Optimization strategy for grafting acidic polypeptide on titanium metal surface

Abstract

The invention discloses an optimization strategy for grafting acidic polypeptide on the surface of titanium metal. The optimization strategy comprises the following steps that (1) the titanium metal is sequentially subjected to aqueous alkali treatment, oven drying treatment and ultrasonic cleaning; (2) dissolving a silane coupling agent (Sil-PEG-DBCO) modified by dibenzocyclooctyne by taking an ethanol water solution as a solvent, so as to obtain a Sil-PEG-DBCO solution; (3) a Sil-PEG-DBCO solution is dropwise added to the surface of the titanium metal, and curing treatment is carried out; (4) dropwise adding the azide-modified polypeptide solution to the surface of the titanium metal prepared in the step (3), reacting, and adjusting the pH value of the polypeptide solution to 2-7.4 through water or a PBS (Phosphate Buffer Solution); and (5) cleaning the sample obtained in the step (4), and blow-drying. According to the optimization strategy, the grafting efficiency and the saturated grafting density of the acidic polypeptide on the surface can be effectively improved, and the method can be widely applied to titanium metal medical materials with different shapes.

Description

Technical Field

[0001] The invention relates to the field of titanium metal surface modification, and in particular to an optimization strategy for grafting an acidic polypeptide on a titanium metal surface. Background Art

[0002] Titanium is widely used in clinical practice as orthopedic and dental implants due to its good biocompatibility and elastic modulus similar to that of bone tissue. In order to improve the interfacial bonding performance between titanium alloy and bone tissue and promote bone tissue regeneration and bone integration, researchers often perform surface biofunctionalization design on titanium. On the one hand, surface micro-topography can be constructed, and on the other hand, bioactive molecules can be chemically grafted onto the surface. Among them, peptides are molecules with good biocompatibility, controllable synthesis methods, and rational design. Currently, a variety of peptide sequences have been widely used in tissue repair fields such as osteogenesis, angiogenesis, and neuroplasia, and their biological effectiveness has been confirmed.

[0003] Peptides are composed of amino acids connected by peptide bonds. According to the number and type of amino acid residues, peptides can be divided into acidic peptides, neutral peptides and basic peptides. Since the surface potentials of pure titanium, TC4 alloy and alkali-treated titanium are all negative, the charge difference is conducive to the grafting of positively charged basic peptides on the surface. However, negatively charged acidic peptides are subject to the same charge state, which is not conducive to the contact between the peptide and the surface reaction site, resulting in low grafting efficiency of acidic peptides on the surface.

[0004] In addition, due to the high carboxyl content of acidic peptides, it is conducive to calcium ion deposition and promotes surface mineralization, which is beneficial to mineral deposition and bone integration repair on the implant surface. Common peptides used for bone targeting, such as DSS6 and D6, are rich in aspartic acid and belong to acidic peptides. At present, there are few surface grafting methods for acidic peptides, mainly the grafting of positively charged antimicrobial peptides on the surface of titanium metal. There are two main methods for grafting peptides on the surface of titanium metal. One is through the catalytic reaction of amino and carboxyl groups in the surface dopamine coating in NHS / EDC; the other is to achieve surface grafting through a silane coupling agent modified with a specific group. The first method has a fast reaction efficiency but requires the introduction of a catalyst, which may involve the problem of catalyst residues, and the amino group of the peptide will cause multi-site reaction, thereby affecting the conformation of the surface peptide, thereby affecting the performance of the peptide. The second grafting method has simple reaction conditions and does not require the addition of initiators or catalysts, but the grafting efficiency of acidic peptides is relatively low. The problem of grafting acidic peptides on titanium-based surfaces or other negatively charged surfaces needs to be solved urgently. Summary of the invention

[0005] In order to overcome the deficiencies and shortcomings of the prior art, the object of the present invention is to provide an optimized strategy for grafting acidic polypeptides on the surface of titanium metal.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] An optimization strategy for grafting an acidic peptide onto a titanium metal surface comprises the following steps:

[0008] (1) treating titanium metal with an alkaline solution, drying it in an oven and then ultrasonically cleaning it;

[0009] (2) using an ethanol aqueous solution as a solvent to dissolve a dibenzocyclooctyne-modified silane coupling agent (Sil-PEG-DBCO) to obtain a Sil-PEG-DBCO solution;

[0010] (3) adding Sil-PEG-DBCO solution dropwise onto the titanium metal surface for curing;

[0011] (4) adding the azide-modified polypeptide solution dropwise to the titanium metal surface prepared in step (3) to react, and adjusting the pH of the polypeptide solution to 2-7.4 using water or PBS buffer;

[0012] (5) washing the sample obtained in step (4) and drying it;

[0013] The structural formula of the Sil-PEG-DBCO is as follows:

[0014]

[0015] Preferably, the alkaline solution in step (1) is a 1M-10M sodium hydroxide solution.

[0016] Preferably, the oven drying temperature in step (1) is 50-80° C., and the oven drying time is 2-36 h.

[0017] Preferably, the mass fraction of ethanol in the ethanol aqueous solution in step (2) is 95%.

[0018] Preferably, the mass concentration of the Sil-PEG-DBCO solution in step (2) is 0.5-5 mg / mL.

[0019] Preferably, the molecular weight of PEG in the Sil-PEG-DBCO in step (2) is 100-4000.

[0020] Preferably, in step (3), 1 mL of Sil-PEG-DBCO solution is added per square centimeter of titanium surface, and the solution is reacted at room temperature for 6-24 hours, and then taken out and placed in an oven at 80-120° C. for curing.

[0021] Preferably, the azide-modified polypeptide in step (4) is (N 3 -OEG 4 )-Asp-Asp-Asp,

[0022] (N 3 -OEG 4 )-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp,

[0023] (N 3 -OEG 4 )-Asp-Gly-Glu-Ala, (N 3 -OEG 4 )-Arg-Glu-Asp-Val;

[0024] The (N 3 -OEG 4 )-Asp-Asp-As has the following structural formula:

[0025]

[0026] The (N 3 -OEG 4 )-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp has the following structural formula:

[0027]

[0028] The (N 3 -OEG 4 )-Asp-Gly-Glu-Ala has the following structural formula:

[0029]

[0030] The (N 3 -OEG 4 The structural formula of )-Arg-Glu-Asp-Val is as follows:

[0031]

[0032] Preferably, the azide-modified polypeptide may also be statherin peptide (DDDEEK) or hirudin (GDFEEIPEEYLQ).

[0033] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0034] The optimization strategy of grafting the acidic polypeptide on the titanium metal surface of the present invention can effectively improve the grafting efficiency and saturated grafting density of the acidic polypeptide on the surface. After the acidic polypeptide is grafted on the surface, it can significantly promote the deposition of calcium and phosphorus in simulated body fluid (SBF), promote the bone integration and repair process of titanium-based implants, and is suitable for pure titanium surfaces and TC4 titanium alloy surfaces. The grafting reaction has no requirements on oxygen concentration, temperature and humidity, and solvent type, and does not require the introduction of catalysts and initiators. The preparation and cleaning operations are simple and convenient, and can be widely used in titanium medical materials with different shapes. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Schematic diagram of polypeptide grafting on the titanium metal surface of the present invention;

[0036] Figure 2 The grafting density (a) and element ratio (b) of polypeptide 1 (D3) of Example 1 and Comparative Example 1 are shown; it can be seen that after optimizing the grafting conditions, the grafting density of polypeptide 1 increased significantly by 9.26 times, and the C and N contents in the element ratios of the XPS results were also relatively increased;

[0037] Figure 3 XRD (a) and element ratio (b) of calcium deposition on the surface of polypeptide 1 (D3) of Example 1 and Comparative Example 1; the mineralization process of the surface was observed by immersing the samples before and after the optimization of the grafting conditions in simulated body fluids. The results showed that the grafted surface of the optimized polypeptide 2 had a strong calcium deposition promoting performance, indicating that the surface carboxyl content increased with the increase of polypeptide grafting density. The grafting strategy can effectively improve the grafting efficiency of acidic polypeptides and achieve better surface biological properties.

[0038] Figure 4 The grafting density (a) and element ratio (b) of polypeptide 2 (D9) of Example 2 and Comparative Example 2 show that after optimizing the grafting conditions, the grafting density of polypeptide 2 increased significantly by 14.55 times, and the C and N contents in the element ratio of the XPS results were also relatively increased;

[0039] Figure 5 XRD (a) and element ratio (b) of calcium deposition on the surface of polypeptide 2 (D9) in Example 2 and Comparative Example 2; and Figure 3 The same operation showed that peptide 2 had a higher aspartic acid ratio than peptide 1, and the amount of calcium and phosphate deposition after optimization was also significantly increased compared with that before optimization;

[0040] Figure 6 is the ratio of elements on the grafted surface of polypeptide 3 (DGEA) in Example 3 and Comparative Example 3; the results show that polypeptide 3 having two acidic amino acids can also achieve optimization of grafting efficiency when grafted using this strategy;

[0041] Figure 7is the ratio of elements on the grafted surface of polypeptide 4 (REDV) of Example 4 and Comparative Example 4; the results show that polypeptide 4 having 2 acidic amino acids and 1 basic amino acid can also achieve optimization of grafting efficiency when grafted using this strategy;

[0042] Figure 8 The grafting density of peptide 1 (D3) and peptide 2 (D9) on the TC4 surface of Example 5 and Comparative Example 5; the results confirm that the application of the grafting strategy can be extended to the grafting of acidic peptides on the surface of TC4 alloy;

[0043] Fig. 9 The grafting density of (a) polypeptide 1 (D3) and (b) polypeptide 2 (D9) in the PBS system of Example 6 and Comparative Example 6; the results show that when the solvent is changed to PBS buffer, the grafting can also be promoted by changing the pH value of the solvent. DETAILED DESCRIPTION

[0044] The present invention will be further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

[0045] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial channels.

[0046] The different polypeptide sequences used in the following examples are as follows:

[0047] (1)(N 3 -OEG 4 )-Asp-Asp-Asp, peptide 1;

[0048] (2)(N 3 -OEG 4 )-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp, polypeptide 2;

[0049] (3)(N 3 -OEG 4 )-Asp-Gly-Glu-Ala, polypeptide 3;

[0050] (4)(N 3 -OEG 4 )-Arg-Glu-Asp-Val, peptide 4.

[0051] The structural formula of the polypeptide 1 is

[0052]

[0053] The structural formula of the polypeptide 2 is

[0054]

[0055] The structural formula of the polypeptide 3 is

[0056]

[0057] The structural formula of the polypeptide 4 is

[0058]

[0059] The above polypeptides 1, 2, 3 and 4 are preferred solutions provided by the present invention, but the solutions applicable to the present invention are not limited to the above 4 polypeptides, and may also be other acidic polypeptides such as statherin peptide (DDDEEK) and hirudin (GDFEEIPEEYLQ).

[0060] Example 1

[0061] (1) placing titanium metal in a 1 M sodium hydroxide solution and placing it in a 60° C. oven for reaction for 36 h, followed by ultrasonic cleaning with water;

[0062] (2) dissolving the DBCO-modified silane coupling agent in an ethanol / water solution to obtain a 0.5 mg / mL silane coupling agent solution, wherein the PEG molecular weight of the silane coupling agent is 4000;

[0063] (3) Add 1 mL of silane coupling agent per square centimeter of titanium sheet, place at room temperature to react for 24 hours, take out and place in a 120°C oven for curing;

[0064] (4) adding the polypeptide 1 solution to the titanium metal surface prepared in step (3) to react, wherein the solvent is water with a pH of 4 (HCl is used to adjust the pH);

[0065] (5) The sample in step (4) was thoroughly cleaned with deionized water and blown dry with nitrogen.

[0066] Comparative Example 1

[0067] (1) placing titanium metal in a 1 M sodium hydroxide solution and placing it in a 60° C. oven for reaction for 36 h, and then adding water for ultrasonic cleaning after the reaction;

[0068] (2) dissolving the DBCO-modified silane coupling agent in an ethanol / water solution to obtain a 0.5 mg / mL silane coupling agent solution, wherein the PEG molecular weight of the silane coupling agent is 4000;

[0069] (3) Add 1 mL of silane coupling agent per square centimeter of titanium sheet, place at room temperature to react for 24 hours, take out and place in a 120°C oven for curing;

[0070] (4) adding the polypeptide 1 solution to the titanium metal surface prepared in step (3) to react, wherein the solvent is deionized water;

[0071] (5) The sample in step (4) was thoroughly cleaned with deionized water and blown dry with nitrogen.

[0072] Example 2

[0073] (1) placing titanium metal in a 5M sodium hydroxide solution and placing it in a 60°C oven for reaction for 12 hours, and then adding water for ultrasonic cleaning after the reaction;

[0074] (2) dissolving the DBCO-modified silane coupling agent in an ethanol / water solution to obtain a 5 mg / mL silane coupling agent solution, wherein the PEG molecular weight of the silane coupling agent is 4000;

[0075] (3) Add 1 mL of silane coupling agent per square centimeter of titanium sheet, place it at room temperature to react for 12 hours, take it out and place it in a 100°C oven for curing;

[0076] (4) adding the polypeptide 2 solution to the titanium metal surface prepared in step (3) to react, wherein the solvent is water with a pH of 3 (HCl is used to adjust the pH);

[0077] (5) The sample in step (4) was thoroughly cleaned with deionized water and blown dry with nitrogen.

[0078] Comparative Example 2

[0079] (1) placing titanium metal in a 5M sodium hydroxide solution and placing it in a 60°C oven for reaction for 12 hours, and then adding water for ultrasonic cleaning after the reaction;

[0080] (2) dissolving the DBCO-modified silane coupling agent in an ethanol / water solution to obtain a 5 mg / mL silane coupling agent solution, wherein the PEG molecular weight of the silane coupling agent is 4000;

[0081] (3) Add 1 mL of silane coupling agent per square centimeter of titanium sheet, place it at room temperature to react for 12 hours, take it out and place it in a 100°C oven for curing;

[0082] (4) adding the polypeptide 2 solution to the titanium metal surface prepared in step (3) to react, wherein the solvent is deionized water;

[0083] (5) The sample in step (4) was thoroughly cleaned with deionized water and blown dry with nitrogen.

[0084] Example 3

[0085] (1) placing titanium metal in a 10M sodium hydroxide solution and placing it in an oven at 80°C for 2 hours, followed by ultrasonic cleaning with water;

[0086] (2) dissolving the DBCO-modified silane coupling agent in an ethanol / water solution to obtain a 1 mg / mL silane coupling agent solution, wherein the PEG molecular weight of the silane coupling agent is 400;

[0087] (3) Add 1 mL of silane coupling agent per square centimeter of titanium sheet, place it at room temperature to react for 24 hours, take it out and place it in a 100°C oven for curing;

[0088] (4) adding the polypeptide 3 solution to the titanium metal surface prepared in step (3) to react, wherein the solvent is water with a pH of 3 (HCl is used to adjust the pH);

[0089] (5) The sample in step (4) was thoroughly cleaned with deionized water and blown dry with nitrogen.

[0090] Comparative Example 3

[0091] (1) placing titanium metal in a 10M sodium hydroxide solution and placing it in an oven at 80°C for 2 hours, followed by ultrasonic cleaning with water;

[0092] (2) dissolving the DBCO-modified silane coupling agent in an ethanol / water solution to obtain a 1 mg / mL silane coupling agent solution, wherein the PEG molecular weight of the silane coupling agent is 400;

[0093] (3) Add 1 mL of silane coupling agent per square centimeter of titanium sheet, place it at room temperature to react for 24 hours, take it out and place it in a 100°C oven for curing;

[0094] (4) adding the polypeptide 3 solution to the titanium metal surface prepared in step (3) to react, wherein the solvent is deionized water;

[0095] (5) The sample in step (4) was thoroughly cleaned with deionized water and blown dry with nitrogen.

[0096] Example 4

[0097] (1) placing titanium metal in a 5M sodium hydroxide solution and placing it in a 60°C oven for reaction for 24 hours, and then adding water for ultrasonic cleaning after the reaction;

[0098] (2) dissolving the DBCO-modified silane coupling agent in an ethanol / water solution to obtain a 2 mg / mL silane coupling agent solution, wherein the PEG molecular weight of the silane coupling agent is 2000;

[0099] (3) Add 1 mL of silane coupling agent per square centimeter of titanium sheet, place it at room temperature to react for 24 hours, take it out and place it in a 100°C oven for curing;

[0100] (4) adding the polypeptide 4 solution to the titanium metal surface prepared in step (3) to react, wherein the solvent is water with a pH of 2 (HCl to adjust the pH);

[0101] (5) The sample in step (4) was thoroughly cleaned with deionized water and blown dry with nitrogen.

[0102] Comparative Example 4

[0103] (1) placing titanium metal in a 5M sodium hydroxide solution and placing it in a 60°C oven for reaction for 24 hours, and then adding water for ultrasonic cleaning after the reaction;

[0104] (2) dissolving the DBCO-modified silane coupling agent in an ethanol / water solution to obtain a 2 mg / mL silane coupling agent solution, wherein the PEG molecular weight of the silane coupling agent is 2000;

[0105] (3) adding 1 mL of silane coupling agent per square centimeter of titanium sheet, placing it at room temperature for reaction for 24 hours, taking it out and placing it in a 100° C. oven for curing; (4) adding the polypeptide 4 solution to the titanium metal surface prepared in step (3) for reaction, wherein the solvent is deionized water;

[0106] (5) The sample in step (4) was thoroughly cleaned with deionized water and blown dry with nitrogen.

[0107] Example 5

[0108] (1) The TC4 sheet was placed in a 1M sodium hydroxide solution and placed in a 60°C oven for reaction for 24 hours. After the reaction, water was added for ultrasonic cleaning;

[0109] (2) dissolving the DBCO-modified silane coupling agent in an ethanol / water solution to obtain a 1 mg / mL silane coupling agent solution, wherein the PEG molecular weight of the silane coupling agent is 3400;

[0110] (3) Add 1 mL of silane coupling agent per square centimeter of titanium sheet, place at room temperature to react for 24 hours, take out and place in a 120°C oven for curing;

[0111] (4) adding the azide-modified polypeptide solution to the titanium metal surface prepared in step (3) to react, wherein the solvent is water with a pH of 3 (HCl is used to adjust the pH);

[0112] (5) The sample in step (4) was thoroughly cleaned with deionized water and blown dry with nitrogen.

[0113] Comparative Example 5

[0114] (1) The TC4 sheet was placed in a 1M sodium hydroxide solution and placed in a 60°C oven for reaction for 24 hours. After the reaction, water was added for ultrasonic cleaning;

[0115] (2) dissolving the DBCO-modified silane coupling agent in an ethanol / water solution to obtain a 1 mg / mL silane coupling agent solution, wherein the PEG molecular weight of the silane coupling agent is 3400;

[0116] (3) Add 1 mL of silane coupling agent per square centimeter of titanium sheet, place at room temperature to react for 24 hours, take out and place in a 120°C oven for curing;

[0117] (4) adding the azide-modified polypeptide solution to the titanium metal surface prepared in step (3) to react, wherein the solvent is deionized water;

[0118] (5) The sample in step (4) was thoroughly cleaned with deionized water and blown dry with nitrogen.

[0119] Example 6

[0120] (1) placing titanium metal in a 5M sodium hydroxide solution and placing it in a 60°C oven for reaction for 24 hours, and then adding water for ultrasonic cleaning after the reaction;

[0121] (2) dissolving the DBCO-modified silane coupling agent in an ethanol / water solution to obtain a 4 mg / mL silane coupling agent solution, wherein the PEG molecular weight of the silane coupling agent is 2000;

[0122] (3) Add 1 mL of silane coupling agent per square centimeter of titanium sheet, place it at room temperature to react for 24 hours, take it out and place it in an oven at 80°C for curing;

[0123] (4) adding the polypeptide 4 solution to the titanium metal surface prepared in step (3) to react, wherein the solvent is a PBS buffer with a pH of 2 and 3;

[0124] (5) The sample in step (4) was thoroughly cleaned with deionized water and blown dry with nitrogen.

[0125] Comparative Example 6

[0126] (1) placing titanium metal in a 5M sodium hydroxide solution and placing it in a 60°C oven for reaction for 24 hours, and then adding water for ultrasonic cleaning after the reaction;

[0127] (2) dissolving the DBCO-modified silane coupling agent in an ethanol / water solution to obtain a 4 mg / mL silane coupling agent solution, wherein the PEG molecular weight of the silane coupling agent is 2000;

[0128] (3) Add 1 mL of silane coupling agent per square centimeter of titanium sheet, place it at room temperature to react for 24 hours, take it out and place it in an oven at 80°C for curing;

[0129] (4) adding the polypeptide 4 solution to the titanium metal surface prepared in step (3) to react, wherein the solvent is deionized water;

[0130] (5) The sample in step (4) was thoroughly cleaned with deionized water and blown dry with nitrogen.

[0131] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. An optimization strategy for grafting acidic polypeptides on titanium metal surfaces, characterized in that: The following steps are involved: (1) treating titanium metal with an alkaline solution, drying it in an oven and then ultrasonically cleaning it; (2) dissolving Sil-PEG-DBCO in an ethanol aqueous solution to obtain a Sil-PEG-DBCO solution; (3) adding Sil-PEG-DBCO solution dropwise onto the titanium metal surface for curing; (4) adding the azide-modified polypeptide solution dropwise to the titanium metal surface prepared in step (3) to react, and adjusting the pH of the polypeptide solution to 2-7.4 using water or PBS buffer; (5) washing the sample obtained in step (4) and drying it; The structural formula of the Sil-PEG-DBCO is as follows:

2. The optimization strategy for grafting acidic polypeptides onto titanium metal surfaces according to claim 1, characterized in that: The alkaline solution in step (1) is a 1M-10M sodium hydroxide solution.

3. The optimization strategy for grafting acidic polypeptides onto titanium metal surfaces according to claim 1, characterized in that: The oven drying temperature in step (1) is 50-80° C., and the oven drying time is 2-36 hours.

4. The optimization strategy for grafting acidic polypeptides onto titanium metal surfaces according to claim 1, characterized in that: The mass fraction of ethanol in the ethanol aqueous solution in step (2) is 95%.

5. The optimization strategy for grafting acidic polypeptides onto titanium metal surfaces according to claim 1, characterized in that: The mass concentration of the Sil-PEG-DBCO solution in step (2) is 0.5-5 mg / mL.

6. The optimization strategy for grafting acidic polypeptides onto titanium metal surfaces according to claim 1, characterized in that: The molecular weight of PEG in the Sil-PEG-DBCO in step (2) is 100-4000.

7. The optimization strategy for grafting acidic polypeptides onto titanium metal surfaces according to claim 1, characterized in that: In step (3), 1 mL of Sil-PEG-DBCO solution is added per square centimeter of titanium surface, and the mixture is allowed to react at room temperature for 6-24 hours. After being taken out, the mixture is placed in an oven at 80-120° C. for curing.

8. The optimization strategy for grafting acidic polypeptides onto titanium metal surfaces according to claim 1, characterized in that: The azide-modified polypeptide in step (4) is one of (N3-OEG4)-Asp-Asp-Asp, (N3-OEG4)-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp, (N3-OEG4)-Asp-Gly-Glu-Ala, and (N3-OEG4)-Arg-Glu-Asp-Val; The structural formula of the (N3-OEG4)-Asp-Asp-As is as follows: The structural formula of the (N3-OEG4)-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp is as follows: The structural formula of the (N3-OEG4)-Asp-Gly-Glu-Ala is as follows: The structural formula of the (N3-OEG4)-Arg-Glu-Asp-Val is as follows:

9. The optimization strategy for grafting acidic polypeptides onto titanium metal surfaces according to claim 1, characterized in that: The azide-modified polypeptide is DDDEEK or GDFEEIPEEYLQ.

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