Disclosure of Invention
In order to solve the defects of the prior art, the invention aims to provide a synthetic method of the somatrum peptide, the method introduces the Depsipeptide Units to solve the problem of difficult sequence synthesis of the somatrum peptide, and then a target product is obtained through ester bond transamidation reaction; the synthesis method of the invention improves the purity and yield of the crude peptide, is beneficial to downstream purification, and is suitable for industrial production.
In order to achieve the above object, the present invention adopts the following technical solutions:
a synthetic method of somaglutide comprises the following steps:
step one, preparing Fmoc-Gly-Wang resin;
step two, preparing the full-protection peptide resin by taking Fmoc-Gly-Wang resin, protected amino acid and octadecanedioic acid (OtBu) -gamma-Glu-OtBu-PEG-PEG-OH as raw materials, wherein the full-protection peptide resin comprises a Depsipeptide Units structure,
the chemical structural formula of the Depsipeptide Units is as follows:
wherein R comprises: h, CH3(Ser,Thr);
Step three, performing TFA cleavage on the peptide resin to obtain crude peptide;
and step four, the pH of the crude peptide solution obtained in the step three is demodulated, and a crude product of the somaglutide is obtained after ester bond-transamidation reaction.
The second step of the synthetic method of the somaglutide is to prepare a full-protection peptide resin;
step a, preparing a protected amino acid fragment,
the chemical structural formula of the protected amino acid fragment is:
wherein R comprises: h, CH3(Ser,Thr);
Step b, adding Fmoc-Gly-Wang resin into a solid phase reactor;
c, adding the single-protection amino acid and the protection amino acid fragments into a solid phase reactor one by one for coupling reaction, and synthesizing according to the reverse sequence of the amino acid sequence of the Somalou peptide;
mono-protected amino acids include: Fmoc-Gly-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu (otBu) -OH, Fmoc-Lys (X) -OH, Fmoc-Gln (Trt) -OH, Fmoc-Tyr (tBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Asp (otBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Aib-OH, Boc-His (Trt) -OH;
protected amino acid fragments include: Boc-Thr5(Fmoc-Gly4)-OH,Boc-Thr7(Fmoc-Phe6)-O,Boc-Ser8(Fmoc-Thr7(tBu))-OH,Boc-Ser11(Fmoc-Val10)-OH,Boc-Ser12(Fmoc-Ser11(tBu))-OH;
Step d, adding octadecanedioic acid (OtBu) -gamma-Glu-OtBu-PEG-PEG-OH, carrying out condensation reaction to obtain peptide resin with Depsipeptide Units,
the chemical structural formula of the Depsipeptide Units is as follows:
wherein R comprises: h, CH
3(Ser,Thr)。
The synthesis method of the sumatriptan is as follows,
step two, preparing full-protection peptide resin;
step a, adding Fmoc-Gly-Wang resin into a solid phase reactor;
step b, solid phase Synthesis method comprises the steps of sequencing Fmoc-Arg (Pbf) -OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu (otBu) -OH, Fmoc-Lys (X) -OH, Fmoc-Ala-OH, Fmoc-Gln (Trt) -OH, Fmoc-Gly-OH, Fmoc-Glu (otBu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Ser (tBu) -OH, Boc-Ser-OH, Fmoc-Val, Fmoc-Asp (otBu) -OH, Fmoc-Ser (tBu) (OH), (Fmoc-Thru) -OH, Thr-OH, Fmoc-Phe-OH, Fmoc-Thr (tBu) -OH, Fmoc-Gly-OH, Fmoc-Glu (otBu) -OH, Fmoc-Aib-OH, Boc-His (Trt) -OH was coupled to Fmoc-Gly-Wang;
depsipeptide Units were introduced during coupling of the mono-protected amino acid to Fmoc-Gly-Wang, and sites at which the Depsipeptide Units could be introduced included: gly4-Thr5,Phe6-Thr7,Thr7-Ser8,Val10-Ser11,Ser11-Ser12;
Step c, adding Pal-gamma-Glu-OtBu, carrying out condensation reaction to obtain peptide resin with the Depsipeptide Units,
the chemical structural formula of the Depsipeptide Units is as follows:
wherein R comprises: h, CH3(Ser,Thr)。
In the aforementioned synthesis method of somaglutide, X in Fmoc-lys (X) -OH comprises: alloc, Dde, ivdde, Mtt, octadecanedioic acid (OtBu) - γ -Glu-OtBu-PEG-PEG-OH; if Lys20The side chains of (A) are Alloc, Dde, ivdde and Mtt, then octadecanedioic acid (OtBu) -gamma-Glu-OtBu-PEG-PEG-OH needs to be coupled after removing the protecting groups.
In the synthesis method of the somaglutide, the Fmoc removal reagent adopted in the coupling reaction is a v/v 25% piperidine/DMF solution.
In the synthesis method of the somaglutide, the condensing agent used in the condensation reaction comprises: DIC/HOBt, HBTU/HOBT/DIEA, PyBop/HOBT/DIEA.
In the synthesis method of the somaglutide, the reaction solvent adopted in the condensation reaction includes: DCM, DMF, NMP, DMSO.
The synthesis method of the sumatriptan is as follows,
step three, performing TFA cleavage on the peptide resin to obtain crude peptide;
the proportion of reagents used for cracking is as follows: TFA-thioanisole-anisole-EDT-H2O=90:3:3:2:2。
The method for synthesizing the somaglutide comprises a fourth step of dissolving the crude peptide obtained in the third step in acetonitrile/water solution, adjusting the pH value to 7.5-9.5 by using an alkaline reagent, reacting for 5-7 hours, and carrying out ester bond-transamidation reaction to obtain a crude somaglutide product.
In the synthesis method of the somaglutide, the alkaline reagent comprises: ammonia water, sodium carbonate, sodium phosphate, sodium hydroxide, triethylamine and pyridine.
The invention has the advantages that:
the method comprises the steps of introducing Depsipeptide Units to obtain full-protection peptide resin, and then performing cracking and ester bond transamidation reaction to obtain a target product; the introduction of the Depsipeptide Units can solve the problem of difficult sequence synthesis of the somataltide;
the invention adopts dipeptide form to carry out coupling, destroys the secondary structure of straight-chain peptide, solves the problem of difficult coupling, and removes the missing peptide generated by the process, thereby reducing the feeding and the cost;
the crude peptide synthesized by the method has excellent purity and yield, is beneficial to downstream purification, and is suitable for industrial production.
Detailed Description
The invention is described in detail below with reference to the figures and the embodiments.
There are two protocols for the preparation of somaglutide:
scheme one as shown in figure 1;
a synthetic method of somaglutide comprises the following steps:
step one, preparing Fmoc-Gly-Wang resin;
step two, preparing full-protection peptide resin;
step a, preparing a protected amino acid fragment,
the chemical structural formula of the protected amino acid fragment is:
whereinR comprises: h, CH3(Ser, Thr); is Thr in the peptide sequence5、Thr7、Ser8、Ser11、Ser12。
Written as chemical formula: Boc-Thr5(Fmoc-Gly4)-OH,Boc-Thr7(Fmoc-Phe6)-OH,Boc-Ser8(Fmoc-Thr7(tBu))-OH,Boc-Ser11(Fmoc-Val10)-OH,Boc-Ser12(Fmoc-Ser11(tBu))-OH。
Step b, adding Fmoc-Gly-Wang resin into a solid phase reactor;
c, adding the single-protection amino acid and the protection amino acid fragments into a solid phase reactor one by one for coupling reaction, and synthesizing according to the reverse sequence of the amino acid sequence of the Somalou peptide;
mono-protected amino acids include: Fmoc-Gly-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu (otBu) -OH, Fmoc-Lys (X) -OH, Fmoc-Gln (Trt) -OH, Fmoc-Tyr (tBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Asp (otBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Aib-OH, Boc-His (Trt) -OH;
protected amino acid fragments include: Boc-Thr5(Fmoc-Gly4)-OH,Boc-Thr7(Fmoc-Phe6)-O,Boc-Ser8(Fmoc-Thr7(tBu))-OH,Boc-Ser11(Fmoc-Val10)-OH,Boc-Ser12(Fmoc-Ser11(tBu))-OH;
Step d, adding octadecanedioic acid (OtBu) -gamma-Glu-OtBu-PEG-PEG-OH, carrying out condensation reaction to obtain peptide resin with Depsipeptide Units,
the chemical structural formula of the Depsipeptide Units is as follows:
step three, performing TFA cleavage on the peptide resin, wherein the cleavage reagent comprises TFA, thioansole, Anisole, EDT and H2O is 90:3:3:2: 2; cleavage to give a crude intermediate containing Val10-Ser11Somatocride constituting 1 DepsipediunitsA precursor;
step four, dissolving the somaglutide precursor obtained in the step three in acetonitrile/water solution, adjusting the pH to 7.5-9.5 by using an alkaline reagent, reacting for 5-7h, and carrying out ester bond-amide bond transfer reaction on the precursor crude product to obtain a somaglutide crude product, as shown in figure 2; the alkaline agent comprises: ammonia water, sodium carbonate, sodium phosphate, sodium hydroxide, triethylamine and pyridine.
Scheme two;
a synthetic method of somaglutide comprises the following steps:
step one, preparing Fmoc-Gly-Wang resin;
step two, preparing full-protection peptide resin;
step a, adding Fmoc-Gly-Wang resin into a solid phase reactor; the Fmoc removal reagent used for the coupling reaction was a v/v 25% piperidine/DMF solution.
Step b, solid phase Synthesis method comprises the steps of sequencing Fmoc-Arg (Pbf) -OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu (otBu) -OH, Fmoc-Lys (X) -OH, Fmoc-Ala-OH, Fmoc-Gln (Trt) -OH, Fmoc-Gly-OH, Fmoc-Glu (otBu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Ser (tBu) -OH, Boc-Ser-OH, Fmoc-Val, Fmoc-Asp (otBu) -OH, Fmoc-Ser (tBu) (OH), (Fmoc-Thru) -OH, Thr-OH, Fmoc-Phe-OH, Fmoc-Thr (tBu) -OH, Fmoc-Gly-OH, Fmoc-Glu (otBu) -OH, Fmoc-Aib-OH, Boc-His (Trt) -OH was coupled to Fmoc-Gly-Wang;
it should be noted that: wherein X in Fmoc-Lys (X) -OH comprises: alloc, Dde, ivdde, Mtt, octadecanedioic acid (OtBu) - γ -Glu-OtBu-PEG-OH; if Lys20The side chain of (1) is Alloc, Dde, ivdde, Mtt, then octadecanedioic acid (OtBu) -gamma-Glu-OtBu-PEG-PEG-OH needs to be coupled after removing the protecting groups.
Depsipeptide Units were introduced during coupling of the mono-protected amino acid to Fmoc-Gly-Wang, and sites at which the Depsipeptide Units could be introduced included: gly4-Thr5,Phe6-Thr7,Thr7-Ser8,Val10-Ser11,Ser11-Ser12(ii) a A roadmap for the synthesis of Depsipeptide Units is shown in FIG. 3.
Step c, adding Pal-gamma-Glu-OtBu, carrying out condensation reaction to obtain peptide resin with the Depsipeptide Units,
the chemical structural formula of the Depsipeptide Units is as follows:
wherein R comprises: h, CH3(Ser,Thr);
The condensing agent used in the condensation reaction includes: DIC/HOBt, HBTU/HOBT/DIEA, PyBop/HOBT/DIEA (N, N-diisopropylethylamine). The reaction solvent used for the condensation reaction includes: DCM, DMF, NMP, DMSO.
Step three, performing TFA cleavage on the peptide resin, wherein the cleavage reagent comprises TFA, thioansole, Anisole, EDT and H2O is 90:3:3:2: 2; obtaining crude peptide;
step four, dissolving the crude peptide obtained in the step three in acetonitrile/water solution, adjusting the pH to 7.5-9.5 by using an alkaline reagent, reacting for 5-7h, and carrying out ester bond-transamidation reaction on the precursor crude product to obtain a crude product of the somaglutide, as shown in figure 2. As an example, the alkaline agent includes: ammonia water, sodium carbonate, sodium phosphate, sodium hydroxide, triethylamine and pyridine.
To demonstrate the excellent purity and yield of the process of the invention, the following validation experiments were performed:
the following synthesis experiment one was performed according to scheme one:
step one, preparing Fmoc-Gly-Wang resin;
weigh Wang resin 17.0g (Sub ═ 0.61mmol/g) into the solid phase reactor and add 150mL DCM swell resin for 0.5 h. The solvent is drained, Fmoc-Gly-OH 9.02g, HOBt5.6g and DIC 5.6mL of 160mLDMF solution are added, and the mixture is stirred and reacted for 5 hours. The solvent was drained, 11mL of acetic anhydride, 8mL of pyridine, 160mL of DMF were added, and the mixture was blocked for 2 h. DCM was washed 3 times with 170mL portions of methanol. After drying in vacuo, the Fmoc-Gly-Wang resin obtained a Sub ═ 0.55mmol/g as determined by uv spectrophotometry.
Step two, Boc-Ser11(Fmoc-Val10) -preparation of OH;
weighing 35g of CTC-Resin, adding into a solid phase reactor, adding 160ml of DMF to wash the Resin for 10min, weighing 23.2g of Fmoc-Ser-OH and 25.1g of DIEA, adding DMF90mL to dissolve, activating in ice bath and stirring for 8 min. Adding the activated solution into a reactor, reacting for 3h, and draining the solvent. DMF was added and the mixture was washed 3 times with 90mL portions.
Weighing 18.7g of Fmoc-Val-OH, 6.3g of HOBt, 6.3mL of CDI and 0.6g of DMAP0.6g, adding DMF100mL to dissolve ice and activate for 8min, adding into a reactor, reacting for 2h, and draining off the solvent. Adding DMF to wash for 3 times, each time 100 mL; washing with DCM for 3 times, each time with 100 mL; the washed resin was dried and weighed to obtain Boc-Ser (Fmoc-Val) -CTC-Resin78.9 g.
Putting 78.9g of Boc-Ser (Fmoc-Val) -CTC-Resin obtained in the previous step into 700ml of a cold DCM solution containing 1% TFA, and cleaving for 2 hours; filtering to obtain 692ml of lysate; after the cracking rotary evaporation is concentrated to one third of the molecule volume, 3 times of DCM is supplemented for continuous rotary evaporation, liquid is circularly added for three times and then the mixture is evaporated to dryness, and 33.7g of Boc-Ser (Fmoc-Val) -OH crude product is obtained.
Step three, preparing full protection peptide;
20.0g of Fmoc-Gly-Wang resin was weighed into a solid phase reactor, and 90mL of DCM was added to swell the resin for 0.5 h. The solvent was drained and 2 times 90mL v/v 20% piperidine/DMF solutions were added for 5min and 15min, respectively. The solvent was drained and washed 4 times with 90mL of DMF. The result of indene test is positive.
The following protected amino acids 19.8g, HOBt5.6g, and DIC 5.6mL were weighed, dissolved in DMF90mL, and activated for 8min on ice. And adding the activated solution into a reactor, reacting for 1h, and draining the solvent after an indene detection result shows negative. DMF was added and the mixture was washed 3 times with 90mL portions.
Repeating the steps to protect the amino acid: Fmoc-Arg (Pbf) -OH, Fmoc-Gly-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu (otBu) -OH, Fmoc-Lys (alloc) -OH, Fmoc-Ala-OH, Fmoc-Gln (Trt) -OH, Fmoc-Gly-OH, Fmoc-Glu (otBu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Ser (tBu) -OH, Boc-Ser (Fmoc-Val) -OH, Fmoc-Asp (oAsp) (Ot) -OH, Fmoc-Leu, Fmoc-Ser (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Phe-OH, Fmoc-Thr (tBu) -OH, Fmoc-Gly-OH, Fmoc-Glu (otBu) -OH, Fmoc-Aib-OH, Boc-His (Trt) -OH.
Weighing 12.2g of tetratriphenylphosphine palladium, 1mL of morpholine and 100mL of THF, adding into a solid phase reactor, reacting for 3h, washing with DMF for 3 times, adding 100mL each time, and detecting with indene to be positive.
Octadecanedioic acid (OtBu) -gamma-Glu-OtBu-PEG-PEG-OH 21.6g, HOBt5.6g, and DIC 5.6mL were weighed, dissolved in 90mL of DMF, and activated for 8min in ice bath. And adding the activated solution into a reactor, reacting for 3 hours, and draining the solvent after an indene detection result shows negative. DMF was added and the mixture was washed 3 times with 90mL portions. DCM was washed 3 times with alternating washes of 90mL each time and dried in vacuo.
Step four, cracking;
500mL of the cleavage reagent was prepared as TFA Thioanisole: Anisole: EDT: H2Adding the mixture into the fully protected peptide under the ice-bath condition, returning to the room temperature after 0.5h, continuing to react for 2.5h, and adding anhydrous ether for precipitation after the reaction is finished. The precipitate was centrifuged 3 times and 3L of diethyl ether was added each time. The crude peptide is dried. 47.8g of the crude peptide was obtained.
Step five, performing ester bond transamidation reaction;
and (3) dissolving 47.8g of the crude peptide product obtained in the previous step in a 5% acetonitrile aqueous solution, adjusting the pH of the solution by using 30% ammonia water, and reacting for 3 hours to obtain the somaglutide.
The solution contains 40.2g of the somaglutide quantitatively through a reference substance, the total synthesis yield is 84.1%, and the purity of a crude product is 80.0% (shown as HPLC spectrograms shown in figures 4 and 5). It can be seen that the crude somaltulipide synthesized according to scheme one is excellent in both yield and purity.
According to the method of the second scheme, the following second synthesis experiment is carried out;
step one, preparing Fmoc-Gly-Wang resin;
weigh Wang resin 17.0g (Sub ═ 0.61mmol/g) into the solid phase reactor and add 150mL DCM swell resin for 0.5 h. The solvent is drained, Fmoc-Gly-OH 9.02g, HOBt5.6g and DIC 5.6mL of 160mLDMF solution are added, and the mixture is stirred and reacted for 5 hours. The solvent was drained, 11mL of acetic anhydride, 8mL of pyridine, 160mL of DMF were added, and the mixture was blocked for 2 h. DCM was washed 3 times with 170mL portions of methanol. After drying in vacuo, the Fmoc-Gly-Wang resin obtained a Sub ═ 0.55mmol/g as determined by uv spectrophotometry.
Step two, preparing full protection peptide;
20.0g of Fmoc-Gly-Wang resin was weighed into a solid phase reactor, and 90mL of DCM was added to swell the resin for 0.5 h. The solvent was drained and 2 times 90mL v/v 20% piperidine/DMF solutions were added for 5min and 15min, respectively. The solvent was drained and washed 4 times with 90mL of DMF. The result of indene test is positive.
19.8g of the following protected amino acid, HOBt5.6g, and DIC 5.6mL were dissolved in DMF90mL and activated on ice for 8 min. And adding the activated solution into a reactor, reacting for 1h, and draining the solvent after an indene detection result shows negative. DMF was added and the mixture was washed 3 times with 90mL portions. Wherein Fmoc-Val10As a coupling reagent for-OH, HOBt5.6g, DIC 5.6mL, DIEA0.52g were used.
Repeating the steps to protect the amino acid: Fmoc-Arg (Pbf) -OH, Fmoc-Gly-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu (otBu) -OH, Fmoc-Lys (alloc) -OH, Fmoc-Ala-OH, Fmoc-Gln (Trt) -OH, Fmoc-Gly-OH, Fmoc-Glu (otBu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Ser (tBu) -OH, Boc-Ser (tBu) (OH), Fmoc-Val-Ser (tBu) -OH, Fmoc-Arg (pro), Fmoc-Asp (OtBu) -OH, Fmoc-Asp (oAsp), Fmoc-Ser (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Phe-OH, Fmoc-Thr (tBu) -OH, Fmoc-Gly-OH, Fmoc-Glu (otBu) -OH, Fmoc-Aib-OH, Boc-His (Trt) -OH. It should be noted that: in the synthesis method compared to scheme one, Boc-Ser (Fmoc-Val) -OH was not added, but Fmoc-Val-OH was added.
Weighing 12.2g of tetratriphenylphosphine palladium, 1mL of morpholine and 100mL of THF, adding into a solid phase reactor, reacting for 3h, washing with DMF for 3 times, adding 100mL each time, and detecting with indene to be positive.
Octadecanedioic acid (OtBu) -gamma-Glu-OtBu-PEG-PEG-OH 21.6g, HOBt5.6g, and DIC 5.6mL were weighed, dissolved in 90mL of DMF, and activated for 8min in ice bath. And adding the activated solution into a reactor, reacting for 3 hours, and draining the solvent after an indene detection result shows negative. DMF was added and the mixture was washed 3 times with 90mL portions. DCM was washed 3 times with alternating washes of 90mL each time and dried in vacuo.
Step three, cracking;
500mL of the cleavage reagent was prepared as TFA Thioanisole: Anisole: EDT: H2Adding the mixture into the fully protected peptide under the ice-bath condition, returning to the room temperature after 0.5h, continuing to react for 2.5h, and adding anhydrous ether for precipitation after the reaction is finished. The precipitate was centrifuged 3 times and 3L of diethyl ether was added each time. The crude peptide is dried. 45.3g of the crude peptide was obtained.
Step four, ester bond transamidation reaction;
and (3) dissolving 45.3g of the crude peptide product obtained in the previous step in a 5% acetonitrile aqueous solution, adjusting the pH of the solution by using 30% ammonia water, and reacting for 5 hours to obtain the somaglutide.
The solution contains 37.1g of the somaglutide quantitatively by a reference substance, the total synthesis yield is 81.9%, and the purity of the crude product is 75.4% (as shown in HPLC spectrograms shown in figures 6 and 7).
It can be seen that the crude soxhlet peptide synthesized according to scheme one or scheme two has excellent yield and purity.
The method comprises the steps of introducing Depsipeptide Units to obtain full-protection peptide resin, and then performing cracking and ester bond transamidation reaction to obtain a target product; the introduction of the Depsipeptide Units can solve the problem of difficult sequence synthesis of the somataltide; the dipeptide is adopted for coupling, the secondary structure of the linear chain peptide is destroyed, the problem of difficult coupling is solved, and the missing peptide generated by the process is removed, so that the feeding is reduced, and the cost is reduced; the crude peptide synthesized by the method has excellent purity and yield, is beneficial to downstream purification, and is suitable for industrial production.
The foregoing illustrates and describes the principles, general features, and advantages of the present invention. It should be understood by those skilled in the art that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by using equivalent alternatives or equivalent variations fall within the scope of the present invention.