CN1452632A - Extended native chemical ligation - Google Patents

Abstract

The invention is directed to methods and compositions for chemical ligation of components comprising a first component having a carboxythioester, and preferable an alpha-carboxythioester, moiety and a second component having an N-substituted, and preferably an N alpha-substituted, 2 or 3 carbon chain alkyl or aryl thiol to give a ligation product having an N-substituted amide bond at the ligation site. The reactants of the invention are chemoselective, and the alkyl or aryl thiol moiety is removable from the ligation product. Removal of the alkyl or aryl thiol gives a native amide bond at the ligation site. The methods and compositions of the invention are particularly useful for ligation of peptides and polypeptides. The ligation system of the invention is applicable to a wide variety of molecules, and thus can be exploited to generate peptides, polypeptides and other amino acid containing polymers having a native amide bond at the ligation site.

Description

The native chemical of expansion connects

Cross reference to related application

The application requires to have benefited from the U.S. Provisional Application 60/231,339 of registration on September 8th, 2000.Thank you

The present invention partly is subjected to the support of National Institutes of Health's post-doctoral research fund GN190402.United States Government can enjoy some right.

Technical field

The present invention relates to be used for technological expansion that native chemical is connected to the method and composition that can couple together the peptide of wide range, polypeptide, other polymkeric substance and other molecule by an amido linkage.

Background

Chemistry connects to relate between first kind of chemical composition and second kind of chemical composition and forms a kind of optionally covalent linkage.Can use the functional group that reacts to each other that is present in the uniqueness on first kind of component and the second kind of component to make ligation have chemo-selective.For example, the chemistry of peptide and polypeptide is connected the peptide of the amino-acid residue that relates to the terminal and N-end of the C-that reacts to each other that has compatible uniqueness or the chemo-selective of polypeptide fragments reacts.Had several chemical processes to be used to this purpose, the example comprises that natural chemistry connects (people such as Dawson, Science (1994) 266:776-779; People such as Kent, WO96/34878; People such as Kent, WO98/28434), the chemistry that forms oxime connects (Rose, Deng the people, J.Amer.Chem.Soc. (1994), 116:30-34), form the connection (Schnolzer of thioester, Deng the people, Science (1992) 256:221-225), form the connection (Englebretsen of thioether, Deng the people, Tet.Letts. (1995) 36 (48): 8871-8874), form the connection (people such as Gaertner of hydrazone, Bioconj.Chem. (1994) 5 (4): 333-338) with form thiazolidine be connected and the connection of Xing Cheng oxazolidine (people such as Zhang, Proc.Nat1.Acad.Sci. (1998) 95 (16): 9184-9189; People such as Tam, WO95/00846; US5,589,356).

In these methods, have only natural chemical connection process can access the connection product that has natural amido linkage (being peptide bond) at the connection site place.Verified, initial natural chemical connection process (people such as Dawson, supra; And WO96/34878) is a kind of strong method that produces natural amido linkage at the connection site place.Natural chemistry connects and relates to first kind of peptide or polypeptide fragments with the terminal α of C--carboxyl thioester part and react with chemo-selective between second kind of peptide with N-terminal cysteine residue or the polypeptide.The mercaptan permutoid reaction has produced the intermediate that initial thioester connects, and this intermediate is spontaneously reset, and produces natural amido linkage at the connection site place, and is simultaneously born again the mercaptan of cysteine side chain.The defective of initial natural chemical connection method is that it needs a N-terminal cysteine, i.e. its permission contains the peptide of halfcystine and being connected of polypeptide fragments at the connection site place.

Although this defective is arranged, the report (WO98/28434) of the native chemical connection that contains the peptide with-terminal amino acid except that halfcystine is arranged still.In the method, use contains first kind of peptide or polypeptide fragments of the terminal α of a C--carboxyl thioester and contains the terminal formula HS-CH of a N- ₂-CH ₂-O-NH-[peptide] auxiliary group that replaces of the N-{ mercaptan of representative second kind of peptide or polypeptide fragments connect.After the connection, by fracture HS-CH ₂-CH ₂-O-auxiliary group and remove the auxiliary group that N-{ mercaptan replaces, produce natural amido linkage at the connection site place.A limitation of this method is to use sulfydryl oxyethyl group auxiliary group can successfully only form amido linkage at the glycine residue place.This has produced a kind of connection product, and this connects product behind cleavage reaction, and the amino acid whose position that replaces at the N-of second kind of peptide or polypeptide fragments produces a glycine residue.For this reason, have only when hope makes the connection product of reaction contain a glycine residue in this position, implement this method be only suitable, and under any circumstance, even the stability of the rate of practicing midwifery, parent all is debatable with the ability aspect of removing the auxiliary group that O-is connected.Yet can use other auxiliary group, for example HSCH ₂CH ₂The NH-[peptide], and can reaction be limited in the connection at glycine residue place, such auxiliary group can not be removed from connecting product.

Therefore, need a kind of extensively that be suitable for and strong chemical linked system that natural chemistry connection can be expanded to various amino-acid residue, peptide, polypeptide, polymkeric substance and other molecule, this chemistry linked system is utilized a kind of auxiliary group of effectively removing easily that contains mercaptan, and this molecule is coupled together by natural amido linkage at the connection site place.

Summary of the invention

The present invention is directed to the method and composition of the natural chemistry connection that relates to expansion.Natural chemical connection process of the present invention comprises: the initial connection product that the acid amides that the N-of a following formula of generation replaces connects:

J1-C (O)-N (C1 (R1)-C2-SH)-J2 I or

(C1 (R1)-C2 (R2)-C3 (R3)-SH)-J2 II wherein J1 is peptide or the polypeptide that contains one or more optionally protected amino acid side chains to J1-C (O)-N, or such peptide or polypeptide portion, polymkeric substance, dyestuff, suitable functionalized surface, linker or detectable marker, or be connected other compatible compound part with the chemosynthesis of peptide or the natural chemistry of expansion; R1, R2 and R3 are independently for H or a kind of and C1 conjugated electron-donating group: condition is that among R1, R2 and the R3 comprises one and C1 conjugated electron-donating group at least; And J2 is peptide or the polypeptide that contains one or more optionally protected amino acid side chains, or such peptide or polypeptide portion, polymkeric substance, dyestuff, suitable functionalized surface, linker or detectable marker or be connected other compatible compound part with the chemosynthesis of peptide or the natural chemistry of expansion.

Connect product and be by first kind of component of the carboxyl thioester that comprises formula J1-C (O) SR and the C2 of the sour stable N-replacement that comprises a kind of following formula or second kind of component of C3 chain aminoalkyl group or alkyl sulfhydryl

HS-C2-C1 (R1)-HN-J2 III or

HS-C3 (R3)-C2 (R2)-C1 (R1)-HN-J2 IV wherein J2, R1, R2 and R3 such as front defines, connect, the method for C2 or C3 alkyl group or aryl mercaptan of removing the connection product of the amide linkage that optionally replaces from N-is then produced.A kind of preferred embodiment in, made things convenient for this fracture by the positively charged ion that under the failure condition compatible, forms a kind of resonance stabilized at the C1 place with peptide.

Remove alkyl sulfhydryl or aryl mercaptan chain from N, obtain the final connection product of following formula:

J1-C (O)-HN-J2 V wherein J1, J2, R1, R2 and R3 such as front defines.The present invention is also at the natural chemistry composition that connects and the cartridge case and the test kit that comprise them that are used to carry out this expansion.Said composition comprise a kind of shielded fully, part is shielded or fully not protected sour stable N-replaces, preferably C2 or the C3 chain aminoalkyl group or the aryl mercaptan of the N alpha-substitution of following formula:

SX2-C2-C1 (R1)-X1 N-CH (Z2)-C (O)-J2 VI or

SX2-C3 (R3)-C2 (R2)-C1 (R1)-X1N-CH (Z2)-C (O)-J2 VII wherein X1 is H or amino protecting group; X2 is H or mercaptan protecting group; J2, R1, R2 and R3 such as front define; And Z2 is connected compatible any compound part (including, but are not limited to a kind of amino acid side chain) with the chemosynthesis of peptide or the natural chemistry of expansion.The present invention is also at the chirality form of these compounds that are substantially free of racemic modification or diastereomer.

The present invention also at the solution of producing this shielded fully, part is shielded or not protected fully N-replaces C2 or C3 chain aminoalkyl group or aryl mercaptan mutually and the method for solid phase.The method of producing these compounds comprises the aminoalkyl groupization, reductive amination of halogen mediation and the N α-protection compatible with the solid phase method of peptide synthesis, and N-is alkylating, and S-protects, aminoalkyl group-or the preparation of aryl-mercaptan amino acid parent.

The J1 of carboxyl thioester component part can comprise the compatible any compound part of reaction conditions that is connected with the natural chemistry that is used to expand with the carboxyl thioester, and the component that N-of the present invention replaces can be provided separately or it with the compound of wide range partly (comprise amino acid, peptide, polypeptide, nucleic acid or other compound partly for example the polymkeric substance of dyestuff, haptens, sugar, lipid, solid carrier, biocompatibility or other polymkeric substance etc.) be connected.The natural chemical connection process of expansion of the present invention is strong, can carry out under certain temperature range condition under near neutral pH condition in a kind of water-based system.The method of the component that production N-of the present invention replaces also is strong, is that also it can provide the synthetic route of wide range with the productive rate of wonderful Gao Erchun to these new compounds.N α of the present invention-protection, N-is alkylating, S-protection, aminoalkyl group-or aryl-mercaptan amino acid parent be particularly useful for using conventional peptide synthetic and other organic synthesis strategy carries out fast automatic synthetic in.And the component that shielded N-of the present invention replaces can expand to the purposes that chemistry connects in the polycomponent connectivity scenario; for example relate to multiple connection strategy when the production of polypeptide, for example the segment connectivity scenario more than three kinds or three kinds or compile when connecting synthetic schemes.For example, the component that method and composition of the present invention allows you to use first pair of carboxyl thioester and N-to replace is synthesized the first part of the molecule of wanting, the other parts of using other that component of carboxyl thioester and N-replacement is come synthetic molecules.Can connect product to each such synthetic then and couple together (after suitable deprotection and/or modification), form desired molecule.

Correspondingly, method and composition of the present invention has greatly enlarged the scope that native chemical connects, and raw material of the present invention, intermediate and final product have found purposes widely.

Brief description of the drawings:

Fig. 1 describes the present invention in detail by the ability that the natural chemistry of peptides that shows mediation expansion of the present invention connects; Can use same scheme to carry out the connection of any suitable molecule.As shown in the figure, contain a kind of formula J1-HN-CH (Z1)-α CO-SR α-carboxyl thioester first kind of component and contain the C2 alkyl group of sour stable N α replacement of N-end of a kind of formula HS-C2-C1 (R1)-NH α CH (Z2)-C (O)-J2 or second kind of component of aryl mercaptan.Component J1 and J2 can be any compound parts compatible with the ligation of chemo-selective, for example a kind of shielded or not protected amino acid, peptide, polypeptide, other polymkeric substance, dyestuff, linker etc.Z1 is any side-chain radical compatible with α CO-SR thioester, for example shielded or not protected amino acid side chain.Z2 is any side-chain radical compatible with the amino acid of N alpha-substitution, for example shielded or not protected amino acid side chain.R1 is a kind of benzyl moiety (be meant benzyl in C1, and be meant phenyl in other cases) that is replaced by an electron-donating group (preferably C1 ortho position or contraposition); Or picolyl (unsubstituted or at the ortho position of C1 or contraposition replaced by hydroxyl or mercaptan).

The mercaptan exchange occurs between α COSR thioester component and amino N-{ alkyl sulfhydryl } component.The intermediate that exchange produces a kind of thioester bonding connects product, connecting product spontaneously resets, through a kind of 5 yuan of ring intermediates, produce a kind of C2 alkyl group of a removable N α replacement or formula J1-HN-CH (Z1)-C (the O)-N α of aryl mercaptan [HS-C2-C1 (R1)-] (first kind of connection product of C1 (R1)-C2-SH)-CH (Z2)-C (O)-J2 of containing at the connection site place.

The C2 alkyl group or the aryl mercaptan [HS-C2-C1 (R1)-] that replace at the N at connection site place α are easy to remove under the condition compatible with peptide, produce a kind of final connection product that contains formula J1-HN-CH (Z1)-CO-NH-CH (Z2) CO-J2 of natural amido linkage at the connection site place.

Fig. 2 comes detailed description the present invention by the ability that the natural chemistry of peptides that shows mediation expansion of the present invention connects; Can use same scheme to carry out the connection of any suitable molecule.As shown in the figure, contain a kind of formula J1-HN-CH (Z1)-α CO-SR α-carboxyl thioester first kind of component and contain the C3 alkyl group of sour stable N α replacement of a kind of formula HS-C3 (R3)-C2 (R2)-C1 (R1)-NH α CH (Z2)-C (O)-J2 or second kind of component of aryl mercaptan.Component J1 and J2 can be any compound parts compatible with the ligation of chemo-selective, for example a kind of shielded or not protected amino acid, peptide, polypeptide, other polymkeric substance, dyestuff, linker etc.Z1 is any side-chain radical compatible with α CO-SR thioester, for example shielded or not protected amino acid side chain.Z2 is any side-chain radical compatible with the amino acid of N alpha-substitution, for example shielded or not protected amino acid side chain.When R1 was not H, R2 and R3 were H, and R1 be unsubstituted at the ortho position of C1 or contraposition by a kind of phenyl moiety of electron donating group-substituted; Picolyl (unsubstituted or at the ortho position of C1 or contraposition replaced by hydroxyl or mercaptan); Methyl mercaptan; Or thionyl methyl.When R2 and R3 were not H, R1 was H, and R2 and R3 form one at the ortho position of C1 or contraposition by the benzyl of an electron donating group-substituted; Or picolyl (unsubstituted or at the ortho position of C1 or contraposition replaced by hydroxyl or mercaptan).

The mercaptan exchange occurs between COSR thioester component and the amino amineothiot component.The intermediate that exchange produces a kind of thioester bonding connects product, connecting product spontaneously resets, through a kind of 6 yuan of ring intermediates, produce a kind of C3 alkyl group of a removable N α replacement or formula J1-HN-CH (Z1)-C (the O)-N α of aryl mercaptan [HS-C3 (R3)-C2 (R2)-C1 (R1)-] (first kind of connection product of C1-C2 (R2)-C3 (R3)-SH)-CH (Z2)-J2 of containing at the connection site place.

C3 chain aryl mercaptan [HS-C3 (R3)-C2 (R2)-C1 (R1)-] in the N at connection site place alpha-substitution is easy to remove under the condition compatible with peptide, produces a kind of final connection product that has formula J1-HN-CH (Z1)-CO-NH-CH (the Z2)-CO-J2 of natural amido linkage at the connection site place.

Fig. 3 describes a kind of natural chemical connectivity scenario of multi-component expansion in detail.According among Fig. 1 specifically described like that, make C2 alkyl group or aryl mercaptan and a kind of reactive polypeptide that contains a N-terminal cysteine residue of N-terminal polypeptide N alpha-substitution of N α-Bao expansion of peptide species α-carboxyl thioester and a kind of formula HS-C2-C1 (R1)-N α (PG1)-CH (Z2)-C (O)-J2.R1 is a phenyl unsubstituted or that replaced by an electron-donating group (preferably C1 ortho position or contraposition); Or picolyl (unsubstituted or at the ortho position of C1 or contraposition replaced by hydroxyl or mercaptan).Protecting group (PG1) can be any suitable protecting group, for example a kind of alkyl-carbonyl protecting group (for example benzyloxycarbonyl (Z), Boc, Bpoc, Fmoc etc.), a kind of trityl group protecting group (Trt), a kind of 2-nitrophenyl sulfinyl protecting group (Nps) etc.After the first step ligation, remove protecting group.

As shown in fig. 1; the natural chemical ligation of the first step is carried out between the polypeptide α-carboxyl thioester of the C2 alkyl group of the N-terminal polypeptide N of the N α-protection that contains formula HS-C2-C1 (R1)-N α (PG1)-CH (Z2)-C (O)-J2 alpha-substitution or aryl mercaptan and the terminal Cys peptide of N-, and the first step that produces a kind of formula HS-C2-C1 (R1)-N α (PG1)-CH (Z2) C (O)-peptide 2-peptide 3 connects product.Remove protecting group PG1 then, obtain the connection product of a kind of formula HS-C2-C1 (R1)-N α (H)-CH (Z2)-C (O)-peptide 2-peptide 3.Make this material and the third component reaction that contains the sulfo-acid esters then.The mercaptan exchange takes place between COSR thioester component and amino N-{ alkyl sulfhydryl } component.The intermediate that exchange produces a kind of thioesters bonding connects product, the latter spontaneously resets, through one 5 yuan ring intermediates, produce a kind of C2 alkyl group of removable N α replacement or formula peptide 1-C (the O)-N α of aryl mercaptan [HS-C2-C1 (R1)-] (second step connection product of C1 (R1)-C2-SH)-CH (Z2)-C (O) peptide 2-Cys-peptide 3 of containing at second connection site place.C2 alkyl group that replaces at the N at second connection site place α or aryl mercaptan [HS-C2-C1 (R1)-] are easy to remove under the condition compatible with peptide and produce and a kind ofly have the final product that is connected of formula peptide 1-C (O)-N α H-CH (Z2)-C (O) the peptide 2-Cys-peptide 3 of natural amido linkage at first with second connection site place.

Fig. 4 describes the general connection strategy of two kinds of different 1-phenyl-2-mercaptoethyl auxiliary agents of a kind of use of the present invention in detail.

Fig. 5 A and 5B show high performance liquid chromatography (HPLC) analytical results of the ligation of a kind of N α-1-of use (4-p-methoxy-phenyl)-cytochrome b5 62 that 2-mercaptoethyl auxiliary agent carries out of describing among the embodiment 21.Fig. 5 A demonstration time is 0 o'clock ligation situation.Fig. 5 B shows the latter linked situation of spending the night of reacting.Also show among Fig. 5 B, observe the connection product at the achirality center at two kinds of C1 places that result from N α-1-(4-methoxyphenol)-2-mercaptoethyl auxiliary agent.

Fig. 6 A shows the electrospray mass spectrum (MS) that is connected the reconstruction that is connected product cytochrome b5 62 residue 1-106 that forms with the terminal segment of the N-of a kind of N α-{ 1-(4-p-methoxy-phenyl)-2 mercapto ethanol } modification by the natural chemistry that uses expansion with 6B.The cytochrome b5 62 residue 1-63 that have the terminal thioester of C-and the 64-106 of cytochrome b5 62 residues that have the terminal N α of N--{ 1-(4-p-methoxy-phenyl)-2 mercapto ethanol } glycine are coupled together.Fig. 6 A is presented at the mass spectrum reconstruction that the initial connection product of a removable N α-{ 1-(4-p-methoxy-phenyl)-2 mercapto ethanol } group is contained at the connection site place.Fig. 6 B is presented at hydrogen fluoride (HF) and handles to remove N α-{ 1-(4-p-methoxy-phenyl)-2 mercapto ethanol } group after the connection site place produces natural amido linkage, and the mass spectrum that connects product is rebuild.Observed quality is 11948 ± 1Da (before HF handles) and 11781 ± 1Da (after HF handles), promptly loses 167 ± 2Da, coincide well with the expected loss 166Da that removes 1-(4-p-methoxy-phenyl)-2 mercapto ethanol auxiliary group.

The canonical analysis result (Fig. 7 A) of the HPLC of cytochrome b5 62 materials of the linearity described in Fig. 7 A and the 7B further explanatory drawings 6B and folding after the ion-exchange chromatography figure (Fig. 7 B) of this material.

The description of specific embodiment

The present invention is directed to the natural chemical connection process and the composition that relate to expansion.In general, this method relates to comprising a kind of carboxyl thioester, the N-that first kind of component of more preferably a kind of α-carboxyl thioester is stable with comprising a kind of acid replaces, and the C2 of preferred N alpha-substitution or second kind of component of C3 chain aminoalkyl group or aryl mercaptan couple together.

Chemo-selective reaction between the C2 that the carboxyl thioester of first kind of component and the N-of second kind of component replace or the mercaptan of C3 alkyl group or aryl mercaptan is undertaken by a kind of intermediate of thioesters bonding, is decomposed into a kind of initial connection product.More specifically, the intermediate that occurs in a kind of thioesters bonding of mercaptan exchange generation between COSR thioester component and the aminoalkyl group thiol component connects product, this connects product and takes place to reset automatically, and the first step that produces a kind of following formula of amide linkage through 5 yuan or 6 yuan of ring intermediates connects product:

J1-C (O)-N (C1 (R1)-C2-SH)-J2 I or

(C1 (R1)-C2 (R2)-C3 (R3)-SH)-J2 II wherein J1, J2, R1, R2 and R3 such as front defines J1-C (O)-N.

The N-at connection site place replace C2 or C3 alkyl group or aryl mercaptan [HS-C2-C1 (R1)-] or [HS-(C3 (R3)-C2 (R2)-C1 (R1)-] is easy to remove under the condition compatible with peptide and can produce infringement to product, produce a kind of final connection product of following formula:

J1-C (O)-HN-J2 V wherein J1, J2, R1, R2 and R3 such as front defines.Final connection product contains a natural amido linkage at the connection site place.

More specifically, the natural chemical connection process of expansion of the present invention comprises that following component is carried out chemistry to be connected: the first kind of component that (i) comprises α-carboxyl thioester of formula J1-C (O) SR, ((C2 that the sour stable N-of C1 (R1)-C2 (R2)-C3 (R3)-SH)-J2 (II) replaces or second kind of component of C3 chain aminoalkyl group or aryl mercaptan, wherein J1, J2, R1, R2 and R3 such as front define for C1 (R1)-C2-SH)-J2 (I) or J1-C (O)-N (ii) to comprise formula J1-C (O)-N.

Select R1, R2 and R3 group with the convenient N-C1 key that under the failure condition compatible, ruptures, for example can use electron-donating group, particularly when with the C1 conjugation, to form the positively charged ion of the resonance stabilized of being convenient to rupture at the C1 place with peptide.The chemistry ligation preferably comprises a kind of mercaptan catalyzer and is carrying out under the mixing condition of water-based or organic-water-based as vehicle and under about neutral pH condition.First kind of chemistry with second kind of component is connected and can be undertaken by a kind of 5 yuan or 6 yuan of rings, and the latter spontaneously resets, and obtains the connection product of the amide linkage that a kind of N-replaces.When first kind and second kind of component were peptide or polypeptide, ((C1 (R1)-C2 (R2)-C3 (R3)-SH)-CH (Z2)-C (O)-J2 (IX) wherein J1, J2 and R1, R2, R3 and Z2 such as front was defined the formula of the connection product of the amide linkage that N-replaces for C1 (R1)-C2-SH)-CH (Z2)-C (O)-J2 (VIII) or J1-C (O)-N α by J1-C (O)-N α.

Conjugated electron-donating group R1, the R2 of the connection product of the amide linkage that N replaces or R3 help to rupture the N-C1 key and remove C2 or C3 alkyl group or aryl mercaptan from the connection product of the amide linkage of N-replacement.Under the cancellation condition compatible, remove the alkyl or aryl mercaptan chain of N, produce a kind of connection product that contains natural amido linkage at the connection site place with peptide.If first kind and second kind of component are peptide or polypeptide, then connect product and will have following formula:

J1-CONαH-CH(Z2)-C(O)-J2???????????????(X)

Be connected with former chemistry and compare, method of the present invention has a plurality of advantages.Wherein several such advantages relate to the C2 of N-replacement of the present invention or the meticulous Harmony of C3 alkyl group or aryl mercaptan component.At first, not having the component of the N-replacement of connection is stable to acidic conditions, and this allows it to carry out strong synthetic and storage.Secondly, it can take place optionally react and produces a kind of initial product that is connected that has the amido linkage that a N-replaces at the connection site place with carboxyl thioester component.Thirdly; place, the N α position regenerated alkyl or aryl thiol moiety of the initial connection site that connects product can with condition not protected, that part is shielded or complete shielded peptide, polypeptide or other parts are compatible fully under by optionally cancellation, promptly can cancellation alkyl or aryl thiol moiety and can not damage desired connection product.According to the difference that connects the alkyl or aryl thiol moiety that selected specific N-replaces, optionally cleavage reaction can be easily under the failure condition compatible with peptide of standard, for example carries out under acid, photodissociation or reductive condition.Therefore, another advantage of the present invention is the difference according to desired end-use, be positioned on the rest part that connects component one or more groups if present, can be not protected, part is shielded or shielded fully.And, the C2 that known carboxyl thioester and N-replace or the chemo-selective and the solvability of C3 alkyl group or aryl mercaptan, ligation can be carried out fast and neatly, is reaching very high product yield about pH7 under aqueous conditions about room temperature.This makes the present invention have king-sized handiness when being used under mild conditions connection portion or not protected fully peptide, polypeptide or other polymkeric substance.

For the C2 alkyl group that comprises the N-replacement or the peptide composition of the present invention of aryl mercaptan component, this compound has following formula:

Shown in HS-C2-C1 (R1)-NH α-CH (Z2)-C (O)-following tabulation 1 of J2 (XI).J2 and R2 such as front define; Z2 is the compatible side-chain radical of amino acid that replaces with N-, for example is a kind of amino acid whose side chain.R1 be preferably at the ortho position of C1 or contraposition by the phenyl of an electron donating group-substituted; Or picolyl (unsubstituted or at the ortho position of C1 or contraposition replaced by hydroxyl or mercaptan).

Table I

In order to keep electron conjugated promoting to be connected the fracture of back N-C1 key with C1 carbon, phenyl and picolyl to electron substituent group R1 ', R3 ' and R5 ' is positioned at the ortho position or contraposition is necessary.

The electron-donating group of preferred R1 ', R3 ' and R5 ' comprises for example methoxyl group (OCH of strong electron-donating group ₃), thiol (SH), hydroxyl (OH), methylthio group (SCH ₃) and medium electron donor(ED) methyl (CH for example ₃), ethyl (CH ₂CH ₃), propyl group (CH ₂-CH ₂-CH ₃), sec.-propyl (CH ₂(CH ₃) ₃).Condition is any one among R1 ', R3 ' and the R5 ' or all can is H.A general viewpoint is the susceptibility that fracture takes place after connection for strong electron-donating group enhancing C2 alkyl group or aryl mercaptan.When having one electron-donating group as R1 ', R3 ' or R5 ' substituting group, ligation can be carried out with faster rate, and failure condition then slower or that needs are strict more ruptures.When having two or more electron-donating groups as R1 ', R3 ' or R5 ' substituting group, ligation will be slower, and failure condition then faster or that needs are not too strict ruptures.Therefore, can correspondingly select specific electron-donating group.

Another embodiment of the invention relates to the C2 chain compound that N-replaces, and it comprises the substituting group of a mercaptan as the R1 of R1 ' and R5 ' position.Except as with C1 conjugated electron-donating group, the place in these positions or two places introduce a mercaptan can make compound be connected in 6 yuan of rings (and be connected in 5 yuan rings by N α-C2 chain alkyl thioalcohol) through the mediation of R1 group.It has also increased the partial concn that is used for the available mercaptan of α-carboxyl thioester reaction, and with regard to structural limitations. other conformation is provided, and this can promote to connect.

Mention the C3 alkyl group or the aryl mercaptan component of N alpha-substitution of the present invention, this compound has formula HS-C3 (R3)-C2 (R2) C1 (R1)-NH α-CH (Z2)-C (O)-J2, shown in the following tabulation II

Table II

As previously described, J2 closes with the chemistry of peptide or the natural chemistry expanded is connected compatible any compound part, and Z2 is the compatible side-chain radical of amino acid that replaces with N-, for example is a kind of amino acid whose side chain.When R1 was not H, then R2 and R3 were H, and R1 be unsubstituted more preferably at the ortho position of C1 or contraposition by the phenyl moiety of an electron donating group-substituted; Or picolyl (unsubstituted or at the ortho position of C1 or contraposition replaced by hydroxyl or mercaptan).When R2 and R3 were not H, R1 was H, and R3 and R2 form one at the ortho position of C1 or contraposition by the benzyl of an electron donating group-substituted; Or picolyl (unsubstituted or at the ortho position of C1 or contraposition replaced by hydroxyl or mercaptan).

The C2 chain compound that replaces as for N-, in order to keep with the electron conjugated of C1 carbon so that the N α-C1 key that after being connected, can effectively rupture, phenyl and picolyl give electron substituent group R1 ', R3 ' and R5 ' is positioned at the ortho position or contraposition is necessary.Yet when R2 and R3 and C2 and C3 formed a benzyl, among R1 ' and the R3 ' comprised a strong electron-donating group at least, wherein R1 ' or R3 ' be selected from methoxyl group (OCH3), thiol (SH), hydroxyl (OH) and methylthio group (SCH3).For wherein R2 and R3 all is the C3 chain mercaptan that the N-of H replaces, and R1 comprises a phenyl or picolyl, and wherein R1 ', R3 ' and R5 ' comprise strong or medium electron-donating group or its combination.The C2 alkyl group that replaces as N-or the situation of aryl mercaptan, strong electron-donating group can strengthen C3 alkyl group or aryl mercaptan the susceptibility that ruptures takes place after connection.Therefore, can correspondingly select specific electron-donating group or its combination.

Similar to the C2 chain compound that N-replaces, when mercaptan can be used for being used to replace in interesting structure, the C3 chain compound that N-of the present invention replaces can comprise the substituting group of a kind of mercaptan as the R1 of R1 ' and R5 ' position.Equally, give thiol group and the C1 conjugation of electronics, it is incorporated into makes compound have two routes that can form 6 yuan of ring connections on these positions.It has also increased the partial concn that is used for the available mercaptan of α-carboxyl thioester reaction, and with regard to structural limitations, provides other conformation, and this can promote to connect.

Amino acid whose synthetic can the carrying out of C2 that the N-of N-end of the present invention replaces or C3 alkyl group or aryl mercaptan according to said method here, for example carry out according to scheme I and scheme II, embodiment and according to the technique of organic chemistry of standard as known in the art, referring to for example " Advanced Organic Chemistry; Reactions; Mechanisms, andStructure, " 4th Edition, J.March (Ed.), John Wiley ﹠amp; Sons, NewYork, NY, 1992; " Comprehensive Organic Transformations, AGuide to Functional Group preparations, " R.Larock (Ed.), VCHPublishers, New York, NY, 1989.They can be in solution, synthesize by synthetic method or its combination of polymkeric substance load.The aminoalkyl group of the alkylating S-protection of the N that preferable methods uses N α to protect-or aryl-mercaptan amino acid parent.Synthetic used reagent can obtain from a plurality of commercial channel.And, should understand well, can be feed composition and various intermediate, for example single amino acid derivative stores for future use, provides with test kit etc.

When the C2 for preparing the terminal N alpha-substitution of N-of the present invention or C3 alkyl group or aryl mercaptan amino acid, adopt the protecting group strategy.In various synthesis strategies the preferred protecting group of using (PG) general to synthesize (" SPPS ") compatible with solid-phase peptide.In some cases, also be necessary to use the orthogonally protect base that can under different conditions, can be removed.Known have many protecting groups of this purpose that are applicable to (referring to for example " protecting groups in Organic Synthesis ", 3rd Edition, T.W.Greene and P.G.M.Wuts, Eds., John Wiley﹠amp; Sons, Inc., 1999; NovaBiochem Catalog 2000; " SyntheticPeptides, A User ' s Guide, " G.A.Grant, Ed., W.H.Freeman ﹠amp; Company, New York, NY, 1992; " Advanced Chemtech Handbook ofCombinatorial ﹠amp; Solid Phase Organic Chemistry, " W.D., Bennet, J.W.Christensen, L.K.Hamaker, M.L.Peterson, M.R.Rhodes, and H.H.Saneii, Eds., Advanced Chemtech, 1998; " Principlesof Peptide Synthesis, 2nd ed., " M.Bodanszky, Ed., Springer-Verlag, 1993; " The Practice of Peptide Synthesis, 2nded., " M.Bodanszky and A.Bodanszky, Eds., Springer-Verlag, 1994; And " protecting groups, " P.J.Kocienski, Ed., GeorgThieme Verlag, Stuttgart, Germany, 1994). example comprises benzyloxycarbonyl (Z), Boc, Bpoc, Trt, Nps, FmocCI-Z, Br-Z, NSC, MSC, Dde etc.For the sulphur part, the example of suitable protecting group includes, but are not limited to benzyl, 4-methyl-benzyl, 4-methoxy-benzyl, trityl, Acm, TACAM, xanthenyl, disulfide derivatives, picolyl and phenacyl.

More specifically, the C2 of N alpha-substitution of the present invention or C3 alkyl group or aryl mercaptan can prepare according to scheme I (solid phase preparation of the parent of N alpha-substitution), scheme II (solution of the parent of N alpha-substitution prepares mutually).In scheme I; the methodology of organic synthesis of the polymkeric substance-load of use standard directly is assemblied in the C2 of N alpha-substitution or C3 alkyl group or aryl mercaptan on the solid phase; use the couling process of standard to make N α-protection of scheme II simultaneously; N-is alkylating, aminoalkyl group or the aryl mercaptan amino acid parent and the resin coupling of S-protection.In scheme I; X is a halogen; R1 and R2 as previously described and can be used as shielded or not protected part and operate or elaborate on resin, and J2 preferably links to each other with halogen as the form of X-CH (R)-J2-resin, wherein R is H or other side chain.Should be understood that J2 can be multiple group, for example wherein halogen X and J2-resin by more than one carbon atom separately, for example β-or gamma-amino acid or similar molecule synthetic in.Wherein use oxoethanoic acid part (HC (O)-C (O) J2-resin), the side chain R that obtains is H.In scheme II, X is a kind of halogen, and R1 and R2 can be used as shielded or not protected part and operate as previously described, perhaps elaborates in solution or on the resin, wherein R is H or other side chain.(HC (O)-C (O)-OH), the side chain R of generation is H wherein to use the oxoethanoic acid part.As described above, you will be appreciated that scheme I and 11 can be used in the alkyl group of C3 or synthesizing of aryl mercaptan.When producing racemic modification or diastereomer product, had necessity before the natural chemistry that is used to expand connects, by standard method these compound separation are come.

Scheme I

Scheme II

The carboxyl thioester of first kind of used component part in the natural chemical connection process with regard to expansion of the present invention, this component is formula J1-CO-SR.Preferred carboxyl thioester component comprises α-carboxyl thioester amino acid of a kind of formula J1-NH-C (Z1)-CO-SR.Group J1 can be any compound part compatible with the ligation of chemo-selective, for example is shielded or not protected amino acid, peptide, polypeptide, other polymkeric substance, dyestuff, linker etc.Z1 is any side-chain radical compatible with α CO-SR thioester, for example a kind of amino acid whose side chain.R is any group compatible with the thioester group, includes, but are not limited to aryl, benzyl and alkyl.The example of R comprises that 3-carboxyl-4-nitrophenyl thioester, benzyl thioester and thiohydracrylic acid leucine thioester are (referring to people such as for example Dawson, Science (1994) 266:776-779; People such as Canne, Tetrahedron Lett. (1995) 36:1217-1220; People such as Kent, WO96/34878; People such as Kent, WO98/28434; People such as Ingenito, JACS (1999) 121 (49): 11369-11374; With people such as Hackeng, Proc.Natl.Acad.Sci.U.S.A. (1999) 96:1006 8-10073).Other example comprises dithiothreitol dithio or alkyl or aryl thioester, they can be produced by the biotechnology of intein-mediation, such biotechnology also is well-known (referring to people such as for example Chong, Gene (1997) 192:277-281; People such as Chong, Nucl.Acids Res. (1998) 26:5109-5115; People such as Evans, Protein Science (1998) 7:2256-2264; With people such as Cotton, Chemistry ﹠amp; Biology (1999) 6 (9): 247-256).

α-carboxyl thioester can be by chemistry or biological method according to standard technique production as known in the art, and method for example described here comprises the method described in the embodiment.For chemosynthesis, can be in solution or from the synthetic α of the resin that produces thioester-carboxyl thioester peptide, these technology be know (referring to people such as for example Dawson, supra; People such as Canne, supra; People such as Hackeng, supra, Hojo H, Aimoto, S. (1991) BullChem Soc Jpn 64:111-117).For example the thioester peptide of chemosynthesis can make from corresponding peptide α-thioic acid sulfoacid, and the latter can synthesize on a kind of thioester-resin or in solution, but the preferred resin method.Can change peptide-α-thioic acid sulfoacid in corresponding 3-carboxyl-4 nitrophenyl thioester, corresponding benzyl ester or the multiple alkylthio acid esters any.All these thioesters can both provide satisfied leavings group to ligation, wherein the speed of reaction that the 3-carboxyl-4-nitrophenyl thioester is showed is higher than corresponding benzyl thioester, and the reactivity of benzyl thioester is than alkylthio acid esters height.As another example, can use the resin that can produce the associating thiohydracrylic acid leucine of trityl thioester make up the terminal thioester of C-(people such as Hackeng, supra).Can also use a kind of 3-carboxylic acid third sulphonamide safety to capture linker, by with diazomethane or iodomethyl cyanide activation, replace and carry out the synthesizing of the terminal thioester of C-(people such as Ingenito, supra then with a kind of suitable mercaptan; People such as Shin, (1999) J.Am.Chem.Soc., 121,11684-11689).

Also can use biological method to prepare peptide or peptide C-terminal α-carboxyl thioester.For example can use and contain or do not contain for example intein expression system of affinity tag of marker, utilize the derivable active of " intein " albumen splice elements, in the presence of a kind of suitable mercaptan, produce terminal thioester peptide of C-or polypeptide fragments from fracture.Particularly, intein is at mercaptan for example in the presence of DTT, beta-mercaptoethanol, β-sulfydryl ethyl sulfonic acid or the halfcystine, takes place specificly from fracture, produces a kind of peptide segment that has C-end thioester, referring to people such as for example Chong, (1997) supra; People such as Chong, (1998) supra; People such as Evans, Supra; With people such as Cotton, supra.

C2 that N-of the present invention is replaced or C3 alkyl group or aryl mercaptan component are connected with first kind of carboxyl thioester component, produce a kind of connection product that contains the amido linkage of N-replacement at the connection site place, as shown in Fig. 1,2 and 3.The condition of contact of selective reaction is with the C2 that keeps thioester and N-and replace or C3 alkyl group or aryl mercaptan part reactivity optionally.A kind of preferred embodiment in, ligation is at a kind of pH6-8, carries out in the buffered soln that preferred pH scope is 6.5-7.5.This buffered soln can be aqueous solution, organic solution or its mixture.Ligation can also comprise one or more catalyzer and/or one or more reductive agents, lipid, stain remover, other denaturing agent or solubilizing agent etc.The example of preferred catalyzer has mercaptan and contains phosphine groups, for example thiophenol, benzyl mercaptan, TCEP and alkylphosphines.The example of denaturing agent and/or solubilizing agent comprise guanidine, urea water or organic solvent for example among TFE, HFIP, DMF, the NMP, be mixed with the solution in the acetonitrile of water or be mixed with guanidine and the aqueous solution of urea.Can also utilize temperature to regulate the speed of ligation, between 5 ℃ and 55 ℃, preferred temperature is between 15 ℃ and 40 ℃ usually for temperature.As an example, in the reaction system of a 6M guanidine that contains 2% thiophenol, under the pH of 6.8-7.8, ligation is carried out finely.

For C2 alkyl group or the aryl mercaptan that N-replaces, connection results from the mercaptan exchange that occurs between COSR thioester component and the aminoalkyl group thiol component.The intermediate that exchange produces a kind of thioesters bonding connects product, the latter spontaneously resets, produce by one 5 yuan ring intermediates and a kind ofly to contain C2 alkyl group that the N that can be removed replaces or formula J1-HN-CH (Z1)-C (the O)-N α of aryl mercaptan [HS-C2-C1 (R1)-] at the connection site place (the connection product of C1 (R1)-C2-SH)-CH (Z2)-J2, wherein substituting group such as front define.C2 alkyl group that replaces at the N at connection site place or aryl mercaptan [HS-C2-C1 (R1)-] are easy to remove under the condition compatible with peptide and produce the final product that is connected of a kind of formula J1-HN-CH (Z1)-CO-NH-CH (Z2)-CO-J2 that has natural amido linkage at the connection site place.

C3 alkyl group or aryl mercaptan for the N-replacement, the intermediate that mercaptan exchange between COSR thioester component and the aminoalkyl group thiol component produces a kind of thioesters bonding connects product, the latter spontaneously resets, and produces a kind of C3 alkyl group of the N replacement that can be removed or formula J1-HN-CH (Z1)-C (O)-N α (connection product of C1-C2 (R2)-C3 (R3)-SH)-CH (Z2)-J2 of aryl mercaptan [HS-C3 (R3)-C2 (R2)-C1 (R1)-] of containing at the connection site place by one 6 yuan ring intermediates.The C3 chain aryl mercaptan [HS-C3 (R3)-C2 (R2)-C1 (R1)-] that replaces at the N at connection site place is easy to remove under the condition compatible with peptide and produces the final product that is connected of a kind of formula J1-HN-CH (Z1)-CO-NH-CH (Z2)-CO-J2 that has natural amido linkage at the connection site place.

The removing of the alkyl or aryl thiol group that N-replaces preferably promotes fracture N-C1 key and carries out, obtain a kind of stable unsubstituted amido linkage at the connection site place under acidic conditions." failure condition compatible with peptide " is meant the physical-chemical condition compatible with peptide that is applicable to the alkyl or aryl thiol moiety of fracture N-bonding from be connected product.The failure condition compatible with peptide generally partly selected according to used N alpha-alkyl or aryl mercaptan, it can easily deduce out (referring to for example " Protecting Groups in OrganicSynthesis " from synthetic route and the method for knowing, 3rd Edition, T.W.Greene and P.G.M.Wuts, Eds., John Wiley ﹠amp; Sons, Inc., 1999; NovaBiochem Catalog 2000; " Synthetic Peptides, A User ' s Guide, " G.A.Grant, Ed., W.H.Freeman ﹠amp; Company, New York, NY, 1992; " Advanced ChemtechHandbook of Combinatorial ﹠amp; Solid Phase Organic Chemistry, " W.D., Bennet, J.W.Christensen, L.K.Hamaker, M.L.Peterson, M.R.Rhodes, and H.H.Saneii, Eds., Advanced Chemtech, 1998; " Principles of Peptide Synthesis, 2nd ed., " M.Bodanszky, Ed., Springer-Verlag, 1993; " The Practice of Peptide Synthesis, 2nded., " M.Bodanszky and A.Bodanszky, Eds., Springer-Verlag, 1994; And " Protecting Groups, " P.J.Kocienski, Ed., GeorgThieme Verlag, Stuttgart, Germany, 1994).

For example, R1 ' wherein, R2 ' or R3 ' substituting group comprise methoxyl group, hydroxyl, thiol or methylthio group, methyl etc., the more general method that is used for cancellation relates to the typical acid failure condition that is used for the peptide synthetic chemistry, this is included under the strong acidic condition or water-sour condition under, fracture N-C1 key under the situation that is with or without reductive agent and/or scavenging agent system (for example acid, for example anhydrous hydrogen fluoride (HF), trifluoroacetic acid (TFA) or trifluoromethanesulfonic acid (TFMSA) etc.).

For given structure, can select to have more the fracture that specific acid rupture System is come optimization N α-C1 key, to remove aryl or alkyl sulfhydryl part.Such condition be know and be compatible with the integrity that keeps peptide.Another kind of fracture method relates to and comprises a kind of mercaptan scavenging agent, and wherein tryptophane is present in peptide or the peptide sequence to avoid tryptophane side chain and free aryl or alkyl sulfhydryl partial reaction.The example of mercaptan scavenging agent comprises ethylene glycol, halfcystine, beta-mercaptoethanol and methylthio phenol.Therefore, another embodiment of the invention is to add a kind of mercaptan scavenging agent when removing aryl or alkyl sulfhydryl part at fracture N-C1 key.

When picolyl during as substituting group, other concrete failure condition comprises light or reductibility failure condition.As an example, when R1 or R2 and R3 substituting group comprise a picolyl part, can use photodissociation (for example UV-light), zinc/acetate or electrolytic reduction to rupture according to standard method.When the R1 of the C2 chain mercaptan that replaces as N-contains a methylthio group at the R1 place, then can use mercury (11) or HF to rupture.Can also use rupture System fracture and/or when first kind is connected component with second kind and comprises other protecting group from solid carrier simultaneously, rupture System can be used as deprotecting regent.For example, can contain activatory zinc and (remove the N-picolyl in～0.5g/ml) 10% the acetic acid/water solution by polypeptide is dissolved in.Sulphomethyl for example 2-sulfydryl, 1-methylsulfinyl ethyl (fracture that HS-C2-C1 (S (O)-CH3)-N α) can mediate by reduction and divalence mercury, sulfydryl after connection be removed.As an example; by polypeptide being dissolved in 3% the acetic acid aqueous solution (for example solution of 1mg polypeptide in 0.5ml acetic acid/water and 0.05mlMMA) that contains N-methyl mercapto ethanamide (MMA); be reduced to sulfo-methane form, spending the night in reaction then, the back is freezing removes the methylsulfinyl ethyl with this mixture of lyophilize.Then can be at a kind of mercuric acetate (Hg (OAc) that contains ₂) 3% acetic acid aqueous solution in remove the reductive auxiliary agent and (for example use acetic acid aqueous solution and the 10mg Hg (OAc) of 0.5ml ₂Handled about 1 hour), add beta-mercaptoethanol (for example 0.2ml beta-mercaptoethanol) then.Then can by standard method for example reversed-phase HPLC (RPHPLC) come purified product.

Be understandable that, can also in rupture System, use one or more catalyzer and/or vehicle, for example one or more scavenging agents, stain remover, solvent, metal etc.In general, existing amino acid is depended in the selection of specific scavenging agent.For example, the carbon ion that can utilize the existence of scavenging agent to be suppressed to produce in the breaking-down process damage effect that might produce some amino acid (for example Met, Cys, Trp and Tyr).Can also use other additive such as stain remover, polymkeric substance, salt, organic solvent etc. to promote fracture by regulating solvability.Catalyzer or other chemical reagent that use can be regulated redox system also are favourable.Understand easily that also for example buffer system, pH and temperature are come the given rupture System of optimization can to regulate multiple other physical and chemical condition by conventional methods.

The present invention also provides C2 or the C3 alkyl group or the aryl mercaptan of the N alpha-substitution of the present invention of protected form.These compounds are particularly useful for automatic peptide connection strategy synthetic and orthogonal and that compile.These components comprise a kind of formula (PG2) S-C2-C1 (R1)-N α (PG1)-CH (Z2)-C (O) J2 or (PG2) S-C3 (R3)-C2 (R2)-C1 (R1)-N α (PG1)-CH (Z2)-C (O)-J2 shielded fully, part is shielded or C2 or the C3 chain aminoalkyl group or the aryl mercaptan of fully not protected sour stable N alpha-substitution, as shown in Table III and Table IV.Especially, R1, one or more containing and C1 conjugated electron-donating group among R2 and the R3, it can form a kind of positively charged ion of resonance stabilized at the C1 place and be convenient to fracture N α-C1 key under the failure condition compatible with peptide behind the acid amides alkyl or aryl mercaptan that the aminoalkyl group or the aryl mercaptan of N alpha-substitution changes the N alpha-substitution into.PG1 and PG2 are the blocking groups that exists separately or together; perhaps do not exist; and can be identical or different; wherein Z2 is connected compatible any compound part with the chemosynthesis of peptide or the natural chemistry of expansion, and wherein J2 is connected compatible any compound part with the chemosynthesis of peptide or the natural chemistry of expansion.

PG1 (or X1) is a kind of protecting group of amine.PG2 (or X2) is a kind of protecting group of mercaptan.Many such protecting groups are known and are applicable to that this purpose is (referring to for example " Protecting Groups in Organic Synthesis ", 3rd Edition, T.W.Greene and P.G.M.Wuts, Eds., John Wiley ﹠amp; Sons, Inc., 1999; NovaBiochem Catalog 2000; " Synthetic Peptides, A User ' sGuide, " G.A.Grant, Ed., W.H.Freeman ﹠amp; Company, New York, NY, 1992; " Advanced Chemtech Handbook of Combinatorial ﹠amp; SolidPhase Organic Chemistry, " W.D., Bennet, J.W.Christensen, L.K.Hamaker, M.L.Peterson, M.R.Rhodes, and H.H.Saneii, Eds., Advanced Chemtech, 1998; " Principles of PeptideSynthesis, 2nd ed., " M.Bodanszky, Ed., Springer-Verlag, 1993; " The Practice of Peptide Synthesis, 2nd ed., " M.Bodanszky and A.Bodanszky, Eds., Springer-Verlag, 1994; And " Protecting Groups, " P.J.Kocienski, Ed., Georg Thieme Verlag, Stuttgart, Germany, 1994).

Table III

The example of preferred PG1 and X1 protecting group includes, but are not limited to [Boc (tertiary butyl carbamate), Troc (2; 2; 2;-trichlorine ethyl carbamate), Fmoc (9-fluorenyl methyl carbamate), Br-Z or C1-Z (Br-or C1-benzylamino manthanoate), Dde (4; 4;-dimethyl-2,6-dioxo cyclohexylene), MsZ (4-methylsulfinyl benzylamino manthanoate), Msc (2-methyl sulfo group ethyl carbamate), Nsc (4-nitrophenyl ethylsulfonyl-ethoxy carbonyl].Preferred PG1 and X1 protecting group are selected from " Protective Groups inOrganic Synthesis, " Green and Wuts, Third Edition, Wiley-Interscience, (1999), wherein most preferably Fmoc and Nsc.The example of preferred PG2 protecting group include, but are not limited to [Acm (acetylamino methyl), MeOBzl or Mob (to methoxy-benzyl), MeBzl (to methyl-benzyl), Trt (trityl), Xan (xanthenyl), tButhio (tertiary butyl sulfenyl), Mmt (to the methoxyl group trityl), 2 or 4-picolyl (2 or 4-pyridyl)), Fm (9-fluorenyl methyl), tBu (tertiary butyl), Tacam (trimethyl-acetyl methyl)].Preferred PG2 and X2 protecting group are selected from " Protective Groupsin Organic Synthesis, " Green and Wuts, Third Edition, Wiley-Interscience, (1999), wherein most preferably Acm, Mob, MeBzl, picolyl.

The orthogonally protect scheme relates to two classes or the multiclass or the group protecting group that can be removed by different chemisms thereby can be removed with any order under the situation that other class protecting group exists.Orthogonal scheme provides the possibility of gentle overall conditions basically, because can be according to the different selectivity that reach of chemical constitution and character rather than speed of reaction.

Table IV

The protected form of the C2 of N alpha-substitution of the present invention or C3 alkyl group or aryl mercaptan can prepare according to the method described in such scheme I and the II.

Any in the aminoalkyl groupization that compound of the present invention can be by comprising halogen mediation, the several different methods of reductive amination and by the compatible N α-protection of preparation and solid phase or solution amino acid or peptide synthetic method; N-is alkylating, the shielded aminoalkyl group of S--or aryl-mercaptan amino acid parent produce.When needing, can split the compound that obtains having the acceptable chiral purity to racemic modification or the diastereomer of producing by standard method.

As above-mentioned institute, in some cases, preferably have C2 or the C3 alkyl group or the aryl mercaptan of the N alpha-substitution of acceptable chiral purity.As shown in embodiment 21 and Fig. 5 B, N α-1-(4-p-methoxy-phenyl)-2-mercaptoethyl auxiliary agent is used to prepare cytochrome b5 62, obtain two kinds of connection products (diastereomer) with eclipsed purifying curve.Have to a kind of primary product although remove N α-auxiliary agent, in final product, will have the seldom unwanted elimination products and the side reaction product of percentage ratio.For example, the reductive amination synthetic route at synthetic used N α-1-(4-the p-methoxy-phenyl)-2-mercaptoethyl auxiliary agent that is used for synthetic cell pigment b562 described in the embodiment 4-6 can produce two kinds of epimers at chiral centre C1 place in essence.As mentioned above, when needing, can split the compound that obtains having the acceptable chiral purity to racemic modification or the diastereomer that produces by standard method.

The standard method that is used to obtain to have the N α-auxiliary agent of the present invention of acceptable chiral purity is: (1) chiral chromatography (2) chirality synthetic (3) uses diastereoisomeric conjugated body of covalent bonding and (4) crystallization or other conventional separation method to obtain the chiral auxiliary(reagent) of enantiomer-pure (referring to for example Ahuja, Satinder. ' Chiral separations.Anoverview. ' ACS Symp.Ser. (1991), 471 (Chiral Sep.Liq.Chromatogr.), 1-26; Collet, Andre. " Separation andPurification of Enantiomers by Crystallization Methods ", In:Enantiomer (1999) 4:157-172; Lopata et al., J.Chem.Res.Minipprint (1984) 10:2930-2962; Lopata et al., J.Chem.Res. (1984) 10:2953-2973; Ahuja, Satinder. ' Chiral separations andtechnology:an overview. ' Chiral Sep. (1997), 1-7; ChiralSeparations:Applications and Technology.Ahuja, Satinder; Editor.USA. (1997), 349pp.Publisher:(ACS, Washington, D.C.)).All these standard methods all can be used for resolution of racemates or diastereomer and obtain having the compound of acceptable chiral purity.For example, can use crystallization to come enantiomorph is carried out optical resolution.For chiral chromatography, well-known, can use Chiral Media raceme mixture to be separated into the enantiomorph of chiral purity by the chromatogram of preparation property.Therefore, can use used reductive amination route is produced in chirality N α-auxiliary agent total synthetic raceme mixture to prepare the enantiomorph of every kind of chiral purity, amino acid auxiliary agent for example as described below (for example wherein R is an amino acid side chain):

About 1: 1 of～mol ratio

Each enantiomorph can obtain with the form of chiral purity, or two kinds of enantiomorphs can obtain with the form of chiral purity.Each enantiomorph all can be used for forming the component of the additive modification of chiral purity, peptide segment (i.e. the epimer of two kinds of chiral purity) for example, and they can be strict purifying, are not subjected to the interference of the existence of other epimer and impurity.Note, unless prepare to use two kinds of enantiomorphs, 50% in the auxiliary agent total amount will be wasted.For example, can be used for the peptide segment of the additive modification of these two kinds of chiral purity to separate the reaction of ENCL then, obtain the connection product mixtures of the additive modification of chiral purity.After separation and purification, from epimer connection product, remove auxiliary agent group (carry out separately or after merging, carry out), obtain having the connection product of same natural structure, then connecting product purification.

Synthetic for chirality, preferable methods is used a kind of chiral raw material of enantiomer-pure, N α-C2 auxiliary agent that p-methoxyphenyl as follows replaces: [PG1=Boc or Fmoc; PG2=(4-methyl) benzyl or (4-methoxyl group) benzyl]

Can use the parent compound of the chiral purity of gained to prepare a kind of shielded (N-replaces) amino acid then, that is: Perhaps be directly used in " inferior monomer " peptide synthetic route, that is: On polymer support, form the peptide of additive modification.Deprotection/fracture then obtains the peptide segment of the additive modification of chiral purity, that is:

N α-the auxiliary agent of chiral purity of the present invention be can prepare from the known phenylglycocoll that is easy to obtain, thereby the chirality and the chiral purity of the auxiliary agent of gained pre-determined with para-orientation of chirality.

In addition, another preferred embodiment uses the synthetic method of the use asymmetric reduction with enantio-selectivity to produce auxiliary agent, and is for example as follows:

R=-H, or-CH ₃, or-CH ₂COOH

(or relative enantiomorph)

Can also be used for the synthetic amino acid of producing N α-additive modification of enantio-selectivity to asymmetric reduction, for example for glycine as follows, that is:

(or other enantiomorph)

Though the asymmetric synthesis meeting of implementing with a kind of suitable manner produces excessive greatly a kind of enantiomorph (with respect to another kind), yet will there be other enantiomorph of some amount in expection.Can use a kind of chirally purified step to tackle this situation to obtain pure main enantiomorph.The major advantage of the synthetic route of enantio-selectivity is that the chiral chromatography separation is more prone to, and can not abandon (waste) a large amount of material.

Another kind of preferred standard technique is to use diastereoisomeric conjugated body of covalency to split.In general, this method uses the amino acid (for example Ala) of chirality to come the racemic modification agent mixture is carried out modification, and passes through the separating obtained diastereomer of (achiral) chromatographic process of standard, and is as follows.For example, mix the mixture that second chiral centre can change racemic auxiliary agent 1 into diastereomer by covalency:

The enantiomorph diastereomer

(raceme mixture)

In this case, by making raceme mixture and (R) 2-Br-propionic acid a kind of SN2 nucleophilic reaction (counter-rotating) taking place, obtains a pair of diastereomer shown in the figure.In fact, we use a kind of chiral auxiliary(reagent) that contains mercaptan of N-bonding to make L-Ala.

As diastereomer, these two kinds of compounds will typically have different chromatographic behaviors under achiral chromatographic condition, thereby can separate under the preparation condition of reality and obtain pure different epimer.

After N α is partly carried out suitable protection, can use not protected or part that each compound production is contained the terminal Ala residue of N-to be protected the peptide segment of additive modification of the chiral purity of expansion.

The component of shielded N alpha-substitution of the present invention is particularly useful for adopting conventional peptide to synthesize and the fast automatic of other organic synthesis strategy synthesized.They also expand the purposes that chemistry connects to the polycomponent connectivity scenario, for example when producing a kind of polypeptide of the connection synthetic schemes that relates to for example three or more segment connectivity scenarios of quadrature connection strategy or compile.

For example, can combine use to the method for the stable thioester of the natural chemical connection process of expansion of the present invention and composition and production nucleophilicity and the safe capture method of thioester, for example in the common pending application PCT application number [also not specifying] of registration on August 31 calendar year 2001 with in former mercaptan ester and the carboxylicesters mercaptan described in U.S. Provisional Patent Application number 60/229,295 (being incorporated herein for reference) of registration on September 1st, 2000.Briefly, the compound that can produce the stable thioester of nucleophilicity comprises a kind of former mercaptan ester or carboxylicesters mercaptan; These compounds have purposes widely in organic synthesis, comprise the production peptide-, polypeptide-and other polymer-sulfur for acid esters.The compound that can produce the stable thioester of nucleophilicity is particularly useful for for example using Fmoc SPPS and needing (have benefited from using) to give the compatible selective protection method's of interested specificity ligation orientation multistep connects or the conjugation scheme prepares peptide or polypeptide from the parent production activatory thioester for preparing under the condition of using strong nucleophilicity material.The stable former mercaptan ester formula of nucleophilicity is X-C (OR ') ₂-S-R; wherein X is the interested target molecule that optionally comprises one or more nucleophilicity protecting groups that rupture; R ' is the stable protecting group of a nucleophilicity that can rupture under the failure condition of non-nucleophilicity, and R is any group compatible with former mercaptan ester-C (OR ')-S-.The resin of the former mercaptan ester thioester that can produce nucleophile-stable also is provided, and this resin formula is X-C (OR ') ₂-S-R-linker-resin or X-C ((OR1 '-linker-resin) (OR2 '))-SR, wherein X, R ' and R are as previously described, and wherein linker and resin are any nucleophile-stable linker and the resins that is applicable to the solid phase organic synthesis, comprise that can change nucleophile-unsettled safety that is used to rupture subsequently into captures linker.Can change nucleophile-stable former mercaptan ester into active thioester by for example acid hydrolysis condition of condition of multiple non-nucleophilicity.The carboxylicesters thiol that nucleophilicity is stable is X-C (O)-O-CH (R ")-(CH ₂) _η-S-R ; wherein X is the interested target molecule that comprises the unsettled protecting group of one or more nucleophiles; R " being a group that non-nucleophile is stable; n is 1 or 2; preferred n=1, and R " ' is H, protecting group or a kind of acid that links to each other with resin-or reduction-unsettled or the linker of safety capture or the blocking group that can remove under non-nucleophilic condition.Resin based on the production thioester of the carboxylicesters mercaptan of nucleophile-stable also is provided, and this resin formula is X-C (O)-O-CH (R ")-CH2 _η-S-linker-resin or X-C (O)-O-CH (R " linker-resin)-CH2 _η-S-R " ', wherein X, R ", n and R " ' define as the front, and linker wherein and resin are any nucleophile-stable linker and resins that is applicable to the solid phase organic synthesis.Can by add a kind of mercaptan catalyzer for example thiophenol nucleophile-stable carboxylicesters mercaptan is changed into active thioester.Therefore; can be used for the how pulsating interconnection technique of compiling to the natural chemical connection process of expansion of the present invention and composition; wherein target compound end can have shielded or not protected N α-C2 of the present invention or C3 alkyl group or aryl mercaptan; and another end has a former mercaptan ester or carboxylicesters thiol moiety, is used for changing active thioester subsequently into and being connected.

Being to be further appreciated that, can being used in combination N α-C2 of the present invention or C3 alkyl group or aryl mercaptan with other method of attachment, for example is that natural chemistry connects that (Dawson waits the people, Science (1994) 266:776-779; Kent, Deng the people, WO96/34878), the general chemistry of expansion connects (Kent, Deng the people, WO98/28434), the chemistry that forms oxime connects (Rose, Deng the people, J.Amer.Chem.Soc. (Schn lzer waits the people (1994) 116:30-33), to form the connection of thioester, Science (1992) 256:221-225), (Englebretsen waits the people, and Tet.Letts. (1995) 36 (48): 8871-8874) in the connection of formation thioether, form the connection (Gaertner of hydrazone, Deng the people, Bioconj.Chem. (1994) 5 (4): 333-338) with the connection that is connected and forms oxazolidine that forms thiazolidine (people such as Zhang, Proc.Natl.Acad.Sci. (1998) 95 (16): 9184-9189; Tam, Deng the people, WO95/00846) or other method (Yan, L.Z.and Dawson, P.E., " Synthesis of Peptide and Proteins without CysteineResidues by Native Chemical Ligation Combined withDesulfurization; " J.Am.Chem.Soc.2001,123,526-533 is incorporated herein for reference; People such as Gieselnan., Org.Lett.20013 (9): 1331-1334; Saxon, people such as E., " Traceless " Staudinger Ligation for theChemoselective Synthesis of Amide Bonds.Org.Lett.2000,2,2141-2143).The present invention also considers to replace mercaptan sulfur in N α-C2 of the present invention or C3 alkyl group or the aryl mercaptan with selenium.

Method and composition of the present invention has many purposes.Method and composition of the present invention is particularly useful for connection peptides, polypeptide and other polymkeric substance.Can carry out ability that natural chemistry is connected to the in esse any amino acid that comprises natural amino acid and alpha-non-natural amino acid and derivative thereof connects natural chemistry and has expanded the target molecule that lacks suitable halfcystine connection site to.Except being used for connection peptides or polypeptide fragments, the present invention also can be used for connecting polymkeric substance, when need be at the connection site place containing N α amido linkage that replace or All Pure Nature and connect such part by one.The present invention has also found purposes in the production of the peptide-labeled thing very widely that is used for expressing protein connection (EPL).For example, can be according to the difference of desired end-use, the amido linkage of the alkyl or aryl mercaptan by a N alpha-substitution or the amido linkage of All Pure Nature couple together the thioester polypeptide of producing EPL and peptide very widely.Can also utilize the present invention to produce multiple cyclic peptide and the polypeptide that has natural amido linkage in the site of peptide and polypeptide generation cyclisation.For example this is very important, because microbiotic and other medicines that most of cyclic peptide is for example according to industrial standard produced do not contain the cysteine residues that can be used for forming natural amido linkage at the connection site place of cyclisation (being that head-tail joins).

As if it is for reference that this paper has been taken in all publications mentioned in this manual and patent application, reaches same degree, and each one publication or patent application all specifically and individually show and be introducing for reference.

Embodiment

Provide following preparation and embodiment in order that allow those skilled in the art can more be expressly understood and implement the present invention.Should not regard them as restriction, and should only be illustrative and representational scope of the present invention.

AbbreviationAcm acetylamino methyl Aloc allyloxy carbonyl BOP benzotriazole-1-base oxygen base three (dimethylamino) phosphorus hexafluorophosphate Br, ClZ Br, Cl benzylamino manthanoate DCM methylene dichloride DDE 4,4-dimethyl-2,6-dioxo hexamethylene-1-subunit DIPCDI N, N-di-isopropyl phosphinylidyne diimine DIPEA N, N-diisopropylethylamine DMAP 4-dimethylaminopyridine DMF N, dinethylformamide DMSO dimethyl sulfoxide (DMSO) EtOH ethanol Fmoc 9-fluorenyl methoxy carbonyl FM 9-fluorenyl methyl HATU (N-[(dimethylamino)-1H-1,2,3-triazole [4,5-b] pyridyl

Methylene radical]-N-methyl first ammonium hexafluorophosphate N-oxide compound) N-[(1-H-benzotriazole-1-yl of being named of HBTU front) (dimethyl amine)

Methylene radical]-0-of N-methyl first ammonium hexafluorophosphate-N-oxide compound

(benzotriazole-1-yl)-1,1,3,3-tetramethyl-urea hexafluorophosphate HF hydrofluoric acid HMP resin 4-hydroxymethyl phenoxy resin; To the alkoxybenzyl alcohol resin; Or

Wang resin HOAt 1-hydroxyl-7-azepine benzotriazole HOBt I-hydroxybenzotriazole Mbh dimethoxy diphenyl-methyl mbha resin 4-methyldiphenyl methylamine resin M eb is right-and methyl-benzyl MMA N-methyl mercapto ethanamide Mmt is right-and methoxyl group trityl Mob is right-methoxy-benzyl Msc 2-methyl sulfo group ethyl carbamate Msz 4-methylsulfinyl benzylamino manthanoate Mtr 4-methoxyl group-2; 3; 6-Three methyl Benzene alkylsulfonyl NMM N-methylmorpholine NMP N-Methyl pyrrolidone; N-N-methyl-2-2-pyrrolidone N-Nsc 4-nitrophenyl ethylsulfonyl-ethoxy carbonyl OPfp pentafluorophenyl group ester OtBu tertiary butyl ester PAC peptide acid linker PAL peptide amide linker Pbf 2; 2; 4; 6; 7-pentamethyl-Dihydrobenzofuranes-5-alkylsulfonyl PEG-PS polyethylene glycol-vinylbenzene Picolyl picolyl Pmc 2; 2; 4; 6,8-pentamethyl-benzo dihydropyrane-6-alkylsulfonyl PyAOP 7-azepine benzo triazol-1-yl oxygen base three (pyrrolidyl) phosphorus hexafluoro

Phosphoric acid salt S-tBu tertiary butyl sulfenyl Tacam trimethyl-acetyl methyl tBoc tert-butoxycarbonyl TBTU 0-(benzotriazole-1-yl)-1,1,3,3-tetramethyl-urea tetrafluoro boron

Hydrochlorate tBu tertiary butyl TFA trifluoroacetic acid Tis tri isopropyl silane Tmob 2,4,6-trimethoxy benzyl TMOF tri-methyl ortho formate Troc 2,2,2-trichlorine ethyl carbamate Trt trityl group embodiment 1: general raw material and method

In mode progressively, be used for the original position neutralization/HBTU activation method of Boc chemistry by SPPS, at the PAM resin or produce on the resin of thioesters according to standard method (people such as Hackeng, supra; People such as Schnolzer, (1992) Int.J.Pept.Prot.Res., 40:180-193; And Kent, S.B.H. (1988) Ann.Rev.Biochem.57 957-984) carries out the synthetic of peptide on a kind of improved ABI 430A peptide synthesizer.After chain assembles, the peptide deprotection, to rupture by handling simultaneously with the anhydrous hydrogen fluoride (HF) that contains 5% p-methyl phenol, lyophilize is by the HPLC purifying of preparation property.Obtain the amino acid of Boc protection from PeptidesInternational and Midwest Biotech.Trifluoroacetic acid (TFA) is obtained by Halocarbon.Other chemical reagent obtains from Fluka or Aldrich.The HPLC of analysis and preparation property carries out on a Rainin HPLC system, and the Vydac C4 of operational analysis or preparation property carries out UV detection at the 214nm place.Peptide and proteomic image carry out on a Sciex API-I electrospray mass spectrograph.The preparation of embodiment 2:2 (4 '-methoxy-benzyl sulfenyl) bromotoluene

Make 10mmol, the 2-hydroxymethyl thiophenol of 1.4g at room temperature reacts with 4-methoxy-benzyl chlorine and the 1.75ml DIEA of 10mmol in 10ml DMF.Be reflected within 10 minutes and finish, add the water of 50ml pH=3.Use the ethyl acetate extraction product, dry on sodium sulfate.The thick oil that obtains and 11mmol carbon tetrabromide (3.64g) and 11mmol triphenylphosphine (2.88g) are reacted in 20ml THF.After reaction is spent the night, evaporation THF.By the silica gel chromatography purified product, the hexane/ethyl acetate with 6/1 is as moving phase.Be recovered to 1.8g.The preparation of embodiment 3:N α (2-sulfydryl benzyl) glycine-peptide

In the peptide of the Ala (78mg) that the Boc-that contains the N-end with resin-bonded protects, add pure TFA to remove the Boc group.The chemical process of use standard is coupled to Boc-glycine-O-succimide on the resin.After coupling is finished, remove the Boc group, neutralize for twice, use DMF and DMSO washing resin then with the DMF solution washing of 10% diisopropylethylamine.Add 9mg2-(4 '-methoxy-benzyl sulfenyl) solution of bromotoluene in 0.2ml DMSO and 0.01ml diisopropylethylamine then, mixture at room temperature reacted 12 hours.Use standard method under the HF condition, peptide to be ruptured and deprotection.Correction mass is 2, and the peak of 079Da accounts for 12% (measuring by HPLC) of all peptide materials greatly.HPLC with the partly preparation property of standard comes the gauged peptide of purifying.The preparation of embodiment 4:4 '-methoxyl group-2-(4 '-methyl-benzyl sulfenyl) methyl phenyl ketone

4mmol, 4-methyl-benzyl mercaptan and the 4mmol of 0.542ml, 4 of 916.3mg '-methoxyl group-2-bromoacetophenone is dissolved among the 4ml DMF.The diisopropylethylamine that adds 4mmol0.7ml then.At room temperature stirred the mixture 1 hour.Mixture is poured in the dilute hydrochloric acid, used ethyl acetate extraction, dry on sodium sulfate.Oily matter is dissolved in the ethyl acetate, adds sherwood oil and precipitate, be recovered to the 450mg white solid.The preparation of embodiment 5:1-amino-1-(4-p-methoxy-phenyl)-2-(4-methyl benzylthio-) ethane

1.44mmol, 4 of 411mg '-methoxyl group-2-(4 '-the methyl benzylthio-) methyl phenyl ketone and 4.3mmol, the aminooxy acetate of 941mg is dissolved among the 20ml TMOF, at room temperature adds the 0.047ml methylsulfonic acid as the catalyst agent.After 48 hours, boil off solvent, in ethyl acetate, leach residue, with the washing of 1M sal enixum, dry on sodium sulfate.With silica gel chromatography purifying crude product, obtain the 200mg oxime complex.This oxime complex of 200mg 0.556mmol is dissolved among the 2ml THF, adds 1.67ml 1M BH3/THF title complex then.After 27 hours, do not stay raw material.Add 3ml water and 1.5ml 10N sodium hydroxide.Backflow mixture 1 hour.Use ethyl acetate extraction (4 times) mixture then, dry on sodium sulfate, use silica gel chromatography purifying final product (40mg) then.The preparation of embodiment 6:N α-1-(4-p-methoxy-phenyl)-2-ethane thiol glycine-peptide

In the template peptide of the Ala (78mg) that the Boc-that contains the N-end with resin-bonded protects, add pure TFA to remove the Boc group.The chemical process of use standard makes bromoacetic acid and resin coupling.In resin, add the solution of 17mg1-amino-1-(4 p-methoxy-phenyl)-2-(4-methyl-benzyl sulfenyl) ethane in 0.3ml DMSO and 0.010ml diisopropylethylamine then.After reaction was spent the night, washing resin used the HF method of standard that peptide is ruptured and deprotection.Use the partly desired product of HPLC purifying of preparation property then.The preparation of another kind of N α-1-(4-p-methoxy-phenyl)-2-ethane thiol glycine-peptide

Use the couling process of standard to make of template peptide resin, the bromoacetic acid coupling of 0.1mmol sequence as the S-Y-R-F-L-polymkeric substance.After the coupling, use the DMSO washing resin, add 32.5mg then, the solution of 0.12mmol 1-amino-1-(4-p-methoxy-phenyl)-2-(4-methyl-benzyl sulfenyl) ethane in 0.3ml DMSO and 0.025ml diisopropylethylamine allows mixture reaction spend the night.The HF method of use standard ruptures and deprotection to peptide.The HPLC of thick cleavage product shows that desired product (MW 2,122) accounts for 60% of all products.Find that positive alkyl sulfur alcohol base 97% is stable in HF.Embodiment 7:2 ', 4 '-preparation of dimethoxy-2-(4 '-methyl-benzyl sulfenyl) methyl phenyl ketone

3.94mmol, 0.534ml 4-methyl-benzyl mercaptan and 3.86mmol, 1g 2 ', 4 '-dimethoxy-2-bromoacetophenone is dissolved among the 4mL DMF.Add the 3.94mmol0.688moL diisopropylethylamine then.At room temperature stirred the mixture 24 hours, mixture is poured in the 1M potassium hydrogen sulfate solution, use ethyl acetate extraction, dry on sodium sulfate.After the evaporation, residual oil is dissolved in the ethyl acetate, precipitates, obtain the 616mg white solid by adding sherwood oil.The preparation of embodiment 8:1-amino-1-(2, the 4-Dimethoxyphenyl)-2-(4-methyl-benzyl sulfenyl) ethane

0.526mmol, 166mg 2 ', 4 '-dimethoxy-2-(4 '-the methyl-benzyl sulfenyl) methyl phenyl ketone and 1.59mmol, 345mg amino oxygen guanidine-acetic acid is dissolved among the 6ml TMOF, at room temperature adds the 0.034ml methylsulfonic acid as catalyzer.After 3 hours, boil off solvent, with the ethyl acetate leaching,, dry on sodium sulfate with the washing of 1 M sal enixum.With silica gel chromatography purifying crude product, obtain 126mg (productive rate: oxime complex 61%).126mg, this oxime complex of 0.324mmol is dissolved among the 1.5ml THF, adds 0.973ml 1M BH3/THF title complex then.

Can also find raw material after 54 hours, add 0.5ml 1M BH3/THF title complex.After reacting 3 days again, total coreaction 6 days adds 3ml water and 1ml 10N sodium hydroxide.Backflow mixture 1h.Use ethyl acetate extraction (4 times) mixture then, dry on sodium sulfate.Use the final product of silica gel chromatography purifying (43mg) then.The preparation of embodiment 9:N α-1-(2, the 4-Dimethoxyphenyl)-2-ethane thiol glycine-peptide

Use the couling process of standard to make the 0.1mmol sequence be the S-Y-R-F-L-polymkeric substance and peptide resin-bonded, bromoacetic acid coupling.After the coupling, use the DMSO washing resin, add 36mg then, 0.12mmol 1-amino-1-(2 ', 4 '-Dimethoxyphenyl)-and the solution of 2-(4-methyl-benzyl sulfenyl) ethane in 0.3ml DMSO and 0.025ml diisopropylethylamine, allow mixture reaction spend the night.The HF method of use standard ruptures and deprotection to peptide.The HPLC of thick cleavage product shows that desired product (MW938) accounts for 42% of all products.Find that positive alkyl sulfur alcohol base 92% is stable in HF.The terminal SDF1-L-Ala-thioester of embodiment 10:C-is connected with the Ala-Gly chemistry of the terminal N α of N--(2-sulfydryl benzyl) glycine-peptide

Reset connection in order to carry out 6 yuan, the terminal N α of the N-of the C-terminal alanine thioester segment (MW4429) of 1mg SDF1-α and 0.6mg SDF1-α-(2-sulfydryl benzyl) glycine segment (MW2079) is dissolved in 100 μ l 6M, in the guanidine buffer reagent of pH7.0, add 1 μ l thiophenol.After room temperature (about 25 ℃) is reacted 2 days down, confirm to have formed desired connection product (MW5472) by ES-MS.Cultivated again 24 hours at 40 ℃ of following reaction mixtures then, determine the desired productive rate that is connected product with the terminal pulsating ratio of unreacted C-based on the product that records by the HPLC integration.Observed productive rate is approximately 40%.The terminal SDF1-L-Ala-thioester of embodiment 11:C-is connected with the Ala-Gly chemistry of the terminal N α-1-(4-p-methoxy-phenyl) of N--2-ethane thiol glycine-peptide

Reset connection in order to carry out 5 yuan, the N-terminal peptide template (MW2122) that has a N α-1-(4-p-methoxy-phenyl)-2 ethane thiol groups that the C-terminal alanine thioester segment (MW4429) of 1mg SDF1 and 1mg is contained a terminal glycine is dissolved in 100 μ l6M, in the guanidine buffer reagent of pH7.0 and the 1 μ l thiophenol.Cultivation reaction mixture under room temperature (about 25 ℃), the monitoring ligation.After 8 hours, confirm to have formed desired connection product (MW5515) by ES-MS.After 3 days, be approximately 45% with the determined desired productive rate that is connected product of the terminal pulsating ratio of unreacted C-based on product.After at room temperature cultivating 3 days, cultivated under 40 ℃ 24 hours, productive rate is 65% again.After at room temperature cultivating 3 days, cultivated under 40 ℃ 48 hours, gain in yield is to about 70% again.

Reset connection in order to carry out 5 yuan, the N-terminal peptide segment (MW2122) that has a N α-1-(4-p-methoxy-phenyl)-2 ethane thiol groups that the C-terminal alanine thioester segment (MW4429) of 0.5mg SDF1-α and 0.5mg is contained a terminal glycine is dissolved in 100 μ l6M, in the guanidine buffer reagent of pH8.2 and the 1 μ l thiophenol.Cultivation reaction mixture under room temperature (about 25 ℃) adds 1 μ l thiophenol again after 6 hours, after 24 hours, the productive rate of the product of is approximately 60%.Terminal glycine-the thioester of embodiment 12:C-is connected with the Gly-Gly chemistry of the terminal N α of N--(2-sulfydryl benzyl) glycine-peptide

Reset connection in order to carry out 6 yuan, N-end N α-(2 sulfydryl benzyl) the glycine segment (MW2079) that the terminal glycine thioester segment (MW1357) of the C-of a kind of ten aggressiveness template peptides of 3.5mg and 2mg is contained a kind of template peptide of three HisDnp is dissolved in 200 μ l6M, the guanidine buffer reagent of pH7.9 adds 2 μ l thiophenols.This mixture was cultivated 60 hours at 33 ℃.Confirm to have formed desired connection product (MW2631) by ES-MS.Based on product and the pulsating ratio of unreacted N-terminal, observe productive rate and be approximately 40%.

The terminal glycine of C--thioester peptide is connected with the chemistry of N α-1-(4-p-methoxy-phenyl) 2-ethane thiol glycine-peptide

Reset connection in order to carry out 5 yuan, the terminal glycine thioester segment (MW1357) of 2mgC-and 2.5mg are had the N-end segment (MW2122) that 3 HisDnp contain the template peptide of a N α-μ l (4-p-methoxy-phenyl) 2-ethane thiol base be dissolved in the 6M that 100 μ l contain 1 μ l thiophenol, pH is in 7.0 the guanidine buffer reagent.Cultivation reaction mixture under room temperature (～25 ℃), the monitoring ligation.Confirm the generation (MW2675.9g) of desired connection product by ES-MS.After 24 hours, it is in product and the terminal pulsating ratio of unreacted N-, and the productive rate that connect product is about 40%. and by adding solid sodium bicarbonate pH is elevated to 8.2 then, cultivates reaction mixture again 24 hours, makes productive rate reach 88%.Embodiment 13:Larc-L-Ala-thioester is connected with the Ala-Gly chemistry of N α-1-(4-p-methoxy-phenyl)-2-ethane thiol glycine-peptide

The L-Ala C-terminal peptidyl thiohydantoin acid esters (MW3609) of 3mg mouse Larc1-31 and 1mg template peptide N α-1-(4-p-methoxy-phenyl)-2-ethane thiol glycine-S-Y-R-F-L (MW908) are dissolved in 0.15ml6M, in the guanidine buffer reagent and 0.03ml thiophenol of pH8.2, after stirring is spent the night, connect and finished 81%, after 40 hours, connect and finished 92% (according to the consumption meter of peptidyl thiohydantoin acid esters).The molecular weight of the connection product of expection is 4312Da, and measured value is that the Ala-Gly chemistry of 4312Da. embodiment 14:Larc1-31-L-Ala-thioester and N α-1-(2, the 4-Dimethoxyphenyl)-2-ethane thiol glycine peptide connects

The L-Ala C-terminal peptidyl thiohydantoin acid esters (MW3609) of 3mg mouse Larc1-31 and 1mg template peptide N α-1-(2, the 4-Dimethoxyphenyl)-2-ethane thiol glycine-S-Y-R-F-L (MW938) is dissolved in 0.15ml6M, in the guanidine buffer reagent and 0.03ml thiophenol of pH8.2, after stirring is spent the night, connect and finished 73%, after 40 hours, connect and finished 85% (according to the consumption meter of peptidyl thiohydantoin acid esters).The estimating of molecular weight value of connection product and trial value all are the Gly-Gly chemistry connections for the terminal tripeptides glycine thioester of 4342Da. embodiment 15:C-and the terminal N α-1-(2, the 4-Dimethoxyphenyl) of N--2-ethane thiol glycine-peptide

0.8mg peptide segment FGG-thioester and 1mg template peptide N α-1-(2, the 4-Dimethoxyphenyl)-2-ethane thiol glycine-S-Y-R-F-L (MW938) are dissolved in 0.1ml6M, in the guanidine buffer reagent and 0.02ml thiophenol of pH8.2.After stirring was spent the night, reaction was finished quantitatively.The estimating of molecular weight value and the trial value that connect product are respectively 1199.4Da and 1195.5Da.Embodiment 16: remove 1-(2, the 4-Dimethoxyphenyl)-2-ethane thiol from connect product

The pure connection product of 1mg embodiment 15 is dissolved among 0.95ml TFA and 0.025ml water and the 0.025ml TIS.After 1 hour, boil off solvent, add 50% water/acetonitrile solution, the mixture lyophilize.Recording fracture by HPLC has finished more than 95%.Estimating of molecular weight value and trial value are 1003Da.The terminal tripeptides glycine of embodiment 17:C-thioester is connected with the Gly-Gly chemistry of the terminal N α-1-(4-p-methoxy-phenyl) of N--2-ethane thiol glycine-peptide

A kind of peptide segment thioester of tripeptides, 1.6mg FGG-thioester and 2mg template peptide N α-1-(4-p-methoxy-phenyl)-2-ethane thiol glycine-S-Y-R-F-L (MW908) are dissolved in 0.2ml6M, in the guanidine buffer reagent of pH8.2, add the 0.04ml thiophenol, after stirring was spent the night, reaction was finished quantitatively.The desired value that connects the molecular weight of product is 1169.4Da, and measured value is 1169.5Da. embodiment 18: after the connection, and the cancellation of 1-(4-p-methoxy-phenyl)-2-ethane thiol group

Under-2 ℃, with the pure connection product 1 hour of the p-methyl phenol Processing Example 17 that contains 5%HF.After boiling off HF, connect product with ether sedimentation.Leach thick peptide with 50% the water/acetonitrile that contains 0.1%TFA, be ejected on the HPLC.Main peak＞80% shows it is the expection molecular weight (prospective quality is 1003Da, and measured value is 1003Da) that passes through the peptide of fracture.The His-Gly chemistry of the terminal Histidine-thioester of embodiment 19:C-and the terminal N α-1-(2, the 4-Dimethoxyphenyl) of N--2-ethane thiol glycine-peptide connects

4mg peptide segment TBP-A1-67C α His thioester and 1mg template peptide N α-1-(2, the 4-Dimethoxyphenyl)-2-ethane thiol glycine-S-Y-R-F-L (MW938) are dissolved in 0.1ml6M, in the guanidine buffer reagent and 0.02ml thiophenol of pH8.2.After stirring is spent the night, finished 87% according to the consumption meter reaction of peptidyl thiohydantoin acid esters.The expection molecular weight that connects product is 9220Da, and measured value is 9220Da.Embodiment 20: after the connection, and the removing of 1-(2, the 4-Dimethoxyphenyl)-2-ethane thiol group

2mg is dissolved among 0.95ml TFA and 0.025ml water and the 0.025ml TIS through H-G connection and purified peptide segment.After 1 hour, boil off solvent, in residue, add water/acetonitrile solution of 50%, the mixture lyophilize.Record fracture by HPLC and finished 90%.The molecular weight of expection is 9023Da, and measured value is 9024Da. embodiment 21: the natural chemistry by expansion connects the synthetic cell pigment

The terminal thioester (MW7349 of the C-of 3mg cytopigment 1-63,0.4 μ mol) and the residue 64-106 (MW4970 of the terminal N α-1-(4-p-methoxy-phenyl) of 1.5mg N--2-ethane thiol glycine cytochrome b5 62,0.3 μ mol) be dissolved in the 6M that 0.1ml contains 0.002ml thiophenol catalyzer, the guanidine of pH7 connects in the buffer reagent, referring to Fig. 5 A.After 24 hours, add the 0.025ml2-mercaptoethanol in mixture, sustained reaction 45 minutes adds 15mg TCEP then.Behind the restir 30 minutes, the connection mixture is loaded among the HPLC of half preparation property.Spill that component elutes and to the mixture desalination after, come all components that connect mixture are all eluted by gradient being tilted to 65%B, be collected in the unique bottle.To the HPLC analysis revealed that the material after the desalination carries out, finished more than 90% according to the consumption meter connection of C-terminal peptide.HPLC has shown the molecular weight 11 that has calculating and expect, two main peaks (diastereomer) of 946Da are referring to Fig. 5 B and 6A.

The aminoacid sequence of cytochrome b5 62 (1-106) is as follows:

ADLEDNMETL?NDNLKVIEKA?DNAAQVKDAL?TKMRAAALDA

QKATPPKLED?KSPDSPEMKD?FRHGFDILVG?QIDDALKLAN

EGKVKEAQAA?AEQLKTTRNA?YHQKYR(SEQ?ID?NO：1)

Estimating of molecular weight value (average isotopics) is 11780.3Da

N-end group: H C-end group: free acid

The single isotopic molecule amount of MH+=11774.0088 atomic unit HPLC index=249.80

MH+ molecular-weight average=11781.2781 atomic units

Bull ﹠amp; Breese value=1.5360

Elementary composition: C508 H830 N147 O168 S3

User-defined amino-acid residue: B-HisDNP embodiment 22: remove 1-(4-p-methoxy-phenyl)-2-ethane thiol group and produce natural protein the residue 1-106 of the cytochrome b5 62 after connecting

Before removing 1-(4 p-methoxy-phenyl)-2-ethane thiol group, the solution lyophilize after the desalination.Under-2 ℃, with containing the 95%HF of 5% methyl-phenoxide and 1mmol halfcystine (121mg) to cryodesiccated mass treatment 1 hour.Use standard method to boil off HF, add the buffer reagent B of 100ml50% then, the mixture lyophilize.Use the partly HPLC purified mixture of preparation property then, obtain the natural cytopigment 1-106 that has only a peak (behind the purifying, productive rate is 56%) of 2mg purifying, estimating of molecular weight value and measured value are 11, and 780Da is referring to Fig. 6 B and 7A.

The analogue that has also synthesized the cytochrome b5 62 of wild-type in the same way, called after S1m7 cyt b562.S1m7 cyt b562 mutant is different from wild-type, and it has replaced the methionine(Met) of 7-position with a kind of selenomethionine (sulphur of methionine(Met) is replaced by its consanguinity selenium).Carry out circular dichroism mensuration, shown the content all very high (data do not show) of alpha-helix in edge wild-type b562 far away and edge S1m7 b562 far away.ESMS shows that also edge wild-type b562 far away and edge S1m7 b562 far away have the molecular weight (data do not show) of expection.Rebuild edge protein far away with protoheme (protoheme pH7 NaPi spends the night room temperature), by the protein of ion-exchange FPLC (FPLC purifying Resource Q, Tris HCL pH8, NaCl gradient) purifying gained, for example referring to Fig. 7 B.Find that the proteinic ultraviolet-visible spectrum after rebuilding through protoheme and sulphur or selenium is not to the coordination of Fe consistent (data show).Also prepared a kind of non-coordinate cyt b562 varient that contains homotopic different amount propylhomoserin in the same way.So just use the natural chemistry connection of expansion to make the synthetic cytopigment, their protoheme avtive spot is rebuild, and characterized fully by bio-physical method.Therefore, this embodiment further shows, is not applicable to that the peptide and the protein of the halfcystine of primary natural chemical connection process can make by the natural chemical connection process of expansion and the non-standard amino acid of introducing wherein.For example, folding the and reactivity of many cyt b562 mutant is studied, but the non-natural axial coordination body of edge far away does not also still develop.Natural chemistry in conjunction with expansion connects, and the available alpha-non-natural amino acid of tremendous amount should allow these protein and other proteinic performance are carried out systematic coordination.Embodiment 23:MCP1-35-Methionin-thioester is connected with the Lys-Gly chemistry of N α-1-(4-p-methoxy-phenyl)-2-ethane thiol glycine-peptide

3mg MCP1-35 Methionin C-terminal peptidyl thiohydantoin acid esters and 1mg template peptide N α-1-(4-p-methoxy-phenyl) 2-ethane thiol glycine-S-Y-R-F-L (MW908) are dissolved in 0.15ml6M, in the guanidine buffer reagent of pH7, be adjusted to pH7.2 by adding triethylamine and 0.03ml thiophenol.After stirring is spent the night, connect and finished 69%.After 40 hours, finished 76% according to the consumption meter connection of peptidyl thiohydantoin acid esters.The expection molecular weight that connects product is 4893Da, and measured value is 4893Da embodiment 24: after the connection, remove 1-(4-p-methoxy-phenyl)-2-ethane thiol group

The crude product of embodiment after the cryodesiccated desalination 23 (Lys-Gly connection) is dissolved among 1mg TFA, 25 μ l dithioglycols, the 50 μ l TIS, adds 150 μ l bromo trimethyl silanes then.At room temperature allow reaction carry out 2 hours.Under vacuum, boil off volatile constituent, use 6M, the residual oily matter of guanidine buffer reagent leaching of pH7.5.Use the chloroform extraction organic substance, the HPLC demonstration no longer includes raw material, and therefore, the auxiliary agent group has successfully been removed.

The expection molecular weight of native sequences is 4726Da, and measured value is 4725Da.Embodiment 25: the comparative studies that the connection of GSYRFL peptide is carried out

Embodiment 13-20 and 24 GSYRFL peptide 5 yuan are reset to be connected compare research, concluded among the following tabulation V.

Table V: to the result of study of the connection of GSYRFL peptide

Template reaction	C-terminal peptide (thioester, peptide)	The N-terminal auxiliary agent	Reaction times (h)	Connect productive rate (%)	Auxiliary agent cancellation condition
						1	Phe-Gly-Gly	????I	????16	????＞98％	???HF
2	Phe-Gly-Gly	????II	????16	????＞98	???TFA
						3	TBP-Al- 67(His)	????II	????16	????87	???TFA
4	Mouse Larcl-31 (Ala)	????I	????16 ????40	????81 ????92	???HF
						5	Mouse Larcl-31 (Ala)	????II	????16 ????40	????73 ????85	???TFA
6	MCP1 1-35(Lys)	????I	????16 ????40	????69 ????76	???TFA/TMS ???Br

The terminal auxiliary agent I=N α-1-(4-p-methoxy-phenyl) of attention: N--2-ethane thiol glycine-SYRFL, and the preparation of the terminal auxiliary agent II=N α-1-(2, the 4-p-methoxy-phenyl) of N--2-ethane thiol glycine-SYRFL embodiment 26:Boc glycine N-1-(4 '-p-methoxy-phenyl)-2-(4 '-methyl-benzyl sulfenyl) ethane

2mmol, 572mg 4 '-methoxyl group-2-(4 '-the methyl-benzyl sulfenyl) methyl phenyl ketone and 2mmol, the 139.5mg glycine ethyl ester hydrochloride is suspended among the 15ml DCM.Slowly add 6mmol, 1g DIEA adds 1ml titanium tetrachloride (1M solution) under nitrogen atmosphere.At room temperature sustained reaction is 2 days.Add 6mmol then, the solution of 0.4g sodium cyanoborohydride in the 2.5ml anhydrous methanol.TLC demonstrates the spot of a kind of new product of one about 40%, is accredited as N-1-(4-p-methoxy-phenyl)-2-(4-methyl-benzyl sulfenyl) ethane glycine ethyl ester by NMR behind the purifying.

Then 1mmol 374mg N-1-(4-p-methoxy-phenyl)-2-(4-methyl-benzyl sulfenyl) ethane glycine ethyl ester is dissolved among the 2ml THF, in solution, adds 2mmol, 83mg LiOH hydrate.After stirring was spent the night, this ester was by complete hydrolysis.Remove THF under vacuum, with 2ml DMF leaching product, add 5mmol, 1.1g heavy carbonic dibutylester adds 3mmol DIEA, 0.45ml at last.After reaction is spent the night, add rare aqueous hydrochloric acid, with ethyl acetate extraction (three times) final product.

The present invention has been done sufficient explanation now, it will be apparent to those skilled in the art that and under the situation of spirit that does not depart from appending claims or protection domain, to make many changes and improvements the present invention.

Sequence table＜110〉Gryphon Sciences

Botti，Paolo

Bradburne，James?A.

Kent，Stephen?B.H.

Low; Donald W.＜120〉the natural chemistry of expansion connects＜130〉GRFN035/01 WO 03504.291＜140〉＜141＜150〉60/231,339＜151〉2000-09-08＜160〉1＜170〉patent (version 2 .1)＜210〉1＜211〉106＜212〉PRT＜213〉mankind＜400〉1Ala Asp Leu Glu Asp Asn Met Glu Thr Leu Asn Asp Asn Leu Lys Val 15 10 15Ile Glu Lys Ala Asp Asn Ala Ala Gln Val Lys Asp Ala Leu Thr Lys

20??????????????????25??????????????????30Met?Arg?Ala?Ala?Ala?Leu?Asp?Ala?Gln?Lys?Ala?Thr?Pro?Pro?Lys?Leu

35??????????????????40??????????????????45Glu?Asp?Lys?Ser?Pro?Asp?Ser?Pro?Glu?Met?Lys?Asp?Phe?Arg?His?Gly

50??????????????????55??????????????????60Phe?Asp?Ile?Leu?Val?Gly?Gln?Ile?Asp?Asp?Ala?Leu?Lys?Leu?Ala?Asn?65??????????????????70??????????????????75??????????????????80Glu?Gly?Lys?Val?Lys?Glu?Ala?Gln?Ala?Ala?Ala?Glu?Gln?Leu?Lys?Thr

85??????????????????90???????????????????95Thr?Arg?Asn?Ala?Tyr?His?Gln?Lys?Tyr?Arg

100?????????????????105

Claims (81)

1. the amide compound that replaces of the N-of following formula:

J1-C (O)-N (C1 (R1)-C2-SH)-J2 I or

J1-C (O)-N (C1 (R1)-C2 (R2)-C3 (R3)-SH)-J2 II wherein:

J1 and J2 are connected other compatible compound part for peptide with one or more optionally protected amino acid side chains or polypeptide or such peptide or polypeptide portion, polymkeric substance, dyestuff, functionalized surface, linker or detectable marker or with the chemosynthesis of peptide or the natural chemistry of expansion independently; And

R1, R2 and R3 are H or a kind of and C1 conjugated electron-donating group independently; Condition be at least among R1, R2 and the R3 comprise one with this electron-donating group of C1 conjugated.

2. the amide compound that replaces of the N-of claim 1, wherein said compound has said formula I.

3. the amide compound that replaces of the N-of claim 2, wherein C1 (R1) is selected from A, B and C: Wherein R1 ', R3 ' and R5 ' comprise can be identical or different electron-donating group.

4. the amide compound that replaces of the N-of claim 1, wherein said compound has said formula II.

5. the amide compound that replaces of the N-of claim 4, wherein C1 (R1)-C2 (R2) C3 (R3) is selected from D, E, F, G, H and I: Wherein one or more among R1 ', R3 ' and the R5 ' comprise one can be identical or different electron-donating group.

6. the amide compound that replaces of any one N-of claim 1-5, wherein the N of said replacement is the acid amides of a N alpha-substitution.

7. the amide compound that replaces of claim 3 or 5 N-, wherein among R1 ', R3 ' and the R5 ' comprises a strong electron-donating group at least.

8. the amide compound that replaces of the N-of claim 7, wherein said strong electron-donating group is selected from methoxyl group (OCH ₃), (SH), hydroxyl (OH) and methylthio group (SCH for sulfydryl ₃).

9. the amide compound that replaces of claim 3 or 5 N-, wherein among R1 ', R3 ' and the R5 ' comprises a medium electron-donating group at least.

10. the amide compound that replaces of the N-of claim 8, wherein said medium electron-donating group comprises methyl (CH ₃), ethyl (CH ₂-CH ₃), propyl group (CH ₂-CH ₂-CH ₃) and sec.-propyl (CH ₂(CH ₃) ₃).

11. the amide compound that claim 1,2 or 4 each N-replace, wherein J1 is peptide or the polypeptide with one or more optionally shielded amino acid side chains, or such peptide or polypeptide portion.

12. the amide compound that claim 1,2 or 4 each N-replace, wherein J1 is a kind of polymkeric substance.

13. the amide compound that claim 1,2 or 4 each N-replace, wherein J1 is a kind of dyestuff.

14. the amide compound that claim 1,2 or 4 each N-replace, wherein J1 is a kind of functionalized surface.

15. the amide compound that claim 1,2 or 4 each N-replace, wherein J1 is a linker or detectable marker.

16. the amide compound that claim 1,2 or 4 each N-replace, wherein J2 is peptide or the polypeptide with one or more optionally shielded amino acid side chains, or such peptide or polypeptide portion.

17. the amide compound that claim 1,2 or 4 each N-replace, wherein J2 is a kind of polymkeric substance, dyestuff, functionalized surface, linker or detectable marker or is connected other compatible compound part with the chemosynthesis of peptide or the natural chemistry of expansion.

18. C2 or C3 chain aminoalkyl group or aryl mercaptan compound that the sour stable N-of a formula HS-C2-C1 (R1)-HN-J2 (III) or formula HS-C3 (R3)-C2 (R2)-C1 (R1)-HN-J2 (IV) replaces, wherein:

J1 and J2 are independently for containing the one or more peptides of optionally being protected the amino acid side chain that expands or polypeptide or such peptide or polypeptide portion, polymkeric substance, dyestuff, functionalized surface, linker or detectable marker or being connected other compatible compound part with the chemosynthesis of peptide or the natural chemistry of expansion; And

R1, R2 and R3 are H or a kind of and C1 conjugated electron-donating group independently; Condition be at least among R1, R2 and the R3 comprise one with C1 conjugated electron-donating group.

19. the compound that the sour stable N-of claim 18 replaces, wherein said compound has formula III.

20. the compound that the sour stable N-of claim 19 replaces, wherein C1 (R1) is selected from A, B and C:

Wherein one or more among R1 ', R3 ' and the R5 ' comprise one can be identical or different electron-donating group.

21. the compound that the sour stable N-of claim 18 replaces, wherein said compound has said formula IV.

22. the compound that the sour stable N-of claim 21 replaces, wherein C3 (R3) C2 (R2)-C1 (R1) is selected from D, E, F, G, H and I:

Wherein one or more among R1 ', R3 ' and the R5 ' comprise one can be identical or different electron-donating group.

23. the compound that each sour stable N-of claim 18-22 replaces, wherein the N of said replacement is a kind of compound of N alpha-substitution.

24. the compound that claim 20 or 22 each sour stable N-replace, R1 ' at least wherein, one among R3 ' and the R5 ' comprises a strong electron-donating group.

25. the compound that the sour stable N-of claim 24 replaces, wherein said strong electron-donating group is selected from methoxyl group (OCH ₃), (SH), hydroxyl (OH) and methylthio group (SCH for thiol ₃).

26. the compound that claim 20 or 22 each sour stable N-replace, wherein among R1 ', R3 ' and the R5 ' comprises a medium electron-donating group at least.

27. the compound that the sour stable N-of claim 26 replaces, wherein said medium electron-donating group comprises methyl (CH ₃), ethyl (CH ₂-CH ₃), propyl group (CH ₂-CH ₂-CH ₃) and sec.-propyl (CH ₂(CH ₃) ₃).

28. the compound that claim 18,19 or 21 each sour stable N-replace, wherein J2 is peptide or the polypeptide that contains one or more optionally shielded amino acid side chains, or such peptide or polypeptide portion.

29. the compound that claim 18,19 or 21 each sour stable N-replace, wherein J2 is a kind of polymkeric substance, dyestuff, functionalized surface, linker or detectable marker or is connected other compatible compound part with the chemosynthesis of peptide or the natural chemistry of expansion.

30. a formula J1-C (O)-N (C1 (R1)-C2-SH)-J2 (I) or formula J1-C (O)-N (amide compound that the N-of C1 (R1)-C2 (R2)-C3 (R3)-SH) J2 (II) replaces, wherein:

J1 and J2 are connected other compatible compound part for the peptide that contains one or more optionally protected amino acid side chains or polypeptide or such peptide or polypeptide portion, polymkeric substance, dyestuff, functionalized surface, linker or detectable marker or with the chemosynthesis of peptide or the natural chemistry of expansion independently; And

R1, R2 and R3 are H or a kind of and C1 conjugated electron-donating group independently; Condition be at least among R1, R2 and the R3 comprise one with this electron-donating group of C1 conjugated;

This amide compound is by the method production that second kind of component of the C2 of first kind of component of the α-carboxyl thioester that comprises formula J1-C (O) SR and the sour stable N-replacement that comprises a kind of Formula Il I or IV or C3 chain aminoalkyl group or aryl mercaptan is connected:

HS-C2-C1 (R1)-HN-J2 III or

HS-C3 (R3)-C2 (R2)-C1 (R1)-HN-J2 IV wherein R1, R2 and R3 is H or a kind of and C1 conjugated electron-donating group independently; Condition be at least among R1, R2 and the R3 comprise one with C1 conjugated electron-donating group.

31. the amide compound that the N-of claim 30 replaces, the compound that the stable N-of wherein said acid replaces has formula III.

32. the amide compound that the N-of claim 31 replaces, the C1 (R1) of the compound that the stable N-of wherein said acid replaces is selected from A, B and C.

Wherein one or more among R1 ', R3 ' and the R5 ' comprise one can be identical or different electron-donating group.

33. the amide compound that the N-of claim 30 replaces, wherein said compound has formula IV.

34. the amide compound that the N-of claim 33 replaces, wherein C1 (R1)-C2 (R2) C3 (R3) is selected from D, E, F, G, H and I: Wherein one or more among R1 ', R3 ' and the R5 ' comprise one can be identical or different electron-donating group.

35. the amide compound that any one N-of claim 30-34 replaces, wherein the N of said replacement is a kind of compound of N alpha-substitution.

36. the amide compound that the N-of claim 32 or 34 replaces, wherein among R1 ', R3 ' and the R5 ' comprises a strong electron-donating group at least.

37. the amide compound that the N-of claim 36 replaces, wherein strong electron-donating group is selected from methoxyl group (OCH ₃), (SH), hydroxyl (OH) and methylthio group (SCH for thiol ₃).

38. the amide compound that the N-of claim 32 or 34 replaces, wherein among R1 ', R3 ' and the R5 ' comprises a medium electron-donating group at least.

39. the amide compound that the N-of claim 38 replaces, wherein said medium electron-donating group comprises methyl (CH ₃), ethyl (CH ₂-CH ₃), propyl group (CH ₂-CH ₂-CH ₃) and sec.-propyl (CH ₂(CH ₃) ₃).

40. the amide compound that claim 30,31 or 33 each N-replace, wherein J1 is a kind of peptide or polypeptide or such peptide or polypeptide portion that contains one or more optionally protected amino acid side chains.

41. the amide compound that claim 30,31 or 33 each N-replace, wherein J1 is a kind of polymkeric substance.

42. the amide compound that claim 30,31 or 33 each N-replace, wherein J1 is a kind of dyestuff.

43. the amide compound that claim 30,31 or 33 each N-replace, wherein J1 is a kind of functionalized surface.

44. the amide compound that claim 30,31 or 33 each N-replace, wherein J1 is a kind of linker or detectable marker.

45. the amide compound that claim 30,31 or 33 each N-replace, wherein J1 is a kind of peptide or polypeptide or such peptide or polypeptide portion that contains one or more optionally protected amino acid side chains.

46. the amide compound that claim 30,31 or 33 each N-replace, wherein J1 is a kind of polymkeric substance, dyestuff, functionalized surface, linker or detectable marker or is connected other compatible compound part with the chemosynthesis of peptide or the natural chemistry of expansion.

47. the compound of a formula J1-C (O)-HN-J2 (V), wherein:

J1 and J2 are connected other compatible compound part for peptide with one or more optionally protected amino acid side chains or polypeptide or such peptide or polypeptide portion, polymkeric substance, dyestuff, functionalized surface, linker or detectable marker or with the chemosynthesis of peptide or the natural chemistry of expansion independently;

This compound is produced by following method:

(A) first kind of component of the carboxyl thioester that comprises formula J1-C (O) SR is connected with the C2 of the sour stable N-replacement that comprises a kind of Formula Il I or second kind of component of C3 chain aminoalkyl group or aryl mercaptan:

HS-C2-C1(R1)-HN-J2?????????????????????III

Wherein R1 is a kind of and C1 conjugated electron-donating group;

Thereby the connection product of the amide linkage that the N-that forms a following formula replaces:

J1-C (O)-N (C1 (R1)-C2-SH)-J2 (I) and

(B) remove C2 alkyl group or aryl mercaptan the connection product of the amide linkage that replaces from this N by fracture N-C1 key.

48. the compound of a formula J1-C (O)-HN-J2 (V), wherein:

J1 and J2 are connected other compatible compound part for the peptide that contains one or more optionally protected amino acid side chains or polypeptide or such peptide or polypeptide portion, polymkeric substance, dyestuff, functionalized surface, linker or detectable marker or with the chemosynthesis of peptide or the natural chemistry of expansion independently;

This compound is produced by following method:

(A) first kind of component of the carboxyl thioester that comprises formula J1-C (O) SR is connected with the C2 of the sour stable N-replacement that comprises a kind of following formula I V or second kind of component of C3 chain aminoalkyl group or aryl mercaptan:

HS-C3(R3)-C2(R2)-C1(R1)-HN-J2????????????IV

Wherein R1, R2 and R3 are H or a kind of and C1 conjugated electron-donating group independently; Condition be at least among R1, R2 and the R3 comprise one with C1 conjugated electron-donating group;

Thereby the connection product of the amide linkage that the N-that forms a Formula Il replaces:

J1-C (O)-N (C1 (R1)-C2 (R2)-C3 (R3)-SH)-J2 (II) and

(B) remove C3 alkyl group or aryl mercaptan the connection product of the amide linkage that replaces from said N by fracture N-C1 key.

49. the compound of claim 47, the C1 (R1) of the compound that the stable N of wherein said acid replaces is selected from A, B and C: Wherein one or more among R1 ', R3 ' and the R5 ' comprise one can be identical or different electron-donating group.

50. the compound of claim 48, C3 (R3)-C2 (R2) C1 (R1) of the compound that the stable N of wherein said acid replaces is selected from D, E, F, G, H and I: Wherein one or more among R1 ', R3 ' and the R5 ' comprise one can be identical or different electron-donating group.

51. the compound that any one sour stable N-of claim 47-50 replaces, the compound that wherein said N-replaces is a kind of compound of N alpha-substitution.

52. the compound of claim 49 or 50, wherein among R1 ', R3 ' and the R5 ' comprises a strong electron-donating group at least.

53. the compound of claim 52, wherein said strong electron-donating group is selected from methoxyl group (OCH ₃), (SH), hydroxyl (OH) and methylthio group (SCH for thiol ₃).

54. the amide compound that the N-of claim 49 or 50 replaces, wherein among R1 ', R3 ' and the R5 ' comprises a medium electron-donating group at least.

55. the compound of claim 54, wherein said medium electron-donating group comprises methyl (CH ₃), ethyl (CH ₂-CH ₃), propyl group (CH ₂-CH ₂-CH ₃) and sec.-propyl (CH ₂(CH ₃) ₃).

56. the compound of claim 47 or 48, wherein J1 is a kind of peptide or polypeptide that contains one or more optionally protected amino acid side chains.

57. the compound of claim 47 or 48, wherein J1 is a kind of polypeptide that contains one or more optionally protected amino acid side chains.

58. the compound of claim 47 or 48, wherein J1 is a kind of polymkeric substance.

59. the compound of claim 47 or 48, wherein J1 is a kind of dyestuff.

60. the compound of claim 47 or 48, wherein J1 is a kind of functionalized surface.

61, claim 47 or 48 compound, wherein J1 is a kind of linker or detectable marker.

62. the compound of claim 47 or 48, wherein J2 is a kind of peptide or polypeptide or such peptide or polypeptide portion that contains one or more optionally protected amino acid side chains.

63. the compound of claim 47 or 48, wherein J2 is a kind of polymkeric substance, dyestuff, functionalized surface, linker or detectable marker or is connected other compatible compound part with the chemosynthesis of peptide or the natural chemistry of expansion.

64. the method for a production formula J1-C (O)-HN-J2 (V) compound, wherein:

J1 and J2 are connected other compatible compound part for the peptide that contains one or more optionally protected amino acid side chains or polypeptide or such peptide or polypeptide portion, polymkeric substance, dyestuff, functionalized surface, linker or detectable marker or with the chemosynthesis of peptide or the natural chemistry of expansion independently;

Wherein said method comprises the following steps:

(A) first kind of component of the carboxyl monothioester that comprises formula J1-C (O) SR is connected with the C2 of the sour stable N-replacement that comprises a kind of Formula Il I or second kind of component of C3 chain aminoalkyl group or aryl mercaptan:

HS-C2-C1(R1)-HN-J2????????????????????????III

Wherein R1 is a kind of and C1 conjugated electron-donating group

Thereby the connection product of the amide linkage that the N-that forms a following formula replaces:

J1-C(O)-N(C1(R1)-C2-SH)-J2????????????????I

(B) remove C2 alkyl group or aryl mercaptan the connection product of the amide linkage that replaces from said N-by fracture N α-C1 key.

65. the method for a production formula J1-C (O)-HN-J2 (V) compound, wherein:

J1 and J2 are connected other compatible compound part for the peptide that contains one or more optionally protected amino acid side chains or polypeptide or such peptide or polypeptide portion, polymkeric substance, dyestuff, functionalized surface, linker or detectable marker or with the chemosynthesis of peptide or the natural chemistry of expansion independently;

Wherein said method comprises the following steps:

(A) first kind of component of the carboxyl thioester that comprises formula J1-C (O) SR is connected with the C2 of the sour stable N-replacement that comprises a kind of following formula I V or second kind of component of C3 chain aminoalkyl group or aryl mercaptan:

HS-C3(R3)-C2(R2)-C1(R1)-HN-J2???????????????????IV

Wherein R1, R2 and R3 are H or a kind of and C1 conjugated electron-donating group independently; Condition be at least among R1, R2 and the R3 comprise one with C1 conjugated electron-donating group;

Thereby the connection product of the amide linkage that the N-that forms a following formula replaces:

J1-C(O)-N(C1(R1)-C2(R2)-C3(R3)-SH)-J2????????????(II)

(B) remove C3 alkyl group or aryl mercaptan the connection product of the amide linkage that replaces from said N by fracture N α-C1 key.

66. the method for claim 64, the C1 (R1) of the compound that the stable N-of wherein said acid replaces is selected from A, B and C:

Wherein one or more among R1 ', R3 ' and the R5 ' comprise one can be identical or different electron-donating group.

67. the method for claim 48, C3 (R3)-C2 (R2) C1 (R1) of the compound that the stable N-of wherein said acid replaces is selected from D, E, F, G, H and I:

Wherein one or more among R1 ', R3 ' and the R5 ' comprise one can be identical or different electron-donating group.

68. any one method of claim 64-67, the compound that wherein said N-replaces is a kind of compound of N alpha-substitution.

69. the method for claim 66 or 67, wherein among R1 ', R3 ' and the R5 ' comprises a strong electron-donating group at least.

70. the method for claim 66, the said strong electron-donating group of the compound that wherein said N-replaces is selected from methoxyl group (OCH ₃), (SH), hydroxyl (OH) and methylthio group (SCH for thiol ₃).

71. the method for claim 66 or 67, at least one comprises a medium electron-donating group among R1 ', the R3 ' of the compound that wherein said N-replaces and the R5 '.

72. the method for claim 71, the said medium electron-donating group of the compound that wherein said N-replaces comprises methyl (CH ₃), ethyl (CH ₂-CH ₃), propyl group (CH ₂-CH ₂-CH ₃) and sec.-propyl (CH ₂(CH ₃) ₃).

73. the method for claim 66 or 67, wherein J1 is a kind of peptide that contains one or more optionally protected amino acid side chains or polypeptide or the such peptide or the part of polypeptide.

74. any one method of claim 66 or 67, wherein J1 is a kind of polymkeric substance.

75. the method for claim 66 or 67, wherein J1 is a kind of dyestuff.

76. any one method of claim 66 or 67, wherein J1 is a kind of functionalized surface.

77, claim 66 or any one method of 67, wherein J1 is a kind of linker or detectable marker.

78. any one method of claim 66 or 67, wherein J2 is a kind of peptide or polypeptide or such peptide or polypeptide portion that contains one or more optionally protected amino acid side chains.

79. any one method of claim 66 or 67, wherein J2 is surface, linker or the detectable marker of a kind of polymkeric substance, dyestuff, officialization or is connected other compatible compound part with the chemosynthesis of peptide or the natural chemistry of expansion.

80. any one method of claim 66 or 67, wherein said compound is a synthetic in solution.

81. any one method of claim 66 or 67, wherein said compound are by being fixed to synthetic on a kind of solid carrier.

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