Novel linker, preparation method, and application thereof

Abstract

Provided in the present invention is a linker and a preparation method thereof, wherein one end of the linker may covalently link a small molecule compound and the like and the other end may specifically and covalently link a targeting substance site under the action of Sortase enzyme. The linker of the present invention can be used to prepare a targeting drug conjugate.

Claims

1. A bi-functional linker, wherein the said linker has chemical structure represented by Formula (I) or (II):
PCA1-(LA).sub.a-CCA1(I)
CCA2-(LA).sub.a-PCA2(II) wherein: PCA1 is a receptor substrate recognition sequence of Sortase; PCA2 is a donor substrate recognition sequence of Sortase; each of CCA1 and CCA2 is chemical conjugate region for connecting a payload to be connected, wherein the said CCA1 and CCA2 each has a peptide sequence with 1-200 residues selected from natural amino acids and chemically reactive non-natural amino acids; and LA is a connecting region, to connect PCA and CCA, wherein a is 0 or 1 and the structure of LA is shown in the following formula:
NH.sub.2R1-P-R2-(CO)OH wherein P represents a polyethylene glycol unit with the formula of (OCH.sub.2CH.sub.2).sub.m, wherein m is 0 or an integer of 1-1000, alternatively P represents a peptide with 1-100 residues; R1 and R2 each independently is H, a linear alkyl group having 1 to 6 carbon atoms; a branched or cyclic alkyl group with 3 to 6 carbon atoms; or a linear, branched or cyclic alkenyl or alkynyl group having 2-6 carbon atoms.

2. The linker according to claim 1, wherein the Sortase is a native Sortase, or a genetically engineered novel Sortase, preferably is a native Sortase A, or a genetically engineered novel Sortase A.

3. (canceled)

4. (canceled)

5. The linker according to claim 1, wherein the said PCA1 comprises at least one, preferably 1-100, more preferably 1-20 series connected one or more unit structures selected from the group consisting of: glycine (Gly) and alanine (Ala), and the said PCA2 comprises the structure of X1X2X3X4X5X6, wherein X1 represents leucine (Leu) or asparagine (Asn), X2 represents proline (Pro) or alanine (Ala), X3 represents any amino acid, X4 represents threonine (Thr), X5 represents glycine (Gly), serine (Ser) or asparagine (Asn), X6 represents any an amino acid or absent, preferably the said PCA2 is LPXTG, wherein X represents any amino acid.

6. (canceled)

7. (canceled)

8. (canceled)

9. (canceled)

10. (canceled)

11. (canceled)

12. The linker according to claim 1, wherein the said peptide sequence contains at least one residue selected from lysine (Lys) residue, cysteine residue, a chemically reactive non-natural amino acid residue and a chemically reactive non-natural amino acid residue incorporated via a side-chain group of an amino acid of the peptide sequence.

13. (canceled)

14. (canceled)

15. The linker according to claim 12, wherein the said peptide sequence contains at least two lysine residues, wherein at least one lysine residue forms an amide bond via its ?-amino and the ?-carboxyl group of another lysine residue to form a branched lysine structure.

16. (canceled)

17. The linker according to claim 15, wherein the said branched lysine structure further contains other amino acid residue and/or a non-amino acid structure, wherein the ?- or ?-amino of lysine is connected with the carboxyl group of the said other amino acid residue to form an amide bond, and the non-amino acid structure, preferably an alkyl or a cyclic alkyl, having chemically reactive groups on both ends covalently connectable with an amino group or a carboxyl group.

18. The linker according to claim 17, wherein the said other amino acid residue is glycine residue and/or cysteine residue.

19. (canceled)

20. (canceled)

21. (canceled)

22. (canceled)

23. The linker according to claim 12, wherein the chemically reactive non-natural amino acid residue comprises a reactive group involving in a reaction of: oxime bond formation by reacting with an alkoxy-amine; Cu (I) catalysized Huisgen 1,3-dipolar cycloaddition (Click reaction) by reacting with an alkyne or azide; inverse electron demand hetero Diels-Alder (HDA) reaction; Michael reaction, metathesis reaction; transitional metal catalyzed cross-coupling; oxidative coupling; acyl-transfer reaction or photo click reaction.

24. (canceled)

25. (canceled)

26. (canceled)

27. (canceled)

28. (canceled)

29. (canceled)

30. (canceled)

31. (canceled)

32. (canceled)

33. (canceled)

34. (canceled)

35. (canceled)

36. (canceled)

37. (canceled)

38. (canceled)

39. The linker according to claim 1, wherein in the LA, the said linear alkyl group is selected from methyl, ethyl, propyl, butyl, pentyl and hexyl group; the said branched or cyclic alkyl group having 3-6 carbon atoms is selected from isopropyl, isobutyl, tertiary butyl, pentyl, hexyl, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl group; the said linear alkenyl group having 2 to 6 carbon atoms is selected from ethenyl, propenyl, butenyl, pentenyl and hexenyl; the said branched or cyclic alkenyl group having 2 to 6 carbon atoms is selected from isobutenyl, isopentenyl, 2-methyl-1-pentenyl and 2-methyl-2-pentenyl; the said linear alkynyl group having 2 to 6 carbon atoms is selected from ethynyl, propynyl, butynyl, pentynyl and hexynyl; the said branched or cyclic alkynyl group having up to 6 carbon atoms is selected from 3-methyl-1-butyne, 3-methyl-1-pentynyl and 4-methyl-2-hexynyl.

40. (canceled)

41. A use of the linker according to claim 1 in coupling of targeting moiety with a cytotoxic drug, a toxin, a nucleic acid, a tracer molecule, to achieve the targeted delivery of the coupled compound and/or effective cell transfection.

42. (canceled)

43. A coupling intermediate having the structure of formula (III) or (IV):
PCA1-(LA).sub.a-CCA1-Payload.sub.h(III),
or
Payload.sub.h-CCA2-(LA).sub.a-PCA2(IV), wherein: Payload is a cytotoxic drug, a toxin, a nucleic acid, or a tracer molecule; h is an integer from 1 to 1000; when h>1, Payload is same or different, and PCA1-(LA).sub.a-CCA1 and CCA2-LA-PCA2 are respectively as defined in claim 1.

44. (canceled)

45. The coupling intermediate according to claim 43, wherein the cytotoxic drug selected from the group consisting of: paclitaxel and its derivatives, Auristatins derivatives such as MMAE, MMAF, maytansine and derivatives, epothilones analogues, vinca alkaloids such as vinblastine, vincristine, vindesine, Vinorelbine, vinflunine, vinglycinate, anhydrovinblastine, dolastatin and analoues, halichondrin B, meturedopa, Uredopa, camptothecine and its derivatives, bryostatin, Callystatin, Melphalan, nitrosoureas such as carmustine, fotemustine, Lomustine, Nimustine, Uramustine, Ranimustine, Neocarzinostatin, Dactinomycin, Porfiromycin, Anthramycin, Azaserine, Esorubicin, Bleomycin, Carabicin, Idarubicin, Nogalamycin, Carzinophilin, carminomycin, Dynemicin, Esperamicin, Epirubicin, Mitomycin, olivomycin, Peplomycin, Puromycin, Marcellomycin, Rodorubicin, Streptonigrin, Ubenimex, Zorubicin, Methotrexate, Denopterin, Pteropterin, Trimetrexate, purine analogs such as Thiamiprine, Fludarabine, Thioguanine; pyrimidine analogs such as Ancitabine, azacitidine, Cytarabine, Dideoxyuridine, 5-Deoxy-5-fluorouridine, Enocitabine, Floxuridin, Calusterone, Drostanolone, Epitiostanol, Mepitiostane, Testolactone, Aceglatone, Aldophosphamide Glycoside, Aminolevulinic Acid, Bisantrene, edatrexate, Colchicinamide, Diaziquone, Eflornithine, Elliptinium Acetate, Lonidamine, Mitoguazone, Mitoxantrone, Pentostatin, Betasizofiran, Spirogermanium, Tenuazonic acid, Triaziquone, Verracurin A, Roridin A, Anguidine, Dacarbazine, Mannomustine, Mitolactol, Pipobroman, DNA topoisomerase inhibitors, flutamide, Nilutamide, Bicalutamide, Leuprorelin Acetate and Goserelin, protein kinases and proteasome inhibitors; the said nucleic acid is selected from: single-stranded DNA, double-stranded DNA, RNA and nucleic acid analogues, preferably the said nucleic acid is siRNA; and the said tracer molecule is selected from fluorescent molecules e.g. TMR, Cy3, FITC, Fluorescein and a radionuclide.

46. (canceled)

47. (canceled)

48. (canceled)

49. (canceled)

50. (canceled)

51. (canceled)

52. A targeting drug conjugate, wherein the said conjugate having a structure represented by the formula (V) or (VI):
T-PCA1-(LA).sub.a-CCA1-Payload.sub.h(V) or
Payload.sub.h-CCA2-(LA).sub.a-PCA2-T(VI) wherein: Payload is a cytotoxic drug, a toxin, a nucleic acid, or a tracer molecule; T is a targeting moiety; h is an integer from 1 to 1000, when h>1, Payload is same or different; PCA1-(LA).sub.a-CCA1 and CCA2-(LA).sub.a-PCA2 are respectively as defined in claim 1.

53. (canceled)

54. (canceled)

55. (canceled)

56. The targeting drug conjugate according to claim 52, wherein the cytotoxic drug is selected from the group consisting of: paclitaxel and its derivatives, Auristatins derivatives such as MMAE, MMAF, maytansine and derivatives, epothilones analogues, vinca alkaloids such as vinblastine, vincristine, vindesine, Vinorelbine, vinflunine, vinglycinate, anhydrovinblastine, dolastatin and analoues, halichondrin B, meturedopa, Uredopa, camptothecine and its derivatives, bryostatin, Callystatin, Melphalan, nitrosoureas such as carmustine, fotemustine, Lomustine, Nimustine, Uramustine, Ranimustine, Neocarzinostatin, Dactinomycin, Porfiromycin, Anthramycin, Azaserine, Esorubicin, Bleomycin, Carabicin, Idarubicin, Nogalamycin, Carzinophilin, carminomycin, Dynemicin, Esperamicin, Epirubicin, Mitomycin, olivomycin, Peplomycin, Puromycin, Marcellomycin, Rodorubicin, Streptonigrin, Ubenimex, Zorubicin, Methotrexate, Denopterin, Pteropterin, Trimetrexate; purine analogs such as Thiamiprine, Fludarabine, Thioguanine; pyrimidine analogs such as Ancitabine, azacitidine, Cytarabine, Dideoxyuridine, 5-Deoxy-5-fluorouridine, Enocitabine, Floxuridin, Calusterone, Drostanolone, Epitiostanol, Mepitiostane, Testolactone, Aceglatone, Aldophosphamide Glycoside, Aminolevulinic Acid, Bisantrene, edatrexate, Colchicinamide, Diaziquone, Eflornithine, Elliptinium Acetate, Lonidamine, Mitoquazone, Mitoxantrone, Pentostatin, Betasizofiran, Spirogermanium, Tenuazonic acid, Triaziquone, Verracurin A, Roridin A, Anguidine, Dacarbazine, Mannomustine, Mitolactol, Pipobroman, DNA topoisomerase inhibitors, flutamide, Nilutamide, Bicalutamide, Leuprorelin Acetate and Goserelin, protein kinases and proteasome inhibitors; the said nucleic acid is selected from: single-stranded DNA, double-stranded DNA, RNA and nucleic acid analogues, preferably the said nucleic acid is siRNA; the said tracer molecule is selected from fluorescent molecules e.g. TMR, Cy3, FITC, Fluorescein, and a radionuclide; and the said targeting moiety is capable of binding to a target cell of: a tumor cell, a commonly used genetic engineering transfected cell, a virus-infected cell, a microorganism infected cell or a primary cultured cell; preferably, the said targeting moiety is an antibody, a single chain antibody, a nano-antibody, a single domain antibody, an antibody fragment, analogue, a peptide or a protein/peptide which binds to targeting cells specifically.

57. (canceled)

58. A pharmaceutical composition, wherein the said composition comprises the targeting drug conjugate according to claim 52 and a pharmaceutically acceptable carrier or excipient.

59. A method for treatment of a disease a subject comprising administration of the pharmaceutical composition according to claim 58 in an effective amount to the subject, preferably the said disease is targeting cell antigen related diseases, and more preferably selected from cancers, autoimmune diseases, inflammatory diseases, cardiovascular diseases and neurodegenerative diseases.

60. (canceled)

61. The linker according to claim 12, wherein the said CCA1 and CCA2 each further contains a bifunctional cross-linking agent that connected to a residue to incorporate a maleimido group, a pyridyldithio group, a haloalkyl group, a haloacetyl group, an isocyanate group in to CCA1 or CCA2; preferably, the bifunctional cross-linking agent that connected to ?-amino of the lysine residue and/or thiol of the cysteine residue; and preferably, the said bifunctional cross-linking agent is selected from the group consisting of: N-Succinimidyl 4-(N-maleimidomethyl) cyclo hexane-1-carboxylate (SMCC), SMCC long chain analog N-[alpha-maleimidoacetoxy] Succinimide ester (AMAS), N-gamma-Maleimidobutyryl-oxysuccinimide ester (GMBS), 3-Maleimidobenzoic acid N-hydroxysuccinimide ester (MBS), 6-maleimidohexanoic acid N-hydroxysuccinimide ester (EMCS), N-SucciniMidyl 4-(4-MaleiMidophenyl) butyrate (SMPB), Succinimidyl 6-[(beta-maleimidopropionamido) hexanoate (SMPH), Succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxy-(6-amidocaproate)(LC-SMCC), N-Succinimidyl 11-(maleimido) undecanoate (KMUS), those comprising N-hydroxysuccinimide-(polyethylene glycol)n-maleimide bifunctional crosslinking agents (SM(PEG)n), where n presents 2, 4, 6, 8, 12 or 24; and those containing dithiopyridyl groups including but not limited to: N-Succinimidyl 3-(2-Pyridyldithio) propionate (SPDP), sulfosuccinimidyl-6-[(a-methyl-a-(2-pyridyldithio)toluamido]hexanoate (S-LC-SMPT), Sulfosuccinimidyl-6-[3-(2-pyridyldithio)-propionamido] hexanoate (S-LC-SPDP), Succinimidyl (4-iodoacetyl)aminobenzoate (SIAB), Succinimidyl iodoacetate (SIA), N-Succinimidyl bromoacetate (SBA) and N-Succinimidyl 3-(Bromoacetamido) propionate (SBAP).

62. The linker according to claim 1, wherein the linker with formula (I) is selected from linkers 1-25; ##STR00001## ##STR00002## ##STR00003## ##STR00004## ##STR00005## in the above linkers 1-25, n is an integer of 1-100, m is 0 or an integer 1-1000, X is OH or NH.sub.2; and the said linker of formula (II) is selected from linkers 26-35: ##STR00006## ##STR00007## ##STR00008## in the above structures 26-35, n is an integer of 1-100, m is 0 or an integer 1-1000, X is OH or NH.sub.2.

Description

DESCRIPTION OF THE DRAWINGS

[0073] FIG. 1 A general structure of linker 1 (n=1-100, X is OH or NH2)

[0074] FIG. 2 A general structure of linker 2 (n=1-100, X is OH or NH2)

[0075] FIG. 3 A general structure of linker 3 (n=1-100, m=0, 1-1000, X is OH or NH2)

[0076] FIG. 4 A general structure of linker 4 (n=1-100, m=0, 1-1000, X is OH or NH2)

[0077] FIG. 5 A general structure of linker 5 (n=1-100, m=0, 1-1000, X is OH or NH2)

[0078] FIG. 6 A general structure of linker 6 (n=1-100, m=0, 1-1000, X is OH or NH2)

[0079] FIG. 7 A general structure of linker 7 (n=1-100, m=0, 1-1000, X is OH or NH2)

[0080] FIG. 8 A general structure of linker 8 (n=1-100, m=0, 1-1000, X is OH or NH2)

[0081] FIG. 9 A general structure of linker 9 (n=1-100, m=0, 1-1000, X is OH or NH2)

[0082] FIG. 10 A general structure of linker 10 (n=1-100, m=0, 1-1000, X is OH or NH2)

[0083] FIG. 11 A general structure of linker 11 (n=1-100, m=0, 1-1000, X is OH or NH2)

[0084] FIG. 12 A general structure of linker 12 (n=1-100, m=0, 1-1000, X is OH or NH2)

[0085] FIG. 13 A general structure of linker 13 (n=1-100, X is OH or NH2)

[0086] FIG. 14 A general structure of linker 14 (n=1-100, X is OH or NH2)

[0087] FIG. 15 A general structure of linker 15 (n=1-100, m=0, 1-1000, X is OH or NH2)

[0088] FIG. 16 A general structure of linker 16 (n=1-100, m=0, 1-1000, X is OH or NH2)

[0089] FIG. 17 A general structure of linker 17 (n=1-100, X is OH or NH2)

[0090] FIG. 18 A general structure of linker 18 (n=1-100, m=0, 1-1000, X is OH or NH2)

[0091] FIG. 19 A general structure of linker 19 (n=1-100, X is OH or NH2)

[0092] FIG. 20 A general structure of linker 20 (n=1-100, X is OH or NH2)

[0093] FIG. 21 A general structure of linker 21 (n=1-100, X is OH or NH2)

[0094] FIG. 22 A general structure of linker 22 (n=1-100, X is OH or NH2)

[0095] FIG. 23 A general structure of linker 23 (n=1-100, m=0, 1-1000, X is OH or NH2)

[0096] FIG. 24 A general structure of linker 24 (n=1-100, m=0, 1-1000, X is OH or NH2)

[0097] FIG. 25 A general structure of linker 25 (n=1-100, m=0, 1-1000, X is OH or NH2)

[0098] FIG. 26 A general structure of linker 26 (X is OH or NH2)

[0099] FIG. 27 A general structure of linker 27 (m=0, 1-1000, X is OH or NH2)

[0100] FIG. 28 A general structure of linker 28 (X is OH or NH2)

[0101] FIG. 29 A general structure of linker 29 (X is OH or NH2)

[0102] FIG. 30 A general structure of linker 30 (X is OH or NH2)

[0103] FIG. 31 A general structure of linker 31 (X is OH or NH2)

[0104] FIG. 32 A general structure of linker 32 (X is OH or NH2)

[0105] FIG. 33 A general structure of linker 33 (n=1-100, m=0, 1-1000, X is OH or NH2)

[0106] FIG. 34 A general structure of linker 34 (X is OH or NH2)

[0107] FIG. 35 A general structure of linker 35 (m=0, 1-1000, X is OH or NH2)

[0108] FIG. 36 The preparation process of antibody-drugs and antibody-siRNA conjugates

[0109] FIG. 37 The chemical structure of linker 1

[0110] FIG. 38 The UPLC profile of linker 1

[0111] FIG. 39 The ESI-MS profile of linker 1

[0112] FIG. 40 The UPLC profile of maysteine derivative DM1

[0113] FIG. 41 The ESI-MS profile of maysteine derivative DM1

[0114] FIG. 42 The structure of a coupling intermediate made of maysteine derivative DM1

[0115] FIG. 43 The UPLC-MS profile of an coupling intermediate made of maysteine derivative DM1

[0116] FIG. 44 The chemical structure of linker 26

[0117] FIG. 45 The HPLC profile of linker 26

[0118] FIG. 46 The ESI-MS of linker 26

[0119] FIG. 47 The structure of a coupling intermediate of GAPDH siRNA-linker 26

[0120] FIG. 48 The coupling efficiency of GAPDH siRNA with linker 26, checked by SDS PAGE 1: GAPDH siRNA; 2: the coupling intermediate GAPDH siRNA-linker 26

[0121] FIG. 49 The structure of coupling product: GAPDH siRNA-linker 26-GFP

[0122] FIG. 50 The coupling efficiency of GAPDH siRNA-linker 26 with GFP checked by native PAGE 1: GAPDH siRNA-linker 26; 2: 0 min, 3: 60 min; 4 120 min; *: final product siRNA-GFP; **: the coupling intermediate GAPDH siRNA-linker 26

[0123] FIG. 51 The structure of linker 2

[0124] FIG. 52 The HPLC profile of linker 2

[0125] FIG. 53 The ESI-MS of linker 2

[0126] FIG. 54 The structure of linker 3

[0127] FIG. 55 The HPLC profile of linker 3

[0128] FIG. 56 The ESI-MS of linker 3

[0129] FIG. 57 The structure of linker 9

[0130] FIG. 58 The HPLC profile of linker 9

[0131] FIG. 59 The ESI-MS of linker 9

DETAILED DESCRIPTION

[0132] The present disclosure is further illustrated with the following specific examples, which, however, are not limitations to the present disclosure.

[0133] 1. The Preparation of Linker 1

[0134] When n=5, X is OH, the general formula of linker 1 shown in FIG. 1 is shown in

[0135] FIG. 37. The linker was prepared via solid phase peptide synthesis protocol on Wang resin using Fmoc Chemistry. The ?-amino group of lysine was deprotected, and N-Succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) was chemically coupled to it in DMF. The linker was then cleaved from the resin and all protection groups were removed. The crude linker 1 was purified by HPLC, and characterized by ESI-MS. As shown in FIG. 38, the purity of the linker was 95.49%, and the found MS was 708.5 (M+1) shown in FIG. 39 (expected MW 707). This linker thus obtained will be coupled with small molecules, nucleic acids or tracer molecules.

[0136] 2. The Preparation of a Coupling Intermediate Made of Linker 1 and DM1

[0137] Maytansine derivative DM1 was purchased from Jiangyin Concortis Bio-Technology Co., Ltd. UPLC analysis showed a purity of 91.43% and ESI-MS showed a molecular weight of 738.5 (expected 738). The results were shown in FIGS. 40 and 41.

[0138] The synthetic linker 1 obtained above and the maytansine derivative DM1 were dissolved in a suitable solvent in equimolar ratio, the mixture was incubated at room temperature. The structure of the coupling intermediate is shown in FIG. 42. It was subjected to UPLC-MS analysis, and the results shown in FIG. 43. The coupling efficiency was 100%, expected molecular weight is 1447.9, ESI-MS found 1447 (M-1).

[0139] The product obtained from the above procedure was site-specifically connected to a tumor-specific antibody or antibody analogue. The antibody-drug conjugate thus obtained was highly homogeneous, i.e., the number of drugs and the sites of coupling are highly specific. This highly homogenous ADC drugs can be used in a variety of tumor targeted therapies, including but not limited to breast cancer, stomach cancer, lung cancer, ovarian cancer and leukemia. In comparison with the ADCs already on the market, the highly homogenous new drugs prepared by the current invention, offer many advantages including but not limited to stability, reliability, efficacy and safety.

[0140] 3. The Preparation of Linker 26

[0141] When X is OH, the general linker structure shown in 26 becomes the structure shown in FIG. 44.

[0142] A similar method as used for the preparation of linker 1 was used. The crude product was purified by HPLC, characterized by ESI-MS analysis. As shown in FIG. 45, the purity of linker 26 was more than 99%; the expected molecular weight of 765, ESI-MS found 764 (M-1), as shown in FIG. 46.

[0143] The linker 26 and those alike may be used to react with small molecules, nucleic acids or tracer molecules.

[0144] 4. The Preparation of a Conjugate Intermediate with siRNA as the Payload

[0145] A 5-terminal thiol modified mice GAPDH siRNA was purchased from Genepharm Shanghai Ltd. The sequence of the said siRNA is:

[0146] 5-GUAUGACAACAGCCUCAAGdTdT-3

[0147] 3-dTdTCAUACUGUUGUCGGAGUUC-5

[0148] The modified siRNA and an excess of linker 26 were incubated in 1?PBS buffer (pH7.4) at room temperature for 1-24 h. The extra linker 26 was removed by ultrafiltration to give a GAPDH siRNA-linker intermediate as shown in FIG. 47. SDS PAGE indicated that the coupling efficiency was >90% as shown in FIG. 48.

[0149] 5. Enzyme Catalysed Site Specific Coupling of siRNA and Green Florecein Proten (GFP)

[0150] Recombinant GFP was purified by nickel affinity purification, treated with TEV enzyme to release the polyglycine sequence as the substrate for Sortase, and the resulted GGG-GFP protein was collected.

[0151] Excess amount of GAPDH siRNA linker intermediate 26 and GGG-GFP was site-specifically coupled by a genetically engineered Sortase A in 1?PBS buffer (containing Tris pH8.0, NaCl, CaCl2) at 37? C. for 2 h. Samples were taken at different time intervals. The structure of the final product is shown in FIG. 49. 15% non-denaturing SDS PAGE showed that the coupling efficiency was 80% in 2 h (FIG. 50).

[0152] This result clearly indicated that siRNA was site-specific coupling to a protein. An important application of this method is the site specific coupling of a tumor targeting antibody or antibody analogue with siRNA of therapeutic value, creating a new generation of targeting siRNA drugs. Another important application of this method is the coupling of tumor targeting antibody or antibody analogue with a tracer molecule which offers a new generation of tumor tracing agents.

[0153] 6. The Preparation of Linkers 2, 3 and 9

[0154] When n=3, X is NH2, the structure in formulus 2 become linker 2 (FIG. 51). A similar method as described for linker 1 was used to prepare linker 2. After purification, it was analyzed by ESI-MS. As shown in FIG. 52, the purity of linker 2 is 97.3492%. The expected MS of linker 2 is 535 and found 536 (M+1) (FIG. 53).

[0155] When n=5, m=4, X is OH, the chemical structure of linker 3 was specified and shown in FIG. 54. Similar protocol as described for linker 1 was applied with modification. The crude product was purified by HPLC. After purification, it was analyzed by ESI-MS. As shown in FIG. 55, the purity of linker 3 is 99.3650%. The expected MS of linker 3 is 954 and found 953 (M+?1) (FIG. 56).

[0156] When n=5, m=4, X is OH, the chemical structure of linker 9 was specified and shown in FIG. 57. Similar protocol as described for linker 1 was applied with modification. The crude product was purified by HPLC. After purification, it was analyzed by ESI-MS. As shown in FIG. 58, the purity of linker 3 is 99.3650%. The expected MS of linker 9 is 1249 and found 1248 (M-1) (FIG. 59).

[0157] Linkers 2, 3, 9 thus obtained can be used to couple with small molecules, nucleic acids, or tracer molecules. Linker 9 has two reactive functional groups which can react with two small molecules, nucleic acids or tracer molecules.