CN1317296C - Anti-HIV fusogenic peptide and its production - Google Patents

Anti-HIV fusogenic peptide and its production Download PDF

Info

Publication number
CN1317296C
CN1317296C CNB2005100341365A CN200510034136A CN1317296C CN 1317296 C CN1317296 C CN 1317296C CN B2005100341365 A CNB2005100341365 A CN B2005100341365A CN 200510034136 A CN200510034136 A CN 200510034136A CN 1317296 C CN1317296 C CN 1317296C
Authority
CN
China
Prior art keywords
glu
otbu
boc
lys
trt
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Fee Related
Application number
CNB2005100341365A
Other languages
Chinese (zh)
Other versions
CN1712416A (en
Inventor
张嘉杰
徐伟
吴少瑜
文晓芸
万山河
吴曙光
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Southern Medical University
Original Assignee
Southern Medical University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Southern Medical University filed Critical Southern Medical University
Priority to CNB2005100341365A priority Critical patent/CN1317296C/en
Publication of CN1712416A publication Critical patent/CN1712416A/en
Application granted granted Critical
Publication of CN1317296C publication Critical patent/CN1317296C/en
Expired - Fee Related legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Abstract

The present invention discloses an anti-HIV fusogenic peptide and a preparation method thereof, which relates to the field of peptide and a preparation method thereof. The anti-HIV fusogenic peptide of the present invention is represented by a general formula (I), namely a-WX1EWDX2X3IX4X5YTSX6IX7X8LIX9X10SX11X12QQX13KNEX14ELX15X16-b, wherein a is one of acetyl, 9-fluorenylmethoxy-carbonyl (Fmoc) and Boc-amino (Boc); b is acylamino or carboxyl. The preparation method of the present invention is a polypeptide liquid phase synthesis method. First, intermediate peptide is coupled under the action of activating reagent, a protection group is then eliminated. The anti-HIV fusogenic peptide of the present invention has the advantages of small side effect, high activity, simple and convenient synthesis and purification and low production cost.

Description

Anti-HIV fusogenic peptide and preparation method thereof
Invention field
The present invention relates to peptide and preparation method thereof field, relate in particular to anti-HIV peptide and preparation method thereof field.
Background technology
Acquired immune deficiency syndrome (AIDS) has become the principal disease that threatens human health.The AIDS patient mainly relies on medicine to prolong life at present.The AIDS-treating medicine of approved use is mainly two big classes in the world: hiv reverse transcriptase inhibitor and hiv protease inhibitor.This two classes medicine all acts on the later stage of HIV host cells infected---transcribe and make up the process of new virus.Because the high mutation rate of HIV, resistance has all appearred in above-mentioned two kinds of inhibitor.There is the specificity toxicity relevant with dosage in reverse transcriptase inhibitors, comprises bone marrow depression, vomiting, dysfunction of liver, peripheral neuropathy, myasthenia and pancreatitis etc.The toxic side effect of proteinase inhibitor comprises that dysfunction of liver, gastrointestinal upset, urinary stone disease, mouth are numb, lipid reaches Bewu etc. unusually on every side.
The fusion process that suppresses viral after birth and host's target cell membrane has become anti-HIV and has infected important target spot.The fusion that suppresses virus and target cell membrane can suppress the infection of HIV.The process that HIV and target cell are merged is as follows: the HIV bag is combined with CD4 and accessory receptor molecule (chemokine receptor CCR 5 or CXCR4 etc.) priority on the target cell by glycoprotein surface gp120 subunit, the conformation that causes striding film subunit gp-41 changes, the fusogenic peptide of its N end is inserted in the host cell membrane, start the fusion of peplos and target cell membrane, finish the course of infection that virus enters host cell.The crystalline diffraction analysis is verified: when HIV virus took place to merge with cell, the core of gp-41 was made up of 6 strands of helical bundles, and wherein the N-end becomes 3 " hairpin structures " with the helical arrangement that C-holds, with HIV coating and cytolemma not together.The gp-41 tripolymer may form and merge the hole, makes virus invade host cell.Therefore, the spirane structure formation that suppresses gp-41 just can suppress HIV virus intrusion host cell.
At present, the polypeptide class anti HIV-1 virus fusion inhibitor that acts on the gp-41 subunit is derived from the peptide section of gp-41 subunit C-terminal more, because its structure makes it hold combining of fusion area with N-competitively with the C-end peptide chain of gp-41 with the similarity of HIV virus gp-41 subunit C-terminal, and the helical bundle of viral gp-41 can not be produced." hairpin structure " between HIV peplos and the cytolemma can't form like this, thereby virus can't be invaded.Because the site of action of fusion inhibitor at cytolemma, just can play a role so needn't be released in the cell.And the height conservativeness prompting of hydrophobicity gp-41 core amino acids sequence: virus produces resistance with unlikely to fusion inhibitor.The polypeptide class anti HIV-1 virus fusion inhibitor T-20 that acts on the gp-41 subunit commonly used at present is made up of 36 amino-acid residues, the ratio that comprises hydrophobic amino acid is higher, poorly soluble, in preparation process, there is the low shortcoming of purifying rate of the low and polypeptide of the coupling efficiency of polypeptide chain.
Summary of the invention
The purpose of this invention is to provide a kind of anti-HIV fusogenic peptide.
Another object of the present invention provides a kind of anti-HIV fusogenic peptide preparation method.
Anti-HIV fusogenic peptide of the present invention aminoacid sequence formula (I) expression down:
a-WX 1EWDX 2X 3IX 4X 5YTSX 6X 7X 8LIX 9X 10SX 11X 12QQX 13KNEX 14ELX 15X 16-b (I)
Wherein a is that ethanoyl, 9-fluorenyl methoxy-carbonyl (Fmoc), tert-butoxycarbonyl (Boc) are wherein a kind of,
B is amido or carboxyl.
The aminoacid sequence abbreviation that other letter is an one-letter symbol in the above-mentioned formula (I), the amino-acid residue of representative is defined as follows: S is that Serine, W are that tryptophane, M are that methionine(Met), E are that L-glutamic acid, T are that Threonine, R are that arginine, I are that Isoleucine, N are that Radix Asparagi acid amide, Y are that tyrosine, K are that Methionin, Q are that glu famine, L are that leucine, D are that aspartic acid, Nle are nor-leucine.
X 1-16Represent natural amino acid or alpha-non-natural amino acid or amino acid analogue or vacancy.
The preferred ethanoyl of a; B preferred amide base.
Anti-HIV fusogenic peptide is preferably:
Ac-WMEWDREINNYTSLIHSLIEESQNQQEKNEQELL-NH 2(SEQ ID NO.1)，
Ac-WMEWDRKINNYTSLIHSLIEESQNQQEKNEQELK-NH 2(SEQ ID NO.2)，
Ac-W-Nle-EWDRKINNYTSLIHSLIEESQNQQEKNEQELK-NH 2(SEQ ID NO.3)，
Ac-WMEWDRKIENYTKLIHSLIEESQNQQEKNEQELK-NH 2(SEQ ID NO.4)，
Ac-WMEWDRKIENYTKLIKSLIEESQNQQEKNEQELK-NH 2(SEQ ID NO.5)，
Ac-W-Nle-EWDRKIENYTKLIKSLIEESQNQQEKNEQELK-NH 2(SEQ ID NO.6)，
Ac-WMEWDRKIENYTKLIKSLIEESQEQQEKNEQELK-NH 2(SEQ ID NO.7)，
Ac-WMEWDRKIENYTKLIKSLIEESQEQQEKNEKELK-NH 2(SEQ ID NO.8)，
Ac-WMEWDRKIEEYTKLIKSLIEESQEQQEKNEKELK-NH 2(SEQ ID NO.9)，
Ac-W-Nle-EWDRKIEEYTKLIKSLIEESQEQQEKNEKELK-NH 2(SEQ ID NO.10)，
Ac-WMEWDRKIEEYTKKIKSLIEESQEQQEKNEKELK-NH 2(SEQ ID NO.11)，
Ac-WMEWDRKIEEYTKKIKKLIEESQEQQEKNEKELK-NH 2(SEQ ID NO.12)，
Ac-W-Nle-EWDRKIEEYTKKIKKLIEESQEQQEKNEKELK-NH 2(SEQ ID NO.13)，
Ac-WMEWDRKIEEYTKKIEKLIKESQEQQEKNEKELK-NH 2(SEQ ID NO.14)，
Ac-WMEWDRKIEEYTKKIKELIEKSQEQQEKNEKELK-NH 2(SEQ ID NO.15)，
Ac-WMEWDRKIEEYTKKIEELIKKSQEQQEKNEKELK-NH 2(SEQ ID NO.16)，
Ac-W-Nle-EWDRKIEEYTKKIEELIKKSQEQQEKNEKELK-NH 2(SEQ ID NO.17)，
Ac-WEEWDKKIEEYTKKIEELIKKSEEQQKKNEEELKK-NH 2(SEQ ID NO.18)，
Ac-WMEWDREINNYTSLIHSLIEESQNQQEK-NH 2SEQ ID NO.19)，
Ac-WMEWDRKIEEYTKLIKSLIEESQEQQEK-NH 2(SEQ ID NO.20)，
Ac-W-Nle-EWDRKIEEYTKLIKSLIEESQEQQEK-NH 2(SEQ ID NO.21)，
Ac-WMEWDRKIEEYTKKIKKLIEESQEQQEK-NH 2(SEQ ID NO.22)，
Ac-W-Nle-EWDRKIEEYTKKIKKLIEESQEQQEK-NH 2(SEQ ID NO.23)，
Ac-WEEWDRKIEEYTKLIKSLIEESQEQQEK-NH 2(SEQ ID NO.24)，
Ac-W-Nle-EWDKKIEEYTKKIEELIKKSEEQQKK-NH 2(SEQ ID NO.25)，
Ac-WEEWDKKIEEYTKKIEELIKKSEEQQKK-NH 2(SEQ ID NO.26)，
Ac-W-Nle-EWDKKIEEYTKKIEELIKKSEEQQKK-NH 2(SEQ ID NO.27) is wherein a kind of.
Anti-HIV fusogenic peptide is preferably:
Ac-WMEWDRKIEEYTKKIEELIKKSQEQQEKNEKELK-NH 2(SEQ ID NO.16)，
Ac-WEEWDKKIEEYTKKIEELIKKSEEQQKKNEEELKK-NH 2(SEQ ID NO.18)，
Ac-WMEWDREINNYTSLIHSLIEESQNQQEKNEQELL-NH 2(SEQ ID NO.1)，
Ac-WMEWDRKINNYTSLIHSLIEESQNQQEKNEQELK-NH 2(SEQ ID NO.2)，
Ac-WMEWDRKIENYTKLIKSLIEESQEQQEKNEQELK-NH 2(SEQIDNO.7)，
Ac-WMEWDRKIEEYTKKIKKLIEESQEQQEKNEKELK-NH 2(SEQ ID NO.12)，
Ac-WMEWDREINNYTSLIHSLIEESQNQQEK-NH 2(SEQ ID NO.19)，
Ac-WEEWDRKIEEYTKLIKSLIEESQEQQEK-NH 2(SEQ ID NO.24)，
Ac-WEEWDKKIEEYTKKIEELIKKSEEQQKK-NH 2(SEQ ID NO.26) is wherein a kind of.
The preparation method of anti-HIV fusogenic peptide of the present invention is: following intermediate peptide is utilized the liquid phase synthetic technology, finish the synthetic of total length anti-HIV fusogenic peptide.Wherein the liquid phase synthetic technology is a liquid phase synthetic technology commonly used in the chemistry of peptides, can obtain the synthetic preferred condition of liquid phase according to method known to those skilled in the art.
Intermediate peptide of the present invention is,
Intermediate peptide 1:Ac-WX 1EWDX 2X 3IX 4X 5-OH
Intermediate peptide 2:Fmoc-YTSX 6IX 7X 8LIX 9X 10S-OH
Intermediate peptide 3:H-X 11X 12QQX 13KNEX 14ELX 15X 16-NH 2
Intermediate peptide 4:Ac-WX 1EWDX 2X 3IX 4X 5YTS-OH
Intermediate peptide 5:H-X 6IX 9X 10SX 11X 12QQX 13K-NH 2
Intermediate peptide 6:Ac-WX 1EWDX 2X 3IX 4X 5YTSX 6IX 7X 8LI-OH
Intermediate peptide 7:H-X 9X 10SX 11X 12QQX 13KNEX 14ELX 15X 16-NH 2
X wherein 1-16Represent natural amino acid or alpha-non-natural amino acid or amino acid analogue or vacancy, special in the anti-HIV fusogenic peptide preferred sequence.
Intermediate peptide of the present invention can be the polypeptide fragment of N end and side chain protected.The amino Fomc radical protection of using, the amino-acid residue side chain is protected with standard blocking group such as the tertiary butyl (t-Bu), trityl (Trt) and tert-butoxycarbonyl (Boc).The t-Bu group is the preferred side chain blocking group of amino-acid residue Tyr (Y), Thr (T), Ser (S) and Asp (D); The Trt group is the preferred side chain blocking group of amino-acid residue His (H), Gln (Q) and Asn (N); The Boc group is the preferred side chain blocking group of amino-acid residue Lys (K) and Trp (W); Pmc is the preferred side chain blocking group of Arg (R).
Intermediate peptide 1 of the present invention is preferably:
Ac-Trp(Boc)-Met-Glu(OtBu)-Trp(Boc)-Asp(OtBu)-Arg(pmc)-Glu(OtBu)-Ile-Asn(trt)-Asn(trt)-OH(SEQ IDNO.28)，
Ac-Trp(Boc)-Met-Glu(OtBu)-Trp(Boc-Asp(OtBu)-Arg(pmc)-Lys(Boc)-Ile-Asn(trt)-Asn(trt)-OH(SEQ IDNO.29)，
Ac-Trp(Boc)-Met-Glu(OtBu)-Trp(Boc)-Asp(OtBu)-Arg(pmc)-Lys(Boc)-Ile-Glu(OtBu)-Asn(trt)-OH(SEQ IDNO.30)，
Ac-Trp(Boc)-Met-Glu(OtBu)-Trp(Boc)-Asp(OtBu)-Arg(pmc)-Lys(Boc)-Ile-Glu(OtBu)-Glu(OtBu)-OH(SEQID NO.31)，
Ac-Trp (Boc)-Glu (OtBu)-Glu (OtBu)-Trp (Boc)-Asp (OtBu)-Lys (Boc)-Lys (Boc)-Ile-Glu (OtBu)-Glu (OtBu)-OH (SEQ ID NO.32) is wherein a kind of.
Intermediate peptide 2 of the present invention is preferably:
Fmoc-Tyr(tBu)-Thr(tBu)-Ser(tBu)-Leu-Ile-His(trt)-Ser(tBu)-Leu-Ile-Glu(OtBu)-Glu(OtBu)-Ser(tBu)-OH(SEQID NO.33)，
Fmoc-Tyr(tBu)-Thr(tBu)-Lys(Boc)-Leu-Ile-Lys(Boc)-Ser(tBu)-Leu-Ile-Glu(OtBu)-Glu(OtBu)-Ser(tBu)-OH(SEQID NO.34)，
Fmoc-Tyr(tBu)-Thr(tBu)-Lys(Boc)-Lys(Boc)-Ile-Lys(Boc)-Lys(Boc)-Leu-Ile-Glu(OtBu)-Glu(OtBu)-Ser(tBu)-OH(SEQ ID NO.35)，
Fmoc-Tyr (tBu)-Thr (tBu)-Lys (Boc)-Lys (Boc)-Ile-Glu (OtBu)-Glu (OtBu)-Leu-Ile-Lys (Boc)-Lys (Boc)-Ser (tBu)-OH (SEQ ID NO.36) is wherein a kind of.
Intermediate peptide 3 of the present invention is preferably:
H-Gln(trt)-Asn(trt)-Gln(trt)-Gln(trt)-Glu(OtBu)-Lys(Boc)-Asn(trt)-Glu(OtBu)-Gln(trt)-Glu(OtBu)-Leu-Leu-NH 2(SEQ ID NO.37)，
H-Gln(trt)-Asn(trt)-Gln(trt)-Gln(trt)-Glu(OtBu)-Lys(Boc)-Asn(trt)-Glu(OtBu)-Gln(trt)-Glu(OtBu)-Leu-Lys(Boc)-NH 2(SEQ ID NO.38)，
H-Gln(trt)-Glu(OtBu)-Gln(trt)-Gln(trt)-Glu(OtBu)-Lys(Boc)-Asn(trt)-Glu(OtBu)-Gln(trt)-Glu(OtBu)-Leu-Lys(Boc)-NH 2(SEQ ID NO.39)，
H-Gln(trt)-Glu(OtBu)-Gln(trt)-Gln(trt)-Glu(OtBu)-Lys(Boc)-Asn(trt)-Glu(OtBu)-Lys(Boc)-Glu(OtBu)-Leu-Lys(Boc)-NH 2(SEQ ID NO.40)，
H-Glu (OtBu)-Glu (OtBu)-Gln (trt)-Gln (trt)-Lys (Boc)-Lys (Boc)-Asn (trt)-Glu (OtBu)-Glu (OtBu)-Glu (OtBu)-Leu-Lys (Boc)-Lys (Boc)-NH 2(SEQ ID NO.41) is wherein a kind of.
Intermediate peptide 4 of the present invention is preferably:
Ac-Trp(Boc)-Met-Glu(OtBu)-Trp(Boc)-Asp(OtBu)-Arg(pmc)-Glu(OtBu)-Ile-Asn(trt)-Asn(trt)-Tyr(tBu)-Thr(tBu)-Ser(tBu)-OH(SEQ ID NO.42)，
Ac-Trp(Boc)-Met-Glu(OtBu)-Trp(Boc)-Asp(OtBu)-Arg(pmc)-Lys(Boc)-Ile-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Thr(tBu)-Lys(Boc)OH(SEQ ID NO.43)，
Ac-Trp (Boc)-Glu (OtBu)-Glu (OtBu)-Trp (Boc)-Asp (OtBu)-Lys (Boc)-Lys (Boc)-Ile-Glu (OtBu)-Glu (OtBu)-Tyr (tBu)-Thr (tBu)-Lys (Boc) OH (SEQ ID NO.44) is wherein a kind of.
Intermediate peptide 5 of the present invention is preferably:
H-Leu-Ile-His(trt)-Ser(tBu)-Leu-Ile-Glu(OtBu)-Glu(OtBu)-Ser(tBu)-Gln(trt)-Asn(trt)-Gln(trt)-Gln(trt)-Glu(OtBu)-Lys(Boc)-NH 2(SEQ ID NO.45)，
H-Leu-Ile-Lys(Boc)-Ser(tBu)-Leu-Ile-Glu(OtBu)-Glu(OtBu)-Ser(tBu)-Gln(trt)-Glu(OtBu)-Gln(trt)-Gln(trt)-Glu(OtBu)-Lys(Boc)-NH 2(SEQ ID NO.46)，
H-Lys (Boc)-Ile-Glu (OtBu)-Glu (OtBu)-Leu-Ile-Lys (Boc)-Lys (Boc)-Ser (tBu)-Glu (OtBu)-Glu (OtBu)-Gln (trt)-Gln (trt)-Lys (Boc)-Lys (Boc)-NH 2(SEQ ID NO.47) is wherein a kind of.
Intermediate peptide 6 of the present invention is preferably:
Ac-Trp(Boc)-Met-Glu(OtBu)-Trp(Boc)-Asp(OtBu)-Arg(pmc)-Glu(OtBu)-Ile-Asn(trt)-Asn(trt)-Tyr(tBu)-Thr(tBu)-Ser(tBu)-Leu-Ile-His(trt)-Ser(tBu)-Leu-Ile-OH(SEQ ID NO.48)，
Ac-Trp(Boc)-Met-Glu(OtBu)-Trp(Boc)-Asp(OtBu)-Arg(pmc)-Lys(Boc)-Ile-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Thr(tBu)-Lys(Boc)-Lys(Boc)-Ile-Glu(OtBu)-Glu(OtBu)-Leu-Ile-OH(SEQ ID NO.49)，
Ac-Trp (Boc)-Glu (OtBu)-Glu (OtBu)-Trp (Boc)-Asp (OtBu)-Lys (Boc)-Lys (Boc)-Ile-Glu (OtBu)-Glu (OtBu)-Tyr (tBu)-Thr (tBu)-Lys (Boc)-Lys (Boc)-Ile-Glu (OtBu)-Glu (OtBu)-Leu-Ile-OH (SEQ ID NO.50) is wherein a kind of.
Intermediate peptide 7 of the present invention is preferably:
H-Glu(OtBu)-Glu(OtBu)-Ser(tBu)-Gln(trt)-Asn(trt)-Gln(trt)-Gln(trt)-Glu(OtBu)-Lys(Boc)-Asn(trt)-Glu(OtBu)-Gln(trt)-Glu(OtBu)-Leu-Leu-NH 2(SEQ ID NO.51)，
H-Glu(OtBu)-Glu(OtBu)-Ser(tBu)-Gln(trt)-Glu(OtBu)-Gln(trt)-Gln(trt)-Glu(OtBu)-Lys(Boc)-Asn(trt)-Glu(OtBu)-Lys(Boc)-Glu(OtBu)-Leu-Lys(Boc)-NH 2(SEQ ID NO.52)，
H-Lys (Boc)-Lys (Boc)-Ser (tBu)-Glu (OtBu)-Glu (OtBu)-Gln (trt)-Gln (trt)-Lys (Boc)-Lys (Boc)-Asn (trt)-Glu (OtBu)-Glu (OtBu)-Glu (OtBu)-Leu-Lys (Boc)-Lys (Boc)-NH 2(SEQ ID NO.53) is wherein a kind of.
The preparation method commonly used of anti-HIV fusogenic peptide of the present invention is: the first step activation coupling, and intermediate peptide is dissolved back adding activating reagent carry out coupling in organic solvent, coupled product adds the methanol/water solution stirring and separates out after-filtration.Second step was sloughed blocking group, and above-mentioned product is added the TFA solution that comprises the ion trap agent, precipitated in ether after sloughing blocking group, filtered the final vacuum drying.
The preparation method of anti-HIV fusogenic peptide of the present invention is intermediate peptide 1 and intermediate peptide 2 couplings at first, carry out the liquid phase coupling with intermediate peptide 3 then, perhaps at first with intermediate peptide 2 and intermediate peptide 3 couplings, then with intermediate peptide 1 coupling, the total length of finishing anti-HIV fusogenic peptide is synthetic.
Anti-HIV fusogenic peptide preparation method of the present invention can be with intermediate peptide 4 and intermediate peptide 5 couplings.
Anti-HIV fusogenic peptide preparation method of the present invention can be with intermediate peptide 6 and intermediate peptide 7 couplings.
The organic solvent that uses among the above-mentioned anti-HIV fusogenic peptide preparation method is that polypeptide liquid is combined to solvent commonly used in the field.Preferably NMP, DMF or its mixture.
In the above-mentioned anti-HIV fusogenic peptide preparation method the first step, intermediate and activating reagent can be selected the mixture or 2-(7-azo the benzotriazole)-N of benzotriazole tetramethyl-urea phosphofluoric acid ester (HBTU), I-hydroxybenzotriazole hydrate (HOBt) and diisopropylethylamine (DIEA) for use, N, N ', mixture or the O-benzotriazole-N of N '-tetramethyl-urea phosphofluoric acid ester (HATU), HOBt and DIEA, N, N ', the mixture of N '-tetramethyl-urea Tetrafluoroboric acid ester (TBTU), HOBt and DIEA is preferably selected the mixture of HBTU, HOBt and DIEA for use.Amount ratio is 1: 1～1: 1.5, better is 1: 1～1: 1.3, preferably 1; 1～1: 1.2.In the above-mentioned anti-HIV fusogenic peptide preparation method the first step, the linked reaction time is 3～8 hours, better is 3～6 hours, preferably 3～4 hours.
Above-mentioned anti-HIV fusogenic peptide preparation method is in second step, and ion trap agent polypeptide liquid is combined to ion trap agent commonly used in the field.Preferably use and heavily steam wherein one or more mixtures of phenol, dimercaptoethane, thioanisole, distilled water.
Above-mentioned anti-HIV fusogenic peptide preparation method is in second step, and sloughing the blocking group reaction times is 4～10 hours, better is 4～8 hours, preferably 5～7 hours.
Above-mentioned anti-HIV fusogenic peptide preparation method is in second step, and the ether that uses during precipitation is wherein one or more mixtures of isopropyl ether, ether, methyl butyl ether.
Above-mentioned anti-HIV fusogenic peptide preparation method at room temperature carries out.
The synthetic employing solid phase synthesis process of intermediate peptide.Synthetic method commonly used is as follows: the amino acid that a will protect loads on the resin, as initial substance; B sloughs the amino acid blocking group on the loaded resin; C activates couping amino acids; D carries out coupling with the amino acid (perhaps peptide chain) of sloughing blocking group on activatory amino acid and the resin; E detects amino acid whose coupling efficiency with ninhydrin method, if the result of being positive, then repetition of activation amino acid and coupling process if be negative reaction, then carry out next step reaction; F repetition of activation, coupling process are until finishing last amino acid whose linked reaction; G cuts the synthetic intermediate peptide fall from resin.
The resin that uses among the intermediate peptide synthetic method step a of the present invention is to the tetchy resin of acid, and commonly used have 2-chloro trityl chloride resin, Sieber amide resins or two kinds of mixing to use.Also can buy the amino acid whose commercial resin of load to polypeptide Synesis Company of specialty.
The activating reagent that uses among the intermediate peptide synthetic method step c of the present invention is selected HBTU, HOBT and DIEA for use.
Among the intermediate peptide synthetic method step c of the present invention, portions of resin amino acid (equivalence ratio) is 1: 2～10, preferably 1: 3.Amino acid: activating reagent (equivalence ratio) is 1: 1.5, better is 1: 1.3, preferably 1: 1.1.
Among the intermediate peptide synthetic method step c of the present invention, linked reaction is carried out at normal temperatures, and the reaction times is 30 minutes to 3 hours, better is 30 minutes hours to 2 hours, preferably 1 hour to 1.5 hours.
Among the intermediate peptide synthetic method step b of the present invention, amido protecting group Fomc sloughs, and carries out at N-Methyl pyrrolidone (NMP) solution of 20% piperidines.
In the intermediate peptide synthetic method step g of the present invention, select for use the dichloromethane solution of 1%～2% trifluoroacetic acid (TFA) to carry out polypeptide and cut.
In the intermediate peptide synthetic method of the present invention; when the amido protecting group is Ac, after in the end an amino acid whose linked reaction is finished, remove the Fmoc blocking group; mixing solutions in diacetyl oxide and NMP/DCM (3: 1 volume ratios) carries out coupling, and then polypeptide is cut from resin.
The anti-HIV fusogenic peptide that the present invention relates to only acts on the gp-41 subunit, and with respect to the HIV fusion inhibitor T-20 that has gone on the market, its side effect is littler, and activity is higher.The increase of the hydrophobic amino acid ratio of anti-HIV fusogenic peptide makes that the αLuo Xuanjiegou of its formation is more stable, the ability of resistant protease hydrolysis and increased activity, solvability increases, synthetic, purifying is more simple and convenient, production cost is lower, has the stronger market competitiveness.
For a better understanding of the present invention, with anti-HIV fusogenic peptide pharmacological testing of the present invention and result its purposes in preparation treatment HIV pharmaceutical field is described below.
The MT4 that anti-HIV fusogenic peptide adopts HIV to infect is the screening active ingredients model, with the index of the plasmodial generation of HIV-1 virus induction cell as evaluate efficacy.Because the CD4 acceptor of MT4 cell surface can combine with HIV is specific, so in-vitro screening carries out representative on the MT4 cell.
The screening active ingredients model that adopts is the MT4 that HIV infects, with the plasmodial index that is produced as evaluate efficacy of HIV-1 virus induction cell.Because the CD4 acceptor of MT4 cell surface can combine with HIV is specific, so in-vitro screening carries out representative on the MT4 cell.Concrete grammar is as described below, and concrete outcome sees Table 1.
1. anti-HIV fusogenic peptide is measured virus virulence: with 8 concentration HIV of 10 times of dilutions, observation of cell pathology in nutrient solution RPMI-1640 is calculated TCID 50Be 10 -6
2. the toxicity test of anti-HIV fusogenic peptide pair cell: with MT4 cell 2 * 10 5Individual/mL is inoculated in 96 orifice plates, and every hole is 0.1mL, adds 1000 * TCID respectively 50HIV virus 100 μ L, establish normal cell contrast and virus control simultaneously, the sample and the AZT 100 μ L that add 5 concentration of 2 times of dilutions then respectively, each sample concentration is all established 3 parallel holes, place in 37 ℃, 5%CO2 incubator and cultivate, behind the 72h under inverted microscope observation of cell pathology (CPE).
CPE criterion: no synplasm forms "-", and no synplasm forms but cell irregular " ± ", has or not synplasm to form "+".
The HIV (human immunodeficiency virus)-resistant activity of table 1 anti-HIV fusogenic peptide
In proper order Aminoacid sequence Molecular weight IC 50
1 2 3 4 5 6 7 8 9 Ac-WMEWDREINNYTSLIHSLIEESQNQQEKNEQELL-NH 2 Ac-WMEWDRKINNYTSLIHSLIEESQNQQEKNEQELK-NH 2 Ac-WMEWDRKIENYTKLIKSLIEESQEQQEKNEQELK-NH 2 Ac-WMEWDRKIEEYTKKIKKLIEESQEQQEKNEKELK-NH 2 Ac-WMEWDRKIEEYTKKIEELIKKSQEQQEKNEKELK-NH 2 Ac-WEEWDKKIEEYTKKIEELIKKSEEQQKKNEEELKK-NH 2 Ac-WMEWDREINNYTSLIHSLIEESQNQQEK-NH 2 Ac-WEEWDRKIEEYTKLIKSLIEESQEQQEK-NH 2 Ac-WEEWDKKIEEYTKKIEELIKKSEEQQKK-NH 2 4289.70 4303.77 4365.92 4437.09 4437.09 4536.16 3562.91 3637.05 3665.19 1.21±0.18nm 1.15±0.21nm 1.02±0.20nm 0.85±0.15nm 0.45±0.11nm 0.75±0.15nm 9.89±0.24nm 2.50±0.18nm 1.91±0.19nm
The judgement of IC50: (1) only 1 hole is "+", and two holes are "-" or "-" and " ± " in addition, and this drug level can be considered EC50.(2) 3 holes are respectively "+", "-" and " ± ", and this drug level can be considered EC50.(3) 3 holes are " ± ", and this drug level can be considered EC50.(4) 3 holes are "+", and 3 holes under the next mass action are "-", and the intermediate value of two concentration can be considered IC50.
Embodiment
Below be that embodiments of the invention, reference example can illustrate in greater detail the present invention, but not with fusion form restriction the present invention.
Reference example 1
Preparation intermediate peptide 1
Table 2 intermediate peptide 1 fragment
In proper order Aminoacid sequence (containing blocking group) Molecular weight Productive rate Purity
1 2 3 4 5 Ac-Trp(Boc)-Met-Glu(OtBu)-Trp(Boc)-Asp(OtBu)-Arg(pmc)- Glu(OtBu)-Ile-Asn(trt)-Asn(trt)-OH Ac-Trp(Boc)-Met-Glu(OtBu)-Trp(Boc)-Asp(OtBu)-Arg(pmc)- Lys(Boc)-Ile-Asn(trt)-Asn(trt)-OH Ac-Trp(Boc)-Met-Glu(OtBu)-Trp(Boc)-Asp(OtBu)-Arg(pmc)- Lys(Boc)-Ile-Glu(OtBu)-Asn(trt)-OH Ac-Trp(Boc)-Met-Glu(OtBu)-Trp(Boc)-Asp(OtBu)-Arg(pmc)- Lys(Boc)-Ile-Glu(OtBu)-Glu(OtBu)-OH Ac-Trp(Boc)-Glu(OtBu)-Glu(OtBu)-Trp(Boc)-Asp(OtBu)-Lys(Boc)- Lys(Boc)-Ile-Glu(OtBu)-Glu(OtBu)-OH 2553.07 2596.14 2424.93 2253.73 2113.50 93％ 92％ 95％ 96％ 95％ 83％ 85％ 88％ 87％ 90％
1. Fmoc-Asn (trt)-OH is carried on 2-chloro trityl chloride resin
(5g, 1.0mmol/g) the 100 milliliters of peptide synthesising containers of packing into are with draining methylene dichloride (DCM) after 50 milliliters of DCM washings with 2-chlorine trityl chloride resin.Fmoc-Asn (trt)-OH 4.48 grams (7.5mmol, 1.5 equivalents) and DIEA1.5ml (8.5mmol, 1.7 equivalents) are dissolved in behind the DCM of 50ml in the adding resin.Nitrogen protection was stirred mixture 2 hours down.Drain solvent and use DCM 50ml washing.With terminal reactive site on 40 milliliters of MeOH: DIEA (9: 1 volume ratios) solution sealing resin, the time is 20 minutes.Drain solvent,,, dry up resin with nitrogen at last, obtain the resin of 7.1 gram loads with MeOH 50ml washing 2 times with 50ml DCM washing 3 times.Fmoc-amino acid on the resin is cut the backward resin-carried quantitative analysis of carrying out.Method is a HPLC outer marking quantitative analytical method, and analytical results shows that resin-carried amount is the 0.73mmol/ gram.
Post: Zabarx C18 4.6 * 250mm 100A 5 μ
Flow velocity: 1ml/min
Detect wavelength: 260nm
Moving phase: the A:0.1%TFA aqueous solution/acetonitrile (30/70)
2. the polypeptide fragment that synthesizes side chain protected
(1) the 2-chloro trityl chloride resin 1.0mmol that takes by weighing the load Fmoc-AA-OH of method for preparing places the reaction tubes (as above-mentioned charge capacity is the resin of 0.73mmol/ gram, should take by weighing 1.37 grams) of 55ml.
(2) on Peptide synthesizer, select the program of FastMoc 1.0mmol to carry out the synthetic of peptide chain as follows.A. use NMP swelling and washing resin.B. remove the Fmoc group with 20% piperidines nmp solution, and with NMP with residual piperidines washes clean.C. with the Fmoc amino acid of 3.0mmol, activate with HBTU, the HOBT of NMP dissolving back adding 3.0mmol and the mixing solutions of DIEA.D. the amino acid (perhaps peptide chain) of sloughing the Fmoc group on activatory amino acid and the resin is carried out coupling.E. use the NMP washing resin, excess activation reagent is removed.F. detect amino acid whose coupling efficiency with ninhydrin method, if the result of being positive then repeats c and d two steps.If be positive, then carry out next step reaction.G. repeat b, c, d, f four steps to finishing last amino acid whose linked reaction.H. dry up with the DCM washing, and with nitrogen.
3. prepare intermediate peptide 1 by step 2 synthetic polypeptide fragment
(1) the negative peptide resin of above-mentioned Fmoc-AA1-10-OH is removed the Fmoc group with 20% piperidines nmp solution, and with NMP with residual piperidines washes clean.
(2) add the pyridine of 5mmol after, carry out coupling at the mixing solutions of the acetate glycosides of 5mmol and the NMP/DCM of its 10 times of volumes (3: 1 volume ratios).
(3) use NMP and DCM washing resin respectively, dry up resin or vacuum-drying with nitrogen.
4. peptide resin cuts
Cut reagent and select the DCM solution of the TFA of 1-2% for use.
(1) with the resin of weighing with the DCM solution washing of the 1%TFA of its 2 times of volumes 5 times, each 3-5 minute.And collect washing lotion with the container that contains pyridine (with the volume ratio of TFA in the washing lotion 1: 1).
(2) 2/3 DCM is removed in distillation, adds after 6 volume ethanol and continues distillation until removing DCM (temperature by distillment increase determine).
(3) solution is cooled to 5～10 ℃, the water that added 6 volumes in 30 minutes is to being settled out AcAA1-10-OH.Solid is collected in vacuum filtration, and water (2-3 volume) washing solid.Slurry stirred 30 minutes at 0～5 ℃, collected solid and was dried to constant weight by vacuum filtration.
Reference example 2
Preparation intermediate peptide 4
Synthetic method is with reference example 1.
Table 3 intermediate peptide 4 fragments
In proper order Aminoacid sequence (containing blocking group) Molecular weight Productive rate Purity
1 2 3 Ac-Trp(Boc)-Met-Glu(OtBu)-Trp(Boc)-Asp(OtBu)-Arg(pmc)-Glu(OtBu)-Ile- Asn(trt)-Asn(trt)-Tyr(tBu)-Thr(tBu)-Ser(tBu)-OH Ac-Trp(Boc)-Met-Glu(OtBu)-Trp(Boc)-Asp(OtBu)-Arg(pmc)-Lys(Boc)-Ile- Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Thr(tBu)-Lys(Boc)OH Ac-Trp(Boc)-Glu(OtBu)-Glu(OtBu)-Trp(Boc)-Asp(OtBu)-Lys(Boc)- Lys(Boc)-Ile-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Thr(tBu)-Lys(Boc)OH 3072.75 2912.55 2718.29 90％ 91％ 92％ 81％ 85％ 84％
Reference example 3
Preparation intermediate peptide 6
Synthetic method is with reference example 1.
Table 4 intermediate peptide 6 fragments
In proper order Aminoacid sequence (containing blocking group) Molecular weight Productive rate Purity
1 2 3 Ac-Trp(Boc)-Met-Glu(OtBu)-Trp(Boc)-Asp(OtBu)-Arg(pmc)-Glu(OtBu)-Ile- Asn(trt)-Asn(trt)-Tht(tBu)-Thr(tBu)-Ser(tBu)-Leu-Ile-His(trt)-Ser(tBu)-Leu-Ile-OH Ac-Trp(Boc)-Met-Glu(OtBu)-Trp(Boc)-Asp(OtBu)-Arg(pmc)-Lys(Boc)-I1e- Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Thr(tBu)-Lys(Boc)-Lys(Boc)-Ile-Glu(OtBu)-Glu(O tBu)-Leu-Ile-OH Ac-Trp(Boc)-Glu(OtBu)-Glu(OtBu)-Trp(Boc)-Asp(OtBu)-Lys(Boc)-Lys(Boc)-Ile- Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Thr(tBu)-Lys(Boc)-Lys(Boc)-Ile-Glu(OtBu)- Glu(OtBu)-Leu-Ile-OH 4049.80 3798.50 3658.27 87％ 90％ 91％ 75％ 78％ 80％
Reference example 4
Preparation intermediate peptide 2
Concrete grammar is as follows, and the synthetic fragment sees Table 5.
Table 5 intermediate peptide 2 fragments
In proper order Aminoacid sequence (containing blocking group) Molecular weight Productive rate Purity
1 2 3 4 Fmoc-Tyr(tBu)-Thr(tBu)-Ser(tBu)-Leu-Ile-His(trt)- Ser(tBu)-Leu-Ile-Glu(OtBu)-Glu(OtBu)-Ser(tBu)-OH Fmoc-Tyr(tBu)-Thr(tBu)-Lys(Boc)-Leu-Ile-Lys(Boc)- Ser(tBu)-Leu-Ile-Glu(OtBu)-Glu(OtBu)-Ser(tBu)-OH Fmoc-Tyr(tBu)-Thr(tBu)-Lys(Boc)-Lys(Boc)-Ile-Lys(Boc)- Lys(Boc)-Leu-Ile-Glu(OtBu)-Glu(OtBu)-Ser(tBu)-OH Fmoc-Tyr(tBu)-Thr(tBu)-Lys(Boc)-Lys(Boc)-Ile- Glu(OtBu)-Glu(OtBu)-Leu-Ile-Lys(Boc)-Lys(Boc)-Ser(tBu)-OH 2247.84 2181.78 2381.02 2381.02 90％ 92％ 91％ 93％ 82％ 85％ 86％ 88％
1. intermediate peptide 2 fragments of synthesizing side chain protected
(1) takes by weighing the reaction tubes (as above-mentioned charge capacity is the resin of 0.73mmol/ gram, should take by weighing 1.37 grams) that places 55ml by the 2-chloro trityl chloride resin 1.0mmol of the load Fmoc-AA-OH of the method for embodiment 1 preparation.
(2) on Peptide synthesizer, select the program of FastMoc 1.0mmol to carry out the synthetic of peptide chain as follows.A. use NMP swelling and washing resin.B. remove the Fmoc group with 20% piperidines nmp solution, and with NMP with residual piperidines washes clean.C. with the Fmoc amino acid of 3.0mmol, activate with HBTU, the HOBT of NMP dissolving back adding 3.0mmol and the mixing solutions of DIEA.D. the amino acid (perhaps peptide chain) of sloughing the Fmoc group on activatory amino acid and the resin is carried out coupling.E. use the NMP washing resin, excess activation reagent is removed.F. detect amino acid whose coupling efficiency with ninhydrin method, if the result of being positive then repeats c and d two steps.If be positive, then carry out next step reaction.G. repeat b, c, d, f four steps to finishing last amino acid whose linked reaction.H. dry up with the DCM washing, and with nitrogen.
2. peptide resin cuts
Cut reagent and select the DCM solution of the TFA of 1-2% for use.
(1) with the resin of weighing with the DCM solution washing of the 1%TFA of its 2 times of volumes 5 times, each 3-5 minute.And collect washing lotion with the container that contains pyridine (with the volume ratio of TFA in the washing lotion 1: 1).
(2) 2/3 DCM is removed in distillation, adds after 6 volume ethanol and continues distillation until removing DCM (temperature by distillment increase determine).
(3) solution is cooled to 5～10 ℃, the water that added 6 volumes in 30 minutes is to being settled out AcAAl-10-OH.Solid is collected in vacuum filtration, and water (2-3 volume) washing solid.Slurry stirred 30 minutes at 0～5 ℃, collected solid and was dried to constant weight by vacuum filtration.
Reference example 5
Preparation intermediate peptide 3
1.Fmoc-Leu-OH be carried on the method for Sieber amide resins
(5.0 restrain, and the 100 milliliters of peptide synthesising containers of 0.6mmol/g) packing into draining methylene dichloride (DCM) after 50 milliliters of DCM washings, are removed the Fmoc group with 50ml NMP washing back with 20% piperidines nmp solution again with the Sieber amide resins.Behind the clean residual piperidines of NMP, with Fmoc-Leu-OH 3.18 gram (9mmol, 3.0 HOBt 1.22 gram (9mmol equivalent),, 3.0 O-benzotriazole-N equivalent),, N, N ', the DIEA of N '-tetramethyl-urea Tetrafluoroboric acid ester (TBTU) 2.89 grams (9mmol, 3.0 equivalents), 0.5ml add in the resin after being dissolved in the NMP of 50ml.Under the room temperature, nitrogen protection was stirred mixture 18 hours down.Wash 50ml * 3 with NMP, MeOH washs 50ml * 3, and ether washing 50ml * 3 final vacuum dryings get 5.6 gram resins.Can be with other amino acid of identical method load.In addition, also can on Peptide synthesizer, adopt first amino acid of the direct load peptide chain of fixed routine of coupling amino acid.
Fmoc-amino acid on the resin is cut the backward resin-carried quantitative analysis of carrying out.Method is a HPLC outer marking quantitative analytical method, and analytical results shows that resin-carried amount is the 0.54mmol/ gram.
Post: Zabarx C18 4.6 * 250mm 100A 5 μ
Flow velocity: 1ml/min
Detect wavelength: 260nm
Moving phase: the A:0.1%TFA aqueous solution/acetonitrile (30/70)
Table 6 intermediate peptide 3 fragments
In proper order Aminoacid sequence (containing blocking group) Molecular weight Productive rate Purity
1 2 3 4 5 H-Gln(trt)-Asn(trt)-Gln(trt)-Gln(trt)-Glu(OtBu)-Lys(Boc)-Asn(trt)- Glu(OtBu)-Gln(trt)-Glu(OtBu)-Leu-Leu-NH2 H-Gln(trt)-Asn(trt)-Gln(trt)-Gln(trt)-Glu(OtBu)-Lys(Boc)-Asn(trt)- Glu(OtBu)-Gln(trt)-Glu(OtBu)-Leu-Lys(Boc)-NH2 H-Gln(trt)-Glu(OtBu)-Gln(trt)-Gln(trt)-Glu(OtBu)-Lys(Boc)-Asn(trt)- Glu(OtBu)-Gln(trt)-Glu(OtBu)-Leu-Lys(Boc)-NH2 H-Glr(trt)-Glu(OtBu)-Gln(trt)-Gln(trt)-Glu(OtBu)-Lys(Boc)-Asn(trt)- Glu(OtBu)-Lys(Boc)-Glu(OtBu)-Leu-Lys(Boc)-NH2 H-Glu(OtBu)-Glu(OtBu)-Gln(trt)-Gln(trt)-Lys(Boc)-Lys(Boc)- Asn(trt)-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Leu-Lys(Boc)- Lys(Boc)-NH2 3187.17 3337.31 3165.11 3022.95 3044.01 89％ 87％ 90％ 86％ 89％ 78％ 75％ 81％ 79％ 85％
2. intermediate peptide 3 fragments of synthesizing side chain protected
(1) the Sieber amide resins 1.0mmol that takes by weighing the load Fmoc-AA-OH of method for preparing places the reaction tubes (as above-mentioned charge capacity is the Sieber amide resins of the Fmoc-Leu-OH of 0.59mmol/ gram, should take by weighing 1.69 grams) of 55ml.
(2) on Peptide synthesizer, select the program of FastMoc 1.0mmol to carry out the synthetic of peptide chain as follows.A. use NMP swelling and washing resin.B. remove the Fmoc group with 20% piperidines nmp solution, and with NMP with residual piperidines washes clean.C. with the Fmoc amino acid of 3.0mmol, activate with HBTU, the HOBT of NMP dissolving back adding 3.0mmol and the mixing solutions of DIEA.D. the amino acid (perhaps peptide chain) of sloughing the Fmoc group on activatory amino acid and the resin is carried out coupling.E NMP washing resin is removed excess activation reagent.F. detect amino acid whose coupling efficiency with ninhydrin method, if the result of being positive then repeats c and d two steps.If be positive, then carry out next step reaction.G. repeat b, c, d, f four steps to finishing last amino acid whose linked reaction.H. remove the Fmoc group with 20% piperidines nmp solution, and with NMP with residual piperidines washes clean.G. dry up with the DCM washing, and with nitrogen.
3. peptide resin cuts
Cut reagent and select the DCM solution of the TFA of 1-2% for use.
(1) with the resin of weighing with the DCM solution washing of the 1%TFA of its 2 times of volumes 5 times, each 3-5 minute.And collect washing lotion with the container that contains pyridine (with the volume ratio of TFA in the washing lotion 1: 1).
(2) 2/3 DCM is removed in distillation, adds after 6 volume ethanol and continues distillation until removing DCM (temperature by distillment increase determine).
(3) solution is cooled to 5～10 ℃, the water that added 6 volumes in 30 minutes is to being settled out H-AA23-34-NH2.Solid is collected in vacuum filtration, and water (2-3 volume) washing solid.Slurry stirred 30 minutes at 0～5 ℃, collected solid and was dried to constant weight by vacuum filtration.
Reference example 6
Preparation intermediate peptide 5
Synthetic method is with reference example 5.
Table 7 intermediate peptide 5 fragments
In proper order Aminoacid sequence (containing blocking group) Molecular weight Productive rate Purity
1 2 3 H-Leu-Ile-His(trt)-Ser(tBu)-Leu-Ile-Glu(OtBu)-Glu(OtBu)-Ser(tBu)- Gln(trt)-Asn(trt)-Gln(trt)-Gln(trt)-Glu(OtBu)-Lys(Boc)-NH2 H-Leu-Ile-Lys(Boc)-Ser(tBu)-Leu-Ile-Glu(OtBu)-Glu(OtBu)-Ser(tBu)- Gln(trt)-Glu(OtBu)-Gln(trt)-Gln(trt)-Glu(OtBu)-Lys(Boc)-NHX H-Lys(Boc)-Ile-Glu(OtBu)-Glu(OtBu)-Leu-Ile-Lys(Boc)-Lys(Boc)-Ser(tBu)- Glu(OtBu)-Glu(OtBu)-Gln(trt)-Gln(trt)-Lys(Boc)-Lys(Boc)-NH2 3385.45 3063.08 3122.45 88％ 90％ 93％ 79％ 82％ 83％
Reference example 7
Preparation intermediate peptide 7
Synthetic method is with reference example 5, and the synthetic fragment sees Table 8.
Table 8 intermediate peptide 7 fragments
In proper order Aminoacid sequence (containing blocking group) Molecular weight Productive rate Purity
1 2 3 H-Glu(OtBu)-Glu(OtBu)-Ser(tBu)-Gln(trt)-Asn(trt)-Gln(trt)-Gln(trt)-Glu(OtBu)- Lys(Boc)Asn(trt)-Glu(OtBu)-Gln(trt)-Glu(OtBu)-Leu-Leu-NH2 H-Glu(OtBu)-Glu(OtBu)-Ser(tBu)-Gln(trt)-Glu(OtBu)-Gln(trt)-Gln(trt)-Glu(OtBu)- Lys(Boc)-Asn(trt)-Glu(OtBu)-Lys(Boc)-Glu(OtBu)-Leu-Lys(Boc)-NH2 H-Lys(Boc)-Lys(Boc)-Ser(tBu)-Glu(OtBu)-Glu(OtBu)-Gln(trt)-Gln(trt)-Lys(Boc)- Lys(Boc)-Asn(trt)-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Leu-Lys(Boc)-Lys(Boc)-NH2 3736.57 3624.48 3667.54 89％ 86％ 89％ 75％ 78％ 80％
Embodiment 1
Ac-WMEWDREINNYTSLIHSLIEESQNQQEKNEQELL-NH 2(SEQ ID NO.1)
Molecular weight: 4289.70
Productive rate: 95%
Purity: 68%.
1. be polypeptide fragment with intermediate peptide 2 with intermediate peptide 3 couplings
Stir intermediate peptide 3H-Gln (trt)-Asn (trt)-Gln (trt)-Gln (trt)-Glu (OtBu)-Lys (Boc)-Asn (trt)-Glu (OtBu)-Gln (trt)-Glu (OtBu)-Leu-Leu-NH with intermediate peptide 2Fmoc-Tyr (tBu)-Thr (tBu)-Ser (tBu)-Leu-Ile-His (trt)-Ser (tBu)-Leu-Ile-Glu (OtBu)-Glu (OtBu)-Ser (tBu)-OH, 0.30mmol of 0.30mmol 2, the HOBt of 0.36mmol is dissolved among the NMP of 8ml, and room temperature reaction is 4 hours behind the DIEA of the following adding of cooling 0.50mmol and the HBTU of 0.36mmol.The methanol (1: 1) that drips 30ml is stirred to pulpous state.Filter, solid is measured purity greater than 75% with HPLC with 8ml methanol (1: 1) washing 2 times after the vacuum-drying.
2. intermediate peptide 1 is the anti-HIV fusogenic peptide of side chain protected with the coupling of step 1 synthetic polypeptide fragment
The aforementioned polypeptides fragment is suspended with isopropyl ether/hexanaphthene (3: 1) mixing solutions, stirred 20 hours behind the adding piperidines, mixture is muddy, filters.Solid isopropyl ether washed twice, intermediate peptide 1Ac-Trp (Boc)-Met-Glu (OtBu)-Trp (Boc)-Asp (OtBu)-Arg (pmc)-Glu (OtBu)-Ile-Asn (trt)-Asn (trt)-OH, 1.2 times of normal HOBt of adding equivalent after dried solid is weighed, be dissolved among the NMP of 8ml, cooling added down behind 1.5 times of normal DIEA and the 1.2 times of normal HBTU room temperature reaction 4 hours.The methanol (1: 1) that drips 30ml is stirred to pulpous state.Filter, solid washs 2 times with 8ml methanol (1: 1), vacuum-drying.
3. remove the side chain protected group and prepare the total length anti-HIV fusogenic peptide
Steam the mixing solutions that phenol, 0.5ml dimercaptoethane, 1.0ml thioanisole and 1.0ml distilled water are made into adding under the 2 gained solid cooled, handled 5 hours under the room temperature with 20ml TFA, 1.5 grammes per square metres.The peptide solution of removing the side chain protected group adds refrigerative 200ml isopropyl ether, and vacuum-drying is collected in the sedimentation and filtration isopropyl ether washing back of generation.
Embodiment 2
Ac-WMEWDRKINNYTSLIHSLIEESQNQQEKNEQELK-NH 2(SEQ ID NO.2)
Molecular weight: 4303.77
Productive rate: 94%
Purity: 73%
Synthetic method is with embodiment 1.
Intermediate peptide 2 is:
Fmoc-Tyr(tBu)-Thr(tBu)-Ser(tBu)-Leu-Ile-His(trt)-Ser(tBu)-Leu-Ile-Glu(OtBu)-Glu(OtBu)-Ser(tBu)-OH
Intermediate peptide 3 is:
H-Gln(trt)-Asn(trt)-Gln(trt)-Gln(trt)-Glu(OtBu)-Lys(Boc)-Asn(trt)-Glu(OtBu)-Gln(trt)-Glu(OtBu)-Leu-Lys(Boc)-NH 2，
Intermediate peptide 1 is: Ac-Trp (Boc)-Met-Glu (OtBu)-Trp (Boc)-Asp (OtBu)-Arg (pmc)-Lys (Boc)-Ile-Asn (trt)-Asn (trt)-OH
Embodiment 3
Ac-WMEWDRKIENYTKLIKSLIEESQEQQEKNEQELK-NH 2(SEQ ID NO.7)
Molecular weight: 4365.92
Productive rate: 96%
Purity: 71%
Synthetic method is with embodiment 1.
Intermediate peptide 2 is:
Fmoc-Tyr(tBu)-Thr(tBu)-Lys(Boc)-Leu-Ile-Lys(Boc)-Ser(tBu)-Leu-Ile-Glu(OtBu)-Glu(OtBu)-Ser(tBu)-OH
Intermediate peptide 3 is;
H-Gln(trt)Glu(OtBu)-Gln(trt)-Gln(trt)-Glu(OtBu)-Lys(Boc)-Asn(trt)-Glu(OtBu)-Gln(trt)-Glu(OtBu)-Leu-Lys(Boc)-NH 2
Intermediate peptide 1 is:
Ac-Trp(Boc)-Met-Glu(OtBu)-Trp(Boc)-Asp(OtBu)-Arg(pmc)-Lys(Boc)-Ile-Glu(OtBu)-Asn(trt)-OH
Embodiment 4
Ac-WMEWDRKIEEYTKKIKKLIEESQEQQEKNEKELK-NH 2(SEQ ID NO.12)
Molecular weight: 4437.09
Productive rate: 93%
Purity: 72%
Synthetic method is as follows:
1. be polypeptide fragment with intermediate peptide 2 with intermediate peptide 1 coupling
Intermediate peptide 2Fmoc-Tyr (tBu)-Thr (tBu)-Lys (Boc)-Lys (Boc)-Ile-Lys (Boc)-Lys (Boc)-Leu-Ile-Glu (OtBu)-Glu (OtBu)-Ser (tBu)-OH of 0.30mmol is suspended with isopropyl ether/hexanaphthene (3: 1) mixing solutions, stirred 20 hours after adding piperidines, mixture is muddy, filters.Solid isopropyl ether washed twice, dried solid adds intermediate peptide 1Ac-Trp (Boc)-Met-Glu (OtBu)-Trp (Boc)-Asp (OtBu)-Arg (pmc)-Lys (Boc)-Ile-Glu (OtBu)-Glu (OtBu)-OH, 1.2 times of normal HOBt of equivalent, be dissolved among the NMP of 8ml, cooling added down behind 1.5 times of normal DIEA and the 1.2 times of normal HBTU room temperature reaction 4 hours.The methanol (1: 1) that drips 30ml is stirred to pulpous state.Filter, solid washs 2 times with 8ml methanol (1: 1), vacuum-drying, and purity is greater than 75%.
2. step 1 synthetic polypeptide fragment and intermediate peptide 3 couplings are the anti-HIV fusogenic peptide of side chain protected
The above-mentioned dried solid back of weighing is added intermediate peptide 3H-Gln (trt)-Glu (OtBu)-Gln (trt)-Gln (trt)-Glu (OtBu)-Lys (Boc)-Asn (trt)-Glu (OtBu)-Lys (Boc)-Glu (OtBu)-Leu-Lys (the Boc)-NH of equivalent 2, 1.2 times of normal HOBt, be dissolved among the NMP of 8ml, cooling added behind 1.5 times of normal DIEA and the 1.2 times of normal HBTU room temperature reaction down 4 hours.The methanol (1: 1) that drips 30ml is stirred to pulpous state.Filter, solid washs 2 times with 8ml methanol (1: 1), vacuum-drying.
3. remove the side chain protected group and prepare the total length anti-HIV fusogenic peptide
Steam the mixing solutions that phenol, 0.5ml dimercaptoethane, 1.0ml thioanisole and 1.0ml distilled water are made into adding under the 2 gained solid cooled, handled 5 hours under the room temperature with 20ml TFA, 1.5 grammes per square metres.The peptide solution of removing the side chain protected group adds refrigerative 200ml isopropyl ether, and vacuum-drying is collected in the sedimentation and filtration isopropyl ether washing back of generation.
Embodiment 5
Ac-WMEWDRKIEEYTKKIEELIKKSQEQQEKNEKELK-NH 2(SEQ ID NO.16)
Molecular weight: 4437.09
Productive rate: 92%
Purity: 75%
Synthetic method is with embodiment 4.
Intermediate peptide 1 is:
Ac-Trp(Boc)-Met-Glu(OtBu)-Trp(Boc)-Asp(OtBu)-Arg(pmc)-Lys(Boc)-Ile-Glu(OtBu)-Glu(OtBu)-OH
Intermediate peptide 2 is:
Fmoc-Tyr(tBu)-Thr(tBu)-Lys(Boc)-Lys(Boc)-Ile-Glu(OtBu)-Glu(OtBu)-Leu-Ile-Lys(Boc)-Lys(Boc)-Ser(tBu)-OH
Intermediate peptide 3 is:
H-Gln(trt)-Glu(OtBu)-Gln(trt)-Gln(trt)-Glu(OtBu)-Lys(Boc)-Asn(trt)-Glu(OtBu)-Lys(Boc)-Glu(OtBu)-Leu-Lys(Boc)-NH 2
Embodiment 6
Ac-WEEWDKKIEEYTKKIEELIKKSEEQQKKNEEELKK-NH 2(SEQ ID NO.18)
Molecular weight: 4536.16
Productive rate: 93%
Purity: 73%
Synthetic method is with embodiment 4.
Intermediate peptide 1 is:
Ac-Trp(Boc)-Glu(OtBu)-Glu(OtBu)-Trp(Boc)-sp(OtBu)-Lys(Boc)-Lys(Boc)-Ile-Glu(OtBu)-Glu(OtBu)-OH
Intermediate peptide 2 is:
Fmoc-Tyr(tBu)-Thr(tBu)-Lys(Boc)-Lys(Boc)-Ile-Glu(OtBu)-Glu(OtBu)-Leu-Ile-Lys(Boc)-Lys(Boc)-Ser(tBu)-OH
Intermediate peptide 3 is:
H-Glu(OtBu)-Glu(OtBu)-Gln(trt)-Gln(trt)-Lys(Boc)-Lys(Boc)-Asn(trt)-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Leu-Lys(Boc)-Lys(Boc)-NH 2
Embodiment 7
Ac-WMEWDREINNYTSLIHSLIEESQNQQEK-NH 2(SEQ ID NO.19)
Molecular weight is: 3562.91
Productive rate: 92%
Purity: 81%
Synthetic method is as follows:
1. intermediate peptide 4 is the anti-HIV fusogenic peptide of side chain protected with intermediate peptide 5 couplings
After the intermediate peptide 5H-Leu-Ile-His (trt) of the intermediate peptide 4Ac-Trp (Boc) of 0.30mmol-Met-Glu (OtBu)-Trp (Boc)-Asp (OtBu)-Arg (pmc) Glu (OtBu)-Ile-Asn (trt)-Asn (trt)-Tyr (tBu)-Thr (tBu)-Ser (tBu)-OH and 0.30mmol-Ser (tBu)-Leu-Ile-Glu (OtBu)-Glu (OtBu)-Ser (tBu)-Gln (trt)-Asn (trt)-Gln (trt)-Gln (trt)-Glu (OtBu)-Lys (Boc)-NH2 being dissolved among the NMP of 8ml; The HOBt that adds 1.2 times of equivalents, room temperature reaction is 4 hours behind the DIEA of 1.5 times of equivalents of the lower adding of cooling and the HATU of 1.2 times of equivalents. The methanol (1:1) that drips 30ml is stirred to pulpous state.Filter, solid washs 2 times with 8ml methanol (1:1), vacuum-drying.
2. remove the side chain protected group and prepare the total length anti-HIV fusogenic peptide
Steam the mixing solutions that phenol, 0.5ml dimercaptoethane, 1.0ml thioanisole and 1.0ml distilled water are made into adding under the 1 gained solid cooled, handled 5 hours under the room temperature with 20ml TFA, 1.5 grammes per square metres.The peptide solution of removing the side chain protected group adds refrigerative 200ml isopropyl ether, and vacuum-drying is collected in the sedimentation and filtration isopropyl ether washing back of generation.
Embodiment 8
Ac-WEEWDRKIEEYTKLIKSLIEESQEQQEK-NH 2(SEQ ID NO.24)
Molecular weight is: 3637.05
Productive rate: 93%
Purity: 83%
Synthetic method is as follows:
1. intermediate peptide 4 is the anti-HIV fusogenic peptide of side chain protected with intermediate peptide 5 couplings
After the intermediate peptide 5H-Leu-Ile-Lys (Boc) of the intermediate peptide 4Ac-Trp (Boc) of 0.30mmol-Met-Glu (OtBu)-Trp (Boc)-Asp (OtBu)-Arg (pmc)-Lys (Boc)-Ile-Glu (OtBu)-Glu (OtBu)-Tyr (tBu)-Thr (tBu)-Lys (Boc) OH and 0.30mmol-Ser (tBu)-Leu-Ile-Glu (OtBu)-Glu (OtBu)-Ser (tBu)-Gln (trt)-Glu (OtBu)-Gln (trt)-Gln (trt) Glu (OtBu)-Lys (Boc)-NH2 being dissolved among the DMF of 8ml; The HOBt that adds 1.5 times of equivalents, room temperature reaction is 3 hours behind the DIEA of 1.5 times of equivalents of the lower adding of cooling and the HBTU of 1.3 times of equivalents. The methanol (1:1) that drips 30ml is stirred to pulpous state.Filter, solid washs 2 times with 8ml methanol (1:1), vacuum-drying.
2. remove the side chain protected group and prepare the total length anti-HIV fusogenic peptide
Steam phenol with adding under the 1 gained solid cooled, handled 4 hours under the room temperature with 20ml TFA, 5 grammes per square metres.The peptide solution of removing the side chain protected group adds refrigerative 200ml ether, and vacuum-drying is collected in the sedimentation and filtration ether washing back of generation.
Embodiment 9
Ac-WEEWDKKIEEYTKKIEELIKKSEEQQKK-NH 2(SEQ ID NO.26)
Molecular weight is: 3665.19
Productive rate: 91%
Purity: 85%
Synthetic method is as follows:
1. intermediate peptide 4 is the anti-HIV fusogenic peptide of side chain protected with intermediate peptide 5 couplings
With after intermediate peptide 5H-Lys (Boc)-Ile-Glu (OtBu)-Glu (OtBu)-Leu-Ile-Lys (Boc)-Lys (Boc)-Ser (tBu)-Glu (OtBu)-Glu (OtBu)-Gln (trt)-Gln (trt)-Lys (Boc)-Lys (Boc)-NH2 is dissolved among 4ml DMF and the 4mlNMP of the intermediate peptide 4Ac-Trp (Boc) of 0.30mmol-Glu (OtBu)-Glu (OtBu)-Trp (Boc)-Asp (OtBu)-Lys (Boc)-Lys (Boc)-Ile-Glu (OtBu)-Glu (OtBu)-Tyr (tBu)-Thr (tBu)-Lys (Boc) OH and 0.30mmol; The HOBt that adds 1.2 times of equivalents, room temperature reaction is 8 hours behind the DIEA of 1.1 times of equivalents of the lower adding of cooling and the TBTU of 1.2 times of equivalents. The methanol (1:1) that drips 30ml is stirred to pulpous state.Filter, solid washs 2 times with 8ml methanol (1:1), vacuum-drying.
2. remove the side chain protected group and prepare the total length anti-HIV fusogenic peptide
With adding the mixing solutions that is made into 20ml TFA, 1.5 grammes per square metres steaming phenol, 0.5ml dimercaptoethane under the 1 gained solid cooled, handled 7 hours under the room temperature.The peptide solution of removing the side chain protected group adds refrigerative 200ml methyl butyl ether, and vacuum-drying is collected in the sedimentation and filtration methyl butyl ether washing back of generation.
Embodiment 10
Ac-WMEWDREINNYTSLIHSLIEESQNQQEKNEQELL-NH 2(SEQ ID NO.1)
Molecular weight is: 4289.70
Productive rate: 86%
Purity: 55%
Synthetic method is as follows:
1. intermediate peptide 6 is the anti-HIV fusogenic peptide of side chain protected with intermediate peptide 7 couplings
With after intermediate peptide 7H-Glu (OtBu)-Glu (OtBu)-Ser (tBu)-Gln (trt)-Asn (trt)-Gln (trt)-Gln (trt)-Glu (OtBu)-Lys (Boc)-Asn (trt)-Glu (OtBu)-Gln (trt)-Glu (OtBu)-Leu-Leu-NH2 is dissolved among 4ml DMF and the 4mlNMP of the intermediate peptide 6Ac-Trp (Boc) of 0.30mmol-Met-Glu (OtBu)-Trp (Boc)-Asp (OtBu)-Arg (pmc)-Glu (OtBu)-Ile-Asn (trt)-Asn (trt)-Tyr (tBu)-Thr (tBu)-Ser (tBu)-Leu-Ile-His (trt)-Ser (tBu)-Leu-Ile-OH and 0.30mmol; The HOBt that adds 1 times of equivalent, room temperature reaction is 6 hours behind the DIEA of 1.1 times of equivalents of the lower adding of cooling and the HBTU of 1.3 times of equivalents. The methanol (1:1) that drips 30ml is mixed to pulpous state.Filter, solid washs 2 times with 8ml methanol (1:1), vacuum-drying.
2. remove the side chain protected group and prepare the total length anti-HIV fusogenic peptide
With adding the mixing solutions that is made into 20ml TFA, 1.5 grammes per square metres steaming phenol, 2ml distilled water under the 1 gained solid cooled, handled 7 hours under the room temperature.The peptide solution of removing the side chain protected group adds refrigerative 100ml methyl butyl ether and 100ml ether, and vacuum-drying is collected in the sedimentation and filtration methyl butyl ether of generation, ether mixed solution washing back.
Embodiment 11
Ac-WMEWDRKIEEYTKKIEELIKKSQEQQEKNEKELK-NH 2(SEQ ID NO.16)
Molecular weight is: 4437.09
Productive rate: 89%
Purity: 60%
Synthetic method is as follows:
1. intermediate peptide 6 is the anti-HIV fusogenic peptide of side chain protected with intermediate peptide 7 couplings
0.30mmol6Ac-Trp （ Boc ）-Met-Glu （ OtBu ）-Trp （ Boc ）-Asp （ OtBu ）-Arg （ pmc ）-Lys （ Boc ）-Ile-Glu （ OtBu ）-Glu （ OtBu ）-Tyr （ tBu ）-Thr （ tBu ）-Lys （ Boc ）-Lys （ Boc ）-Ile-Glu （ OtBu ）-Glu （ OtBu ）-Leu-Ile-OH0.30mmol7H-Glu （ OtBu ）-Glu （ OtBu ）-Ser （ tBu ）-Gln （ trt ）-Glu （ OtBu ）-Gln （ trt ）-Gln （ trt ）-Glu （ OtBu ）-Lys （ Boc ）-Asn （ trt ）-Glu （ OtBu ）-Lys （ Boc ）-Glu （ OtBu ）-Leu-Lys （ Boc ）-NH。2
After being dissolved among the DMF of 8ml, add 1.5 times of normal HOBt, cooling added behind 1.5 times of normal DIEA and the 1.3 times of normal HBTU room temperature reaction down 3 hours.The methanol (1: 1) that drips 30ml is stirred to pulpous state.Filter, solid washs 2 times with 8ml methanol (1: 1), vacuum-drying.
2. remove the side chain protected group and prepare the total length anti-HIV fusogenic peptide
Steam phenol with adding under the 1 gained solid cooled, handled 7 hours under the room temperature with 20ml TFA, 5 grammes per square metres.The peptide solution of removing the side chain protected group adds refrigerative 200ml ether, and vacuum-drying is collected in the sedimentation and filtration ether washing back of generation.
Embodiment 12
Ac-WEEWDKKIEEYTKKIEELIKKSEEQQKKNEEELKK-NH 2(SEQ ID NO.18)
Molecular weight is: 4536.16
Productive rate: 90%
Purity: 61%
Synthetic method is as follows:
1. intermediate peptide 6 is the anti-HIV fusogenic peptide of side chain protected with intermediate peptide 7 couplings
0.30mmol6Ac-Trp ( Boc )-Glu ( OtBu )-Glu ( OtBu )-Trp ( Boc )-Asp ( OtBu )-Lys ( Boc )-Lys ( Boc )-Ile-Glu ( OtBu )-Glu ( OtBu )-Tyr ( tBu )-Thr ( tBu )-Lys ( Boc )-Lys ( Boc )-Ile-Glu ( OtBu )-Glu ( OtBu )-Leu-Ile-OH0.30mmol7H-Lys ( Boc )-Lys ( Boc )-Ser ( tBu )-Glu ( OtBu )-Glu ( OtBu )-Gln ( trt )-Gln ( trt )-Lys ( Boc )-Lys ( Boc )-Asn ( trt )-Glu ( OtBu )-Glu ( OtBu )-Glu ( OtBu )-Leu-Lys ( Boc )-Lys ( Boc )-NH28mlDMF； The HOBt that adds 1.5 times of equivalents, room temperature reaction is 5 hours behind the DIEA of 1.2 times of equivalents of the lower adding of cooling and the HBTU of 1.3 times of equivalents. The methanol (1:1) that drips 30ml is mixed to pulpous state.Filter, solid washs 2 times with 8ml methanol (1:1), vacuum-drying.
2. remove the side chain protected group and prepare the total length anti-HIV fusogenic peptide
Steam phenol, 1.0ml thioanisole with adding under the 1 gained solid cooled, handled 8 hours under the room temperature with 20ml TFA, 5 grammes per square metres.The peptide solution of removing the side chain protected group adds refrigerative 100ml ether and 100ml isopropyl ether, and vacuum-drying is collected in the sedimentation and filtration ether washing back of generation.
Sequence table
＜110〉Nanfang Medical Univ
＜120〉anti-HIV fusogenic peptide and preparation method thereof
<160>53
<170>PatentIn Version 3.1
<210>1
<211>34
<212>PRT
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: peptide segment
<400>1
Trp Met Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr Ser Leu Ile His
5 10 15
Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys Asn Glu
20 25 30
Gln Glu Leu Leu
<210>2
<211>34
<212>PRT
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: peptide segment
<400>2
Trp Met Glu Trp Asp Arg Lys Ile Asn Asn Tyr Thr Ser Leu Ile His
5 10 15
Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys Asn Glu
20 25 30
Gln Glu Leu Lys
<210>3
<211>34
<212>PRT
＜213〉artificial sequence
<220>
<222>(2)
<223>Xaa＝Nle
<400>3
Trp Xaa Glu Trp Asp Arg Lys Ile Asn Asn Tyr Thr Ser Leu Ile His
5 10 15
Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys Asn Glu
20 25 30
Gln Glu Leu Lys
<210>4
<211>34
<212>PRT
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: peptide segment
<400>4
Trp Met Glu Trp Asp Arg Lys Ile Glu Asn Tyr Thr Lys Leu Ile His
5 10 15
Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys Asn Glu
20 25 30
Gln Glu Leu Lys
<210>5
<211>34
<212>PRT
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: peptide segment
<400>5
Trp Met Glu Trp Asp Arg Lys Ile Glu Asn Tyr Thr Lys Leu Ile Lys
5 10 15
Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys Asn Glu
20 25 30
Gln Glu Leu Lys
<210>6
<211>34
<212>PRT
＜213〉artificial sequence
<220>
<222>(2)
<223>Xaa＝Nle
<400>6
Trp Xaa Glu Trp Asp Arg Lys Ile Glu Asn Tyr Thr Lys Leu Ile Lys
5 10 15
Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys Asn Glu
20 25 30
Gln Glu Leu Lys
<210>7
<211>34
<212>PRT
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: peptide segment
<400>7
Trp Met Glu Trp Asp Arg Lys Ile Glu Asn Tyr Thr Lys Leu Ile Lys
5 10 15
Ser Leu Ile Glu Glu Ser Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Gln Glu Leu Lys
<210>8
<211>34
<212>PRT
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: peptide segment
<400>8
Trp Met Glu Trp Asp Arg Lys Ile Glu Asn Tyr Thr Lys Leu Ile Lys
5 10 15
Ser Leu Ile Glu Glu Ser Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Lys Glu Leu Lys
<210>9
<211>34
<212>PRT
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: peptide segment
<400>9
Trp Met Glu Trp Asp Arg Lys Ile Glu Glu Tyr Thr Lys Leu Ile Lys
5 10 15
Ser Leu Ile Glu Glu Ser Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Lys Glu Leu Lys
<210>10
<211>34
<212>PRT
＜213〉artificial sequence
<220>
<222>(2)
<223>Xaa＝Nle
<400>10
Trp Xaa Glu Trp Asp Arg Lys Ile Glu Glu Tyr Thr Lys Leu Ile Lys
5 10 15
Ser Leu Ile Glu Glu Ser Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Lys Glu Leu Lys
<210>11
<211>34
<212>PRT
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: peptide segment
<400>11
Trp Met Glu Trp Asp Arg Lys Ile Glu Glu Tyr Thr Lys Lys Ile Lys
5 10 15
Ser Leu Ile Glu Glu Ser Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Lys Glu Leu Lys
<210>12
<211>34
<212>PRT
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: peptide segment
<400>12
Trp Met Glu Trp Asp Arg Lys Ile Glu Glu Tyr Thr Lys Lys Ile Lys
5 10 15
Lys Leu Ile Glu Glu Ser Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Lys Glu Leu Lys
<210>13
<211>34
<212>PRT
＜213〉artificial sequence
<220>
<222>(2)
<223>Xaa＝Nle
<400>13
Trp Xaa Glu Trp Asp Arg Lys Ile Glu Glu Tyr Thr Lys Lys Ile Lys
5 10 15
Lys Leu Ile Glu Glu Ser Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Lys Glu Leu Lys
<210>14
<211>34
<212>PRT
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: peptide segment
<400>14
Trp Met Glu Trp Asp Arg Lys Ile Glu Glu Tyr Thr Lys Lys Ile Glu
5 10 15
Lys Leu Ile Lys Glu Ser Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Lys Glu Leu Lys
<210>15
<211>34
<212>PRT
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: peptide segment
<400>15
Trp Met Glu Trp Asp Arg Lys Ile Glu Glu Tyr Thr Lys Lys Ile Lys
5 10 15
Glu Leu Ile Glu Lys Ser Gln Glu Gln Gln Glu Lys Asn Glu
20 25 30
Lys Glu Leu Lys
<210>16
<211>34
<212>PRT
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: peptide segment
<400>16
Trp Met Glu Trp Asp Arg Lys Ile Glu Glu Tyr Thr Lys Lys Ile Glu
5 10 15
Glu Leu Ile Lys Lys Ser Gln Glu Gln Gln Glu Lys Asn Glu Lys
20 25 30
Glu Leu Lys
<210>17
<211>34
<212>PRT
＜213〉artificial sequence
<220>
<222>(2)
<223>Xaa＝Nle
<400>17
Trp Xaa Glu Trp Asp Arg Lys Ile Glu Glu Tyr Thr Lys Lys Ile Glu
5 10 15
Glu Leu Ile Lys Lys Ser Gln Glu Gln Gln Glu Lys Asn
20 25
Glu Lys Glu Leu Lys
30
<210>18
<211>35
<212>PRT
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: peptide segment
<400>18
Trp Glu Glu Trp Asp Lys Lys Ile Glu Glu Tyr Thr Lys Lys Ile Glu
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>19
<211>28
<212>PRT
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: peptide segment
<400>19
Trp Met Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr Ser Leu Ile His
5 10 15
Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys
20 25
<210>20
<211>28
<212>PRT
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: peptide segment
<400>20
Trp Met Glu Trp Asp Arg Lys Ile Glu Glu Tyr Thr Lys Leu Ile Lys
5 10 15
Ser Leu Ile Glu Glu Ser Gln Glu Gln Gln Glu Lys
20 25
<210>21
<211>28
<212>PRT
＜213〉artificial sequence
<220>
<222>(2)
<223>Xaa＝Nle
<400>21
Trp Xaa Glu Trp Asp Arg Lys Ile Glu Glu Tyr Thr Lys Leu Ile Lys
5 10 15
Ser Leu Ile Glu Glu Ser Gln Glu Gln Gln Glu Lys
20 25
<210>22
<211>28
<212>PRT
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: peptide segment
<400>22
Trp Met Glu Trp Asp Arg Lys Ile Glu Glu Tyr Thr Lys Lys Ile Lys
5 10 15
Lys Leu Ile Glu Glu Ser Gln Glu Gln Gln Glu Lys
20 25
<210>23
<211>28
<212>PRT
＜213〉artificial sequence
<220>
<222>(2)
<223>Xaa＝Nle
<400>23
Trp Xaa Glu Trp Asp Arg Lys Ile Glu Glu Tyr Thr Lys Lys Ile Lys
5 10 15
Lys Leu Ile Glu Glu Ser Gln Glu Gln Gln Glu Lys
20 25
<210>24
<211>28
<212>PRT
＜213〉artificial sequence
<220>
＜223〉artificial sequence description; The peptide segment
<400>24
Trp Glu Glu Trp Asp Arg Lys Ile Glu Glu Tyr Thr Lys Leu Ile Lys
5 10 15
Ser Leu Ile Glu Glu Ser Gln Glu Gln Gln Glu Lys
20 25
<210>25
<211>28
<212>PRT
＜213〉artificial sequence
<220>
<222>(2)
<223>Xaa＝Nle
<400>25
Trp Xaa Glu Trp Asp Lys Lys Ile Glu Glu Tyr Thr Lys Lys Ile Glu
5 10 15
Glu Leu Ile Lys Lys Ser Glu Glu Gln Gln Lys Lys
20 25
<210>26
<211>28
<212>PRT
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: peptide segment
<400>26
Trp Glu Glu Trp Asp Lys Lys Ile Glu Glu Tyr Thr Lys Lys Ile Glu
5 10 15
Glu Leu Ile Lys Lys Ser Glu Glu Gln Gln Lys Lys
20 25
<210>27
<211>28
<212>PRT
＜213〉artificial sequence
<220>
<222>(2)
<223>Xaa＝Nle
<400>27
Trp Xaa Glu Trp Asp Lys Lys Ile Glu Glu Tyr Thr Lys Lys Ile Glu
5 10 15
Glu Leu Ile Lys Lys Ser Glu Glu Gln Gln Lys Lys
20 25
<210>28
<211>10
<212>PRT
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: peptide segment
<400>28
Trp Met Glu Trp Asp Arg Glu Ile Asn Asn
5 10
<210>29
<211>10
<212>PRT
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: peptide segment
<400>29
Trp Met Glu Trp Asp Arg Lys Ile Asn Asn
5 10
<210>30
<211>10
<212>PRT
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: peptide segment
<400>30
Trp Met Glu Trp Asp Arg Lys Ile Glu Asn
5 10
<210>31
<211>10
<212>PRT
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: peptide segment
<400>31
Trp Met Glu Trp Asp Arg Lys Ile Glu Glu
5 10
<210>32
<211>10
<212>PRT
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: peptide segment
<400>32
Trp Glu Glu Trp Asp Lys Lys Ile Glu Glu
5 10
<210>33
<211>12
<212>PRT
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: peptide segment
<400>33
Tyr Thr Ser Leu Ile His Ser Leu Ile Glu Glu Ser
5 10
<210>34
<211>12
<212>PRT
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: peptide segment
<400>34
Tyr Thr Lys Leu Ile Lys Ser Leu Ile Glu Glu Ser
5 10
<210>35
<211>12
<212>PRT
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: peptide segment
<400>35
Tyr Thr Lys Lys Ile Lys Lys Leu Ile Glu Glu Ser
5 10
<210>36
<211>12
<212>PRT
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: peptide segment
<400>36
Tyr Thr Lys Lys Ile Glu Glu Leu Ile Lys Lys Ser
5 10
<210>37
<211>12
<212>PRT
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: peptide segment
<400>37
Gln Asn Gln Gln Glu Lys Asn Glu Gln Glu Leu Leu
5 10
<210>38
<211>12
<212>PRT
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: peptide segment
<400>38
Gln Asn Gln Gln Glu Lys Asn Glu Gln Glu Leu Lys
5 10
<210>39
<211>12
<212>PRT
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: peptide segment
<400>39
Gln Glu Gln Gln Glu Lys Asn Glu Gln Glu Leu Lys
5 10
<210>40
<211>12
<212>PRT
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: peptide segment
<400>40
Gln Glu Gln Gln Glu Lys Asn Glu Lys Glu Leu Lys
5 10
<210>41
<211>13
<212>PRT
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: peptide segment
<400>41
Glu Glu Gln Gln Lys Lys Asn Glu Glu Glu Leu Lys Lys
5 10
<210>42
<211>13
<212>PRT
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: peptide segment
<400>42
Trp Met Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr Ser
5 10
<210>43
<211>13
<212>PRT
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: peptide segment
<400>43
Trp Met Glu Trp Asp Arg Lys Ile Glu Glu Tyr Thr Lys
5 10
<210>44
<211>13
<212>PRT
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: peptide segment
<400>44
Trp Glu Glu Trp Asp Lys Lys Ile Glu Glu Tyr Thr Lys
5 10
<210>45
<211>15
<212>PRT
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: peptide segment
<400>45
Leu Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys
5 10 15
<210>46
<211>15
<212>PRT
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: peptide segment
<400>46
Leu Ile Lys Ser Leu Ile Glu Glu Ser Gln Glu Gln Gln Glu Lys
5 10 15
<210>47
<211>15
<212>PRT
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: peptide segment
<400>47
Lys Ile Glu Glu Leu Ile Lys Lys Ser Glu Glu Gln Gln Lys Lys
5 10 15
<210>48
<211>19
<212>PRT
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: peptide segment
<400>48
Trp Met Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr Ser Leu Ile His
5 10 15
Ser Leu Ile
<210>49
<211>19
<212>PRT
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: peptide segment
<400>49
Trp Met Glu Trp Asp Arg Lys Ile Glu Glu Tyr Thr Lys Lys Ile Glu
5 10 15
Glu Leu Ile
<210>50
<211>19
<212>PRT
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: peptide segment
<400>50
Trp Glu Glu Trp Asp Lys Lys Ile Glu Glu Tyr Thr Lys Lys Ile Glu
5 10 15
Glu Leu Ile
<210>51
<211>15
<212>PRT
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: peptide segment
<400>51
Glu Glu Ser Gln Asn Gln Gln Glu Lys Asn Glu Gln Glu Leu Leu
5 10 15
<210>52
<211>15
<212>PRT
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: peptide segment
<400>52
Glu Glu Ser Gln Glu Gln Gln Glu Lys Asn Glu Lys Glu Leu Lys
5 10 15
<210>53
<211>16
<212>PRT
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: peptide segment
<400>53
Lys Lys Ser Glu Glu Gln Gln Lys Lys Asn Glu Glu Glu Leu Lys lys
5 10 15

Claims (3)

1, a kind of preparation method of anti-HIV fusogenic peptide comprises: (a) intermediate peptide dissolving back is added activating reagent and carry out coupling, coupled product is separated out after-filtration at methanol aqueous solution; (b) coupled product is added the trifluoroacetic acid solution that comprises the ion trap agent and sloughs blocking group, in ether, precipitate then, filter the final vacuum drying, obtain the polypeptide of following aminoacid sequence:
a-WX 1EWDX 2X 3IX 4X 5YTSX 6IX 7X 8LIX 9X 10SX 11X 12QQX 13KNEX 14ELX 15X 16-b (I)
The aminoacid sequence abbreviation that other letter is an one-letter symbol in the aminoacid sequence in the general formula (I), the amino-acid residue of representative is defined as follows:
S is that Serine, W are that tryptophane, E are that L-glutamic acid, T are that Threonine, R are that arginine, I are that Isoleucine, N are that asparagine, Y are that tyrosine, K are that Methionin, Q are that glutamine, L are that leucine, D are that aspartic acid, M are methionine(Met); A is that ethanoyl, 9-fluorenyl methoxy-carbonyl (Fmoc), tert-butoxycarbonyl (Boc) are wherein a kind of, and b is amido or carboxyl; X 1-16Represent natural amino acid or alpha-non-natural amino acid or amino acid analogue or vacancy; Wherein,
Described intermediate peptide is:
Intermediate peptide 1:Ac-WX 1EWDX 2X 3IX 4X 5-OH,
Intermediate peptide 2:Fmoc-YTSX 6IX 7X 8LIX 9X 10S-OH,
Intermediate peptide 3:H-X 11X 12QQX 13KNEX 14ELX 15X 16-NH 2
Perhaps be:
Intermediate peptide 6:Ac-WX 1EWDX 2X 3IX 4X 5YTSX 6IX 7X 8LI-OH,
Intermediate peptide 7:H-X 9X 10SX 11X 12QQX 13KNEX 14ELX 15X 16-NH 2
2, the described preparation method of claim 1, wherein intermediate peptide 1 is
Ac-Trp(Boc)-Met-Glu(OtBu)-Trp(Boc)-Asp(OtBu)-Arg(pmc)-Glu(OtBu)-Ile-Asn(trt)-Asn(trt)-OH(SEQ IDNO.36)，
Ac-Trp(Boc)-Met-Glu(OtBu)-Trp(Boc)-Asp(OtBu)-Arg(pmc)-Lys(Boc)-Ile-Asn(trt)-Asn(trt)-OH(SEQ IDNO.37)，
Ac-Trp(Boc)-Met-Glu(OtBu)-Trp(Boc)-Asp(OtBu)-Arg(pmc)-Lys(Boc)-Ile-Glu(OtBu)-Asn(trt)-OH(SEQ ID NO.38)，
Ac-Trp(Boc)-Met-Glu(OtBu)-Trp(Boc)-Asp(OtBu)-Arg(pmc)-Lys(Boc)-Ile-Glu(OtBu)-Glu(OtBu)-OH(SEQ ID NO.39)，
Ac-Trp (Boc)-Glu (OtBu)-Glu (OtBu)-Trp (Boc)-Asp (OtBu)-Lys (Boc)-Lys (Boc)-Ile-Glu (OtBu)-Glu (OtBu)-OH (SEQ ID NO.40) is wherein a kind of;
Intermediate peptide 2 is
Fmoc-Tyr(tBu)-Thr(tBu)-Ser(tBu)-Leu-Ile-His(trt)-Ser(tBu)-Leu-Ile-Glu(OtBu)-Glu(OtBu)-Ser(tBu)-OH(SEQ ID NO.41)，
Fmoc-Tyr(tBu)-Thr(tBu)-Lys(Boc)-Leu-Ile-Lys(Boc)-Ser(tBu)-Leu-Ile-Glu(OtBu)-Glu(OtBu)-Ser(tBu)-OH(SEQ ID NO.42)，
Fmoc-Tyr(tBu)-Thr(tBu)-Lys(Boc)-Lys(Boc)-Ile-Lys(Boc)-Lys(Boc)-Leu-Ile-Glu(OtBu)-Glu(OtBu)-Ser(tBu)-OH(SEQ ID NO.43)，
Fmoc-Tyr (tBu)-Thr (tBu)-Lys (Boc)-Lys (Boc)-Ile-Glu (OtBu)-Glu (OtBu)-Leu-Ile-Lys (Boc)-Lys (Boc)-Ser (tBu)-OH (SEQ ID NO.44) is wherein a kind of;
Intermediate peptide 3 is
H-Gln(trt)-Asn(trt)-Gln(trt)-Gln(trt)-Glu(OtBu)-Lys(Boc)-Asn(trt)-Glu(OtBu)-Gln(trt)-Glu(OtBu)-Leu-Leu-NH 2(SEQ ID NO.45)，
H-Gln(trt)-Asn(trt)-Gln(trt)-Gln(trt)-Glu(OtBu)-Lys(Boc)-Asn(trt)-Glu(OtBu)-Gln(trt)-Glu(OtBu)-Leu-Lys(Boc)-NH 2(SEQ ID NO.46)，
H-Gln(trt)-Glu(OtBu)-Gln(trt)-Gln(trt)-Glu(OtBu)-Lys(Boc)-Asn(trt)-Glu(OtBu)-Gln(trt)-Glu(OtBu)-Leu-Lys(Boc)-NH 2(SEQ ID NO.47)，
H-Gln(trt)-Glu(OtBu)-Gln(trt)-Gln(trt)-Glu(OtBu)-Lys(Boc)-Asn(trt)-Glu(OtBu)-Lys(Boc)-Glu(OtBu)-Leu-Lys(Boc)-NH 2(SEQ ID NO.48)，
H-Glu (OtBu)-Glu (OtBu)-Gln (trt)-Gln (trt)-Lys (Boc)-Lys (Boc)-Asn (trt)-Glu (OtBu)-Glu (OtBu)-Glu (OtBu)-Leu-Lys (Boc)-Lys (Boc)-NH 2(SEQ ID NO.49) is wherein a kind of;
Wherein tBu is the tertiary butyl, and trt is a trityl.
3, the described preparation method of claim 1, wherein intermediate peptide 6 is
Ac-Trp(Boc)-Met-Glu(OtBu)-Trp(Boc)-Asp(OtBu)-Arg(pmc)-Glu(OtBu)-Ile-Asn(trt)-Asn(trt)-Tyr(tBu)-Thr(tBu)-Ser(tBu)-Leu-Ile-His(trt)-Ser(tBu)-Leu-Ile-OH(SEQ ID NO.56)，
Ac-Trp(Boc)-Met-Glu(OtBu)-Trp(Boc)-Asp(OtBu)-Arg(pmc)-Lys(Boc)-Ile-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Thr(tBu)-Lys(Boc)-Lys(Boc)-Ile-Glu(OtBu)-Glu(OtBu)-Leu-Ile-OH(SEQ ID NO.57)，
Ac-Trp (Boc)-Glu (OtBu)-Glu (OtBu)-Trp (Boc)-Asp (OtBu)-Lys (Boc)-Lys (Boc)-Ile-Glu (OtBu)-Glu (OtBu)-Tyr (tBu)-Thr (tBu)-Lys (Boc)-Lys (Boc)-Ile-Glu (OtBu)-Glu (OtBu)-Leu-Ile-OH (SEQ ID NO.58) is wherein a kind of;
Intermediate peptide 7 is
H-Glu(OtBu)-Glu(OtBu)-Ser(tBu)-Gln(trt)-Asn(trt)-Gln(trt)-Gln(trt)-Glu(OtBu)-Lys(Boc)-Asn(trt)-Glu(OtBu)-Gln(trt)-Glu(OtBu)-Leu-Leu-NH 2(SEQ ID NO.59)，
H-Glu(OtBu)-Glu(OtBu)-Ser(tBu)-Gln(trt)-Glu(OtBu)-Gln(trt)-Gln(trt)-Glu(OtBu)-Lys(Boc)-Asn(trt)-Glu(OtBu)-Lys(Boc)-Glu(OtBu)-Leu-Lys(Boc)-NH 2(SEQ ID NO.60)，
H-Lys (Boc)-Lys (Boc)-Ser (tBu)-Glu (OtBu)-Glu (OtBu)-Gln (trt)-Gln (trt)-Lys (Boc)-Lys (Boc)-Asn (trt)-Glu (OtBu)-Glu (OtBu)-Glu (OtBu)-Leu-Lys (Boc)-Lys (Boc)-NH 2(SEQ ID NO.61) is wherein a kind of;
Wherein tBu is the tertiary butyl, and trt is a trityl.
CNB2005100341365A 2005-04-19 2005-04-19 Anti-HIV fusogenic peptide and its production Expired - Fee Related CN1317296C (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CNB2005100341365A CN1317296C (en) 2005-04-19 2005-04-19 Anti-HIV fusogenic peptide and its production

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CNB2005100341365A CN1317296C (en) 2005-04-19 2005-04-19 Anti-HIV fusogenic peptide and its production

Publications (2)

Publication Number Publication Date
CN1712416A CN1712416A (en) 2005-12-28
CN1317296C true CN1317296C (en) 2007-05-23

Family

ID=35718231

Family Applications (1)

Application Number Title Priority Date Filing Date
CNB2005100341365A Expired - Fee Related CN1317296C (en) 2005-04-19 2005-04-19 Anti-HIV fusogenic peptide and its production

Country Status (1)

Country Link
CN (1) CN1317296C (en)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101235083B (en) * 2007-02-02 2012-05-23 同济大学 HIV-I resisting polypeptide, coded sequence and use thereof
CN102180951B (en) * 2011-05-03 2012-09-26 中国医学科学院病原生物学研究所 Cholesterol modified anti-human immunodeficiency virus (HIV) polypeptide medicament and application thereof

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004103313A2 (en) * 2003-05-20 2004-12-02 The American National Red Cross Methods of reducing vascular permeability in tissue by inhibition of tissue plasminogen activator (tpa) and tpa inhibitors useful therein

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004103313A2 (en) * 2003-05-20 2004-12-02 The American National Red Cross Methods of reducing vascular permeability in tissue by inhibition of tissue plasminogen activator (tpa) and tpa inhibitors useful therein

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
多肽片段连接方法及肽硫酯和肽醛的合成 麻远等，化学进展，第15卷第5期 2003 *

Also Published As

Publication number Publication date
CN1712416A (en) 2005-12-28

Similar Documents

Publication Publication Date Title
CN1132845C (en) Improved method for obtaining isulin precursor having correctly-bonded cystine bond
CN1282656C (en) HIV peptides from conserved regions in GAGP 17 and 924 and their application in e.g. vaccines
CN1756763A (en) Dimerized peptide
CN1832959A (en) Polyethelene glycol link glp-1 compounds
CN1041951A (en) New peptidase inhibitors
CN1535264A (en) Arginine derivatives
CN1065874C (en) Novel peptides, the preparation and use thereof
CN1929860A (en) Site-specific chemical modification of HIV gp41-derived peptides
CN1896105A (en) Compounds and methods of early diagnosis of cervical cancer and genital condyloma with hpv, chsp60 tumor suppressor h-ras, k-ras and pten derived peptides modified
CN1174845A (en) Method for synthesis of analogs of parathyroid hormone and parathyroid hormone related peptide
CN1269837C (en) Serial synthetic antibacterial peptide
CN1865283A (en) Solid phase polypeptide synthesis preparation method for salcatonin
CN1049166A (en) The replacement analogue of Magainin peptide
CN1684972A (en) Conjugates comprised of polymer and HIV gp41-derived peptides and their use in therapy
CN1313491C (en) Cat omega interferon and its coding gene and uses
CN1317296C (en) Anti-HIV fusogenic peptide and its production
CN100339396C (en) Multi-helix protein for inhibiting membrane virus infection, its coding gene and use
CN1016341B (en) Novel spergualin-related compounds and process for producing the same
CN1189477C (en) Bicyclic tachykinins antagonists, preparation thereof and their use in autagonistes composition
CN1778930A (en) Fusion gene of provocative organ anti-Coccidium tenellum infection and its coding protein and use thereof
CN1698881A (en) Long lasting fusion peptide inhibitors of viral infection
CN1846785A (en) Freeze dried composition containing genetic engineering fusion protein
CN1968961A (en) Process for the preparation of peptides
CN1531548A (en) Chimeric chains that code for proteins that induce effects directed against viruses
CN1781933A (en) Thymosin alpha 1 active segment cyclicpeptide analogue and its poly glycol derivative

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C14 Grant of patent or utility model
GR01 Patent grant
C17 Cessation of patent right
CF01 Termination of patent right due to non-payment of annual fee

Granted publication date: 20070523

Termination date: 20100419