemistry or chemistry is come chemosynthesis, generation or modification, be substantially free of chemical drugs precursor or other chemical drugss.Described synthetic peptide can comprise in its aminoacid sequence that one or more conservatives are replaced and/or the sequence of one or more relevant ranges at HIVgp41 in the polymorphism found, maybe can comprise one or more aminoacid and replace, it is added to stablize helical structure and/or to influence oligomerization; Condition is that it has kept the suitable antiviral activity at HIV-1 (for example, at the IC50 of picomole in the micro-molar range).Below be can be according to the present invention and the illustrative example of the bonded HIV gp41 of polymer locus specificity derived peptide.Yet preferably synthetic peptide can be used for the present invention and get rid of preferred synthetic peptide synthetic peptide in addition.It is evident that for those skilled in the art with according to instruction at this, lysine in the aminoacid sequence of synthetic peptide can be with another aminoacid with band free amino (for example, ε amine) side chain (natural generation or non-natural takes place) replacement.Ornithine is this amino acid whose illustrative example that can be used for substituting lysine.
For used according to the invention preferred, for the synthetic peptide that comprises from the sequence in the HR1 zone of HIV-1 gp41, described synthetic peptide comprises the continuous sequence of at least 15 amino acid residues in the aminoacid sequence of SEQ ID NO:1, or its polymorphic body, because have been found that in this part in HR1 zone crucial determinant (for example, as represented by single-letter aminoacid labelling, NNLLRAIEAQQHLLQLTVWG IKQLQARILAVERYLKD, it is that the amino acid residue 18 of SEQ ID NO:1 is to amino acid residue 54) influenced structure described here, and biochemistry and antiviral parameter.Noticing has two lysine residues in this part in HR1 zone, they one or more can be used for that locus specificity ground and polymer link according to the present invention.Illustrated from the HR-1 zone of HIV gp41 and contained the preferred examples of the amino acid whose synthetic peptide that exists in this regional native sequences to have the aminoacid sequence of SEQ ID NO:6.Illustrated from the HR-1 zone of HIV gp41 and contained other examples of the amino acid whose synthetic peptide that exists in this regional native sequences, aminoacid sequence with SEQID NO:7-22, and can further comprise with one or more of SEQ ID NO:6-22 have at least 95%, preferred aminoacid sequence with homogeneity of at least 90%.For used according to the invention preferred, containing one or more aminoacid from the synthetic peptide in the HR1 zone of HIV gp41 (for example replaces, compare with the aminoacid sequence of SEQ ID NO:1), it preferably allows described synthetic peptide oneself (for example to be combined into trimer, the trimer that comprises three molecules of synthetic peptide), as disclosed in detail as institute in the US 20040076637 disclosed common co-pending applications.HR-1 zone from HIV gp41 has been described, has further comprised and allow synthetic peptide oneself to be combined into the example of the synthetic peptide that trimeric one or more aminoacid replaces, aminoacid sequence with SEQ ID NO:23-36, and can further comprise with SEQ ID NO:23-36 any or a plurality ofly have at least 95%, preferred aminoacid sequence with homogeneity of at least 90%.Notice that this synthetic peptide has one or more lysine residues of the inside of this part that is positioned at the HR1 zone; in site-specific chemical modification according to the present invention; they one or more can selectedly be left not shielded (promptly; do not select its side chain reactive group and chemoproection reagent to link), or be selected as chemoprotectant.
For used according to the invention preferred, for the synthetic peptide that comprises from the sequence in the HR2 zone of HIV-1 gp41, described synthetic peptide (for example comprises SEQ ID NO:2, the continuous sequence of amino acid residue at least 43 to 51 QQEKNEQEL), or its polymorphic body, influenced biochemistry described here and antiviral parameter because have been found that the crucial determinant in this part in HR2 zone.Notice an internal lysine residue is arranged in this sequence.Illustrative synthetic peptide from the HR2 zone includes but not limited to, peptide with aminoacid sequence shown in the SEQ ID NO:3,4,5,37 to 63 and 175, can further comprise with SEQ ID NO:3,4,5,37 to 63 and 175 any or a plurality ofly have at least 95% homogeneity, a preferred aminoacid sequence with homogeneity of at least 90%.Notice that this synthetic peptide has one or more internal lysine residues (and/or for SEQID NO:34,39,48 and 175 at carboxyl terminal); in site-specific chemical modification according to the present invention; the one or more of them can selectedly be left not shielded, or are selected as chemoprotectant.For used according to the invention preferred, containing one or more aminoacid from the synthetic peptide in the HR2 zone of HIV gp41 (for example replaces, compare with the relevant portion of the aminoacid sequence of SEQ ID NO:2), it preferably promotes the helicity of described synthetic peptide and/or the biologic activity that spiral stability (" spiral stabilisation peptide ") is given improvement, as more detailed disclosed among the common co-pending application PCT/US2004/042918.The example of the synthetic peptide of this spiral stabilisation has been described, aminoacid sequence with SEQ ID NO:64-92 and 113-174, can further comprise with SEQID NO:64-92 and 113-174 any or a plurality ofly have at least 95% homogeneity, a preferred aminoacid sequence with homogeneity of at least 90%.The helicity that design improves also can comprise SEQ ID NO:93-95 from other examples of the peptide in the HR2 zone of HIV gp41.Notice that this spiral stabilisation peptide has one or more internal lysine residues (sometimes at more than 25% of aminoacid sequence that synthesizes peptide); in site-specific chemical modification according to the present invention; the one or more of them can selectedly be left not shielded, or are selected as chemoprotectant.
According to of the present invention another preferred embodiment in, described synthetic peptide can comprise " hybridization " peptide, its contain from one or more aminoacid sequence of HIV-1, HIV-2 and SIV fusion rotein (referring to, for example, United States Patent (USP) the 6th, 258, No. 782).The example of the synthetic peptide of hybridization has been described, aminoacid sequence with SEQ ID NO:96-112, can further comprise with SEQ ID NO:96-112 any or a plurality ofly have at least 95% homogeneity, a preferred aminoacid sequence with homogeneity of at least 90%.The example that exemplifies of noticing the synthetic peptide of this hybridization has at least two internal lysine residues, and in site-specific chemical modification according to the present invention, the one or more of them can selectedly be left not shielded, or are selected as chemoprotectant.
Term " homogeneity percent ", when for this description and claim, about sequence used according to the invention when this uses, be meant described sequence (" being compared sequence ") with describe or reference sequences (" reference sequences ") compare; Wherein determine homogeneity percent according to following formula:
Homogeneity percentage ratio=[1-(xC/yR)] * 100
Wherein xC is reference sequences and is compared between the sequence to cross over and is compared the number of arranging the difference of correlation length between sequence and the reference sequences, wherein (a) do not have each base or the aminoacid in localized base of corresponding arrangement in the sequence of being compared or the amino acid whose reference sequences, (b) each breach in the reference sequences, and (c) be compared in the sequence each arrangement localized, be different from correlated base of sequence or aminoacid in the reference sequences, form difference; And yR crosses over base or amino acid whose number in the reference sequences that is compared sequence length, and any breach that produces in the reference sequences is also as base or the correlated result of amino acid whose arrangement.The method and the software that are used for arranging between two predetermined sequences are as known in the art.Thereby, for example, reference sequences can be according to any the synthetic peptide among the SEQ ID NO:1-175, be compared sequence and be and reference sequences HIV gp41 derived peptide relatively, to determine any or a plurality of aminoacid sequence with the aminoacid sequence of SEQ ID NO:1-175 with homogeneity of at least 95%.
When being used herein to description and claim, term " chemoproection reagent " is meant chemical part, its: (a) be with amino acid whose unhindered amina have chemically reactive, thereby sealing (" chemoproection ") described amine avoids and the polymer reaction with the reactive functional group of amine; (b) can stand (for example, keeping wanting chemoprotectant amine to carry out chemical reaction) with it well known by persons skilled in the art be used to remove amino acid whose tBU (t-butyl), Fmoc (9-Cong methoxycarbonyl group), Boc (uncle-butoxy carbonyl) or trt (trityl (trityl)) separate the protection step; (c) can remove from the amino acid whose amine of its chemical reaction subsequently, thereby it is unprotected that described amine is become, and be chemically reactive for the reactive functional group of amine.More specifically; described chemoproection reagent can stand by removing Fmoc or Boc for this general reagent that uses in protection this area of separating from peptide; described reagent comprises; for example; one or more: 20%piperdine, 2%DBU (1,8-diazabicyclo[5,4; 0] undec-7-ene), 50% to 90% trifluoroacetic acid, tetramine (for example 4-butyl amine fluoride), or inorganic base potassium carbonate for example.For example; as discussing in more detail at this; described chemoproection reagent can keep, and stable (with the amido reaction) removes described chemoproection reagent up to hope; then subsequently independently separate the protection step in (for example; use 2% hydrazine or other reagent that is fit to) remove described chemoproection reagent, produce unhindered amina.This chemoproection reagent is known in the art; include but not limited to; 1-(4; 4-dimethyl-2; 6-dioxy hexamethylene-1-subunit) ethyl (" Dde "), 1-(4; 4-dimethyl-2,6-dioxy hexamethylene-1-subunit)-3-methyl butyl (ivDde), allyloxy carbonyl (" Alloc "), benzyloxycarbonyl (" Cbz ") and 2-chlorine benzyloxycarbonyl (" 2-Cl-Z ").Preferably; in synthetic peptide; with the free amino of chemoproection reagent reacting be the N-terminal amine of-terminal amino acid; or the amido of the side chain of aminoacid (no matter this seed amino acid is-terminal amino acid, C-end amino acid or internal amino acid) (for example; ε amine); or their combination, as determined by site-specific chemical modification.In preferred embodiment, described chemoproection reagent is stable for amine alkali, and is unsettled for amine alkali Fmoc, Boc, tBu, trt etc.
When in description and claim, use and relate to produce according to the present invention comprise the conjugate of the HIVgp41 derived peptide that is attached to polymer the time; term " basically even matter " be meant generation final conjugate at least 90%; preferred at least 95% contain the orthogonally protect strategy (as describing in more detail in the embodiments herein 5) that adopts by the method according to this invention as the locus specificity be scheduled to be attached to the synthetic peptide (that is, as single kind) of polymer.Can use isolation technics, include but not limited to that chromatographic technique known in the art is further purified described conjugate.
The invention provides the method for the site-specific chemical modification of HIV gp41 derived peptide; and provide and contained chemoprotectant at least one pendant amine and contain unprotected and (for example at least one amine of reactive the responding property of functional group of amine;-terminal amino acid α amine, one or more pendant amine, or their combination) isolating HIVgp41 derived peptide.Isolating then HIV gp41 derived peptide can be selected (wittingly without the protection of chemoproection reagent) with in the banded locus specificity of polymer position (promptly; specific amino acids position in synthetic peptide has unhindered amina) combine (covalency binding) with polymer.Thereby it is one or more free aminos by synthetic peptide that polymer is attached to synthetic peptide, and described free amino can chemical reaction for the polymer with the reactive functional group of amine.Therefore, for example, a molecule covalency of polymer is attached to selected with on the aminoacid with unhindered amina, produces the conjugate of even matter basically that comprises polymer and HIV gp41 derived peptide.Explanation for example, rather than it is restrictive, the exemplary HIV gp41 derived peptide (SEQ ID NO:3) that is called T20 by utilization, the isolating HIV gp41 derived peptide that following sketch map represented the method according to this invention, produce by the method according to this invention and comprise the conjugate of even matter basically according to polymer of the present invention-synthetic peptide.
Sketch map 1: the synthetic peptide that has carried out modifying at the N-end after synthetic has 3 unhindered aminas (" NH2 ") group and can link with the polymer with the reactive functional group of amine:-terminal amino acid α amine and two internal lysine residues (are labeled as K for ease of explanation 1And K 2), each all has the side chain of band ε amine.
Synthetic peptide:
Sketch map 2: illustrate synthetic peptide shown in Figure 1 with have be used for the polymer of the reactive functional group of the banded amine of synthetic peptide (
Figure A20058000734800192
) combination.Heterogeneous conjugate colony may be from cohesive process, and is as follows:
Conjugate:
Figure A20058000734800193
Figure A20058000734800194
Figure A20058000734800201
Sketch map 3: for illustrative purposes, only select internal lysine residue " K 1" unhindered amina be used as can be chemically reactive to the reactive functional group of amine.At first, synthetic peptide has reactive amido of wishing sealing and polymer, by chemoproection reagent protection (" X ") to mix aminoacid, form isolating HIV gp41 derived peptide, have at least one aminoacid (sketch map 3A) that has chemoprotectant pendant amine; Has unique aminoacid (lysine residue K of unhindered amina then 1) locate polymer scale is incorporated into this HIV gp41 derived peptide (sketch map 3B); Remove chemoproection reagent from the aminoacid of chemical reaction subsequently, produce the conjugate (sketch map 3C) of even matter basically.
Sketch map 3A:
Figure A20058000734800202
Sketch map 3B:
Figure A20058000734800203
Sketch map 3C:
Following examples explanation the present invention should not regard restriction the present invention as.
Embodiment 1
Synthetic peptide generally can or the solid phase synthesis technique by the use standard and the Fmoc chemistry of peptides effect of the standard of use or the chemistry of peptides effect of other standards, the enterprising line linearity of peptide synthesizer synthetic come synthetic.Thereby; shown in segmental as described in this synthesizing; the chemistry of peptides effect of solid phase synthesis or other standards can be used for synthesizing described synthetic peptide; wherein chemoprotectant aminoacid (for example; as its pendant amine described here by chemoprotectant aminoacid) can add in the amino acid position of expectation at synthetic time point, this aminoacid of naming a person for a particular job when described is incorporated in the amino acid whose chain to produce synthetic peptide (as in this embodiment 6 explanation).Yet in preferred embodiment, the HIV gp41 derived peptide of experience site-specific chemical modification is to use fragment condensation method synthetic (referring to, for example, Fig. 4-6), as among the embodiment 5 herein in greater detail.Briefly, the individual or more a plurality of fragments of Synthetic 2, each fragment contains the aminoacid sequence that exists in the various piece of synthetic peptide.In segmental synthesizing, if desired, what mix can be the aminoacid that has by the chemoprotectant unhindered amina of chemoproection reagent (for example, pendant amine).Make up these fragments (linking with the order covalency in a certain way) then, thereby produce synthetic peptide (having suitable aminoacid sequence).As previous describe in more detail (referring to, for example, United States Patent (USP) the 6th, 015, No. 881), by the synthetic T20 (SEQ ID NO:3) of fragment condensation method.Briefly, as in Fig. 4, summarizing, the at first synthetic fragment that will be combined into synthetic peptide.Come syntheticly to comprise preceding 16 the amino acid whose fragments of SEQ ID NO:3 by standard solid-phase synthetic (using super acid sensitivity resin), the hydroxyl with the acetylation (" Ac ") of N-end and C-end (OH).By the synthetic fragment of synthesizing the amino acid/11 7-26 that comprises SEQ ID NO:3 of standard solid-phase, have Fmoc at the N-end, have-OH at the C-end.By the synthetic fragment of synthesizing the aminoacid 27-35 that comprises SEQ IDNO:3 of standard solid-phase, have Fmoc at the N-end, have-OH at the C-end.As shown in Figure 4, the fragment that comprises the aminoacid 27-35 of SEQ ID NO:3 links with aminoacid 36 chemistry in liquid phase, and generation comprises aminoacid 27-36, has the amidated fragment of C-end.The fragment chemistry of the aminoacid 27-36 of the fragment of the amino acid/11 7-26 of SEQ ID NO:3 and SEQ ID NO:3 links (after the Fmoc that removes-terminal amino acid 27).The aminoacid sequence of the amino acid/11 7-36 with SEQ ID NO:3 that produces links (after the Fmoc that removes-terminal amino acid 17) with the fragment chemistry of the amino acid/11-16 that comprises SEQ ID NO:3, forms the synthetic peptide of the aminoacid sequence that comprises SEQ ID NO:3.With separating the protection step synthetic peptide is separated protection/decarboxylation (removing tBU, the trt and the Boc that use in each segmental synthesizing), described separate the protection step use trifluoracetic acid/dithiothrietol/ water (volume ratio: mixture 90/5/5), stirred 5 to 6 hours at 30 ℃; Use RPHPLC (reversed-phase high-performance liquid chromatography) method purification then.Confirm the homogeneity of peptide with electrospray ionization mass spectrometry.
Embodiment 2
What illustrate is the embodiment of method of the site-specific chemical modification of HIV gp41 derived peptide, and it can be used to produce (a) and has the isolating HIV gp41 derived peptide of the side chain amido of chemoprotectant one or more internal amino acids; (b) comprise the conjugate of the even basically matter of HIV gp41 derived peptide and polymer.More specifically, what be incorporated into synthetic peptide (as using the fragment assemble method, or its fragment) between synthesis stage is: one or more aminoacid have by chemoproection reagent and have sealed subsequently and the chemically reactive pendant amine reactive functional group of amine; With one or more aminoacid, have unprotected, can with the amine (for example, selecting) of the reactive functional group chemical reaction of amine by the terminal α amine of N-, one or more ε amine and their group that constitutes.Unprotected (" dissociate ") amido by making synthetic peptide and the amine reactivity functional group of polymer carry out chemical reaction; thereby can link with the polymer covalency by this method isolating synthetic peptide that produce, that be used for site-specific chemical modification, produce the conjugate of even matter basically.In this illustrative embodiment; select T20 (SEQ ID NO:3) as exemplary synthetic peptide; select the lysine residue (" K18 ") (aminoacid with pendant amine) of amino acid position 18 to carry out chemoproection by site-specific chemical modification, the lysine residue (" K28 ") that keeps amino acid position 28 is as the internal amino acid with unhindered amina.This unhindered amina can carry out chemical reaction with the reactive functional group of the amine of polymer subsequently, and the lysine on the amino acid position 28 of the aminoacid sequence by synthetic peptide is attached to the polymer covalency on the described synthetic peptide.For Fig. 5, use the synthetic T20 (SEQ ID NO:3) of the previous fragment condensation method of describing among the embodiment 1 herein.
Briefly, summarize as Fig. 5, the solid phase synthesis by standard synthesizes preceding 16 the amino acid whose fragments that comprise SEQ ID NO:3, and the N-terminal amine of amino acid residue 1 (" Y ") is acetylizad (" Ac ").Use Fmoc-Lys-(ivDde) to come the synthetic fragment that comprises the amino acid/11 7-26 of SEQ ID NO:3 by standard solid-phase is synthetic as amino acid residue 18, thus the ε amido that chemoproection reagent ivDde (" X " among Fig. 5) has sealed K18 subsequently with the reaction of the reactive functional group of amine.By the synthetic fragment of synthesizing the aminoacid 27-35 that comprises SEQ ID NO:3 of standard solid-phase, in liquid phase, link with aminoacid 36 chemistry, form the fragment of the aminoacid 27-36 that comprises SEQ ID NO:3.Comprise the fragment (have ivDde protection K18) of the amino acid/11 7-26 of SEQ ID NO:3 and the fragment of the aminoacid 27-36 of SEQ ID NO:3 (containing the lysine that in the position 28 (" K28 ") have free ε amine) chemistry binding.The aminoacid sequence of the amino acid/11 7-36 that comprises SEQ ID NO:3 that produces and the fragment combination that comprises the amino acid/11-16 of SEQ ID NO:3; formation has the isolating HIV gp41 derived peptide of the aminoacid sequence of SEQ ID NO:3; contain at least one aminoacid, described amino acid whose side chain amino is by the chemoproection of chemoproection reagent (having sealed subsequently the chemical reactivity with the reactive functional group of amine).As what in this embodiment 1, describe in more detail, synthetic peptide (SEQ ID NO:3) is separated protection with the blocking group trt, the Boc that remove use in standard solid-phase is synthetic and tBu (and K18 is still chemoprotectant); Decarboxylation; Use the RPHPLC (reversed-phase high-performance liquid chromatography) method to carry out purification then.Then will this isolating HIV gp41 derived peptide be used for the polymer locus specificity be attached to the free ε amine of the K28 of SEQ ID NO:3.
When production comprises the conjugate of even basically matter of HIV gp41 derived peptide and polymer, select mPEG succinyl phosphorons amino propyl acid salt (" mPEG-SPA ") as and the bonded exemplary polymer of T20 (SEQ ID NO:3).The T20 (SEQ ID NO:3) (9.0mg, 2.0 μ mol) that will on the ε of K18 amido, have an ivDde be dissolved in dimethyl formamide (DMF) (0.3ml) in.Add diisopropylethylamine (DIEA) (10 μ l) to reaction, that add then is mPEG-SPA (mean molecule quantity, 5000 dalton (" 5K ") among the DMF (1ml); 20mg, 4.0 μ mol).At room temperature stir mixture, monitor reaction, finish up to PEGization by HPLC.In order to remove chemoproection reagent ivDde from the ε amido of K18, (40 μ l) adds in the reaction with hydrazine, reaches the hydrazine that 3% (v/v) arranged in the reactant mixture.Continue again to stir 30 minutes, or show that up to HPLC separating protection finishes.Water (6.5ml) diluted reaction mixture makes the final concentration of DMF reach 20%, and (0.45 μ m 2ml) filters to use syringe filter then.(10 μ m carry out the HPLC purification on 250*21.2mm) for PRLP-S, 300A, use acetonitrile-water-0.1% trifluoroacetic acid buffer as eluent at the polystyrene/divinylbenzene post.Check the fraction of collecting by HPLC with UV and ELS detector.Pure fraction is concentrated in together lyophilizing two days.After lyophilizing, obtained the conjugate of expectation, comprise the conjugate of even matter basically of the 5K-PEG-T20 at K28 place, be fluffy white solid (5.5mg).
In another variant of this embodiment, the α amine of the-terminal amino acid of SEQ ID NO:1 does not have acetylation, but uses the Fmoc radical protection.The process of synthesizing and combining that peptide is provided as the carrying out that provides in this embodiment 2 with polymer.Thereby the conjugate of even matter basically of generation is included in the 5K-PEG-T20 at K28 place, and just the T20 (SEQ ID NO:3) of conjugate contains the free α amine that-terminal amino acid (Y) is located.
Embodiment 3
What illustrate is another embodiment of method of the site-specific chemical modification of HIV gp41 derived peptide, and it can be used to produce (a) and has isolating HIV gp41 derived peptide with one or more internal amino acids of chemoprotectant pendant amine; (b) comprise the conjugate of the even basically matter of HIV gp41 derived peptide and polymer.In this illustrative embodiment; select T20 (SEQ ID NO:3) as exemplary synthetic peptide; select the lysine residue (" K28 ") of amino acid position 28 to carry out chemoproection by site-specific chemical modification; the lysine residue (" K18 ") that keeps amino acid position 18 is used for to link with polymer by the reactive functional group of the amine of lysine side-chain amine and polymer subsequently as free internal amino acid.For Fig. 6, use basically as the synthetic T20 (SEQ ID NO:3) of the fragment condensation method of describing among the above-mentioned embodiment 1.Briefly, summarize as Fig. 6, the solid phase synthesis by standard synthesizes the fragment that comprises preceding 16 amino acid whose SEQ ID NO:3, and the N-terminal amine of amino acid residue 1 (Tyr) is acetylizad (" Ac ").The fragment of synthesizing the SEQ ID NO:3 that comprises amino acid/11 7-26 by the solid phase synthesis of standard.Use Fmoc-Lys-(ivDde) to come the synthetic fragment that comprises the SEQ ID NO:3 of aminoacid 27-35 by standard solid-phase is synthetic as amino acid residue 28 (" K28 "), thus the ε amido that chemoproection reagent ivDde (" X " among Fig. 6) has sealed K28 subsequently with the chemical reactivity of the reactive functional group of amine.In liquid phase, back one fragment and aminoacid 36 are linked, form the fragment of the aminoacid 27-36 with SEQ ID NO:3.To have the fragment (K18 that contains the free ε amine of band) of amino acid/11 7-26 and fragment (K28 that the has the ivDde protection) combination of aminoacid 27-36.With aminoacid sequence that produces and the fragment combination that comprises aminoacid sequence 1-16, form the synthetic peptide of the aminoacid sequence that comprises SEQ ID NO:3 with amino acid/11 7-36.As what in this embodiment 1, describe in more detail, synthetic peptide (SEQ ID NO:3) is separated protection with the blocking group trt, the Boc that remove use in standard solid-phase is synthetic and tBu (and K28 is still chemoprotectant); Decarboxylation; Use the RPHPLC (reversed-phase high-performance liquid chromatography) method to carry out purification then.Then will this isolating HIV gp41 derived peptide be used for the polymer locus specificity be attached to the free ε amine of the K18 of SEQ ID NO:3.
Select mPEG succinyl phosphorons amino propyl acid salt (" mPEG-SPA ") conduct and the bonded exemplary polymer of T20 (SEQ ID NO:3).The T20 (SEQ ID NO:3) (19.7mg, 4.4 μ mol) that will have ivDde on the ε of K28 amido is dissolved among the DMF (0.5ml).Add DIEA (20 μ l) to reaction, that add then is mPEG-SPA (mean molecule quantity, 5000 dalton (" 5K ") among the DMF (1ml); 50mg, 10 μ mol).At room temperature stir mixture, monitor reaction, finish up to PEGization by HPLC.In order to remove chemoproection reagent ivDde from the ε amido of K28, (45 μ l) adds in the reaction with hydrazine, reaches the hydrazine of 3% (v/v) in the reactant mixture.Continue stirring again and carry out 30 minutes (or separate protection up to the HPLC demonstration and finish).Water (6.5ml) diluted reaction mixture makes the final concentration of DMF reach 20%, and (0.45 μ m 2ml) filters to use syringe filter then.(10 μ m carry out the HPLC purification on 250*21.2mm) for PRLP-S, 300A, use acetonitrile-water-0.1% trifluoroacetic acid buffer as eluent at the polystyrene/divinylbenzene post.Check the fraction of collecting by HPLC with UV and ELS detector.Pure fraction is concentrated in together lyophilizing two days.After lyophilizing, obtained the conjugate of expectation, comprise the conjugate of even matter basically of the 5K-PEG-T20 at K18 place, be fluffy white solid (10.4mg).
In another variant of this embodiment, the α amine of the-terminal amino acid of SEQ ID NO:1 does not have acetylation, but uses the Fmoc radical protection.The process of synthesizing and combining that peptide is provided as the carrying out that provides in this embodiment 3 with polymer.Thereby the conjugate of even matter basically of generation is included in the 5K-PEG-T20 at K18 place, and just the T20 (SEQ ID NO:3) of conjugate contains the free α amine that-terminal amino acid (Y) is located.
Embodiment 4
Explanation is in this embodiment: (a) measure the method for the antiviral activity of the conjugate of even matter basically produced according to the invention; (b) site-specific chemical modification production according to the present invention comprises the necessity with the conjugate of the even basically matter of the banded polymer of HIV gp41 derived peptide covalency.When using analyzed in vitro proof antiviral efficacy, be important to note that the antiviral effect of the synthetic peptide that proves is associated with intravital synthetic peptide antiviral effect in analyzed in vitro.At the antiviral activity of measuring the synthetic peptide-polymer conjugate that produces according to the present invention (for example, a kind of measurement is to suppress the ability that HIV propagates to target cell) time, what use is analyzed in vitro, by the data of use, shown that for the antiviral activity of observing in the body be predictability from the synthetic peptide generation in arbitrary HR zone of HIV gp41.More specifically, use external infectiousness analysis (" analysis of Magi-CCR5 infectiousness "; Referring to, for example United States Patent (USP) the 6th, 258, No. 782) observed antiviral activity shown and the identical HIV gp41 derived peptide suitable relatedness of observed antiviral activity in vivo.In order further to emphasize this point, all proved the powerful antiviral activity of T20 (SEQ ID NO:3) and T1249 (SEQ.ID NO:96) external infectiousness analysis and human clinical trial at HIV.
The CCR5 of infectiousness analysis and utilization indicating clone MAGI or expression derivant cMAGI comes the reduction score for the infective virus titre.Two kinds of cell lines have all been utilized the ability of HIV-1tat transactivation (transactivate) by the expression of the beta galactosidase reporter gene of HIV-LTR driving.Having revised β-gal report thing locatees in nuclear, can detect according to the strong nuclear staining of infecting in a couple of days with the X-gal substrate.If before dyeing, only take turns infection, the number of painted nuclear thereby can be interpreted as equaling to attack the number of infectious virus particle in the inoculum.Use the CCD imager that infected cell is counted, the separator that primary laboratory is revised has all shown viral input and by the linear relationship between the infected cell number of imager range estimation.In MAGI and cMAGI analysis, it is significant that 50% in the infectivity titer reduces (Vn/Vo=0.5), and original boundaries the value (" IC that is used to evaluate antiviral activity is provided 50" be defined as the dilution factor that causes that infective virus titre 50% reduces).Also evaluated second border of Vn/Vo=0.1, be equivalent to 90% of infectivity titer and reduce (" IC 90").The conjugate of the even basically matter of test antiviral activity is diluted to various concentration, comes relative HIV inoculums to test with two parts or three parts, described HIV inoculum is conditioned to produce about 1500-2000 infected cell/hole on the 48 hole microtitration plates.In cMAGI or MAGI cell, add the conjugate (with each dilution factor) of even matter basically, succeeded by the virus inoculation thing; After 24 hours, the mortifier that adds infection and cell fusion (for example, T20) stops the second HIV infection and cell-cell virus of taking turns to be propagated.Cell is cultivated more than 2 days again, fixed and detect the cell of HIV infection then with the X-gal substrate staining.To the number of each contrast, calculate IC with the CCD imager then with the conjugate dilution factor mensuration infected cell of sparing matter basically 50And IC 90(generally representing) with μ g/ml.
In this embodiment, analyzed several antiviral activities of the conjugate of even matter basically that produce separately by method described here, as shown in table 1, mark following." conjugate A " is the synthetic peptide with aminoacid sequence of SEQ ID NO:3, has the 2K PEG (thereby the pendant amine of K18 and K28 is all by chemoproection between synthesis stage) that locus specificity is attached to the N-terminal amine." conjugate B " is the synthetic peptide with aminoacid sequence of SEQ ID NO:3, has the 2K PEG (thereby the pendant amine of N-terminal amine and K28 is all by chemoproection between synthesis stage) that locus specificity is attached to K18." conjugate C " is the synthetic peptide with aminoacid sequence of SEQ ID NO:3, has the 2K PEG (thereby the pendant amine of N-terminal amine and K18 is all by chemoproection between synthesis stage) that locus specificity is attached to K28." conjugate D " is the synthetic peptide with aminoacid sequence of SEQ ID NO:3, has the 5K PEG (thereby the pendant amine of K18 and K28 is all by chemoproection between synthesis stage) that locus specificity is attached to the N-terminal amine." conjugate E " is the synthetic peptide with aminoacid sequence of SEQ ID NO:3, has the 5K PEG (thereby the pendant amine of N-terminal amine and K28 is all by chemoproection between synthesis stage) that locus specificity is attached to K18." conjugate F " is the synthetic peptide with aminoacid sequence of SEQ ID NO:3, has the 5K PEG (thereby the pendant amine of N-terminal amine and K18 is all by chemoproection between synthesis stage) that locus specificity is attached to K28.
Table 1
The molecule of test Antiviral activity (IC50 μ g/ml)
Synthetic peptide (SEQ ID NO:3) ＜0.01
Conjugate A ＜0.02
Conjugate B ＞0.05＜0.1
Conjugate C ＞0.15
Conjugate D ＞0.05＜0.1
Conjugate E ＞0.3
Conjugate F ＞0.4
It is apparent that relatively from this, as 2K (2, when the polymer scale of mean size 000 dalton) was incorporated into the N-terminal amine, contrast is combined in K18 (active low at least 5 times) or K28 (active low at least 10 times) locates, and has kept the antiviral activity of synthetic peptide best.Similarly, be apparent that when the polymer scale of 5K was incorporated into the N-terminal amine, contrast is combined in K18 (active low about 5 times) or K28 (active low about 7 times) locates, and has kept the antiviral activity of synthetic peptide best.Further, can conclude from this embodiment, site-specific chemical modification method according to the present invention can be used for locus specificity ground and polymer scale is incorporated into the selected aminoacid of synthetic peptide, production have aspiration level biologic activity the peptide-polymer conjugate even basically matter conjugate (for example, in this embodiment, the antiviral activity that measures expectation has the IC that is lower than 0.02 μ g/ml 50) (for example, " the conjugate A " of table 1), and avoid using the production of standard P EGization to lack the synthetic peptide-polymer conjugate (for example, the mixture of " the conjugate A " of table 1 and " conjugate B " and " conjugate C ") of multiple class of the biologic activity of aspiration level.
Embodiment 5
In this embodiment, what illustrate is other embodiments of the site-specific chemical modification method of HIV gp41 derived peptide, wherein between synthesis stage, be incorporated into and in the synthetic peptide be: one or more aminoacid, it has by chemoproection reagent and has sealed subsequently chemically reactive pendant amine with the reactive functional group of amine; With one or more aminoacid, its have unprotected, can with the amine (for example, being selected from) of the reactive functional group chemical reaction of amine by the terminal α amine of N-, one or more ε amine and their group that constitutes.The isolating HIV gp41 derivant that will produce then is attached on the polymer, produces the conjugate of even matter basically that comprises polymer and HIV gp-41 derived peptide.
According to the instruction of synthesizing HIV gp41 derived peptide among the embodiment 1 about the fragment condensation method, it will be apparent for a person skilled in the art that, can use the method for this fragment combination, be used to have some synthetic peptide of sequence shown in the SEQ ID NO:3-175 in the method according to the invention.Generally speaking, general synthetic 3 fragments (referring to, Fig. 3 for example): " N-terminal fragment " (aminoterminal 10 to 20 aminoacid that comprise synthetic peptide usually), " C-terminal fragment " (10 to 20 aminoacid that comprise the carboxyl terminal of synthetic peptide usually), " intermediate segment " (comprising 10 to 20 aminoacid that exist between the N-terminal fragment and C-terminal fragment in the synthetic peptide usually), then with their combinations to produce complete synthetic peptide.Yet, depend on the amino acid whose numbering and the position that have pendant amine in the length, aminoacid sequence, aminoacid sequence of specific synthetic peptide, can Synthetic 2 to 4 fragment, make up then to finish the synthetic of this specific synthetic peptide.
For example, T1249 (SEQ ID NO:96) is used for according to site-specific chemical modification method of the present invention.In this embodiment, use 3 fragments, by the fragment condensation method is synthetic should synthetic peptide: the N-terminal fragment comprises amino acid/11-12 and contains lysine (" K7 ") and acetylizad-terminal amino acid at amino acid position 7; Intermediate segment comprises amino acid/11 3 to 26 and contains the lysine (" K21 ") of amino acid position 21; The C-terminal fragment comprise aminoacid 27 to 36 and contain the lysine (" K28 ") of amino acid position 28 and the lysine (" K31 ") of amino acid position 31 (amino acid position is numbered corresponding to each position among the SEQ ID NO:96, that is the position in the synthetic peptide of combination).Produced individually according to many isolating HIV gp41 derived peptide of the present invention: (a) have the synthetic peptide of the aminoacid sequence of SEQ ID NO:96, on K7, K21, K28 and K31, have chemoproection reagent (only staying the N-terminal amine can combine with polymer subsequently); (b) have the synthetic peptide of the aminoacid sequence of SEQ ID NO:96, have the chemoproection reagent (only staying the K7 pendant amine can combine with polymer subsequently) on N-terminal amine, K21, K28 and the K31; (c) have the synthetic peptide of the aminoacid sequence of SEQ ID NO:96, have the chemoproection reagent (only staying the K21 pendant amine can combine with polymer subsequently) on N-terminal amine, K7, K28 and the K31; (d) have the synthetic peptide of the aminoacid sequence of SEQ ID NO:96, have the chemoproection reagent (only staying the K28 pendant amine can combine with polymer subsequently) on N-terminal amine, K7, K21 and the K31; And the synthetic peptide that (e) has the aminoacid sequence of SEQ ID NO:96, have the chemoproection reagent (only staying the K31 pendant amine can combine with polymer subsequently) on N-terminal amine, K7, K21 and the K28.
Have the isolating HIV gp41 derived peptide and the specific chemical modification method described here of the aminoacid sequence of SEQ ID NO:96 according to these, the PEG of the different sizes of use from 2K dalton (" 2K ") meansigma methods to 20K dalton (" 20K ") meansigma methods produces several conjugates of even matter basically individually.Table 2 has shown these some antiviral activities of certain in conjugates of even matter basically, following marking." conjugate A " is the synthetic peptide with aminoacid sequence of SEQ IDNO:96, has the 2K PEG (thereby the pendant amine of K7, K21, K28 and K31 is all by chemoproection between synthesis stage) that locus specificity is attached to the N-terminal amine." conjugate B " is the synthetic peptide with aminoacid sequence of SEQ ID NO:96, has the 2K PEG (thereby the pendant amine of N-terminal amine and K21, K28 and K31 is all by chemoproection between synthesis stage) that locus specificity is attached to K7." conjugate C " is the synthetic peptide with aminoacid sequence of SEQ ID NO:96, has the 2K PEG (thereby the pendant amine of N-terminal amine and K7, K28 and K31 is all by chemoproection between synthesis stage) that locus specificity is attached to K21." conjugate D " is the synthetic peptide with aminoacid sequence of SEQ ID NO:96, has the 2K PEG (thereby the pendant amine of N-terminal amine and K7, K21 and K31 is all by chemoproection between synthesis stage) that locus specificity is attached to K28." conjugate E " is the synthetic peptide with aminoacid sequence of SEQ ID NO:96, has the 2K PEG (thereby the pendant amine of N-terminal amine and K7, K21 and K28 is all by chemoproection between synthesis stage) that locus specificity is attached to K31." conjugate F " is the synthetic peptide with aminoacid sequence of SEQ ID NO:96, has the 5K PEG that locus specificity is attached to the N-terminal amine." conjugate G " is the synthetic peptide with aminoacid sequence of SEQ ID NO:96, has the 5K PEG that locus specificity is attached to K7." conjugate H " is the synthetic peptide with aminoacid sequence of SEQ IDNO:96, has the 5K PEG that locus specificity is attached to K21." conjugate I " is the synthetic peptide with aminoacid sequence of SEQ ID NO:96, has the 5KPEG that locus specificity is attached to K28." conjugate J " is the synthetic peptide with aminoacid sequence of SEQ ID NO:96, has the 5K PEG that locus specificity is attached to K31.
Table 2
The molecule of test Antiviral activity (IC50 μ g/ml)
Synthetic peptide (SEQ ID NO:96) ＜0.01
Conjugate A ＜0.01
Conjugate B ＜0.01
Conjugate C ＞0.01
Conjugate D ＜0.01
Conjugate E ＞0.01
Conjugate F ＞0.03
Conjugate G ＞0.04
Conjugate H ＞0.04
Conjugate I ＞0.03
Conjugate J ＞0.04
Also consider the result of the PEG of use mean size 2K (2000 dalton) according to this embodiment, can conclude, site-specific chemical modification method according to the present invention can be used for the polymer locus specificity be attached to the selected aminoacid of synthetic peptide, generation have aspiration level biologic activity even basically matter conjugate (for example, in this embodiment, the antiviral activity that measures expectation has the IC that is lower than 0.01 μ g/ml 50) (for example, " the conjugate A " of table 2, " conjugate B " and " conjugate D "), and avoid using the generation of standard P EGization to lack the synthetic peptide-polymer conjugate (for example, the mixture of the conjugate A-E of table 2) of multiple class of the biologic activity of aspiration level.
Embodiment 6
In another embodiment, the synthetic peptide that will have the aminoacid sequence of SEQ ID NO:174 is used for according to site-specific chemical modification method of the present invention.In this embodiment, synthetic peptide contains the lysine (" K30 ") of amino acid position 30 and the lysine (" K39 " of amino acid position 39; The C-end amino acid; The amino acid position numbering is corresponding to the position among the SEQ ID NO:174); come syntheticly by linearity is synthetic, have on the N-terminal amine of-terminal amino acid and the chemoproection reagent (only staying the K39 pendant amine can combine with polymer subsequently) on the pendant amine of K30.Use have the mean size of 2K polymer, have 5K mean size polymer and have the polymer of the mean size of 20K, produce the conjugate of even matter basically individually from this isolating HIV gp41 derived peptide.For example, synthetic peptide (not combining with polymer) itself has the biologic activity of aspiration level (for example, the antiviral activity of measurement has the IC of being less than or equal to 0.02 μ g/ml 50), and the conjugate of even matter basically that has synthesized polymer with 2K mean size has the biologic activity that approximates 0.02 μ g/ml greatly.The conjugate of even matter basically with polymer of mean size 5K or 20K has the biologic activity more much bigger than 0.1 μ g/ml (that is, outside this embodiment desired biological activity scope).
Embodiment 7
The invention provides the conjugate of even matter basically (" synthetic peptide-polymer conjugate ") that comprises locus specificity and be attached to the HIV gp41 derived peptide of polymer.The antiviral activity of this synthetic peptide-polymer conjugate can utilize in suppressing the method that HIV propagates into target cell, comprise to virus and cell interpolation an amount of according to synthetic peptide-polymer conjugate of the present invention, effectively suppress the HIV infection cell, and preferred, suppress the fusion of the HIV mediation between virus and the target cell.This method can be used for treating individuality (therapeutic ground) that HIV infects or treatment and be exposed to (for example, take drugs or high risk sexual behavior) individual (prophylactically) that HIV or existence are exposed to the HIV risk recently.Thereby for example, for the individuality of infected by HIV-1, the synthetic peptide-polymer conjugate of effective dose is the dosage that is enough to reduce HIV virus loads in the individuality that will treat (itself, and/or together with administering mode).It is known to those skilled in the art that the standard method that exists several measurement HIV virus loads, include but not limited to, carry out quantitatively and pass through the measurement of blood plasma HIV RNA by culture peripheral blood lymphocytes.Synthetic peptide-polymer conjugate of the present invention can single administration, off and on, periodically or continuously use, can determine by for example monitoring virus loads by practitioner.Depend on the preparation that contains synthetic peptide-polymer conjugate, and some factors, as be used to form the polymer of synthetic peptide-polymer conjugate and the composition of synthetic peptide, whether further comprise pharmaceutically acceptable carrier, with the character of pharmaceutically acceptable carrier, can or may use in the cycle for more time with a couple of days to several weeks according to synthetic peptide-polymer conjugate of the present invention.Further, when with other antiviral drugs that are used for the treatment of HIV (for example, include but not limited to, other HIV (for example enters mortifier, the CCR5 mortifier, retrocyclin or the like), the hiv integrase mortifier, the reverse transcriptase mortifier (for example, nucleoside or non-nucleoside), protease inhibitor, virus-specific is transcribed mortifier, virus processing mortifier, the ripe mortifier of HIV, the mortifier of uridine phosphorylase, the HIV vaccine, or the like, be well known in the art) when in combination or in therapeutic modality, using (for example, when using simultaneously, or with a kind of medicine circulate get on and when breaking away from another kind of medicine), in antiviral therapy, can use according to synthetic peptide-polymer conjugate of the present invention.
For example, one preferred embodiment in, can be in treatment, thereby improve the effectiveness of treatment and reduce the virus enantiopathy cytotoxic drug that becomes the ability of resistance is arranged with one or more antiviral agent and synthetic peptide-polymer conjugate combination according to the present invention.This compositions can prepare from the antiviral agent (useful treatment HIV infects) of effective dose current approval or that ratify in the future, these include but not limited to, abacavir, AZT, delaviridine, ddC, ddI, efavirenz, FTC, (+) and (-) FTC, Reverset, GS 840, HBY097,3TC, nevirapine, d4T, FLT, emtricitabine, amprenivir, CGP-73547, CGP-61755, DMP-450, indinavir, nelfinavir, PNU-140690, ritonavir, saquinavir, telinavir, tenofovir, adefovir, atazanavir, lopinavir, VX 478, PRO-542, and betulin and dihydrobetulin derivant are (for example, PA-457).The effective dose that can be used for these illustrative antiviral agent of synthetic peptide-polymer conjugate combination according to the present invention is known in the art.This compositions can comprise many antiviral agent, and they can or side by side be used by one or more approach order ground, depend on the pharmacological effect of route of administration and expectation, are conspicuous for those skilled in the art.
The effective dose of of the present invention synthetic peptide-polymer conjugate to be administered can be determined by those processes well known in the art; For example, by measuring effectiveness, biological half-life, bioavailability and toxicity.In preferred embodiment, be used to from well known to a person skilled in the art that the data of studying in the conventional external and body determine effectively to synthesize peptide-polymer conjugate dosage range by those skilled in the art.For example, as the external infectiousness analysis of antiviral activity described here, allow those skilled in the art to determine that (for example, 50% suppresses IC to a certain amount of viral infectivity of blocking-up 50Or 90% suppress IC 90) mean inhibitory concentration (IC) of required synthetic peptide-polymer conjugate.Use can be selected proper dosage from pharmacokinetic data, those skilled in the art of one or more standard animal model, thereby obtain the minimum plasma concentration (C[min]) of described synthetic peptide-polymer conjugate, it is equal to or higher than predetermined IC value.Though dosage range generally depends on the route of administration of selection and the prescription of dosage, can be not less than 0.1 μ g/kg body weight and not be higher than the 10mg/kg body weight according to the exemplary dosage range of synthetic peptide-polymer conjugate of the present invention; The preferred dosage scope is about 0.1-100 μ g/kg body weight; Preferred, about 10mg is to the dosage of the synthetic peptide-polymer conjugate between about 250mg.
Can use synthetic peptide-polymer conjugate of the present invention to individuality by any means that allow active agent to arrive target cell (cell that can be infected by HIV).Thereby, synthetic peptide-polymer conjugate of the present invention can be used by any suitable technology, comprise oral, parenteral (for example, intramuscular, endoperitoneal, intravenous or subcutaneous injection or infusion, Intradermal or implant), nose, pulmonary, vagina, rectum, the Sublingual or surperficial route of administration, can be formulated as the dosage form that is suitable for every kind of route of administration.Concrete route of administration will depend on, for example, individual medical history comprises the side effect of this any known or expection of using and the composition of the conjugate that will use (for example, the polymer that comprised of synthetic peptide-polymer conjugate and the character of synthetic peptide).Most preferred, using is by injection (use, for example, intravenous or subcutaneous means), but also can pass through successive infusion (use, for example, delayed release device or pony pump be osmotic pumps for example, or the like).Synthetic peptide-polymer conjugate according to the present invention can further comprise pharmaceutically acceptable carrier; Can further depend on known other factors of the prescription of expectation, the site of sending, the method for using, the arrangement of using and practitioner.
For illustrative purposes, described the above-mentioned explanation of the specific embodiment of the present invention in detail.In view of this description and illustrative explanation, others skilled in the art can be by using existing knowledge, easily changes and/or revise the present invention to be used for different application and do not deviate from basic notion; Therefore, this modification and/or improvement are determined to be within the meaning and scope of appended claim.
Sequence table
Sequence table
＜110〉Trimeris Inc.
＜120〉site-specific chemical modification of HIV gp41 derived peptide
<130>TRM-008PCT
<150>US 60/553,063
<151>2004-03-15
<160>175
＜170〉the PatentIn translation 3.2
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Arg Ala Ile Glu Ala Gln Gln His Leu Leu Gln Leu Thr Val Trp Gly
1 5 10 15
Ile Lys Gln Leu Gln Ala Arg Ile Leu Ala Val Glu Arg Tyr Leu Lys
20 25 30
Asp Gln
<210>18
<211>41
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>18
Cys Gly Gly Asn Asn Leu Leu Arg Ala Ile Glu Ala Gln Gln His Leu
1 5 10 15
Leu Gln Leu Thr Val Trp Gly Ile Lys Gln Leu Gln Ala Arg Ile Leu
20 25 30
Ala Val Glu Arg Tyr Leu Lys Asp Gln
35 40
<210>19
<211>31
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>19
Asn Asn Leu Leu Arg Ala Ile Glu Ala Gln Gln His Leu Leu Gln Leu
1 5 10 15
Thr Val Trp Gly Ile Lys Gln Leu Gln Ala Arg Ile Leu Ala Val
20 25 30
<210>20
<211>41
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>20
Asn Asn Leu Leu Arg Ala Ile Glu Ala Gln Gln His Leu Leu Gln Leu
1 5 10 15
Thr Val Trp Gly Ile Lys Gln Leu Gln Ala Arg Ile Leu Ala Val Glu
20 25 30
Arg Tyr Leu Lys Asp Gln Gly Gly Cys
35 40
<210>21
<211>44
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>21
Cys Gly Gly Asn Asn Leu Leu Arg Ala Ile Glu Ala Gln Gln His Leu
1 5 10 15
Leu Gln Leu Thr Val Trp Gly Ile Lys Gln Leu Gln Ala Arg Ile Leu
20 25 30
Ala Val Glu Arg Tyr Leu Lys Asp Gln Gly Gly Cys
35 40
<210>22
<211>39
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>22
Leu Ser Gly Ile Val Gln Gln Gln Asn Asn Leu Leu Arg Ala Ile Glu
1 5 10 15
Ala Gln Gln His Leu Leu Gln Leu Thr Val Trp Gly Ile Lys Gln Leu
20 25 30
Gln Ala Arg Ile Leu Ala Val
35
<210>23
<211>41
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>23
Gln Gln Gln Asn Asn Leu Leu Arg Ala Ile Glu Ala Gln Gln His Leu
1 5 10 15
Leu Gln Leu Thr Ala Trp Gly Ile Lys Gln Leu Gln Ala Arg Ile Leu
20 25 30
Ala Val Glu Arg Tyr Leu Lys Asp Gln
35 40
<210>24
<211>41
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>24
Gln Gln Gln Asn Asn Leu Leu Arg Ala Ile Glu Ala Gln Gln His Leu
1 5 10 15
Leu Gln Leu Thr Val Ala Gly Ile Lys Gln Leu Gln Ala Arg Ile Leu
20 25 30
Ala Val Glu Arg Tyr Leu Lys Asp Gln
35 40
<210>25
<211>49
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>25
Gln Ala Arg Gln Leu Leu Ser Gly Ile Val Gln Gln Gln Asn Asn Leu
1 5 10 15
Leu Arg Ala Ile Glu Ala Gln Gln His Ala Leu Gln Ala Thr Val Trp
20 25 30
Gly Ile Lys Gln Leu Gln Ala Arg Ile Leu Ala Val Glu Arg Tyr Leu
35 40 45
Lys
<210>26
<211>51
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>26
Gln Ala Arg Gln Leu Leu Ser Gly Ile Val Gln Gln Gln Asn Asn Leu
1 5 10 15
Leu Arg Ala Ile Glu Ala Gln Gln His Ala Leu Gln Ala Thr Val Trp
20 25 30
Gly Ile Lys Gln Leu Gln Ala Arg Ile Leu Ala Val Glu Arg Tyr Leu
35 40 45
Lys Asp Gln
50
<210>27
<211>49
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>27
Gln Ala Arg Gln Leu Val Ser Gly Leu Val Gln Gln Gln Asn Asn Ile
1 5 10 15
Leu Arg Ala Leu Glu Ala Thr Gln His Ala Val Gln Ala Leu Val Trp
20 25 30
Gly Val Lys Gln Leu Gln Ala Arg Val Leu Ala Leu Glu Arg Tyr Ile
35 40 45
Lys
<210>28
<211>49
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>28
Gln Ile Arg Gln Leu Leu Ser Gly Ile Val Gln Gln Gln Asn Asn Leu
1 5 10 15
Leu Arg Ala Ile Glu Ala Ile Gln His Ala Leu Gln Ala Ile Val Trp
20 25 30
Gly Ile Lys Gln Leu Gln Ala Arg Ile Leu Ala Val Glu Arg Tyr Leu
35 40 45
Lys
<210>29
<211>49
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>29
Gln Ala Arg Gln Leu Val Ser Gly Leu Val Gln Gln Gln Asn Asn Ile
1 5 10 15
Leu Arg Ala Leu Glu Ala Thr Gln His Ala Val Gln Ala Leu Val Trp
20 25 30
Gly Val Arg Gln Leu Gln Ala Arg Val Leu Ala Leu Glu Arg Tyr Ile
35 40 45
Lys
<210>30
<211>51
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>30
Gln Ala Arg Gln Leu Leu Ser Gly Ile Val Gln Gln Gln Asn Asn Leu
1 5 10 15
Leu Arg Ala Ile Glu Ala Thr Gln His Ala Val Gln Ala Leu Val Trp
20 25 30
Gly Val Lys Gln Leu Gln Ala Arg Val Leu Ala Leu Glu Arg Tyr Ile
35 40 45
Lys Asp Gln
50
<210>31
<211>51
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>31
Gln Ala Arg Gln Leu Val Ser Gly Leu Val Gln Gln Gln Asn Asn Ile
1 5 10 15
Leu Arg Ala Leu Glu Ala Gln Gln His Ala Leu Gln Ala Thr Val Trp
20 25 30
Gly Ile Lys Gln Leu Gln Ala Arg Val Leu Ala Leu Glu Arg Tyr Ile
35 40 45
Lys Asp Gln
50
<210>32
<211>51
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>32
Gln Ala Arg Gln Leu Leu Ser Gly Ile Val Gln Gln Gln Asn Asn Leu
1 5 10 15
Leu Arg Ala Ile Glu Ala Gln Gln His Ala Leu Gln Ala Thr Val Trp
20 25 30
Gly Val Lys Gln Leu Gln Ala Arg Ile Leu Ala Val Glu Arg Tyr Leu
35 40 45
Lys Asp Gln
50
<210>33
<211>41
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>33
Gln Gln Gln Asn Asn Leu Leu Arg Ala Ile Glu Ala Gln Gln His Leu
1 5 10 15
Leu Gln Leu Thr Val Phe Gly Ile Lys Gln Leu Gln Ala Arg Ile Leu
20 25 30
Ala Val Glu Arg Tyr Leu Lys Asp Gln
35 40
<210>34
<211>49
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>34
Gln Ala Arg Gln Leu Leu Ser Gly Ile Val Gln Gln Gln Asn Asn Leu
1 5 10 15
Leu Arg Ala Ile Glu Ala Gln Gln His Leu Leu Gln Leu Thr Val Phe
20 25 30
Gly Ile Arg Gln Leu Gln Ala Arg Ile Leu Ala Val Glu Arg Tyr Leu
35 40 45
Lys
<210>35
<211>51
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>35
Gln Ala Arg Gln Leu Leu Ser Gly Ile Val Gln Gln Gln Asn Asn Leu
1 5 10 15
Leu Arg Ala Ile Glu Ala Gln Gln His Leu Leu Gln Ala Thr Val Trp
20 25 30
Gly Ile Lys Gln Leu Gln Ala Arg Ile Leu Ala Val Glu Arg Tyr Leu
35 40 45
Lys Asp Gln
50
<210>36
<211>41
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>36
Gln Gln Gln Asn Asn Leu Leu Arg Ala Ile Glu Ala Gln Gln His Leu
1 5 10 15
Leu Gln Ala Thr Val Trp Gly Ile Lys Gln Leu Gln Ala Arg Ile Leu
20 25 30
Ala Val Glu Arg Tyr Leu Lys Asp Gln
35 40
<210>37
<211>36
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>37
Asn Ala Ser Trp Ser Asn Lys Ser Leu Glu Gln Ile Trp Asn Asn Met
1 5 10 15
Thr Trp Met Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr Ser Leu Ile
20 25 30
His Ser Leu Ile
35
<210>38
<211>36
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>38
Asn Lys Ser Leu Glu Gln Ile Trp Asn Asn Met Thr Trp Met Glu Trp
1 5 10 15
Asp Arg Glu Ile Asn Asn Tyr Thr Ser Leu Ile His Ser Leu Ile Glu
20 25 30
Glu Ser Gln Asn
35
<210>39
<211>36
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>39
Glu Gln Ile Trp Asn Asn Met Thr Trp Met Glu Trp Asp Arg Glu Ile
1 5 10 15
Asn Asn Tyr Thr Ser Leu Ile His Ser Leu Ile Glu Glu Ser Gln Asn
20 25 30
Gln Gln Glu Lys
35
<210>40
<211>36
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>40
Gln Ile Trp Asn Asn Met Thr Trp Met Glu Trp Asp Arg Glu Ile Asn
1 5 10 15
Asn Tyr Thr Ser Leu Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln
20 25 30
Gln Glu Lys Asn
35
<210>41
<211>36
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>41
Ile Trp Asn Asn Met Thr Trp Met Glu Trp Asp Arg Glu Ile Asn Asn
1 5 10 15
Tyr Thr Ser Leu Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln
20 25 30
Glu Lys Asn Glu
35
<210>42
<211>36
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>42
Trp Asn Asn Met Thr Trp Met Glu Trp Asp Arg Glu Ile Asn Asn Tyr
1 5 10 15
Thr Ser Leu Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu
20 25 30
Lys Asn Glu Gln
35
<210>43
<211>36
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>43
Asn Asn Met Thr Trp Met Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr
1 5 10 15
Ser Leu Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys
20 25 30
Asn Glu Gln Glu
35
<210>44
<211>36
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>44
Asn Met Thr Trp Met Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr Ser
1 5 10 15
Leu Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys Asn
20 25 30
Glu Gln Glu Leu
35
<210>45
<211>36
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>45
Thr Trp Met Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr Ser Leu Ile
1 5 10 15
His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys Asn Glu Gln
20 25 30
Glu Leu Leu Glu
35
<210>46
<211>38
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>46
Met Thr Trp Met Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr Ser Leu
1 5 10 15
Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys Asn Glu
20 25 30
Gln Glu Leu Leu Glu Leu
35
<210>47
<211>35
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>47
Met Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr Ser Leu Ile His Ser
1 5 10 15
Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys Asn Glu Gln Glu Leu
20 25 30
Leu Glu Asp
35
<210>48
<211>36
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>48
Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr Ser Leu Ile His Ser Leu
1 5 10 15
Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys Asn Glu Gln Glu Leu Leu
20 25 30
Glu Leu Asp Lys
35
<210>49
<211>36
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>49
Trp Asp Arg Glu Ile Asn Asn Tyr Thr Ser Leu Ile His Ser Leu Ile
1 5 10 15
Glu Glu Ser Gln Asn Gln Gln Glu Lys Asn Glu Gln Glu Leu Leu Glu
20 25 30
Leu Asp Lys Trp
35
<210>50
<211>36
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>50
Asn Tyr Thr Ser Leu Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln
1 5 10 15
Gln Glu Lys Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser
20 25 30
Leu Trp Asn Trp
35
<210>51
<211>36
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>51
Thr Ser Leu Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu
1 5 10 15
Lys Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu Trp
20 25 30
Asn Trp Phe Asn
35
<210>52
<211>36
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>52
Ser Leu Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys
1 5 10 15
Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu Trp Asn
20 25 30
Trp Phe Asn Ile
35
<210>53
<211>36
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>53
Leu Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys Asn
1 5 10 15
Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu Trp Asn Trp
20 25 30
Phe Asn Ile Thr
35
<210>54
<211>43
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>54
Lys Ser Leu Glu Gln Ile Trp Asn Asn Met Thr Trp Met Glu Trp Glu
1 5 10 15
Arg Glu Ile Asp Asn Tyr Thr Ser Leu Ile Tyr Ser Leu Ile Glu Glu
20 25 30
Ser Gln Asn Gln Gln Glu Lys Asn Glu Gln Glu
35 40
<210>55
<211>36
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>55
Asn Asn Met Thr Trp Met Glu Trp Glu Arg Glu Ile Asp Asn Tyr Thr
1 5 10 15
Ser Leu Ile Tyr Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys
20 25 30
Asn Glu Gln Glu
35
<210>56
<211>30
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>56
Glu Trp Glu Arg Glu Ile Asp Asn Tyr Thr Ser Leu Ile Tyr Ser Leu
1 5 10 15
Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys Asn Glu Gln Glu
20 25 30
<210>57
<211>36
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>57
Tyr Thr Asn Thr Ile Tyr Thr Leu Leu Glu Glu Ser Gln Asn Gln Gln
1 5 10 15
Glu Lys Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu
20 25 30
Trp Asn Trp Phe
35
<210>58
<211>36
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>58
Tyr Thr Gly Ile Ile Tyr Asn Leu Leu Glu Glu Ser Gln Asn Gln Gln
1 5 10 15
Glu Lys Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Asn Leu
20 25 30
Trp Asn Trp Phe
35
<210>59
<211>36
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>59
Tyr Thr Ser Leu Ile Tyr Ser Leu Leu Glu Lys Ser Gln Ile Gln Gln
1 5 10 15
Glu Lys Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu
20 25 30
Trp Asn Trp Phe
35
<210>60
<211>36
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>60
Tyr Thr Ser Leu Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln
1 5 10 15
Glu Lys Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu
20 25 30
Phe Asn Phe Phe
35
<210>61
<211>42
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>61
Asp Arg Glu Ile Asn Asn Tyr Thr Ser Leu Ile His Ser Leu Ile Glu
1 5 10 15
Glu Ser Gln Asn Gln Gln Glu Lys Asn Glu Gln Glu Leu Leu Glu Leu
20 25 30
Asp Lys Trp Ala Ser Leu Trp Asn Trp Phe
35 40
<210>62
<211>48
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>62
Met Thr Trp Met Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr Ser Leu
1 5 10 15
Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys Asn Glu
20 25 30
Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu Trp Asn Trp Phe
35 40 45
<210>63
<211>42
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>63
Asn Asn Met Thr Trp Met Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr
1 5 10 15
Ser Leu Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys
20 25 30
Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys
35 40
<210>64
<211>38
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>64
Met Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg
1 5 10 15
Ile Glu Ala Leu Leu Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Ala Ala Leu Arg Glu Leu
35
<210>65
<211>46
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>65
Ala Pro Lys Glu Met Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu
1 5 10 15
Tyr Ala Ala Arg Ile Glu Ala Leu Ile Arg Ala Ala Gln Glu Gln Gln
20 25 30
Glu Lys Asn Glu Ala Ala Leu Arg Glu Leu Lys Gln Gly Ile
35 40 45
<210>66
<211>38
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>66
Met Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg
1 5 10 15
Ile Glu Ala Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Ala Ala Leu Arg Glu Leu
35
<210>67
<211>38
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>67
Thr Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg
1 5 10 15
Ile Glu Ala Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Ala Ala Leu Arg Glu Leu
35
<210>68
<211>38
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>68
Met Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Ala Ala Ala Arg
1 5 10 15
Ile Glu Ala Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Ala Ala Leu Arg Glu Leu
35
<210>69
<211>38
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>69
Met Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg
1 5 10 15
Ile Glu Ala Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Ala Glu
20 25 30
Ala Ala Leu Arg Glu Leu
35
<210>70
<211>42
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>70
Met Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg
1 5 10 15
Ile Glu Ala Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Ala Ala Leu Arg Glu Leu Trp Glu Trp Phe
35 40
<210>71
<211>42
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>71
Thr Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg
1 5 10 15
Ile Glu Ala Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Ala Ala Leu Arg Glu Leu Trp Glu Trp Phe
35 40
<210>72
<211>41
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>72
Thr Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg
1 5 10 15
Ile Glu Ala Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Ala Ala Leu Arg Glu Trp Glu Trp Phe
35 40
<210>73
<211>38
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>73
Thr Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg
1 5 10 15
Ile Glu Ala Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Ala Ala Trp Glu Trp Phe
35
<210>74
<211>48
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>74
Trp Glu Trp Phe Gly Gly Ser Gly Gly Ser Thr Thr Trp Glu Ala Trp
1 5 10 15
Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg Ile Glu Ala Leu Ile Arg
20 25 30
Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu Ala Ala Leu Arg Glu Leu
35 40 45
<210>75
<211>48
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>75
Thr Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg
1 5 10 15
Ile Glu Ala Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Ala Ala Leu Arg Glu Leu Gly Gly Ser Gly Gly Ser Trp Glu Trp Phe
35 40 45
<210>76
<211>45
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>76
Thr Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg
1 5 10 15
Ile Glu Ala Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Ala Ala Leu Arg Glu Leu Gly Gly Ser Gly Gly Ser Trp
35 40 45
<210>77
<211>45
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>77
Trp Gly Gly Ser Gly Gly Ser Thr Thr Trp Glu Ala Trp Asp Arg Ala
1 5 10 15
Ile Ala Glu Tyr Ala Ala Arg Ile Glu Ala Leu Ile Arg Ala Ala Gln
20 25 30
Glu Gln Gln Glu Lys Asn Glu Ala Ala Leu Arg Glu Leu
35 40 45
<210>78
<211>39
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>78
Pro Thr Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala
1 5 10 15
Arg Ile Glu Ala Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn
20 25 30
Glu Ala Ala Leu Arg Glu Leu
35
<210>79
<211>40
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>79
Pro Thr Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala
1 5 10 15
Arg Ile Glu Ala Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn
20 25 30
Glu Ala Ala Leu Arg Glu Leu Pro
35 40
<210>80
<211>38
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>80
Thr Thr Trp Glu Ala Trp Asp Lys Ala Ile Ala Glu Tyr Ala Ala Lys
1 5 10 15
Ile Glu Ala Leu Ile Lys Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Ala Ala Leu Lys Glu Leu
35
<210>81
<211>38
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>81
Thr Thr Trp Glu Ala Trp Asp Arg Ala Trp Gln Glu Trp Glu Gln Lys
1 5 10 15
Ile Glu Ala Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Ala Ala Leu Arg Glu Leu
35
<210>82
<211>38
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>82
Thr Thr Trp Ala Ala Trp Asp Ala Ala Ile Ala Glu Tyr Ala Ala Arg
1 5 10 15
Ile Glu Ala Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Ala Ala Leu Arg Glu Leu
35
<210>83
<211>38
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>83
Thr Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Ala Tyr Ala Ala Ala
1 5 10 15
Ile Glu Ala Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Ala Ala Leu Arg Glu Leu
35
<210>84
<211>38
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>84
Thr Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg
1 5 10 15
Ile Ala Ala Leu Ile Ala Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Ala Ala Leu Arg Glu Leu
35
<210>85
<211>38
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>85
Thr Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg
1 5 10 15
Ile Glu Ala Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn Ala
20 25 30
Ala Ala Leu Ala Glu Leu
35
<210>86
<211>38
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>86
Thr Thr Trp Glu Glu Trp Asp Arg Glu Ile Asn Glu Tyr Ala Ala Arg
1 5 10 15
Ile Glu Ala Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Ala Ala Leu Arg Glu Leu
35
<210>87
<211>38
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>87
Thr Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Thr Ser Arg
1 5 10 15
Ile Glu Ser Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Ala Ala Leu Arg Glu Leu
35
<210>88
<211>38
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>88
Thr Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg
1 5 10 15
Ile Glu Ala Leu Ile Arg Ala Ala Gln Asn Gln Gln Glu Lys Asn Glu
20 25 30
Ala Ala Leu Arg Glu Leu
35
<210>89
<211>38
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>89
Thr Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg
1 5 10 15
Ile Glu Ala Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Ala Ala Leu Leu Glu Leu
35
<210>90
<211>38
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>90
Thr Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg
1 5 10 15
Ile His Ala Leu Ile Glu Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Ala Ala Leu Arg Glu Leu
35
<210>91
<211>38
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>91
Thr Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Asn Tyr Ala Ala Leu
1 5 10 15
Ile Glu Ala Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Ala Ala Leu Arg Glu Leu
35
<210>92
<211>38
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>92
Glu Thr Trp Lys Glu Trp Asp Arg Ala Ile Glu Glu Tyr Lys Lys Arg
1 5 10 15
Ile Glu Glu Leu Ile Lys Ala Ala Glu Asn Gln Gln Glu Lys Asn Lys
20 25 30
Glu Ala Leu Arg Glu Leu
35
<210>93
<211>34
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>93
Trp Met Glu Trp Asp Arg Lys Ile Glu Glu Tyr Thr Lys Lys Ile Lys
1 5 10 15
Lys Leu Ile Glu Glu Ser Gln Glu Gln Gln Glu Lys Asn Glu Lys Glu
20 25 30
Leu Lys
<210>94
<211>34
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>94
Trp Met Glu Trp Asp Arg Lys Ile Glu Glu Tyr Thr Lys Lys Ile Glu
1 5 10 15
Glu Leu Ile Lys Lys Ser Gln Glu Gln Gln Glu Lys Asn Glu Lys Glu
20 25 30
Leu Lys
<210>95
<211>35
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>95
Trp Glu Glu Trp Asp Lys Lys Ile Glu Glu Tyr Thr Lys Lys Ile Glu
1 5 10 15
Glu Leu Ile Lys Lys Ser Glu Glu Gln Gln Lys Lys Asn Glu Glu Glu
20 25 30
Leu Lys Lys
35
<210>96
<211>39
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>96
Trp Gln Glu Trp Glu Gln Lys Ile Thr Ala Leu Leu Glu Gln Ala Gln
1 5 10 15
Ile Gln Gln Glu Lys Asn Glu Tyr Glu Leu Gln Lys Leu Asp Lys Trp
20 25 30
Ala Ser Leu Trp Glu Trp Phe
35
<210>97
<211>36
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>97
Trp Gln Glu Trp Glu Gln Lys Val Arg Tyr Leu Glu Ala Asn Ile Thr
1 5 10 15
Ala Leu Leu Glu Gln Ala Gln Ile Gln Gln Glu Lys Asn Glu Tyr Glu
20 25 30
Leu Gln Lys Leu
35
<210>98
<211>46
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>98
Trp Gln Glu Trp Glu Gln Lys Val Arg Tyr Leu Glu Ala Asn Ile Thr
1 5 10 15
Ala Leu Leu Glu Gln Ala Gln Ile Gln Gln Glu Lys Asn Glu Tyr Glu
20 25 30
Leu Gln Lys Leu Asp Lys Trp Ala Ser Leu Trp Asn Trp Phe
35 40 45
<210>99
<211>50
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>99
Asn Asn Met Thr Trp Gln Glu Trp Glu Gln Lys Val Arg Tyr Leu Glu
1 5 10 15
Ala Asn Ile Thr Ala Leu Leu Glu Gln Ala Gln Ile Gln Gln Glu Lys
20 25 30
Asn Glu Tyr Glu Leu Gln Lys Leu Asp Lys Trp Ala Ser Leu Trp Asn
35 40 45
Trp Phe
50
<210>100
<211>36
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>100
Trp Asn Trp Phe Ile Thr Ala Leu Leu Glu Gln Ala Gln Ile Gln Gln
1 5 10 15
Glu Lys Asn Glu Tyr Glu Leu Gln Lys Leu Asp Lys Trp Ala Ser Leu
20 25 30
Trp Asn Trp Phe
35
<210>101
<211>46
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>101
Trp Gln Glu Trp Asp Arg Glu Ile Ser Asn Tyr Thr Ser Leu Ile Thr
1 5 10 15
Ala Leu Leu Glu Gln Ala Gln Ile Gln Gln Glu Lys Asn Glu Tyr Glu
20 25 30
Leu Gln Lys Leu Asp Glu Trp Ala Ser Leu Trp Glu Trp Phe
35 40 45
<210>102
<211>40
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>102
Trp Gln Glu Trp Glu Arg Glu Ile Ser Ala Tyr Thr Ser Leu Ile Thr
1 5 10 15
Ala Leu Leu Glu Gln Ala Gln Ile Gln Gln Glu Lys Ile Glu Tyr Glu
20 25 30
Leu Gln Lys Leu Glu Trp Glu Trp
35 40
<210>103
<211>39
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>103
Trp Gln Glu Trp Asp Arg Glu Ile Thr Ala Leu Leu Glu Gln Ala Gln
1 5 10 15
Ile Gln Gln Glu Lys Asn Glu Tyr Glu Leu Gln Lys Leu Asp Lys Trp
20 25 30
Ala Ser Leu Trp Asn Trp Phe
35
<210>104
<211>39
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>104
Trp Gln Glu Trp Asp Arg Glu Ile Thr Ala Leu Leu Glu Gln Ala Gln
1 5 10 15
Ile Gln Gln Glu Lys Asn Glu Tyr Glu Leu Gln Lys Leu Asp Glu Trp
20 25 30
Ala Ser Leu Trp Glu Trp Phe
35
<210>105
<211>35
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>105
Trp Gln Glu Trp Asp Arg Glu Ile Thr Ala Leu Leu Glu Gln Ala Gln
1 5 10 15
Ile Gln Gln Glu Lys Asn Glu Tyr Glu Leu Gln Lys Leu Asp Glu Trp
20 25 30
Glu Trp Phe
35
<210>106
<211>35
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>106
Trp Gln Glu Trp Glu Arg Glu Ile Thr Ala Leu Leu Glu Gln Ala Gln
1 5 10 15
Ile Gln Gln Glu Lys Ile Glu Tyr Glu Leu Gln Lys Leu Ile Glu Trp
20 25 30
Glu Trp Phe
35
<210>107
<211>35
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>107
Trp Gln Glu Trp Glu Arg Glu Ile Thr Ala Leu Leu Glu Gln Ala Gln
1 5 10 15
Ile Gln Gln Glu Lys Asn Glu Tyr Glu Leu Gln Lys Leu Ile Glu Trp
20 25 30
Glu Trp Phe
35
<210>108
<211>35
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>108
Trp Gln Glu Trp Glu Arg Glu Ile Thr Ala Leu Leu Glu Gln Ala Gln
1 5 10 15
Ile Gln Gln Glu Lys Ile Glu Tyr Glu Leu Gln Lys Leu Asp Glu Trp
20 25 30
Glu Trp Phe
35
<210>109
<211>39
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>109
Trp Gln Glu Trp Glu Gln Lys Ile Thr Ala Leu Leu Glu Gln Ala Gln
1 5 10 15
Ile Gln Gln Glu Lys Asn Glu Tyr Glu Leu Gln Lys Leu Asp Lys Trp
20 25 30
Ala Ser Leu Trp Asn Trp Phe
35
<210>110
<211>39
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>110
Trp Gln Glu Trp Glu Gln Lys Ile Thr Ala Leu Leu Glu Gln Ala Gln
1 5 10 15
Ile Gln Gln Glu Lys Asn Glu Tyr Glu Leu Gln Lys Leu Asp Lys Trp
20 25 30
Ala Gly Leu Trp Glu Trp Phe
35
<210>111
<211>39
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>111
Trp Gln Glu Trp Glu Gln Lys Ile Thr Ala Leu Leu Glu Gln Ala Gln
1 5 10 15
Ile Gln Gln Glu Lys Asn Glu Tyr Glu Leu Gln Lys Leu Ala Glu Trp
20 25 30
Ala Gly Leu Trp Ala Trp Phe
35
<210>112
<211>35
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>112
Trp Gln Glu Trp Glu Gln Lys Ile Thr Ala Leu Leu Glu Gln Ala Gln
1 5 10 15
Ile Gln Gln Glu Lys Ile Glu Tyr Glu Leu Gln Lys Leu Ile Glu Trp
20 25 30
Glu Trp Phe
35
<210>113
<211>38
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>113
Thr Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Ala Ala Ala Arg
1 5 10 15
Ile Glu Ala Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Ala Ala Leu Arg Glu Leu
35
<210>114
<211>38
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>114
Thr Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg
1 5 10 15
Ala Glu Ala Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Ala Ala Leu Arg Glu Leu
35
<210>115
<211>38
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>115
Thr Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg
1 5 10 15
Ile Glu Ala Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn Ala
20 25 30
Ala Ala Leu Ala Glu Leu
35
<210>116
<211>48
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>116
Ala Lys Glu Ala Ala Gln Arg Ala Asn Ala Thr Thr Trp Glu Ala Trp
1 5 10 15
Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg Ile Glu Ala Leu Ile Arg
20 25 30
Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu Ala Ala Leu Arg Glu Leu
35 40 45
<210>117
<211>48
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>117
Asn Lys Glu Leu Glu Gln Arg Trp Asn Asn Thr Thr Trp Glu Ala Trp
1 5 10 15
Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg Ile Glu Ala Leu Ile Arg
20 25 30
Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu Ala Ala Leu Arg Glu Leu
35 40 45
<210>118
<211>48
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>118
Glu Lys Ala Ala Arg Gln Ala Glu Asn Ala Ala Arg Trp Glu Ala Trp
1 5 10 15
Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg Ile Glu Ala Leu Ile Arg
20 25 30
Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu Ala Ala Leu Arg Glu Leu
35 40 45
<210>119
<211>48
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>119
Glu Lys Ser Leu Arg Gln Ile Glu Asn Asn Thr Arg Trp Glu Ala Trp
1 5 10 15
Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg Ile Glu Ala Leu Ile Arg
20 25 30
Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu Ala Ala Leu Arg Glu Leu
35 40 45
<210>120
<211>48
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>120
Thr Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg
1 5 10 15
Ile Glu Ala Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Ala Ala Leu Arg Glu Leu Ala Ala Arg Glu Ala Ala Trp Arg Trp Phe
35 40 45
<210>121
<211>48
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>121
Thr Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg
1 5 10 15
Ile Glu Ala Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Ala Ala Leu Arg Glu Leu Asp Lys Arg Glu Ala Leu Trp Arg Trp Phe
35 40 45
<210>122
<211>48
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>122
Thr Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg
1 5 10 15
Ile Glu Ala Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Ala Ala Leu Arg Glu Leu Asp Lys Arg Glu Ser Leu Trp Arg Trp Phe
35 40 45
<210>123
<211>49
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>123
Gly Ala Lys Glu Ala Ala Gln Arg Ala Asn Ala Thr Thr Trp Glu Ala
1 5 10 15
Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg Ile Glu Ala Leu Ile
20 25 30
Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu Ala Ala Leu Arg Glu
35 40 45
Leu
<210>124
<211>49
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>124
Gly Glu Lys Ala Ala Arg Gln Ala Glu Asn Ala Ala Arg Trp Glu Ala
1 5 10 15
Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg Ile Glu Ala Leu Ile
20 25 30
Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu Ala Ala Leu Arg Glu
35 40 45
Leu
<210>125
<211>37
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>125
Thr Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg
1 5 10 15
Ile Glu Ala Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Ala Ala Leu Arg Glu
35
<210>126
<211>36
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>126
Thr Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg
1 5 10 15
Ile Glu Ala Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Ala Ala Leu Arg
35
<210>127
<211>35
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>127
Thr Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg
1 5 10 15
Ile Glu Ala Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Ala Ala Leu
35
<210>128
<211>33
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>128
Thr Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg
1 5 10 15
Ile Glu Ala Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Ala
<210>129
<211>38
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>129
Thr Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg
1 5 10 15
Ile Glu Ala Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Ala Ile Leu Arg Glu Leu
35
<210>130
<211>38
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>130
Thr Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg
1 5 10 15
Ile Glu Ala Leu Ile Arg Ala Leu Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Ala Ala Leu Arg Glu Leu
35
<210>131
<211>38
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>131
Thr Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg
1 5 10 15
Ile Glu Ala Leu Ile Arg Ala Leu Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Ala Ile Leu Arg Glu Leu
35
<210>132
<211>38
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>132
Thr Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg
1 5 10 15
Ile Glu Ala Leu Ile Arg Ala Ala Gln Glu Leu Gln Glu Lys Asn Glu
20 25 30
Ala Ala Leu Arg Glu Leu
35
<210>133
<211>38
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>133
Thr Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg
1 5 10 15
Ile Glu Ala Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Leu Glu
20 25 30
Ala Ala Leu Arg Glu Leu
35
<210>134
<211>38
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>134
Thr Thr Trp Glu Ala Trp Asp Arg Ala Ala Ala Glu Tyr Ala Ala Arg
1 5 10 15
Ile Glu Ala Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Ala Ala Leu Arg Glu Leu
35
<210>135
<211>38
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>135
Thr Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg
1 5 10 15
Ile Glu Ala Ala Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Ala Ala Leu Arg Glu Leu
35
<210>136
<211>38
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>136
Thr Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg
1 5 10 15
Ile Glu Ala Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Ala Ala Leu Arg Glu Ala
35
<210>137
<211>36
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>137
Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg Ile Glu
1 5 10 15
Ala Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu Ala Ala
20 25 30
Leu Arg Glu Leu
35
<210>138
<211>37
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>138
Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg Ile Glu
1 5 10 15
Ala Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu Ala Ala
20 25 30
Leu Arg Glu Leu Ala
35
<210>139
<211>38
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>139
Thr Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg
1 5 10 15
Ile Glu Ala Leu Ile Arg Ala Ala Gln Glu Ala Gln Glu Lys Asn Glu
20 25 30
Ala Ala Leu Arg Glu Leu
35
<210>140
<211>38
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>140
Glu Thr Trp Lys Glu Trp Asp Arg Ala Ile Glu Glu Tyr Lys Lys Arg
1 5 10 15
Ile Glu Glu Leu Ile Lys Ala Ala Glu Asn Gln Gln Glu Lys Asn Lys
20 25 30
Glu Ala Leu Arg Glu Leu
35
<210>141
<211>38
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>141
Met Ala Trp Met Glu Trp Asp Arg Arg Ile Glu Ala Tyr Ala Arg Leu
1 5 10 15
Ile Ala Glu Leu Ile Ala Arg Ala Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Ala Ala Leu Arg Glu Leu
35
<210>142
<211>41
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>142
Thr Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg
1 5 10 15
Ile Glu Ala Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Gln Gln Leu Arg Glu Trp Glu Trp Phe
35 40
<210>143
<211>41
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>143
Thr Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg
1 5 10 15
Ile Glu Ala Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Ala Ala Leu Arg Glu Trp Glu Trp Ile
35 40
<210>144
<211>38
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>144
Thr Thr Trp Asp Ala Trp Asp Arg Ala Ile Ala Asp Tyr Ala Ala Arg
1 5 10 15
Ile Asp Ala Leu Ile Arg Ala Ala Gln Asp Gln Gln Glu Lys Asn Asp
20 25 30
Ala Ala Leu Arg Glu Leu
35
<210>145
<211>41
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>145
Thr Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg
1 5 10 15
Ile Glu Ala Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Ala Glu
20 25 30
Ala Ala Leu Arg Glu Trp Glu Trp Phe
35 40
<210>146
<211>52
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>146
Trp Ala Ser Leu Trp Glu Trp Phe Gly Gly Ser Gly Gly Ser Thr Thr
1 5 10 15
Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg Ile Glu
20 25 30
Ala Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu Ala Ala
35 40 45
Leu Arg Glu Leu
50
<210>147
<211>52
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>147
Thr Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg
1 5 10 15
lle Glu Ala Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Ala Ala Leu Arg Glu Leu Gly Gly Ser Gly Gly Ser Trp Ala Ser Leu
35 40 45
Trp Glu Trp Phe
50
<210>148
<211>38
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>148
Thr Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg
1 5 10 15
Ile Glu Ala Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Gln Glu Leu Arg Glu Leu
35
<210>149
<211>38
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>149
Thr Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg
1 5 10 15
Ile Glu Ala Leu Ile Arg Ala Ala Gln Glu Ala Gln Glu Lys Asn Glu
20 25 30
Ala Ala Leu Arg Glu Leu
35
<210>150
<211>41
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>150
Thr Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg
1 5 10 15
Ile Glu Ala Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Ala Ala Leu Arg Glu Trp Trp Trp Trp
35 40
<210>151
<211>47
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>151
Thr Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg
1 5 10 15
Ile Glu Ala Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Ala Ala Leu Arg Glu Leu Asp Lys Trp Ser Leu Trp Arg Trp Phe
35 40 45
<210>152
<211>47
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>152
Thr Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg
1 5 10 15
Ile Glu Ala Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Ala Ala Leu Arg Ala Leu Asp Lys Trp Glu Ala Leu Trp Arg Phe
35 40 45
<210>153
<211>41
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>153
Thr Thr Trp Glu Ala Trp Asp Arg Ala Trp Gln Glu Trp Glu Gln Lys
1 5 10 15
Ile Glu Ala Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Ala Ala Leu Arg Glu Trp Glu Trp Phe
35 40
<210>154
<211>38
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>154
Leu Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg
1 5 10 15
Ile Glu Ala Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Ala Ala Leu Arg Glu Leu
35
<210>155
<211>38
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>155
Thr Thr Trp Met Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg
1 5 10 15
Ile Glu Ala Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Ala Ala Leu Arg Glu Leu
35
<210>156
<211>55
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>156
Thr Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg
1 5 10 15
Ile Glu Ala Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Ala Ala Leu Arg Glu Leu Gly Gly Ser Gly Gly Ser Gly Gly Ser Trp
35 40 45
Ala Ser Leu Trp Glu Trp Phe
50 55
<210>157
<211>38
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>157
Thr Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Ala Ala Ala Arg
1 5 10 15
Ile Glu Ala Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Ala Ala Leu Arg Glu Leu
35
<210>158
<211>58
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>158
Gly Ala Lys Glu Ala Ala Gln Arg Ala Asn Ala Thr Thr Trp Glu Ala
1 5 10 15
Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg Ile Glu Ala Leu Ile
20 25 30
Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu Ala Ala Leu Arg Glu
35 40 45
Leu Asp Lys Trp Ala Ser Leu Trp Trp Phe
50 55
<210>159
<211>39
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>159
Pro Ala Asn Trp Lys Ala Trp Glu Ala Gln Ile Gln Lys Tyr Gln Arg
1 5 10 15
Gln Ile Ala Glu Leu Ile Ala Asn Ala Lys Lys Gln Gln Glu Gln Asn
20 25 30
Glu Lys Ala Leu Arg Glu Leu
35
<210>160
<211>38
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>160
Met Thr Trp Met Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr Ser Leu
1 5 10 15
Ile His Ser Leu Ile Glu Glu Ile Gln Asn Gln Gln Glu Lys Asn Glu
20 25 30
Gln Glu Leu Leu Glu Leu
35
<210>161
<211>38
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>161
Thr Thr Trp Glu Glu Trp Asp Arg Glu Ile Asn Glu Tyr Thr Ser Arg
1 5 10 15
Ile Glu Ser Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn Ala
20 25 30
Ala Ala Leu Ala Glu Leu
35
<210>162
<211>38
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>162
Met Thr Trp Met Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr Ser Leu
1 5 10 15
Ile His Ser Leu Ile Glu Glu Ile Gln Asn Ile Gln Glu Lys Asn Glu
20 25 30
Gln Glu Leu Leu Glu Leu
35
<210>163
<211>38
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>163
Met Thr Trp Met Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr Ser Leu
1 5 10 15
Ile His Ser Leu Ile Glu Glu Ile Gln Asn Ile Gln Glu Lys Ile Glu
20 25 30
Gln Glu Leu Leu Glu Leu
35
<210>164
<211>38
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>164
Met Thr Trp Met Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr Ser Leu
1 5 10 15
Ile His Ser Leu Ile Glu Glu Ile Gln Asn Ile Gln Glu Lys Asn Glu
20 25 30
Gln Ile Leu Leu Glu Leu
35
<210>165
<211>38
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>165
Met Thr Trp Met Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr Ser Leu
1 5 10 15
Ile His Ser Leu Ile Glu Glu Ala Gln Asn Gln Gln Glu Lys Asn Glu
20 25 30
Gln Ala Leu Leu Glu Leu
35
<210>166
<211>42
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>166
Pro Ala Asn Trp Lys Ala Trp Glu Ala Gln Ile Gln Lys Tyr Gln Arg
1 5 10 15
Gln Ile Ala Glu Leu Ile Ala Asn Ala Lys Lys Gln Gln Glu Gln Asn
20 25 30
Glu Lys Ala Leu Arg Glu Trp Glu Trp Phe
35 40
<210>167
<211>38
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>167
Ala Asn Trp Lys Ala Trp Glu Ala Gln Ile Gln Lys Tyr Gln Arg Gln
1 5 10 15
Ile Ala Glu Leu Ile Ala Asn Ala Lys Lys Gln Gln Glu Gln Asn Glu
20 25 30
Lys Ala Leu Arg Glu Leu
35
<210>168
<211>38
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>168
Thr Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg
1 5 10 15
Ile Glu Ala Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Ala Val Leu Arg Glu Leu
35
<210>169
<211>38
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>169
Thr Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg
1 5 10 15
Ile Glu Ala Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Val Glu
20 25 30
Ala Ala Leu Arg Glu Leu
35
<210>170
<211>38
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>170
Thr Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg
1 5 10 15
Ile Glu Ala Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Ile Glu
20 25 30
Ala Ala Leu Arg Glu Leu
35
<210>171
<211>38
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>171
Thr Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg
1 5 10 15
Ile Glu Ala Leu Ile Arg Ala Ala Gln Glu Leu Gln Glu Lys Asn Glu
20 25 30
Ala Ile Leu Arg Glu Leu
35
<210>172
<211>38
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>172
Thr Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg
1 5 10 15
Ile Glu Ala Leu Ile Arg Ala Leu Gln Glu Leu Gln Glu Lys Asn Glu
20 25 30
Ala Ala Leu Arg Glu Leu
35
<210>173
<211>38
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>173
Thr Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg
1 5 10 15
Ile Glu Ala Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Ala Leu Leu Arg Glu Leu
35
<210>174
<211>39
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>174
Thr Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg
1 5 10 15
Ile Glu Ala Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Ala Ala Leu Arg Glu Leu Lys
35
<210>175
<211>36
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic
<400>175
Leu Thr Trp Ile Glu Trp Asp Arg Glu Ile Asn Lys Tyr Thr Ser Leu
1 5 10 15
Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys Asn Glu
20 25 30
Gln Glu Leu Lys
35

Claims (34)

1. the method for the site-specific chemical modification of HIV gp41 derived peptide between the synthesis stage of peptide, wherein synthetic peptide has one or more aminoacid with pendant amine, and described method is included between synthesis stage and mixes in described peptide or its fragment:
(a) at least one aminoacid, its selected so that its pendant amine and chemoproection reagent generation chemical reaction, described chemoproection reagent protect described pendant amine to avoid subsequently and the reactive functional group generation of amine chemical reaction; With
(b) at least one aminoacid, its have unprotected and can with the amine of the reactive functional group reaction of amine, wherein unhindered amina is selected from by N-terminal amine, pendant amine and their group that constitutes.
2. the method for claim 1, wherein said HIV gp41 derived peptide link two or more fragments by covalency and produce described synthetic peptide and synthesize; And wherein to be incorporated at least one described fragment be the aminoacid of the pendant amine that had with chemoproection reagent chemical reaction.
3. the method for claim 1, wherein said HIV gp41 derived peptide be have SEQ ID NO:1-175 any aminoacid sequence or with any or a plurality of peptide of SEQ ID NO:1-175 with aminoacid sequence of at least 95% homogeneity.
4. the method for claim 1; wherein said chemoproection reagent is selected from by 1-(4; 4-dimethyl-2; 6-dioxy hexamethylene-1-subunit) ethyl, 1-(4; 4-dimethyl-2,6-dioxy hexamethylene-1-subunit)-group that 3-methyl butyl, allyloxy carbonyl, benzyloxycarbonyl and 2-chlorine benzyloxycarbonyl constitute.
5. as each described method in the claim 1,2,3 or 4, wherein the aminoacid of its pendant amine and chemoproection reagent generation chemical reaction is lysine.
6. as each described method in the claim 1,2,3 or 4, wherein the pendant amine with chemoproection reagent generation chemical reaction is a ε amine.
7. isolating HIV gp41 derived peptide, it has one or more aminoacid that contain pendant amine, wherein chemical reaction has taken place at least one amino acid whose pendant amine and chemoproection reagent, and described chemoproection reagent protects described pendant amine to avoid subsequently and the reactive functional group generation of amine chemical reaction; Have unprotected with at least one aminoacid of described peptide and can with the amine of the reactive functional group reaction of amine, wherein unhindered amina is selected from by N-terminal amine, pendant amine and their group that constitutes.
8. HIV gp41 derived peptide as claimed in claim 7, wherein said peptide be have SEQ ID NO:1-175 any aminoacid sequence or with any or a plurality of peptide of SEQ ID NO:1-175 with aminoacid sequence of at least 95% homogeneity.
9. HIV gp41 derived peptide as claimed in claim 7; wherein said chemoproection reagent is selected from by 1-(4; 4-dimethyl-2; 6-dioxy hexamethylene-1-subunit) ethyl, 1-(4; 4-dimethyl-2,6-dioxy hexamethylene-1-subunit)-group that 3-methyl butyl, allyloxy carbonyl, benzyloxycarbonyl and 2-chlorine benzyloxycarbonyl constitute.
10. as each described HIV gp41 derived peptide in the claim 7,8 or 9, wherein the aminoacid of its pendant amine and chemoproection reagent generation chemical reaction is lysine.
11. as each described HIV gp41 derived peptide in the claim 7,8 or 9, wherein the pendant amine with chemoproection reagent generation chemical reaction is a ε amine.
12. a production comprises the method for the conjugate of even matter basically of HIV gp41 derived peptide and polymer, described method comprises:
(a) synthetic HIV gp41 derived peptide, it has one or more aminoacid that contain pendant amine, its pendant amine and chemoproection reagent generation chemical reaction thereby at least one aminoacid is selected, described chemoproection reagent protects described pendant amine to avoid subsequently and the reactive functional group generation of amine chemical reaction, have unprotected with at least one aminoacid of described peptide and can with the amine of the reactive functional group reaction of amine, wherein unhindered amina is selected from by N-terminal amine, pendant amine and their group that constitutes; With
(b) the free amino generation chemical reaction by reactive functional group of the amine that makes polymer and described HIV gp41 derived peptide; the polymer covalency is attached on the described HIV gp41 derived peptide; wherein said polymer only covalency is attached on one or more aminoacid with unhindered amina; and be not linked at least one aminoacid of being protected by chemoproection reagent, produce the described conjugate of even matter basically.
13. method as claimed in claim 12, wherein said conjugate comprise and surpass the banded HIV gp41 of a polymer molecule covalency derived peptide, wherein each polymer molecule is attached on the aminoacid of described HIV gp41 derived peptide.
14. further comprising from the described conjugate of even matter basically, method as claimed in claim 12, wherein said method remove chemoproection reagent.
15. method as claimed in claim 12, wherein said HIV gp41 derived peptide links two or more fragments by covalency and synthesizes to produce described synthetic peptide; And wherein to be incorporated at least one described fragment be the aminoacid of the pendant amine that had with chemoproection reagent chemical reaction.
16. method as claimed in claim 12, wherein said HIV gp41 derived peptide be have SEQ ID NO:1-175 any aminoacid sequence or with any or a plurality of peptide of SEQ ID NO:1-175 with aminoacid sequence of at least 95% homogeneity.
17. method as claimed in claim 12; wherein said chemoproection reagent is selected from by 1-(4; 4-dimethyl-2; 6-dioxy hexamethylene-1-subunit) ethyl, 1-(4; 4-dimethyl-2,6-dioxy hexamethylene-1-subunit)-group that 3-methyl butyl, allyloxy carbonyl, benzyloxycarbonyl and 2-chlorine benzyloxycarbonyl constitute.
18. method as claimed in claim 12, wherein said polymer comprises Polyethylene Glycol.
19. as each described method in the claim 12,13,14,15,16,17 or 18, wherein the aminoacid of its pendant amine and chemoproection reagent generation chemical reaction is lysine.
20. as each described method in the claim 12,13,14,15,16,17 or 18, wherein the pendant amine with chemoproection reagent generation chemical reaction is a ε amine.
21. the conjugate of producing as each described method in the claim 12,13,14,15,16,17 or 18, comprise HIV gp41 derived peptide and polymer of even matter basically.
22. the conjugate of even matter basically that comprises HIV gp41 derived peptide and polymer as claimed in claim 21 further comprises pharmaceutically acceptable carrier.
23. the purposes that comprises the conjugate of sparing matter basically of HIV gp41 derived peptide and polymer as active treatment material in the treatment of HIV infection as claimed in claim 21.
24. the purposes that comprises the conjugate of sparing matter basically of HIV gp41 derived peptide and polymer as active treatment material in the treatment of HIV infection as claimed in claim 22.
25. as each described purposes of the conjugate of even matter basically that comprises HIV gp41 derived peptide and polymer in claim 23 or 24, the wherein said conjugate of even matter basically is used as the part of therapeutic modality, and described therapeutic modality contains one or more and is used for the treatment of other Anti-virus agents that HIV infects.
26. the purposes of the conjugate of even matter basically that comprises HIV gp41 derived peptide and polymer as claimed in claim 21 is used to make medicine, described medicine is used to comprise the therapeutic application of HIV treatment.
27. a pharmaceutical composition comprises the conjugate of the even basically matter of the HIV of comprising gp41 derived peptide as claimed in claim 21 and polymer.
28. a pharmaceutical composition comprises the conjugate of the even basically matter of the HIV of comprising gp41 derived peptide as claimed in claim 22 and polymer.
29. a method that suppresses HIV to cell propagation comprises the conjugate of even matter basically of using an amount of HIV of comprising gp41 derived peptide as claimed in claim 21 and polymer to viral and cell, effectively suppresses HIV and infects described cell.
30. a method that suppresses HIV to cell propagation comprises to virus and cell and uses an amount of pharmaceutical composition as claimed in claim 27, effectively suppresses HIV and infects described cell.
31. method as claimed in claim 29, the conjugate of wherein said even matter basically assigns to add as the one-tenth of therapeutic modality.
32. method as claimed in claim 30, wherein said pharmaceutical composition assigns to add as the one-tenth of therapeutic modality.
33. one kind is suppressed the method that HIV merges, and is included in the conjugate of even matter basically of the viral and an amount of HIV of comprising gp41 derived peptide as claimed in claim 21 and polymer is contacted, and effectively suppresses HIV and merges.
34. one kind is suppressed the method that HIV merges, and is included in viral and an amount of pharmaceutical composition as claimed in claim 27 is contacted, and effectively suppresses HIV and merges.
CNA2005800073482A 2004-03-15 2005-03-08 Site-specific chemical modification of HIV gp41-derived peptides Pending CN1929860A (en)

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US60/553,063 2004-03-15

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WO (1) WO2005089796A1 (en)

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WO2005089805A2 (en) * 2004-03-15 2005-09-29 Nektar Therapeutics Al, Corporation Polymer-based compositions and conjugates of hiv entry inhibitors
CN103965300A (en) * 2005-11-02 2014-08-06 Ambrx公司 Biosynthetic Polypeptide Fusion Inhibitors
CN1793170A (en) * 2005-12-14 2006-06-28 中国人民解放军军事医学科学院生物工程研究所 Polypeptide of inhibiting HIV virus fusion and application thereof
NZ570300A (en) * 2006-02-02 2011-08-26 Trimeris Inc HIV fusion inhibitor peptides with improved biological properties
EP2139526A4 (en) * 2007-04-03 2010-07-14 Trimeris Inc Novel formulations for delivery of antiviral peptide therapeutics
WO2011095989A2 (en) * 2010-02-04 2011-08-11 Matrix Laboratories Ltd An improved process for the preparation of enfuvirtide
JP2013522352A (en) * 2010-03-24 2013-06-13 メディカル リサーチ カウンシル Method
EP2571510B1 (en) 2010-05-21 2018-08-08 XL-protein GmbH Biosynthetic proline/alanine random coil polypeptides and their uses
CN104788548B (en) * 2014-01-20 2018-06-05 中国人民解放军军事医学科学院毒物药物研究所 New AntiHIV1 RT activity infection polypeptide and its utilization

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EP1734994A1 (en) 2006-12-27
WO2005089796A1 (en) 2005-09-29

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