CN117603294A - Novel Fmoc deprotection method in polypeptide solid-phase synthesis - Google Patents

Abstract

The invention relates to a novel Fmoc deprotection method in polypeptide solid-phase synthesis, which comprises the steps of adding a DMF solution containing 0.2 mol/L1, 5, 7-triazabicyclo [4.4.0] dec-5-ene and 2.0mol/L tetrahydroisoquinoline into resin loaded with Fmoc protected amino acid or polypeptide in the deprotection step of polypeptide solid-phase synthesis, oscillating for 15 minutes at room temperature, and filtering to obtain resin loaded with amino acid or polypeptide, wherein Fmoc protecting groups of the resin are removed. The invention takes 1,5, 7-triazabicyclo [4.4.0] dec-5-ene and tetrahydroisoquinoline as components of Fmoc deprotection agent for the first time, and replaces the traditional pyridine. The new deprotection method does not use chemicals easy to prepare, has the advantages of cheap and easily available raw materials, low toxicity, low volatility and low corrosion, excellent Fmoc removal capability, mild reaction conditions, simplified polypeptide production flow, reduced polypeptide preparation cost and provides a great improvement space for the design of a synthesis strategy of the polypeptide containing the alkaline sensitive side chain protecting group.

Description

Novel Fmoc deprotection method in polypeptide solid-phase synthesis

The technical field is as follows:

the invention relates to preparation of polypeptide medicines, in particular to a method for solid-phase synthesis of leupeptin, belonging to the technical field of solid-phase synthesis of polypeptides.

The background technology is as follows:

leu-enkephalin is an endogenous enkephalin and consists of five amino acids and has the sequence H-tyrosine-glycine-phenylalanine-leucine (Tyr-Gly-Gly-Phe-Leu-OH). It belongs to the endorphin family, and is mainly found in the central nervous system of humans or various animals, especially in the forebrain region. Leupeptin has analgesic and sedative effects, and produces pharmacological effects by binding to mu-and delta-opioid receptors. It is a natural analgesic agent that is involved in regulating pain transmission and emotional response. Synthesis and release of human pre-enkephalin is regulated by a number of factors including neurotransmitters, stress and pain stimuli, etc. Its presence in the nervous system plays an important role in pain regulation and mood stabilization. Studies have shown that leupeptin is associated with analgesic drugs and drug abuse, and its regulatory function is of great importance for understanding the pain mechanism and drug abuse behavior.

The solid-phase synthesis method of the 9-fluorenylmethoxycarbonyl protecting group (Fmoc) polypeptide has the advantage of rapid, efficient and controllable aspects of polypeptide synthesis. It is widely used in the fields of medicine research and development, biomedical research, bioengineering and the like. Through reasonable design and optimization of reaction conditions, high-yield and high-purity polypeptide synthesis can be realized, and a feasible route is provided for synthesizing complex peptide compounds and medicines. The Fmoc polypeptide solid-phase synthesis method has the advantages of simple operation, mild reaction conditions, high synthesis efficiency and the like, and becomes the most commonly used method for chemically synthesizing the polypeptide at present. Fmoc deprotection not only requires that the deprotection agent be suitably basic such that the 9-H adjacent to the Fmoc fluorene ring system is removed, but also requires the removal of the resulting dibenzofulvene by-product 9-methylenefluorene (DBF) while maintaining a gentle deprotection environment to ensure the side chain protecting group safety of the polypeptide. Conventional Fmoc deprotection uses 20-50% by volume of a high concentration piperidine solution, resulting in a certain impediment to the use of base-sensitive protecting groups in part of the synthesis. It should be noted that piperidine itself has high biotoxicity and environmental toxicity, and belongs to easily-made toxic compounds in multiple countries (including China) worldwide, and is strictly limited in purchase and use by enterprises and universities under the strict control of government parts.

Generally, the more basic/nucleophilic the amine compound is, the more toxic it is often. Tetrahydroisoquinoline (TIQ), a natural alkaloid that is widely found in body fluids and/or tissues of the human body, including the brain, is a low toxicity secondary amine. TIQ is limited by the space of the benzene ring part of the TIQ, the molecule is generally close to a plane, and a chair-type conformation is not generated like piperidine, so that nitrogen atoms can be more easily added with DBF, and deprotection byproducts are efficiently removed. However, removal of Fmoc protecting groups by TIQ alone proved to be inefficient due to its weaker basicity. 1,5, 7-triazabicyclo [4.4.0] dec-5-ene (TBD) is a strong base with bridged cyclic guanidine groups, which is strong enough to rapidly deprotect Fmoc groups even at low concentrations, but which has a weak ability to scavenge DBF. Compared with most other strong organic alkali, TBD has the advantages of low toxicity, no volatilization, no malodor, stable property and the like. According to the invention, TBD is matched with TIQ, the advantages of the two components are combined, the advantages are improved, the effects of removing Fmoc protecting groups and removing 9-methylene fluorene (DBF) can be greatly improved, the substitution of piperidine in polypeptide synthesis is provided, and the risk of easy toxicity preparation and management is avoided.

The invention comprises the following steps:

the invention aims to provide a method for solid-phase synthesis of leupeptin by Fmoc method.

The technical scheme adopted by the invention is as follows:

a method for solid phase synthesis of leucinrphin by Fmoc method, which is characterized in that: the Fmoc deprotection agent used in the method is a mixed solution of TBD and TIQ.

Further, the TBD has the structure of

Further, the TIQ has the structure that

Further, the solvent of the mixed solution of TBD and TIQ is N, N-Dimethylformamide (DMF).

Further, the molar concentrations of TBD and TIQ in the above-mentioned mixed solution of TBD and TIQ were 0.2mol/L and 2.0mol/L, respectively.

Further, the first amino acid tyrosine in this method uses Fmoc-Tyr-OH as a starting material without the need for a side chain protecting group.

Further, the method comprises the following basic steps: in the deprotection step of polypeptide solid phase synthesis, DMF solution containing 0.2mol/L TBD and 2.0mol/L TIQ is added to resin loaded with Fmoc protected amino acid or polypeptide, and after shaking for 15 minutes at room temperature, the resin loaded with amino acid or polypeptide is filtered to obtain Fmoc protected group removed resin.

The Fmoc deprotection process using a mixed solution of TBD and TIQ may be:

r is any resin-loaded polypeptide moiety other than an N-terminal amine group, resulting inIs an N-dibenzofulvene-tetrahydroisoquinoline adduct (DBF-TIQ).

Compared with the traditional method using piperidine deprotection agent, the method for synthesizing the leucinrphine peptide by the solid phase has the following innovation and advantages:

TBD and TIQ are more stable than physical and chemical properties of piperidine, low in toxicity and volatilization, safer to use and low in price; TBD and TIQ do not belong to chemicals easy to be made and exploded, are not regulated by the regulations of safety management of dangerous chemicals and the regulations of management of easy to be made chemicals, and can be easily obtained in the purchasing and using processes of scientific research institutions and enterprises.

TBD only needs one tenth molar concentration of the dosage of organic alkali such as traditional piperidine, and Fmoc deprotection can be completed, and the low concentration has little influence on the stability of the whole deprotection system.

And 3, the TIQ effectively compensates a short plate with TBD and DBF removing capability, DBF-TIQ with an ultraviolet chromophore is preferentially generated in the deprotection process, no other adducts are generated, the excellent linearity of ultraviolet detection absorbance and concentration is ensured, and the reliability of the deprotection detection method is improved.

4. The invention realizes solid-phase synthesis of the leupeptin without any side chain protecting group by using a mixed solution of TBD and TIQ, and does not find possible byproducts such as tyrosine side chain acylation and the like caused by the traditional piperidine method. The crude product of the leucinrphine peptide obtained by the novel method has high purity, greatly reduces the cost and difficulty of subsequent purification, embodies the superiority of the method in solid phase synthesis of the leucinrphine peptide, and has great reference significance for production and preparation of other polypeptides.

5. The method for synthesizing the leucinrphin has no obvious difference from the conventional Fmoc synthesis method in operation steps and process flows, does not need additional training on the existing operators, and has no additional updating requirement on the existing equipment.

Description of the drawings

FIG. 1 Fmoc standard curve of Fmoc-alanine (Fmoc-L-Ala) deprotection with a DMF solution of TBD and TIQ mixture.

FIG. 2 shows HPLC comparison of polypeptide products of example 2 (solid phase synthesis of leupeptin using piperidine as the deprotecting agent component (no protecting group on Tyr side chain)), 3 (solid phase synthesis of leupeptin using piperidine as the deprotecting agent component (protecting group on Tyr side chain)), example 4 (solid phase synthesis of leupeptin using a mixed solution of TBD and TIQ as the deprotecting agent component (no protecting group on Tyr side chain)).

The specific embodiment is as follows:

the experimental materials, reagents, etc. used in the examples described below are all commercially available or known experimental methods. The technical solutions of the present invention will be clearly and completely described below with reference to specific embodiments and drawings, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Example 1 deprotection method test and preparation of Fmoc standard curve

Step 1. Using Fmoc-alanine (Fmoc-L-Ala) as a standard substance, 62.2mg of Fmoc-L-Ala was accurately weighed and treated with 2.0mL of a DMF solution containing 0.2mol/L TBD (55.7 mg) and 2.0mol/L TIQ (532.8 mg) for 15 minutes. Taking 1.0mL of the mixture, separating each product by thin layer chromatography, and comparing the product with an alanine standard sample to determine a reaction end point; identification of the product alanine ([ M+H) by mass spectrometry] ⁺ = 90.0513) and by-product DBF-TIQ ([ m+h)] ⁺ = 312.1750). The remaining 1.0mL of the 0.1mL solution was diluted to 10.0mL.

And 2, continuing to dilute the diluted solution obtained in the step 1 into standard solutions with the concentration of 23, 46, 66, 85, 107 and 120 mu mol/L. The absorbance of the comparative DMF solution blank sample (λ=289 nm) was measured by uv-vis spectrophotometer as: 0.23, 0.36, 0.48, 0.60, 0.75 and 0.83.

Step 3. A standard curve for Fmoc was prepared with a linear regression equation of Y=0.0062X+0.0769 (X-axis: concentration (. Mu. Mol/L), Y-axis: absorbance A, linear correlation coefficient: R2=0.9989) (FIG. 1).

EXAMPLE 2 solid phase synthesis of Lespeptin Using piperidine as a deprotection agent component (Tyr side chain has no protecting group)

Preparation of the preloaded resin: 200.4mg of Wang resin (loading 0.82 mmol/g) was weighed into a syringe reactor with a sieve plate. The resin was sequentially swollen with 5.0mL of Dichloromethane (DCM) and 5.0mL of Dimethylformamide (DMF). 87.0mg Fmoc-L-Leu (1.5 eq) and 128.3mg 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (PyBOP, 1.5 eq) were completely dissolved in 5.0mL DMF, 86. Mu. L N, N-diisopropylethylamine (DIPEA, 3.0 eq) were added, transferred into a reactor and mixed thoroughly with the swollen resin and then reacted with shaking for 40 minutes. After the reaction was completed, the resin was filtered. A DCM solution containing 31. Mu.L acetic anhydride (2.0 eq.) and 57. Mu.L DIPEA (2.0 eq.) was added. The unreacted hydroxyl groups on the resin were blocked by stirring for an additional 30 minutes at room temperature. Subsequently, the resin was filtered and washed 3 times sequentially with DMF, 2 times with DCM, 2 times with DMF, and finally 3 times with DCM. The resin was dried in vacuo.

Step 2, removing Fmoc protecting groups of the preloaded Fmoc-L-Leu on the resin: 5.0mL of 20% (v/v) piperidine in DMF was added, and after shaking at room temperature for 15 min, filtration was performed. Comparing 20% (v/v) piperidine in DMF blank (lambda=289 nm), measuring absorbance of the deprotected sample to confirm progress of the reaction, and taking very little resin for ninhydrin detection to appear dark blue, demonstrating complete deprotection.

Step 3. Loading amino acid Phe, gly, gly and Tyr and Fmoc deprotection thereof in sequence: washing the preloaded resin with DMF 5 times; 95.3mg Fmoc-L-Phe (1.5 eq), 86. Mu.L DIPEA (3.0 eq) and 128.3mg PyBOP (1.5 eq) were mixed, dissolved in 5.0mL DMF, transferred into a reactor and mixed thoroughly with the resin and reacted by shaking for 40 minutes. After the reaction was completed, the resin was filtered. Wash 3 times with DMF, 2 times with DCM, and 3 times with DMF. 5.0mL of 20% piperidine in DMF was added, shaken at room temperature for 15 min and then filtered. Comparing 20% (v/v) piperidine in DMF blank (lambda=289 nm), measuring absorbance of the deprotected sample to confirm progress of the reaction, and taking very little resin for ninhydrin detection to appear dark blue, demonstrating complete deprotection. The above procedure was repeated, with 73.2mg Fmoc-L-Gly (1.5 eq), 73.2mg Fmoc-L-Gly (1.5 eq) and 99.4mg Fmoc-L-Tyr (1.5 eq) being used as reactants to load the remaining amino acids L-Gly, L-Gly and L-Tyr onto the resin. Ninhydrin detection showed that deprotection was complete after loading of each amino acid by treatment with 20% (v/v) piperidine in DMF. After all amino acid loading and deprotection was completed, 3 washes with DMF, 2 washes with DCM, 3 washes with DMF and 5 washes with DCM. And (5) freeze-drying the resin.

Step 4. Cleavage of the polypeptide from the resin: adding 95% trifluoroacetic acid (TFA) water solution to the lyophilized resin, shaking for 1 hour, filtering the resin, and collecting liquid phase part solution (about 3 mL); the solution was precipitated in 20mL of cold diethyl ether; centrifuging the separated polypeptide completely with a centrifuge capable of reaching 3000 rpm to precipitate the polypeptide completely; washing three times with diethyl ether to wash out residual trifluoroacetic acid and other impurities, adding a small amount of cyclohexane, and lyophilizing to obtain crude product of enkephalin 77.3mg (HPLC purity 69.17%).

Step 5, purifying the polypeptide: the crude leupeptin was dissolved in a small amount of 90% acetonitrile in water (. About.2.0 mL), centrifuged and filtered through a 0.2 μm filter. Injecting into a preparative RP-HPLC column for purification. The mobile phase was 0.1% tfa in water and acetonitrile. The flow rate was 10mL/min, the gradient was 30% -50% (100 min), and the wavelength was set at 220nm. Isolation and lyophilization gave 16.3mg of the enkephalin product (final yield 17.9%, HPLC purity 90.9%).

EXAMPLE 3 solid phase Synthesis of Lespeptin Using piperidine as a deprotection agent component (Tyr side chain with protecting group)

Preparation of the preloaded resin: 200.4mg of Wang resin (loading 0.82 mmol/g) was weighed into a syringe reactor with a sieve plate. The resin was sequentially swollen with 5.0mL of Dichloromethane (DCM) and 5.0mL of Dimethylformamide (DMF). 87.0mg Fmoc-L-Leu (1.5 eq.) and 128.3mg PyBOP (1.5 eq.) were completely dissolved in 5.0mL DMF, 86. Mu.L DIPEA (3.0 eq.) was added, transferred into the reactor and mixed thoroughly with the swollen resin and reacted for 40 minutes with shaking. After the reaction was completed, the resin was filtered. A DCM solution containing 31. Mu.L acetic anhydride (2.0 eq.) and 57. Mu.L DIPEA (2.0 eq.) was added. The unreacted hydroxyl groups on the resin were blocked by stirring for an additional 30 minutes at room temperature. Subsequently, the resin was filtered and washed 3 times sequentially with DMF, 2 times with DCM, 2 times with DMF, and finally 3 times with DCM. The resin was dried in vacuo.

Step 2, removing Fmoc protecting groups of the preloaded Fmoc-L-Leu on the resin: 5.0mL of 20% (v/v) piperidine in DMF was added, and after shaking at room temperature for 15 min, filtration was performed. Comparing 20% (v/v) piperidine in DMF blank (lambda=289 nm), measuring absorbance of the deprotected sample to confirm progress of the reaction, and taking very little resin for ninhydrin detection to appear dark blue, demonstrating complete deprotection.

Step 3. Loading amino acid Phe, gly, gly and Tyr and Fmoc deprotection thereof in sequence: washing the preloaded resin with DMF 5 times; 95.3mg Fmoc-L-Phe (1.5 eq), 86. Mu.L DIPEA (3.0 eq) and 128.3mg PyBOP (1.5 eq) were mixed, dissolved in 5.0mL DMF, transferred into a reactor and mixed thoroughly with the resin and reacted by shaking for 40 minutes. After the reaction was completed, the resin was filtered. Wash 3 times with DMF, 2 times with DCM, and 3 times with DMF. 5.0mL of 20% piperidine in DMF was added, shaken at room temperature for 15 min and then filtered. Comparing 20% (v/v) piperidine in DMF blank (lambda=289 nm), measuring absorbance of the deprotected sample to confirm progress of the reaction, and taking very little resin for ninhydrin detection to appear dark blue, demonstrating complete deprotection. The above procedure was repeated, and the remaining amino acids L-Gly, L-Gly and L-Tyr (tBu) were loaded onto the resin with 73.2mg Fmoc-L-Gly (1.5 eq), 73.2mg Fmoc-L-Gly (1.5 eq) and 113.1mg Fmoc-L-Tyr (tBu) (1.5 eq) in sequence as reactants. Ninhydrin detection showed that deprotection was complete after loading of each amino acid by treatment with 20% (v/v) piperidine in DMF. After all amino acid loading and deprotection was completed, 3 washes with DMF, 2 washes with DCM, 3 washes with DMF and 5 washes with DCM. And (5) freeze-drying the resin.

Step 4. Cleavage of the polypeptide from the resin: adding 95% trifluoroacetic acid (TFA) water solution to the lyophilized resin, shaking for 1 hour, filtering the resin, and collecting liquid phase part solution (about 3 mL); the solution was precipitated in 20mL of cold diethyl ether; centrifuging the separated polypeptide completely with a centrifuge capable of reaching 3000 rpm to precipitate the polypeptide completely; washing three times with diethyl ether to wash out residual trifluoroacetic acid and other impurities, adding a small amount of cyclohexane, and lyophilizing to obtain crude product of enkephalin 74.5mg (HPLC purity 81.3%).

Step 5, purifying the polypeptide: the crude leupeptin was dissolved in a small amount of 90% acetonitrile in water (. About.2.0 mL), centrifuged and filtered through a 0.2 μm filter. Injecting into a preparative RP-HPLC column for purification. The mobile phase was 0.1% tfa in water and acetonitrile. The flow rate was 10mL/min, the gradient was 30% -50% (100 min), and the wavelength was set at 220nm. After isolation and lyophilization 70.2mg of the enkephalin product was obtained (final yield 76.3%, HPLC purity 98.9%).

EXAMPLE 4 solid phase Synthesis of Lespeptin Using a Mixed solution of TBD and TIQ as the deprotection agent component (Tyr side chain has no protecting group)

Preparation of the preloaded resin: 200.4mg of Wang resin (loading 0.82 mmol/g) was weighed into a syringe reactor with a sieve plate. The resin was sequentially swollen with 5.0mL of Dichloromethane (DCM) and 5.0mL of Dimethylformamide (DMF). 87.0mg Fmoc-L-Leu (1.5 eq) and 128.3mg 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (PyBOP, 1.5 eq) were completely dissolved in 5.0mL DMF, 86. Mu. L N, N-diisopropylethylamine (DIPEA, 3.0 eq) were added, transferred into a reactor and mixed thoroughly with the swollen resin and then reacted with shaking for 40 minutes. After the reaction was completed, the resin was filtered. A DCM solution containing 31. Mu.L acetic anhydride (2.0 eq.) and 57. Mu.L DIPEA (2.0 eq.) was added. The unreacted hydroxyl groups on the resin were blocked by stirring for an additional 30 minutes at room temperature. Subsequently, the resin was filtered and washed 3 times sequentially with DMF, 2 times with DCM, 2 times with DMF, and finally 3 times with DCM. The resin was dried in vacuo.

Step 2, removing Fmoc protecting groups of the preloaded Fmoc-L-Leu on the resin: 5.0mL of DMF solution containing 0.2mol/L TBD and 2.0mol/L TIQ was added, and the mixture was stirred at room temperature for 15 minutes and then filtered. Comparing DMF solution blank samples (lambda=289 nm) containing 0.2mol/L TBD and 2.0mol/L TIQ, measuring absorbance of the deprotected samples to confirm the progress of the reaction, and taking very little resin for ninhydrin detection to appear dark blue, thus proving that the deprotection is complete.

Step 3. Loading amino acid Phe, gly, gly and Tyr and Fmoc deprotection thereof in sequence: washing the preloaded resin with DMF 5 times; 95.3mg Fmoc-L-Phe (1.5 eq), 86. Mu.L DIPEA (3.0 eq) and 128.3mg PyBOP (1.5 eq) were mixed, dissolved in 5.0mL DMF, transferred into a reactor and mixed thoroughly with the resin and reacted by shaking for 40 minutes. After the reaction was completed, the resin was filtered. Wash 3 times with DMF, 2 times with DCM, and 3 times with DMF. 5.0mL of DMF solution containing 0.2mol/L TBD and 2.0mol/L TIQ was added, and the mixture was stirred at room temperature for 15 minutes and then filtered. Comparing DMF solution blank samples (lambda=289 nm) containing 0.2mol/L TBD and 2.0mol/L TIQ, measuring absorbance of the deprotected samples to confirm the progress of the reaction, and taking very little resin for ninhydrin detection to appear dark blue, thus proving that the deprotection is complete. The above procedure was repeated, with 73.2mg Fmoc-L-Gly (1.5 eq), 73.2mg Fmoc-L-Gly (1.5 eq) and 99.4mg Fmoc-L-Tyr (1.5 eq) being used as reactants to load the remaining amino acids L-Gly, L-Gly and L-Tyr onto the resin. Ninhydrin detection showed that deprotection was complete after each amino acid loading by treatment with DMF solution containing 0.2mol/L TBD and 2.0mol/L TIQ. After all amino acid loading and deprotection was completed, 3 washes with DMF, 2 washes with DCM, 3 washes with DMF and 5 washes with DCM. And (5) freeze-drying the resin.

Step 4. Cleavage of the polypeptide from the resin: adding 95% trifluoroacetic acid (TFA) water solution to the lyophilized resin, shaking for 1 hour, filtering the resin, and collecting liquid phase part solution (about 3 mL); the solution was precipitated in 20mL of cold diethyl ether; centrifuging the separated polypeptide completely with a centrifuge capable of reaching 3000 rpm to precipitate the polypeptide completely; washing three times with diethyl ether to wash out residual trifluoroacetic acid and other impurities, adding a small amount of cyclohexane, and lyophilizing to obtain 88.8mg (HPLC purity 87.1%) of crude enkephalin.

Step 5, purifying the polypeptide: the crude leupeptin was dissolved in a small amount of 90% acetonitrile in water (. About.2.0 mL), centrifuged and filtered through a 0.2 μm filter. Injecting into a preparative RP-HPLC column for purification. The mobile phase was 0.1% tfa in water and acetonitrile. The flow rate was 10mL/min, the gradient was 30% -50% (100 min), and the wavelength was set at 220nm. Isolation and lyophilization gave 63.5mg of the enkephalin product (final yield 75.2%, HPLC purity 98.1%).

Even though the side chain is free of protecting groups, the actual yield of example 4 is still about four times higher than that of example 2, and the yield and purity of the final product are also comparable to those of example 3 (FIG. 2) of the conventional process requiring additional side chain protecting groups.

The applicant states that the detailed method of the present invention is illustrated by the above examples, but the present invention is not limited to the detailed method described above, i.e. it does not mean that the present invention must be practiced in dependence upon the detailed method described above. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc. are included in the scope of the present invention and the scope of disclosure.

Claims (1)

1. A method for Fmoc deprotection in polypeptide solid-phase synthesis is characterized in that: the Fmoc deprotection method in polypeptide solid-phase synthesis comprises the following basic steps: in the deprotection step of the solid-phase synthesis of the leucinrphine, DMF solution containing 0.2 mol/L1, 5, 7-triazabicyclo [4.4.0] dec-5-ene and 2.0mol/L tetrahydroisoquinoline is added to resin loaded with Fmoc-protected amino acid or polypeptide, and after shaking for 15 minutes at room temperature, the resin loaded with amino acid or polypeptide is filtered to obtain the Fmoc-protected resin.