WO1998028434A1

WO1998028434A1 - General chemical ligation

Info

Publication number: WO1998028434A1
Application number: PCT/US1996/020610
Authority: WO
Inventors: Stephen B. H. Kent; Tom W. Muir; Philip E. Dawson; Steven J. Bark; Lynne Canne
Original assignee: The Scripps Research Institute
Priority date: 1996-12-24
Filing date: 1996-12-24
Publication date: 1998-07-02
Also published as: JP4358307B2; CA2273071A1; CA2273071C; JP2001507027A

Abstract

Oligopeptides are ligated to form ligation peptide products. In the first step, two starting oligopeptides are ligated to form an intermediate having an aminothioester linkage. In the second step, the aminothioester linkage undergoes a rearrangement to form a peptide having an N-substituted amide linkage. In an optional third step, the N-substitution of the amide linkage is chemically removed to form a native peptide linkage.

Description

GENERAL CHEMICAL LIGATION

Description

Technical Field of the Invention:

The present invention relates to methods for chemically ligating oligopeptides. More particularly, the invention relates to methods for chemically ligating oligopeptides end to end using a peptide bond.

Government Rights: This invention was made with government support under Grants No. R01 GM 48897 and P01 GM 48870 awarded by the National Institutes of Health. The U.S. government has certain rights in the invention.

Background of the Invention:

The synthesis of peptides by conventional stepwise solid phase methodologies is limited by low yields when synthesizing long peptides. (Merrifield et al. J . Am . Chem . Soc . 1963, 85 , 2149-2154; Kent et al. Ann . Rev . Biochem . 1988, 57 , 957-989.) To overcome this limitation, smaller synthetic peptides may be joined to one another by chemical ligation to produce longer peptide products.

A method for chemically ligating peptides was disclosed by Schnδlzer et al.. (Schnδlzer et al., Science 1992, 256 , 221-225; Rose et al. J . Am . Chem . Soc . 1994, 116 , 30-34; Liu et al. Proc . Natl . Acad . Sci . USA 1994, 91 , 6584-6588). The methodology disclosed by Schnδlzer involves a chemoselective reaction of unprotected peptide segments to give a product with an unnatural backbone structure at the site of ligation. This methodology makes possible the synthesis of peptides of greater size than attainable by standard methods of peptide synthesis. (Canne et al. J . Am . Chem . Soc . 1995, 117 , 2998-3007; Baca et al. J. Am . Chem . Soc .

1995, 117 , 1881-1887; Williams et al. J. Am . Chem . Soc .

1994, 116, 10797-10798). This methedology also makes possible the synthesis of peptides of unusual structure and topology. (Dawson et al. J. Am . Chem . Soc . 1993,

115 , 7263-7266; Rose et al. J. Am . Chem . Soc . 1994, 116,

30-34; Muir et al. Biochemistry 1994, 33 , 7701-7708;

Canne et al. J. Am . Chem . Soc . 1995, 117, 2998-3007) .

The combined use of conventional stepwise solid phase peptide synthesis together with chemical ligation enables chemists to routinely make unprotected peptides of up to 60 amino acid residues in good yield and purity. (Schnδlzer et al. Int . J. Pept . Protein Res .

1992, 40 , 180-193). The combination of these two methodologies may also be employed to achieve a total chemical synthesis of proteins.

Another chemical ligation technique has been reported for the preparation of proteins having a native backbone structure (Dawson et al. Science 1994, 266 , 776-779) . This mode of chemical ligation is termed "native ligation." In this technique, an unprotected synthetic peptide bearing a C-terminal α-thioester is reacted in a chemoselective manner with an unprotected peptide containing an W-terminal Cys residue. Thiol exchange reaction yields an intial thioester-linked intermediate which spontaneously rearranges to give a native amide bond at the ligation site joining the two peptide segments, in the process regenerating the Cys side chain thiol. This version of native ligation uses chemistry first described by Wieland for reacting amino acids. (Wieland et al. Liehigs Ann . Chem . 1953, 583 , 129-149.) As originally described, native ligation is restricted to joining peptide segments at an X-Cys bond. (Dawson et al. Science 1994, 266 , 116-119 . ) What is needed is a general method for joining a C- terminal α-thioester peptide segment to an W-terminal amino acid peptide segment, wherein the W-terminal amino acid peptide segment need not have an W-terminal cysteine.

Summary of the Invention:

The invention is directed to a method for chemically ligating unprotected oligopeptides to form a product having all peptide . linkages. In the first step, two oligopeptides are ligated to form a ligation product having an aminothioester linkage. In the second step, the aminothioester linkage rearranges to form a product having an W-substituted amide linkage. In an optional third step, the substitution on the amide bond is removed by facile treatment with Zn in acidic medium, to give a native peptide bond at the ligation site.

The method employs two starting oligopeptides, viz., a first starting oligopeptide and a second starting oligopeptide. The first oligopeptide has a C- terminal auxiliary group with a thioester moiety, i.e., [peptide^COSR, where R is selected from the group consisting of 3-carboxy 4-nitrophenyl and benzyl. The second oligopeptide has an W-terminal auxiliary functional group with an unoxidized sulfhydryl moiety, i.e., HSCH₂CH₂(0) -N^α[peptide₂] . When the first and second starting oligopeptides are admixed under conditions promoting thioester exchange, they condense with one another to form an intermediate oligopeptide product wherein the first and second oligopeptides are linked via an amino-thioester bond. The amino-thioester bond then spontaneously rearranges intra olecularly to form a ligation product linked by an W-substituted amide bond. During the intramolecular rearrangement, the amino group of the W-terminal auxiliary functional group atacks the thioester to form an amide bond with the attached N- linked auxiliary functional group containing a displaced sulfhydryl moiety. The W-linked auxiliary functional group containing the displaced sulfhydryl moiety may then be optionally removed by chemical means to form a product having all native peptide linkages.

One aspect of the invention is directed to a method for ligating a first oligopeptide with a second oligopeptide end to end for producing an oligopeptide product. More particularly, the method comprises two steps with an optional third step.

The first step involves condensing the C-terminal thioester of a first peptide with the unoxidized sulfhydryl moiety of a second oligopeptide for producing an intermediate oligopeptide linking the first and second oligopeptides with an δ or y - amino-thioester bond. The first oligopeptide includes the C-terminal thioester on a C-terminal residue and the second oligopeptide includes the W-terminal auxiliary functional group on an W-terminal residue having an unoxidized sulfhydryl moiety. If the C-terminal residue on the first oligopeptide is non glycine then the N- terminal residue on the second oligopeptide is glycine and if the W-terminal residue on the second oligopeptide is non glycine then the C-terminal residue on the first oligopeptide is glycine with the proviso that the non glycine residue is a non β-branched amino acid.

The second step rearranges the δ or y - aminothioester bond of the intermediate oligopeptide via intramolecular attack of the δ or y - amino group onto the thioester moiety and displaces a sulfhydryl moiety as a byproduct from the thioester moiety thereby producing an oligopeptide product linking the first and second oligopeptides with an amide bond. The nitrogen of the amide bond contains the auxiliary functional group with the displaced sulfhydryl moiety.

An optional third step involves the removal of the auxiliary functional group on the amide nitrogen, from the oligopeptide product with a reducing agent for producing a native peptide bond. The amide nitrogen auxiliary functional group is N-α-0-(CH₂)_n-SH wherein l<n<2 and the reducing agent is Zinc (underlined nitrogen represents the coupled amide nitrogen) .

Another apect of the invention includes the oligopeptide intermediate comprising a first oligopeptide segment including a C-terminal thioester, a second oligopeptide segment including an W-terminal auxiliary functional group having an unoxidized sulfhydryl moiety, and an aminothioester linkage unit linking the C-terminal thioester and the sulfhydryl group of the auxiliary functional group.

Description of Figures:

Figure 1 illustrates the generalized native chemical ligation of unprotected peptide segments wherein R is a non β-branched amino acid residue with the following conditions: if the C-terminal residue on the first oligopeptide is non glycine then the N- terminal residue on the second oligopeptide is glycine and if the W-terminal residue on the second oligopeptide is non glycine then the C-terminal residue on the first oligopeptide is glycine. R' is selected from the group consisting of 3-carboxy 4-nitrophenyl and benzyl.

Figure 2 illustrates the chemical synthesis of N^α(substituted) peptide segments 2a and 2b. Figure 3 illustrates the chemical synthesis of compound 8b. Figure 4 shows a table with summaries of model ligations for Examples #1-5. Superscript is as follows: (a) Thioester peptides where -SNB=3-carboxy-4- nitrophenyl thioester arid -Sbzl = benzyl thioester; (b) N^α(substituted) peptides where etsh=N^α(ethanethiol) - and oetsh=N (oxyethanethiol)-; (c) Based on" Peptide 2, estimated from analytical reverse phase HPLC (peak areas) and ESMS; (d) Adjusted to pH 4.5 after the indicated hrs at pH 7.5.

Figures 5A-5C illustrate the model ligation,'Example #2 (infra), to form amide

compound 4b carried out in 6 M guanidine«HCl, 0.1 M Na₂HPO_,, pH 7.5. (A) -(C) represent HPLC plots as follows: (A) Analytical HPLC (10-50%B over 30 rain) at t = 0; peak a thioester peptide, 1, LYRA£-^αCOSC₆H₃(3-C0₂H-4-N0₂) ; peak b, N^α(oxyethanethiol) peptide, 2b, [HSCH₂CH₂0]- GRNTATIMMQRGNFR-^αCONH₂ ; (B) Analytical HPLC (10-50%B over 30 min) at t = 1 h; peak c, non-peptide impurity; peak d, LYRAG-^αCOSC₆H₅, resulting from transthioesterification of 1 (peak a) with thiophenol; peak e, intermediate ligation product, 4b, LYRAGG(N^ot- OCH₂CH₂SH)RNTATIMMQRGNFR-^αCONH₂ and a minor amount of unreacted [HSCH₂CH₂0]-£RNTATIMMQRGNFR-^aCONH₂ (2b), as determined by electrospray ionization MS. (C)

Analytical HPLC (15-40%B over 30 min) of peak e after HPLC-purification and treatment with Zn in acidic HPLC solvent overnight; peak f, final ligation product, 5b, LYRA≤GRNTATIMMQRGNFR-^αCONH₂ ; peak g, non-peptide impurity.

Figures 6A-6C illustrate the model ligation. Example #3 (infra) to form a -Phe(N^α-OCH₂CH₂SH) Gly- linked product 4b. Ligation and rearrangement carried out at 37 °C. (A) -(C) represent HPLC plots as follows: (A)

Analytical HPLC (20-40%B over 30 min) after 11.5 h in 6 M guanidine»HCl, 0.1 M Na₂HP0₄, pH 7.5; peak a, N^α(oxyethanethiol) peptide, 2b, [HSCH₂CH₂0]- £RNTATIMMQRGNFR-^aCONH₂; peak b, unrearranged intermediate ligation product, 3b, LYRAZ-[^αCOSCH₂CH₂0]- <3RNTATIMMQRGNFR-^αCONH₂; peak c, rearranged intermediate ligation product, 4b, LYRA_E£(N°'-OCH₂CH₂SH)RNTATIMMQRGNFR- ^αCONH₂; peak d, thioester peptide, 1, LYRA∑-^αCOSCH₂C₆H₅. (B) Analytical HPLC (20-40%B over 30 min) after 10 h in 6 M guanidine»HCl, 0.1 M NaCH₃C0₂, pH 4.5. (C) Analytical HPLC (0-67%B over 30 min) of HPLC-purified peak c after treatment with Zn in acidic HPLC solvent overnight; peak f, final ligation product, 5b, LYRAF_£RNT_ATIMMQRGNFR-⁰⁽CONH₂.

Detailed Description of the invention The invention is directed to a two step method for chemically ligating together two seperate peptide fragments. More particularly, the invention relates to a general methodology for native (amide-forming) chemical ligation of unprotected peptide segments to produce a native peptide bond.

Step 1 comprises the condensation of a C-terminal thioester of a first peptide with an unoxidized sulfhydryl moiety of a second oligopeptide and produces an intermediate oligopeptide which links the first and second oligopeptides with an δ or y - amino-thioester bond. If the C-terminal residue on the first oligopeptide is non glycine then the N-terminal residue on the second oligopeptide is glycine and if the N- terminal residue on the second oligopeptide is non glycine then the C-terminal residue on the first oligopeptide is glycine with the proviso that the non glycine residue is a non β-branched amino acid.

Step 2 comprises the rearrangement of the δ or y - amino-thioester bond of the intermediate oligopeptide via intramolecular attack of the δ or y - amino group onto the thioester moiety which displaces a sulfhydryl moiety as a byproduct from the thioester moiety and produces an oligopeptide product linking the first and second oligopeptides with a native amide bond. The nitrogen of the amide bond, however, contains the auxiliary functional group with the displaced sulfhydryl moiety.

An optional third step comprises the removal of the auxiliary functional group on the amide nitrogen, from the oligopeptide product with a reducing agent and produces a native peptide bond. The amide nitrogen auxiliary functional group is W-α-0-(CH₂)W-SH wherein l≤n<2 and the reducing agent is Zinc (underlined nitrogen represents the coupled amide nitrogen) .

The method allows for the synthesis of peptides and proteins with either native or modified backbone structures. Figure 1 outlines the procedure. The peptide α-carboxythioester (1) reacts, via thiol exchange, with either an N^α(ethanethiol) peptide (2a) or an N^α(oxyethanethiol) peptide (2b) to produce the ligation product 3. This thioester-linked intermediate rearranges through a favorable geometric arrangement involving a 5- or 6-membered ring to give the amide- linked product 4, containing a secondary amide in 4a or the analogous W-oxyalkyl compound in 4b. The resulting N-^a (substituted) amides also have potentially favorable solubility properties (Quibell et al. J . Am . Chem . Soc . 1995, 117 , 11656-11668) the W-^α(0-alkyl) amide ligation product has the further advantage of being stable to HF cleavage conditions, yet is simply removed under mild conditions. In the case of 4b, zinc dust can be added directly to the reverse phase HPLC-purified peptide in the acidic eluant to reduce the W-0 bond of the O- alkoxyhydroxamate and give the native backbone structure of ligation product 5b. All peptide segments were synthesized in stepwise fashion by established solid phase methods using in situ neutralization/2- (llf-benzotriazol-l-yl) -1,1,3,3- tetramethyluronium hexafluorophosphate (HBTU) activation protocols for tert-butoxycarbonyl (Boc) chemistry, purified by preparative reverse phase HPLC, and characterized by electrospray mass spectrometry (ESMS) (Schnδlzer et al. Int . J. Pept . Protein Res . 1992, 40 , 180-193) . Peptide-α-thioesters (1) were generated from the corresponding peptide-α-thioacids, which in turn, were synthesized on a thioester resin as described in Canne et al. Tetrahedron Lett . 1995, 36 , 1217-1220). The peptide-α-thioacids were converted to either the corresponding 3-carboxy-4-nitrophenyl thioesters in 6 M guanidine»HCl, 0.1 M Na acetate, pH 5.0-6.5, by reaction with 1.5 equivalents of 5, 5 '-dithio-bis(2-nitrobenzoic acid) or to the corresponding benzyl ester in 6M guanidine»HCl, 0.1 M Na acetate, pH 4.0, using 10 equivalents of benzyl bromide (Dawson et al. Science 1994, 266, 776-779). Both these thioesters provide satisfactory leaving groups for the ligation reactions, with the 3-carboxy-4-nitrophenyl thioesters demonstrating a somewhat faster reaction rate than the corresponding benzyl thioesters (Dawson et al. Science 1994, 266 , 776-779).

The syntheses of N^α(ethanethiol) - (2a) and N^oxyethanethiol) - peptides (2b) are illustrated in Figure 2. The appropriate α-bromocarboxylic acid, activated as the symmetric anhydride (0.5 equivalents 1, 3-diisopropylcarbodiimde (DIC) in dichloromethane) (Robey et al. Anal . Biochem . 1989, 177 , 373-377) was coupled to the deprotected W-terminal amino acid of peptide-resin 6 to give bromoacyl-peptide-resin 7. The bromide was then displaced, with inversion of stereochemistry, by the amine function of either structure 8a or 8b in DMSO to give peptide-resin 9. Deprotection and cleavage from the resin in anhydrous HF gave 2b directly or 2a still in the form of the disulfide which was reduced to the free thiol.

The aminoethanethiol derivative 8a was synthesized in one step from the reaction of 2-aminoethanethiol and 5,5'-dithio-bis(2-nitrobenzoic acid) in 80% acetonitrile in water. The synthesis of the aminooxyethanethiol derivative 8b was more involved and is illustrated in Figure 3. Bromide 11 was produced from the reaction of W-hydroxyphthalimide (10) with a large excess of 1,2- dibromoethane (Bauer J . Org. Chem . 1963, 28 , 1604-1608). Bromide 11 was then converted to the protected aminooxyethanethiol derivative 12 with 4-methylbenzyl mercaptan in the presence of the base 1,8-

Diazabicylco[5.4.0]undeC-7-ene (DBU) . The phthalimide group of 12 was removed in a two step process (Osby et al. Tetrahedron Lett . 1984, 25 , 2093-2096) involving reduction with NaBH followed by treatment with acetic acid to give the desired aminooxyethanethiol derivative 8b.

Compared with the original native chemical ligation at X-Cys sites (Dawson et al. Science 1994, 266, 776- 779) , the most notable feature of the ligation chemistry of this invention is the slower rearrangement of the initial thioester ligation product. Intramolecular attack by the α-NH(O-alkyl) group via a six-membered ring intermediate in the current instance is considerably less favored (Mandolini et al. J. Am . Chem . Soc. 1978, 200, 550-554) than the facile five-membered ring mediated attack of the unsubstituted α-NH₂ group in the X-Cys ligation (Dawson et al. Science 1994, 266 , 776-779) . Except for the Gly-Gly ligation, one is able to isolate the thioester-linked initial ligation product of the present invention (Figure 4) , which rearranged only slowly. Remarkably, for C^α-substituted amino acids - li ¬

on both sides of the ligation site, one is unable to observe susbsequent rearrangement to an amide bond: the thioester-linked intermediate was indefinitely stable under the conditions of reaction. These slow rearrangements are similar to those observed in an (N- substituted) amide-forming ligation chemistry previously described (Liu et al. Proc . Natl . Acad . Sci . USA 1994, 91 , 6584-6588).

The use, in the invention, of a temporary auxiliary functional group in amide-forming ligation is reminiscent of the 'thiol capture' strategy proposed by Kemp (Fotouhi et al. J. Org. Chem . 1989, 54 , 2806- 2817) . Both native chemical ligation and the thiol- capture method have as their stated goal the use of unprotected peptides in a segment condensation strategy to achieve the synthesis of long polypeptide chains. The key aspect of the native chemical ligation approach is reaction under conditions that promote exchange of the thiol moiety of the initial thioester-linked intermediate products, to give regioselective ligation at the W-terminal Cys even in the presence of other Cys residues in both segments (Dawson et al. Science 1994, 266 , 776-779). By contrast, in the 'thiol capture¹ strategy and related methods (Liu et al. Tetrahedron

Lett . 1996, 37 , 933-936) regiospecific protection of Cys side chain functionalities prior to the ligation reaction is a necessity (Kemp et al. Peptides : Proceedings of the 11th American Peptide Symposium Rivier, J.E.; Marshall, G.R., Eds, ESCOM: Lieden, 1990; pp 920-922) , thus frustrating their original intent.

Similarly, the use, in the current invention, of a carbonyl-activated α-thiocarboxyl function (i.e. an α- COSR) is reminiscent of chemistry previously used in peptide segment condensation (Blake et al. Proc . Natl . Acad . Sci . USA 1981, 78 , 4055-4058; Yamashiro et al. Int . J. Pept . Protein Res . 1988, 31 , 322-334; Hojo et al. Peptide Chemistry Okada, Y., Ed, Protein Research Foundation: Osaka, 1994; pp 9-12) However, these syntheses were based on conventional noW-chemoselective attack by the α-amine nucleophile of the second segment on a Ag⁺-activated peptide-COSR, and thus necessitated regiospecific (re)protection of all other α- and e- amine functional groups in both segments.

By contrast, thioester-mediated amide-forming ligation chemistry is compatible with the use of completely unprotected peptide segments with the full range of side chain functionalities found in nature, including thiols (Baca et al. J. Am . Chem . Soc . 1995, 117 , 1881-1887; Dawson et al. Science 1994, 266 , 776- 779) . For this reason, native chemical ligation is simple, practical, and is a general approach to the total chemical synthesis of proteins provided they contain appropriate ligation sites.

This invention increases the utility of the native chemical ligation method reported by Dawson, et al (Dawson et al. Science 1994, 266 , 776-779) by extending the number of dipeptide sequences that can be used as ligation sites. In addition to the X-Cys ligation site of that methodology, it is disclosed herein how make use of X-Gly and Gly-X ligation sites (i.e. the C-terminal residue being glycine if the tf-terminal residue is non- glycine or conversely, the W-terminal residue being glycine if the C-terminal residue is non-glycine) . In the preferred mode, X is any of the non-β-branched amino acids. However, our previous studies have shown that X can be any amino acid, including β-branched such as Val; (Dawson et al. Science 1994, 266, 776-779) .

The invention potentially extends the number of suitable sites for native chemical ligation by a factor of three, to more than 50 of the 400 dipeptide sequences found in proteins. Because there is considerable latitude in choosing a ligation site in a target sequence which contains a glycine residue, this extended applicability will render most polypeptides accessible by native chemical ligation.

Native chemical ligation in its original form (Dawson et al. Science 1994, 266 , 776-779) or in the form described here provides direct synthetic access to polypeptide chains the size of typical protein domains. Other ligation chemistries (Schnδlzer et al. Science 1992, 256, 221-225; Rose et al. Am . Chem . Soc . 1994, 116 , 30-34; Liu et al. Proc . Natl . Acad . Sci . USA 1994, 91 , 6584-6588) can be used to join synthetic domains in a modular fashion to produce large (i.e. >20 kilodalton) , fully functional synthetic proteins (Canne et al. J. Am . Chem . Soc . 1995, 117 , 2998-3007; Baca et al. J. Am . Chem . Soc . 1995, 117 , 1881-1887). In its most general form, incorporating all suitable chemistries (Dawson et al. Science 1994, 266 , 776-779) the chemical ligation approach represents the next stage in the evolution of methods for the chemical synthesis of polypeptides and, for the first time, provides for repoducible, practical total chemical synthesis of proteins.

Example Liαations 1-4

Results of example ligations are summarized in Figure 4. All ligations were run at concentrations ranging from 4 mgs/mL to 8 mgs/mL of each peptide in either 8M Urea, 0.1 M Na₂HP0₄, pH 7.0 or 6M guanidine»HCl, 0.1 M Na₂HP0₄, pH 7.5. Immediately after solvation of the peptide segments, 2-5% benzyl mercaptan (example ligation #1) or thiophenol (example ligations #2-5) , by volume of ligation buffer, was added to keep thiol functions in the reduced form. This had the added consequence of exchanging a significant amount of the original peptide-^αthioester (1) to a peptide-^αthiobenzyl ester or peptide-^αthiophenyl ester, both of which are still capable of reacting in the desired fashion with peptide 2. The ligation reactions were followed by analytical reverse-phase HPLC and the products identified by ESMS. In the following examples, the amino acid residues involved in the ligation are underlined.

Example 1

The peptide-°'thio(3-carboxy-4-nitro)phenyl ester 1 had the sequence LYRAG-^αCOSC₆H₃(3-C0₂H-4-N0₂) (SEQ ID NO 1) (observed mass 760 Da, calcd 760 Da) . The N^α(ethanethiol) peptide 2a, protected as the 5-thio-2-

(nitrobenzoic acid) disulfide, had the sequence [ (3-C0₂H- 4-N0₂)-C₆H₃-S-SCH₂CH₂]-GAGPAGD-^αCONH₂ (SEQ ID NO 2) (observed mass 800 Da, calcd 800 Da) which was reduced to [HSCH₂CH₂]-£AGPAGD-^αCONH₂ (SEQ ID NO 2) (observed mass 603 Da, calcd 603 Da) in the ligation mixture. After one hour, the product of the ligation, 4a, [LYRAGG(N^α- CH₂CH₂SH)AGPAGD-^αCONH₂ (SEQ ID NO 3) (observed mass 1163±1 Da, calcd 1163 Da) ] had already formed to a significant degree. After purification of the ligation product by reverse phase HPLC, it was treated with 5,5'-dithio- bis(2-nitrobenzoic acid) for one hour in 6 M urea, 0.1 M phosphate, pH 6.0 to yield the expected -S-nitrobenzoic acid disulfide product, LYRAfifi[N^α-CH₂CH₂S-SC₆H₃(3-C0₂H-4- N0₂) ]AGPAGD-^αCONH₂ (SEQ ID NO 3) (observed mass 136011 Da, calcd 1360 Da) . This confirmed the rearrangement of 3a to form 4a, since 3a would have been unreactive towards 5, 5 ' -dithio-bis (2-nitrobenzoic acid). The results are summarized in Figure 4.

Example 2

Figures 5A-5C show the course of example ligation #2. The peptide-^αthioester segment 1 (peak a) had the sequence LYRA_5-^αCOSC₆H₃(3-C0₂H,4-N0₂) (SEQ ID NO 1) (observed mass 760 Da, calcd 760 Da) . The N^α (oxyethanethiol) peptide 2b (peak b) consisted of the sequence [HSCH₂CH₂0]-£RNTATIMMQRGNFR-^αCONH₂ (SEQ ID NO 4) (observed mass 182711 Da, calcd 1828 Da) . After one hour reaction at room temperature in the presence of added thiophenol, the ligation product, LYRAGG(N^α- OCH₂CH₂SH) RNTATIMMQRGNFR-^αCONH₂ (SEQ ID NO 5) (4b, observed mass 238811 Da, calcd 2389 Da) had formed to a significant degree, coeluting with unreacted

N^α (oxyethanethiol) peptide 2b (peak e) . After further reaction overnight at room temperature, the ligation product was purified by HPLC and zinc dust was added directly to the collected peptide in HPLC eluant and stirred overnight at room temperature. Under these conditions, the zinc effectively reduced the N-0 bond. Reductions of this type are possible through a variety of reagents (Keck et al. Tetrahedron Lett . 1995, 36, 7419-7922) . The resulting peptide (peak f) gave a mass consistent with reduction of the N-0 bond to produce

LYRA£GRNTATIMMQRGNFR-^αCONH₂ (SEQ ID NO 5) (5b, observed mass 231311 Da, calcd 2313 Da) . This confirmed the rearrangement of 3b to 4b as shown in Figure 1, since cleavage of the iV-0 bond in unrearranged 3b would have resulted in the formation of two distinct peptides of significantly lower masses.

Example 3

Figures 6A-6C show the course of example ligation #3. The peptide-^αthioester segment 1 (peak d) consisted of a peptide with a C-terminal phenylalanine thioester [LYRA£-^αCOSCH₂C₆H₅ (SEQ ID NO 6) (observed mass 775 Da, calcd 775 Da) ] , thus providing a more sterically hindered model than the previous cases (example ligations #1 and #2) which involved C-terminal glycine thioesters. The N^α(oxyethanethiol) peptide 2b (peak a) consisted of the sequence [HSCH₂CH₂0] -GRNTATIMMQRGNFR- ^αCONH₂ (SEQ ID NO 7) (observed mass 182711 Da, calcd 1828 Da) . The presence of the more sterically hindered thioester slowed the reaction relative to the unhindered models. However, heating at 37 °C was found to accelerate the rate of initial ligation. Figure 6A illustrates the ligation reaction after 11.5 hours at 37 °C. The rate of rearrangement of 3b to 4b was slowed enough to observe the unrearranged product 3b, LYRAZ- [^αCOSCH₂CH₂0]-GRNTATIMMQRGNFR-^αCONH₂ (SEQ ID NO 8) (peak b, observed mass 247811 Da, calcd 2478 Da) , eluting slightly before the rearranged product 4b, LYRAFG(N^α- OCH₂CH₂SH)RNTATIMMQRGNFR-^αCONH₂ (SEQ ID NO 9) (peak c, observed mass 247811 Da, calcd 2478 Da) . Having identical masses, the unrearranged and rearranged products, 3b and 4b, were identified by their zinc reduction products. Intermediate 3b, peak b, gave two peptides upon zinc reduction with masses of 669 Da and 1753 Da corresponding to the peptide sequences LYRAZ- "COOH (thioester hydrolysis) and £RNTATIMMQRGNFR-^αCONH₂, respectively. It was subsequently determined that lowering the pH to 4.5 after initial ligation by diluting the crude reaction mixture (to five times the volume) with 6M guanidine»HCl, 0.1 M Na acetate, pH 4.0 accelerated the rate of rearrangement. Figure 6B shows that rearrangement of the initial ligation product 3b to 4b (peak c) was complete after 10 hours at pH 4.5 at 37°C. HPLC purification of this peak and subsequent zinc reduction gave a peptide (peak f) of the expected mass, LYRAZSRNTATIMMQRGNFR-^αCONH₂ (SEQ ID NO 9) (5b, observed mass 240311 Da, calcd 2404 Da) .

Example 4

LYRA£-^αCOSCH₂C₆H₅ 1 (SEQ ID NO 1) (observed mass 685 Da, calcd 685 Da) was ligated to [HSCH₂CH₂0]- AARHTVHQRHLHG-^αCOOH 2b (SEQ ID NO 10) (observed mass 159511 Da, calcd 1596 Da) at pH 7.5. This example provided steric hindrance, in the form of an Ala residue, on the opposite side of the ligation site from example ligation #3. The rate of reaction was similiar to that of example 3, the rate being significantly slower than the unhindered examples (example ligations #1 and #2), but enhanced with heating at 37 °C. Both unrearranged (3 , LYRA£-^α[COSCH₂CH₂0] -AARHTVHQRHLHG-^αCOOH) (SEQ ID NO 11) and rearranged (4b, LYRAGΔ(N^α- OCH₂CH₂SH)ARHTVHQRHLHG-^αCOOH) (SEQ ID NO 12) ligation products were formed, observed mass (both unrearranged and rearranged) 215611 Da, calcd 2157 Da. However, unlike example ligation #3, the rate of rearrangement was not enhanced by lowering the pH to 4.5 after initial ligation, though rearrangment was nearly complete after 2 days at 37 °C regardless of pH as shown by successful reduction to the amide. It should also be noted that the presence of a significant number of histidines in the final ligation product 5b (LYRAfiΔARHTVHQRHLHG-^αCOOH (SEQ ID NO 12), observed mass 208011 Da, calcd 2080 Da) resulted in binding of the peptide to the zinc. EDTA had to be added to the HPLC buffer/Zn mixture to free the peptide from the zinc after reduction of the N-0 bond. The results are summarized in Figure 4.

Example 5 Example 5 provides an example with steric bulk on both sides of the ligation site. LYRAZ-^αCOSCH₂C₆H₅ 1 (SEQ ID NO 6) (observed mass 775 Da, calcd 775 Da) was ligated to [HSCH₂CH₂0]-AARHT QRHLHG-^aCOOH 2b (SEQ ID NO 13) (observed mass 159511 Da, calcd 1596 Da) at pH 7.5. Though the initial (i.e. unrearranged) ligation product 3b was observed, LYRAZ-^α[COSCH₂CH₂0]-AARHTVHQRHLHG-^αCOOH (SEQ ID NO 14) (observed mass 224611 Da, calcd 2247 Da) , there was no evidence of rearrangement over time, even at lower pH. The presence of side chains on both sides of the ligation site apparently provided too much steric hindrance for the rearrangement to occur via a 6- membered ring intermediate under the conditions used. The results are summarized in Figure 4.

The above examples emphasize the extraordinary facility of our original native ligation chemistry (Dawson et al. Science 1994, 266 , 716-119 ) which recently was independently repeated in essentially identical form in model ligation studies (Tam et al. Proc . Natl . Acad . Sci . USA 1995, 92 , 12485-12489). We have used this original native peptide bond-forming ligation reaction in the chemical synthesis of a number of proteins with full biological activity, including the chemokine IL-8 (Dawson et al. Science 1994, 266, 776- 779) , the enzymes HIV-l protease and barnase, the serine proteinase inhibitors turkey ovomucoid third domain and eglin C, and a b/HLH transcription factor. This ligation reaction was based on principles enunciated by Max Brenner (Brenner M. Peptides . Proceedings of the Eighth European Peptide Symposium Beyerman, H.C., Ed., North Holland: Amsterdam, 1976; pp 1-7) and made use of chemistry first described by Wieland (Wieland et al. Liebigs Ann . Chem . 1953, 583 , 129-149).

EXPERIMENTAL PROTOCALS General

Machine-assisted solid-phase peptide syntheses were carried out on a custom-modified Applied Biosystems 430A peptide synthesizer (Schnδlzer et al. Int . J . Pept . Protein Res . 1992, 40 , 180-193). Reverse-phase HPLC was performed on a Rainin HPLC system with 214-nm UV detection, using Vydac C-18 analytical (5 μm, 0.46 X 15 cm) and semipreparative (10 μm, 1.0 X 25cm) columns. Chromatographic separations were achieved using linear gradients of buffer B in A (A = 0.1% TFA in water, B= 90% CH₃CN/10%water containing 0.09% TFA) over 30-60 min at 1 mL/min (analytical) or 3 mL/min (semipreparative) . Mass spectra of all peptide segments were obtained with a Sciex API-Ill electrospray quadrupole mass spectrometer; observed masses were derived from the experimental m/z values for all observed protonation states of a molecular species, using the program MacSpec (Sciex) . Calculated masses were based on average isotope composition and were derived using the program MacProMass (Terry Lee and Sunil Vemuri, Beckman Research Institute, Duarte, CA) . All other mass spectrometry was performed at The Scripps Research Institute Mass Spectrometry Facility. ^XH NMR spectra were recorded on a Bruker 250 MHz spectrophotometer and chemical shifts are reported in parts per million downfield from Me₄Si. Microanalyses were performed at The Scripps Research Institute X-ray Crystallographic Facility and agreed with calculated values 10.4%. BoC-L-amino acids and

HBTU were purchased from Novabiochem (La Jolla, CA) . 4- Hydromethylphenylacetamidomethyl (PAM) resins and diisopropylethylamine (DIEA) were obtained from Applied Biosystems (Foster City, CA) , and methylbenzhydrylamine (MBHA) resin was obtained from Peninsula Laboratories,

Inc (San Carlos, CA) . Synthesis grade dimethylformamide (DMF) was obtained from Baker, and AR grade CH₂C1₂ and HPLC-grade CH₃CN obtained from Fisher. Trifluoroacetic acid (TFA) was obtained from Halocarbon (New Jersey) . HF was purchased from Matheson Gas. 4-Methylbenzyl mercaptan was obtained from Lancaster. All other reagents were AR grade or better and were obtained from Aldrich Chemical or from Fisher.

Peptide Segment Synthesis Chain Assembly

Peptides were synthesized in stepwise fashion by established machine-assisted or manual solid-phase methods using in situ neutralization/HBTU activation protocols for Boc chemistry (Schnδlzer et al. Int . J. Pept . Protein Res . 1992, 40 , 180-193). Side chain protection was as follows: BoC-Arg(p-toluenesulfonyl) - OH, BoC-Asn(xanthyl) -OH, BoC-Asp(O-cyclohexyl) -OH, BoC- His(dinitrophenyl) -OH, BoC-Thr (benzyl) -OH, and BoC- Tyr (2-bromobenzyloxycarbonyl)-OH. BoC-GlN-OH and BoC- Met-OH were used without side chain protection. Coupling reactions were monitored by quantitative ninhydrin assay (Sarin et al Anal. Biochem . 1981, 117 , 147-157) and were typically >99%. After chain assembly was complete, peptides were deprotected and simultaneously cleaved from the resin by treatment with HF containing 5% p-cresol for 1 h at 0 °C to give the peptide-^αCOSH, -^αCONH₂, or -^αC0₂H. After removal of the HF under reduced pressure, the crude peptide was precipitated in anhydrous Et₂0 (diethyl ether) , dissolved in HPLC buffer (40-50% B) and lyophilized.

Synthesis of Peptide^thioesters (1) as shown in Figure 1 Thioacid peptides were synthesized on the appropriate BoC-aminoacyl-S-Resins, made by coupling [4- [α-(IΫ-t-BoC-aminoacyl-S)benzyl]phenoxy]acetic acid, DCHA salt (Canne et al. Tetrahedron Lett . 1995, 36 , 1217- 1220), (2.0 equiv) and aminomethyl-resin (1 equiv, washed with 10% DIEA in DMF; Aldrich) with HBTU (1.6 equiv; Aldrich) added as an activating agent and DIEA (1 equiv; Aldrich) , in DMF (dimethylformamide) . Peptide- ^αCOSC₆H₃(3-C0₂H-4-N0₂) thioesters were generated by dissolving the crude peptide-^αCOSH (15-20 mgs) in 6 M guanidine»HCl, 0.1 M Na Acetate, pH 5.0-6.5, to which was added 1.5 equivalents of 5, 5 '-dithio-bis(2- nitrobenzoic acid) (Dawson et al. Science 1994, 266 , 776-779) . The mixture was vortexed briefly and purified after 10 min. Identity of the peptide- ^otCOSC₆H₃(3-C0₂H-4-N0₂) ester was unambigously confirmed by precise electrospray mass measurements, in contrast with Liu et al. Tetrahedron Lett . 1996, 37 , 933-936. Peptide-^αCOSCH₂C₆H₅ thioesters were generated by dissolving the crude peptide-^αCOSH (15-20 mgs) in 6M guanidine»HCl, 0.1 M Na acetate, pH 4.0, to which was added 10 equivalents of benzyl bromide (Dawson et al. Science 1994, 266, 776-779) . The mixture was vortexed briefly and purified after 1 h. LYRAG-^αCOSC₆H₃(3-C0₂H-4- N0₂) (observed mass 760 Da, calcd 760 Da) was purified by semipreparative HPLC (20-60%B over 40 min) to give 20- 30% yield. LYRAG.-^aCOSCH₂C₆H₅ (observed mass 685 Da, calcd 685 Da) was purified by semipreparative HPLC (15-45%B over 60 min) to give 25-30% yield. LYRAZ-^αCOSCH₂C₆H₅ (observed mass 775 Da, calcd 775 Da) was purified by semipreparative HPLC (30-60%B over 60 min) to give 25- 30% yield. Most of the loses in yield arose simply from HPLC recoveries.

Synthesis of N^α(ethanethiol^ and N^αroxyethanethiol peptides (2 ) as illustrated in Figure 2 These peptides were synthesized on either MBHA

(Sigma) or the appropriate BoC-aminoacyl-OCH₂-PAM-resins (Sigma) . After chain assembly was complete and the N^αBoc group removed with neat TFA (two 1 min treatments) and neutralized with 10% DIEA in DMF (two 1 min treatments) , the peptide was bromoacetylated by the method of Robey (Robey et al. Anal . Biochem . 1989, 177 , 373-377) . Bromoacetic acid (2.0 mmol) was dissolved in CH₂C1₂ (2 mL) to which was added DIC (1 mmol; 2- dimethylaminoisopropyl chloride hydrochloride; Aldrich or Sigma) . After activation for 10-15 min to form the symmetric anhydride, the mixture was diluted with DMF (2 mL) , added to the peptide-resin and coupled for 30 min. The resin was then washed with DMSO and 8a (2 M in DMSO) or 8b (1 M in DMSO) was added and allowed to react with the bromoacetylated peptide-resin for 8-23 h. The peptides were purified without further modifcation after cleavage from the resin. [ (3-C0₂H-4-N0₂) -C₆H₃-S-SCH₂CH₂]- AGPAGD-^αCONH₂ (2a) (observed mass 800 Da, calcd 800 Da) was purified by semipreparative HPLC (0-67%B over 60 min) to give ^"24% yield. [HSCH₂CH₂0]-GRNTATIMMQRGNFR- ^αCONH₂ (2b) (observed mass 182711 Da, calcd 1828 Da) was purified by semipreparative HPLC (15-40%B over 60 min) to give "25% yield. [HSCH₂CH₂0]-AARHTVHQRHLHG-αCOOH (2b) was synthesized by the above method using the racemate of 2-bromoproprionic acid instead of bromoacetic acid. The resulting crude lyophilized product was a mixture of the desired peptide and the peptide that results from elimination of HBr from the bromopropionylated peptide (CH₂=CHC0-ARHTVHQRHLHG-^aC00H, observed mass 150211 Da, calcd 1502 Da) . The mixture was purified by semipreparative HPLC (10-40%B over 60 min) to give [HSCH₂CH₂0]-AARHTVHQRHLHG-αCOOH (observed mass 1595+1 Da, calcd 1596 Da) in "10% yield.

Synthesis of S-f t3-Carboxy-4-nitro henylthio] -2- aroinpethanethiol (8a) as shown in Figure 2 2-Aminoethanethiol (1.0 g, 13.0 mmol) and 5,5*- dithio-bis(2-nitrobenzoic acid) (1.75 g, 4.4 mmol) were combined in acetonitrile (100 mL) and water (25 mL) and stirred at room temperature for 14 hr. The reaction mixture was diluted with water (450 mL) and purified by preparative reverse phase HPLC on a Waters Delta Prep 4000 with a Vydac 5.0 x 2.5 cm preparative C-18 column to give 8a (1.0 g, 40%) ^αH NMR (D₂0) : δ 7.95 (d, 1H, J=8.7 Hz), 7.61 (dd, 1H, J=8.7 , 2.2 Hz), 7.58 (d, 1H, J=2.2 Hz), 3.18 (t, 2H, J=6.5 Hz) , 2.92 (t, 2H, J=6.7 Hz) .

Synthesis of iV- f2-Bromoethoxy)phthalimide (111 as illustrated in Figure 3.

W-(2-Bromoethoxy)phthalimide (11) was synthesized by a modification of the procedure of Bauer and Sureshl (Bauer et al. J. Org. Chem . 1963, 28 , 1604-1608). N- Hydroxyphthalimide (16.0 g, 98 mmol; Aldrich), triethylamine (30 mL, 215 mmol), and 1,2-dibromoethane (35 mL, 406 mmol; Aldrich) were combined in DMF (115 mL) and stirred at room temperature overnight. Solids were filtered and washed with DMF. The filtrate was diluted with water (800 mL) and the resulting precipitate filtered, dissolved in EtOAc (200 mL) and washed with 1 N HCl (2 x 50 mL) , water (1 x 50 mL) , saturated NaCl (1 x 50 mL) and dried over MgS0₄. Volatiles were removed in vacuo and the resulting solid was recrystallized from 95% EtOH to give 11 as a white solid (16.6 g, 63%). ^αH NMR (CDC1₃) : δ 7.82 (m, 4H) , 4.49 (t, 2H, J=6.9 Hz), 3.65 (t, 2H, J=6.9 Hz) ; FAB MS (sodium ion): calc for [C₁₀H₈BrNO₃, H⁺] 291.9585, found 291.9579.

Synthesis of N- [ 2- [ S- t 4-Methylbenzyl ) ]mercapto]ethoxy] phthalimide (12) as illustrated in Figure 3

Bromide 11 (16.6, 62 mmol), 4-methylbenzyl mercaptan (8.5 mL, 63 mmol), and DBU (9.5 mL, 64 mmol; 1,8- diazabicyclo[5.4.0]undeC-7-ene; Aldrich) were combined in benzene (150 mL) and stirred at room temperature for 8 hr. Solids were filtered, washed with benzene and the filtrate washed with 1 N HCl (2 x 35 mL) , water (1 x 35 mL) , saturated NaCl (1 x 35 mL) and dried over MgS0₄. Volatiles were removed in vacuo and the resulting solid recrystallized from EtOAc/hexane to yield 12 as a white solid (14.8 g, 74%) ^XH NMR (CDC1₃) : δ 7.80 (m, 4H) , 7.18 (d, 2H, J=8.0 Hz), 7.04 (d, 2H, J=8.0 Hz) , 4.22 (t, 2H, J=7.4 Hz), 3.75 (s, 2H) , 2.79 (t, 2H, J=7.4 Hz) , 2.27 (s, 3H) ; FAB MS: calc for [C₁₈H₁₇N0₃S, H⁺] 328.1007, found 328.1016. Anal. Calcd for C₁₈H₁₇N0₃S C, 66.03; H, 5.23; N, 4.28; S, 9.79. Found C, 66.04; H, 4.95; N, 4.30; S, 9.58.

Synthesis of S-C4-MethylbenzyH -2-aminooxyethanethiol fδb as illustrated in Figure 3

S- (4-methylbenzyl) -2-aminooxyethanethiol was synthesized by the method of Osby (Osby et al. Tetrahedron Lett . 1984, 25 , 2093-2096). The N- substituted phthalimide 12 (7.4 g, 23 mmol) was suspended in isopropanol (203 mL) and water (35 mL) to which was added NaBH₄ (3.5 g, 92 mmol). The mixture was stirred at room temperature overnight. Acetic acid (25 mL) was slowly added till bubbling ceased, the flask stoppered and heated to 50 °C for 2-3 hr. Volatiles were removed in vacuo , the resulting solution diluted with 1 N NaOH and extracted with EtOAc (4 x 50 mL) . The combined EtOAc extractions were washed with saturated NaCl (1 x 50 mL) and dried over MgS0₄. Volatiles were removed in vacuo and the resulting oil purified by flash chromatography (1:1 hexane:EtOAc) to yield 8b as a clear, colorless oil (3.2 g, 72%) ²H NMR (CDC1₃) : δ 7.21 (d, 2H, J=8.0 Hz) , 7.12 (d, 2H, J=8.0 Hz), 5.40 (br s, 2H, D₂0 ex.), 3.77 (t, 2H, J=6.5 Hz), 3.71 (s, 2H) , 2.64 (t, 2H, J=6.5 Hz), 2.33 (s, 3H) ; FAB MS: calc for [C₁₀H₁₅NOS, H⁺] 198.0953, found 198.0958. Anal. Calcd for C₁₀H₁₅NOS C, 60.88; H, 7.66; N, 7.10; S, 16.25. Found C, 60.79; H, 7.88; N, 7.03; S, 16.11.

Claims

What is claimed is:

1. A method for ligating a first oligopeptide with a second oligopeptide end to end for producing an oligopeptide product, the method comprising the following steps: Step A: admixing the first and second oligopeptides in a reaction solution including a catalytic thiol, the first oligopeptide including a C-terminal residue having a C-terminal thioester, the second oligopeptide including an _V-terminal residue having an W-terminal auxiliary functional group with an unoxidized sulfhydryl moiety, the C-terminal residue being glycine if the W-terminal residue is non- glycine, the N-terminal residue being glycine if the C-terminal residue is non-glycine; Step B: condensing the unoxidized sulfhydryl moiety of the W-terminal auxiliary functional group with the C-terminal thioester for producing an intermediate oligopeptide linking the first and second oligopeptides with an aminothioester bond; and then Step C: rearranging the aminothioester bond of the intermediate oligopeptide of said Step B for producing the oligopeptide product linking the first and second oligopeptides with an amide bond having a W-linked auxiliary functional group.

2. A method as described in Claim 1 wherein, the catalytic thiol is selected from the group consisting of benzyl mercaptan and thiophenol.

3. A method as described in claim 2 wherein the N- terminal auxiliary functional group is W-╬▒-(CH₂)W-SH where l<nΓëñ3.

4. A method as described in claim 2 comprising the following additional step: Step D: Removing the N-terminal auxiliary functional group from the oligopeptide product of said step C with a reducing agent for producing a native peptide bond.

5. A method as described in claim 4 wherein the N- terminal auxiliary functional group is is _V-╬▒-0-(CH₂)W-SH where lΓëñnΓëñ2.

6. A method as described in claim 5 wherein the reducing agent is Zinc.

7. An oligopeptide intermediate comprising: a first oligopeptide segment including a C-terminal thioester, a second oligopeptide segment including a _V-terminal auxiliary functional group having an unoxidized sulfhydryl moiety, and an aminothioester linkage unit linking the C- terminal thioester and the sulfhydryl group of the auxiliary functional group, said aminothioester linkage unit spontaneously rearranging intramolecularly to form an amide bond linking said first and second oligopeptides segments end to end.

8. A linkage unit for joining two peptide sequences, i.e. an amino terminal peptide sequence and a carboxyl terminal peptide sequence, the linkage unit comprising: a dipeptide having a first amino acid residue (aa₂) , a second amino acid resiude (aa₂) , and an exploded linkage between said first and second amino acid residues, said first amino acid residue having a backbone carbonyl group, i.e. (aai-CO-) , said second amino acid residue having a backbone amino group, i.e. (-NR-aa₂) , wherein R is an auxiliary moiety selected from the group consisting of ╬▒-0-(CH₂)iY- SH and ╬▒-(CH₂)W-SH, wherein l<nΓëñ2, said carbonyl group on the first amino acid residue having a single bond linking said second amino acid residue to the first amino acid at the sulfydryl group of said auxiliary moiety of the second amino acid, said exploded linkage between said first and said second amino acid residue being represented by the formula

(aa₁-CO)-S-(CH₂)W-X-(W-aa₂) wherein l<nΓëñ2 and X is selected from the group consisting of oxygen and methylene, whereby said first amino acid residue (aa_╬▒) of said dipeptide being employable for forming a peptide linkage with said amino terminal peptide sequence and said second amino acid residue (aa) of said dipeptide being employable for forming a peptide linkage with said carboxyl terminal peptide sequence, thereby linking said amino terminal peptide sequence to said carboxyl terminal peptide sequence with said exploded linkage.