WO2018057470A1 - Synthèse rapide de peptides à températures élevées - Google Patents

Synthèse rapide de peptides à températures élevées Download PDF

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Publication number
WO2018057470A1
WO2018057470A1 PCT/US2017/052099 US2017052099W WO2018057470A1 WO 2018057470 A1 WO2018057470 A1 WO 2018057470A1 US 2017052099 W US2017052099 W US 2017052099W WO 2018057470 A1 WO2018057470 A1 WO 2018057470A1
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minutes
resin
peptide
mmol
synthesis
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PCT/US2017/052099
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English (en)
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Dario SLAVAZZA
Heng Wei CHANG
Jason Chang
Wei Wang
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C S Bio Co.
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Publication of WO2018057470A1 publication Critical patent/WO2018057470A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C07K1/045—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers using devices to improve synthesis, e.g. reactors, special vessels
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
    • C07K1/08—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using activating agents

Definitions

  • the present invention relates to the field of peptide synthesis, and particularly, to methods of rapidly synthesizing peptides at elevated temperatures.
  • Peptides are widely used for biological and clinical research. Peptides are routinely used as epitopes for generating vaccines, and several peptides are approved for clinical therapy. Typically, peptide vaccine epitopes are 6 to 40 amino acids long. In general, it takes three to five days (72 to 120 hours) for an automated peptide synthesizer to produce a 40-mer peptide using Fmoc chemistry. Shortening the overall synthetic time would therefore provide higher productivity and would lower synthesis costs.
  • HATU/DIEA hexafluorophosphate/diisopropylethylamine
  • Microwave synthesizers can accelerate the completion of solid phase peptide synthesis (SPPS) coupling and deblocking reactions in a shorter time, as microwave-assisted SPPS reactions are faster than those under traditional SPPS conditions.
  • SPPS solid phase peptide synthesis
  • both the microwave power and reaction temperature need to be controlled very carefully, and usually after >10 coupling cycles, microwave-assisted SPPS gives a peptide-resin in low yield with a dark color.
  • the microwave energy stimulates molecular vibrations among the solvent molecules, the resin, and the anchored peptides, overheating can occur during the coupling and deFmoc reactions, and some of the growing peptide chains can be lost during synthesis.
  • the harsh conditions of microwave-assisted SPPS also generate more undesired by-products from side reactions involving amino acid side chains.
  • HATU/DIEA and [(6-chlorobenzotriazol-l-yl)oxy-(dimethylamino)methylidene]- dimethylazanium hexafluorophosphate (HCTU)/DIEA are more efficient peptide coupling reagents, and the coupling reactions are usually complete in a short time.
  • the efficacy of HBTU coupling is usually high after one hour, and reaction times of 1 to 3 hours are recommended to provide sufficient mixing of the Fmoc amino acid solution and the resin.
  • the efficacy of HATU coupling is usually high after 45 minutes, and reaction times of 20 to 45 minutes are
  • uronium salts such as HBTU, HATU, and HCTU are expensive, especially for the large-scale peptide synthesis needed to produce vaccine epitopes.
  • Diisopropyl carbodiimide (DIC) and HOBt are mild coupling reagents, and much less expensive than HBTU, HATU, and HCTU.
  • the coupling efficacy of DIC and HOBt is typically good after 2-3 hours at room temperature.
  • the longer coupling reaction time needed for good coupling efficacy with DIC/HOBt leads to longer synthesis times and increased expense.
  • the invention provides methods for rapid solid-phase peptide synthesis at elevated temperatures, which can be used for both small-scale and large-scale synthesis of peptides.
  • Elevated temperatures provide numerous advantages during the rapid heated peptide synthesis methods. Reaction rates are accelerated, and are completed in less time, improving the overall efficiency of the process and reducing the time needed for synthesis. Intrachain or interchain structures which may form during peptide synthesis, such as beta turns or aggregating peptide chains, are disrupted at elevated temperatures and pose less hindrance to completion of coupling. Elevated temperatures also provide for better mixing and penetration of solvents and reagents into the resin, which also enhances coupling efficiency and reduces synthesis time. The methods for synthesis described herein also minimize undesired side reactions which have affected past attempts at using elevated temperatures, while at the same time preserving the advantages of the elevated temperatures. In contrast to earlier methods, the methods of the invention result in good stereochemical purity and lower amounts of other side reactions, as well as good overall yield.
  • Peptides with sensitive residues such as Cys, Met, and Trp
  • the methods of the invention also facilitate non-standard reactions, such as peptide modification including PEGylation or coupling with palmitic acid, stearic acid, etc., because the elevated temperatures increase their molecular motion and effective concentration, leading to better reaction yields.
  • the invention embraces a method for rapid solid-phase peptide synthesis of a desired peptide sequence on a resin, comprising:
  • the contacting with a coupling solution can be carried out at a temperature between about 50 °C and about 70 °C.
  • the invention embraces a method for rapid solid-phase peptide synthesis of a desired peptide sequence on a resin, comprising: [0017] a) contacting a resin-bound peptide or amino acid having a protected N-terminus with a deblocking solution comprising a deblocking reagent, to form an N-terminal-unprotected resin- bound peptide or amino acid, for between about 4 and about 20 minutes at a temperature between about 40 °C and about 80 °C;
  • the contacting with the deblocking solution can be performed for between about 4 minutes to about 10 minutes,
  • the contacting with the deblocking solution can be performed for between about 7 minutes to about 20 minutes, and
  • the contacting with the deblocking solution can be performed either for between about 4 minutes to about 10 minutes or between about 7 minutes to about 20 minutes;
  • the contacting with the coupling solution can be performed for between about 10 minutes to about 30 minutes,
  • the contacting with the coupling solution can be performed for between about 20 minutes to about 60 minutes, and
  • the contacting with the coupling solution can be performed either for between about 10 minutes to about 30 minutes or between about 20 minutes to about 60 minutes;
  • the contacting with a coupling solution can be carried out at a temperature between about 50 °C and about 70 °C.
  • the carbodiimide activating reagent can comprise a reagent selected from the group consisting of diisopropylcarbodiimide (DIC), dicyclohexylcarbodiimide (DCC), and l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).
  • the carbodiimide activating reagent can comprise diisopropylcarbodiimide (DIC).
  • the active ester-forming reagent can comprise a reagent selected from the group consisting of 1-hydroxybenzotriazole (HOBt) and 1- hydroxy-7-azabenzotriazole (HO At).
  • HOBt 1-hydroxybenzotriazole
  • HO At 1- hydroxy-7-azabenzotriazole
  • the N-protected amino acid and the protected N-terminus can be protected with 9-fluorenylmethoxycarbonyl (Fmoc) groups.
  • the resin can comprise a resin selected from the group consisting of 4-methylbenzhydrylamine (MB HA) resin, benzhydrylamine (BHA) resin, Wang resin, or aminomethyl (AM) resin.
  • the resin can comprise a resin selected from the group consisting of 4-methylbenzhydrylamine (MBHA) resin, Wang resin, or aminomethyl (AM) resin.
  • the deblocking solution can comprise a dipolar aprotic solvent
  • the coupling solution independently can comprise a dipolar aprotic solvent
  • the deblocking solution can comprise a solvent selected from the group consisting of dimethylformamide (DMF) and N-methyl-2- pyrrolidone (NMP), and the coupling solution independently can comprise a solvent selected from the group consisting of dimethylformamide (DMF) and N-methyl-2-pyrrolidone (NMP).
  • DMF dimethylformamide
  • NMP N-methyl-2- pyrrolidone
  • the concentrations of N-protected amino acid, carbodiimide activating reagent, and active ester-forming reagent can be independently between about 0.2 molar and 0.4 molar.
  • the solution of N-protected amino acid, carbodiimide activating reagent, and active ester-forming reagent can comprise approximately equal molar amounts of N-protected amino acid, carbodiimide activating reagent, and active ester-forming reagent.
  • the deblocking reagent of the deblocking solution can comprise piperidine.
  • the deblocking solution can comprise about 20% piperidine.
  • the method can further comprise aa) washing the resin after a); bb) washing the resin after b); and repeating aa) and bb) each time a) and b) are repeated.
  • the washing of the resin can comprise washing the resin with a dipolar aprotic solvent.
  • contacting the resin-bound peptide or amino acid having a protected N-terminus with the deblocking solution can be performed at least twice before contacting the resin with the coupling solution, and can comprise: al) contacting the resin-bound peptide or amino acid having a protected N-terminus with a first portion of deblocking solution comprising the deblocking reagent; a2) draining the deblocking solution from the resin; a3) optionally washing the resin; a4) contacting the resin-bound peptide or amino acid having a protected N-terminus with a second portion of deblocking solution comprising a deblocking reagent; and a5) draining the deblocking solution from the resin.
  • the amount of time for contacting the resin-bound peptide or amino acid having a protected N-terminus with the deblocking solution can be about 2 minutes to about 6 minutes for al), and about 2 minutes to about 20 minutes or about 4 minutes to 10 minutes for a4).
  • contacting the resin-bound peptide or amino acid having a protected N-terminus with the deblocking solution can be performed at least twice before contacting the resin with the coupling solution, and can comprise: al) contacting the resin-bound peptide or amino acid having a protected N-terminus with a first portion of deblocking solution comprising the deblocking reagent; a2) draining the deblocking solution from the resin; a3) optionally washing the resin; a4) contacting the resin-bound peptide or amino acid having a protected N-terminus with a second portion of deblocking solution comprising a deblocking reagent; and a5) draining the deblocking solution from the resin.
  • the amount of time for contacting the resin-bound peptide or amino acid having a protected N-terminus with the deblocking solution can be: i) about 2 minutes to about 5 minutes for al) and about 2 minutes to about 10 minutes for a4) when the scale of synthesis is at or below about 1 mmol of peptide; ii) about 3 minutes to about 5 minutes for al) and about 7.5 minutes to about 12.5 minutes for a4) when the scale of synthesis is above about 5 mmol of peptide; and iii) either about 2 minutes to about 5 minutes for al) and about 2 minutes to about 10 minutes for a4), or about 3 minutes to about 5 minutes for al) and about 7.5 minutes to about 12.5 minutes for a4), when the scale of synthesis is between about 1 mmol of peptide to about 5 mmol of peptide.
  • the peptide synthesis can be performed on a scale to yield about 50 mg to about 20 grams of crude peptide, about 50 mg to about 15 grams of crude peptide, about 50 mg to about 10 grams of crude peptide, about 50 mg to about 5 grams of crude peptide, about 50 mg to about 2 grams of crude peptide, about 50 mg to about 1 gram of crude peptide, about 50 mg to about 750 mg of crude peptide, about 50 mg to about 500 mg of crude peptide, about 50 mg to about 250 mg of crude peptide, about 50 mg to about 200 mg of peptide, or about 50 mg to about 100 mg of peptide.
  • the crude peptide can have a purity of at least about 20% as determined by analytical HPLC, or of at least about 30%, preferably at least about 40%.
  • the peptide synthesis can be performed on a scale to yield about 0.2 mmol to about 0.4 mmol of peptide.
  • contacting the N-terminal-unprotected resin-bound peptide or amino acid with the coupling solution can be conducted under neutral conditions, non-basic conditions, or without added base.
  • the peptide synthesis can be performed without microwave irradiation.
  • the peptide synthesis can be performed without added salts.
  • FIG. 1 depicts the purity of various synthesized peptides, versus the temperature of synthesis.
  • FIG. 2 depicts the weight gain of the peptide-resins (resin weight after synthesis minus resin weight before synthesis) for the various synthesized peptides.
  • deblock refers to removal of the protecting group of the amine group (-NH 2 ) at the N-terminus of a peptide attached to a resin during solid phase peptide synthesis (also referred to as “deprotect”).
  • deprotect also referred to as "deprotect”
  • deFmoc solid phase peptide synthesis
  • Lame peptide refers to peptide product after cleavage of the peptide chain from the resin and removal of protecting groups from the peptide chain, and after removal of volatile cleavage reagents and volatile cleavage products, but before purification.
  • “approximately 50° C” includes both the disclosure of 50° C itself, as well as values close to 50° C.
  • the phrases “about X” or “approximately X” include a description of the value X itself. If a range is indicated, such as “approximately 50° C to 60° C” or “about 50° C to 60° C,” it is understood that both the values specified by the endpoints are included, and that values close to each endpoint or both endpoints are included for each endpoint or both endpoints; that is, “approximately 50° C to 60° C” (or “about 50° C to 60° C") is equivalent to reciting both “50° C to 60° C” and “approximately 50° C to approximately 60° C” (or “about 50° C to about 60° C”).
  • any disclosed upper limit for a component or parameter may be combined with any disclosed lower limit for that component or parameter to provide a range (provided that the upper limit is greater than the lower limit with which it is to be combined).
  • Each of these combinations of disclosed upper and lower limits are explicitly envisaged herein. For example, if ranges for the amount of a particular component or parameter are given as 10% to 30%, 10% to 12%, and 15% to 20%, the ranges 10% to 20% and 15% to 30% are also envisaged, whereas the combination of a 15% lower limit and a 12% upper limit is not possible and hence is not envisaged.
  • relative percentages in a composition assumes that the combined total percentages of all components in the composition add up to 100. It is further understood that relative percentages of one or more components may be adjusted upwards or downwards such that the percent of the components in the composition combine to a total of 100, provided that the percent of any particular component does not fall outside the limits of the range specified for that component.
  • the device, composition, or system either does not contain any other elements which do materially affect the condition being treated other than those elements expressly listed (for compositions for treating systems) or does not contain any other elements which do materially affect the properties of the device or system; or, if the device, composition, or system does contain extra elements other than those listed which may materially affect the condition being treated or the properties of the system, the device, composition or system does not contain a sufficient concentration or amount of those extra elements to materially affect the condition being treated by the composition or the properties of the device or system.
  • the method contains the steps listed, and may contain other steps that do not materially affect the condition being treated by the method or the properties of the device, composition, or system produced by or used by the method, but the method does not contain any other steps which materially affect the condition being treated by the method or the device, composition, or system produced or used other than those steps expressly listed.
  • the methods of the present invention provide for rapid synthesis of peptides at elevated temperatures, with good resulting yield, purity, and stereochemical purity.
  • an automated or semi-automated peptide synthesizer is used to perform the solid-phase peptide synthesis (SPPS).
  • [0065] 2 attaching the initial N-protected amino acid to the resin or other solid support.
  • the initial amino acid attached to the resin is the C-terminal amino acid of the desired peptide sequence.
  • Various solid- phase peptide synthesis resins with the first protected amino acid attached are widely available commercially for use as starting material. Thus, purchasing such a protected amino acid-resin completes 1) and 2);
  • Solid-phase peptide synthesis provides a convenient way of isolating the synthetic intermediates, as they are attached to the solid resin.
  • the reagent solutions can be removed simply by draining them away from the resin, for example, on a fritted glass filter. Washing is typically performed before and after deblocking and coupling, in order to remove traces of deblocking or coupling reagents, and isolating the resin from the washing solution is similarly accomplished conveniently simply by draining the solution from the resin.
  • a typical procedure for rapid heated peptide synthesis uses a glass reaction vessel jacketed with a water bath for heating of the reaction vessel.
  • the water bath can heat the reagents in the reaction vessel quickly to or near the temperature of the water bath, typically in about 15 to about 20 seconds.
  • Temperature used for the synthesis about 40°C to about 80°C, about 45 °C to about
  • the protected amino acid (amino acid to be coupled to peptide-resin) is used in an amount at least equal to one equivalent of the initial loading of the resin. Preferably, about 1 to about 3 equivalents, or about 1.5 to about 3 equivalents, of protected amino acid are used per equivalent of peptide (or amino acid) bound to the resin.
  • the protected amino acid is typically dissolved in a polar aprotic solvent, such as DMF or NMP.
  • Coupling reagents DIC/HOBt; about 0.2 to about 0.4 M DIC in a polar aprotic solvent, such as DMF or NMP; about 0.2 to about 0.4 M HOBt in a polar aprotic solvent, such as DMF or NMP.
  • DIC/HOBt a polar aprotic solvent
  • HOBt a polar aprotic solvent
  • Approximately equimolar amounts of DIC and HOBt are used, and an approximately equimolar amount of DIC and HOBt are used with respect to the protected amino acid to be activated by the activating and active ester-forming reagents.
  • Coupling time about 10 minutes to about 30 minutes, for synthesis on a scale below about 1 mmol, such as about 0.1 to about 1 mmol. About 20 minutes to about 60 minutes for synthesis on a scale between about 1 mmol and about 5 mmol. About 40 minutes to about 60 minutes for synthesis on a scale above about 5 mmol, such as about 5 mmol to about 7.5 mmol.
  • Washing solvent after coupling DMF.
  • Washing after coupling about 2 to about 3 washes for about 20 to about 40 seconds per each wash.
  • Kaiser ninhydrin test (Kaiser, E. et al., Analytical Biochemistry 34(2):595 ( 1970)) can be used. In some embodiments of the methods of the invention, a Kaiser test is performed after each coupling.
  • a Kaiser test is performed only after a difficult coupling, such as a coupling step known to proceed in less than desired yield based on prior experience with the synthesis, or a coupling step which is predicted to proceed in less than desired yield based on literature reports (examples of difficult couplings are coupling of a beta-branched residue to another beta- branched residue, or syntheses where the peptide sequence is highly hydrophobic or known to be prone to aggregation).
  • a second coupling (“double-coupling") is performed if coupling is incomplete (less than about 99.5% complete, less than about 99% complete, less than about 98% complete, or less than about 97% complete, as desired).
  • Acetylation after coupling is optional, but typically not necessary.
  • Deblocking/DeFmoc reagent about 20% piperidine in DMF.
  • Deblocking/DeFmoc time two deblock steps. For synthesis below about 1 mmol, such as about 0.1 mmol to about 1 mmol, deblocking times of about 2 minutes and about 5 minutes sequentially are used. For synthesis on a scale between about 1 mmol and about 5 mmol, deblocking times of about 2 minutes and about 8 minutes sequentially are used. For synthesis on a scale above about 5 mmol, such as about 5 mmol to about 7.5 mmol, deblocking times of about 4 minutes and about 10 minutes sequentially are used. Alternatively, one deblock step, for the combined time of the two steps, can be used.
  • three deblock steps for the combined time of the two steps, can be used, such as about 1 minute, about 2 minutes, and about 4 minutes for synthesis below about 1 mmol, such as about 0.1 mmol to about 1 mmol; about 2 minutes, about 2 minutes, and about 6 minutes for synthesis on a scale between about 1 mmol and about 5 mmol; and about 2 minutes, about 4 minutes, and about 8 minutes for synthesis on a scale above about 5 mmol, such as about 5 mmol to about 7.5 mmol.
  • the three deblock steps can be of equal length; that is, each of the three deblock steps can be for a period of about the total length of the two combined steps, divided by three. Other patterns of deblock times can be used for a three-step deblocking process.
  • Washing solvents after deblocking DMF/DCM.
  • Washing time after deblocking about 5 to about 7 washes for about 30 seconds to about 40 seconds per each wash.
  • Total cycle time about 30 minutes to about 35 minutes.
  • the N-protecting group of the last amino acid to be coupled (which is the N-terminal amino acid of the desired sequence) is removed. Then the peptide -resin is treated with appropriate cleavage reagents, liberating the free peptide.
  • the cleavage reaction also preferably deprotects the side chains of the amino acids.
  • Side chain protecting groups are well-known; see, for example, M Bodanszky and A Bodanszky , "The Practice of Peptide Synthesis," 2 Ed, Springer- Verlag, 1994, J Jones, "The Chemical Synthesis of Peptides," Clarendon Press, 1991. and Dryland et al , 1986, J Chem Soc . Perkin Trans 1 125- 137).
  • Peptides synthesized using Fmoc chemistry are typically cleaved from the resin using acidic reagents, including, but not limited to, trifluoroacetic (TFA) acid, trifluoromethanesulfonic acid, hydrogen bromide, hydrogen chloride, hydrogen fluoride, etc.
  • acidic cleavage solution further comprises one or more scavengers, including, but not limited to, ethanedithiol (EDT), triisopropylsilane (TIS), phenol, and thioanisole.
  • the acidic cleavage solution comprises TFA. In some embodiments, the acidic cleavage solution comprises water. In some embodiments, the acidic cleavage solution comprises EDT. In some embodiments, the acidic cleavage solution comprises TIS. In some embodiments, the acidic cleavage solution comprises TFA, EDT, TIS and water.
  • Suitable concentrations of TFA in the acidic cleavage solution include, but are not limited to, at least about any of 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or more as measured by volume. In some embodiments, the acidic cleavage solution comprises about 94% of TFA by volume.
  • Suitable concentration of a scavenger (such as EDT or TIS) in the acidic cleavage solution include, but are not limited to, no more than about any of 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2.5%, 2%, 1.5%, 1 % or less.
  • the acidic cleavage solution comprises about 2% of EDT by volume.
  • the acidic cleavage solution comprises about 2% of TIS by volume.
  • the acidic cleavage solution comprises about 2% of EDT and about 2% of TIS by volume.
  • Suitable concentration of water in the acidic cleavage solution include, but are not limited to, no more than about any of 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2.5%, 2%, 1.5%, 1 % or less.
  • the acidic cleavage solution comprises about 2% of water by volume.
  • the acidic cleavage solution comprises about 94% of TFA, 2% of EDT, 2% of TIS and about 2% of water by volume.
  • exemplary methods for purifying peptides include, but are not limited to, reverse- phase chromatography (such as using a C4, C8 or C18 column), ion exchange chromatography, and size exclusion chromatography.
  • Peptides can be synthesized on a scale to provide about 50mg to about 100 mg of peptide with a purity of greater than about 85% after purification. For a purification yield of at least about 10%, about 500 mg to 1,000 mg of crude peptide can be synthesized. These parameters are provided for an approximately 20-residue-long peptide having a MW between about 2000 to about 3000 Dalton, and can be adjusted as needed for peptides of different length or molecular weight, different amounts of total purified peptide desired, different percent yields after purification, or different desired purity levels. Resins for use in rapid heated peptide synthesis
  • any solid-phase peptide synthesis resin, gel, or other solid support can be used in rapid heated peptide synthesis which results in good crude peptide yield and purity.
  • resins useful in rapid heated peptide synthesis are 4-methylbenzhydrylamine (MB HA) resin, benzhydrylamine (BHA) resin, Wang resin, and aminomethyl (AM) resin.
  • rapid heated peptide synthesis is performed using 4-methylbenzhydrylamine (MBHA) resin, benzhydrylamine (BHA) resin, Wang resin, or aminomethyl (AM) resin.
  • rapid heated peptide synthesis is performed using 4-methylbenzhydrylamine (MBHA) resin, Wang resin, or aminomethyl (AM) resin.
  • rapid heated peptide synthesis is performed using 4-methylbenzhydrylamine (MBHA) resin. In one embodiment, rapid heated peptide synthesis is performed using benzhydrylamine (BHA) resin. In one embodiment, rapid heated peptide synthesis is performed using Wang resin. In one embodiment, rapid heated peptide synthesis is performed using aminomethyl (AM) resin.
  • MBHA 4-methylbenzhydrylamine
  • BHA benzhydrylamine
  • AM aminomethyl
  • Solid-phase peptide synthesis resins and gels, along with other peptide synthesis reagents, are available from a wide variety of suppliers, including Sigma- Aldrich (St. Louis, Missouri, USA), Bachem (San Carlos, California, USA), or Novabiochem (part of EMD
  • Rapid heated peptide synthesis can be performed between about 40°C and about 80°C. Preferred ranges include between about 45 °C and about 75 °C, between about 50°C and about 70°C, or between about 55 °C and about 65 °C. A preferred temperature for rapid heated peptide synthesis is about 60°C.
  • Additional ranges for rapid heated peptide synthesis include between about 40°C and about 70°C, between about 50°C and about 80°C, between about 40°C and about 60°C, between about 60°C and about 80°C, between about 40°C and about 50°C, between about 50°C and about 60°C, between about 60°C and about 70°C, between about 70°C and about 80°C, between about 45°C and about 55°C, and between about 65°C and about 75°C.
  • the reaction vessel heating element such as a water jacket or heating mantle, is kept at the desired temperature or temperature range.
  • Solvents and solutions can be introduced at room temperature (for example, from a reservoir at room temperature), or can be pre -heated to the desired temperature in a warming chamber before introduction into the reaction vessel containing the peptide-resin. Alternatively, solvents and solutions can be kept in a heated reservoir at the desired temperature.
  • the water jacket of the CS 136H Automated Synthesizer used in the Examples can heat solvents and solutions to the desired temperature within about 15 to about 20 seconds, and typically the solvents and solutions are introduced at room temperature, with subsequent heating of the solvents or solutions to the desired temperature.
  • Coupling reagents activating reagents/active ester-forming reagents for rapid heated peptide synthesis
  • Bases such as diisopropylethylamine (DIEA) or triethylamine (TEA), are often added to peptide coupling reactions as promoters. These hindered bases act to increase the completion rate of coupling. However, the presence of base also increases the danger of racemization of the activated amino acids.
  • the current invention minimizes the racemization side reaction, by excluding the addition of base during the coupling reaction. That is, the coupling reactions of the methods of the present invention are run under neutral conditions, without adding base to the mixture of solvent, resin, protected amino acid, activating reagent, and activated ester-forming reagent.
  • the free amino groups of the peptide chains on the resin which are present during coupling are of course basic, but the methods of the current invention refer to exclusion of any added bases to the reaction mixture.
  • the activating reagent comprises a carbodiimide activating reagent.
  • the carbodiimide activating reagent comprises a reagent selected from the group consisting of diisopropylcarbodiimide (DIC), dicyclohexylcarbodiimide (DCC), and l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).
  • the carbodiimide activating reagent comprises
  • the active ester-forming reagent used with the activating reagent comprises a benzotriazole or azabenzotnazole reagent.
  • the active ester-forming reagent used with the activating reagent comprises a reagent selected from the group consisting of 1-hydroxybenzotriazole (HOBt) and l-hydroxy-7- azabenzotriazole (HO At).
  • the active ester-forming reagent used with the activating reagent comprises 1-hydroxybenzotriazole (HOBt).
  • the activating reagent comprises
  • DIC diisopropylcarbodiimide
  • HOBt 1-hydroxybenzotriazole
  • the coupling reaction is conducted under neutral conditions. In any of the above embodiments, the coupling reaction is conducted under non- basic conditions. In any of the above embodiments, the reaction mixture used for coupling excludes added bases.
  • the protected amino acid, the activating reagent, and the active-ester forming reagent can be used in approximately equimolar ratios to each other in the coupling solution.
  • the protected amino acid, the activating reagent, and the active-ester forming reagent can be used in ratios of about 1 equivalent to about 5 equivalents, about 1 equivalent to about 4 equivalents, about 1 equivalent to about 3 equivalents, about 1 equivalent to about 2 equivalents, about 1 equivalent to about 1.5 equivalents, or about 1 equivalent to about 1.2 equivalents with respect to the peptide-resin or amino acid-resin to which the protected amino acid is to be coupled.
  • the protected amino acid, the activating reagent, and the active-ester forming reagent can be used in ratios of about 1.2 equivalents to about 5 equivalents, about 1.2 equivalents to about 4 equivalents, about 1.2 equivalents to about 3 equivalents, about 1.2 equivalents to about 2 equivalents, about 1.2 equivalents to about 1.5 equivalents, or about 1 equivalents to about 1.4 equivalents, with respect to the peptide-resin or amino acid-resin to which the protected amino acid is to be coupled.
  • the protected amino acid, the activating reagent, and the active-ester forming reagent can be used in approximately equimolar ratios to each other in the coupling solution.
  • the protected amino acid, the activating reagent, and the active-ester forming reagent can be used in concentrations of about 0.05 M, about 0.1 M, about 0.2 M, about 0.3 M, about 0.4 M, about 0.5 M, about 0.6 M, about 0.7 M, about 0.8 M, about 0.9 M, about 1 M, about 1.25 M, about 1.5 M, or about 2 M in solution.
  • the protected amino acid, the activating reagent, and the active-ester forming reagent can be used in concentrations of about 0.05 M to about 2 M, about 0.1 M to about 2 M, about 0.2 M to about 2 M, about 0.3 M to about 2 M, about 0.4 M to about 2 M, about 0.5 M to about 2 M, about 0.6 M to about 2 M, about 0.7 M to about 2 M, about 0.8 M to about 2 M, about 0.9 M to about 2 M, about 1 M to about 2 M, about 1.25 M to about 2 M, or about 1.5 M to about 2 M in solution.
  • the protected amino acid, the activating reagent, and the active-ester forming reagent can be used in concentrations of about 0.05 M to about 1 M, about 0.1 M to about 1 M, about 0.2 M to about 1 M, about 0.3 M to about 1 M, about 0.4 M to about 1 M, about 0.5 M to about 1 M, about 0.6 M to about 1 M, about 0.7 M to about 1 M, about 0.8 M to about 1 M, or about 0.9 M to about 1 M in solution.
  • the protected amino acid, the activating reagent, and the active-ester forming reagent can be used in concentrations of about 0.05 M to about 0.6 M, about 0.1 M to about 0.6 M, about 0.2 M to about 0.6 M, about 0.3 M to about 0.6 M, about 0.4 M to about 0.6 M, or about 0.5 M to 0.6 M in solution.
  • the protected amino acid, the activating reagent, and the active-ester forming reagent can be used in concentrations of about 0.05 M to about 0.4 M, about 0.1 M to about 0.4 M, about 0.2 M to about 0.4 M, or about 0.3 M to about 0.4 M in solution.
  • the concentration ranges used are preferably at or below 1 M.
  • the protected amino acid, the activating reagent, and the active-ester forming reagent can be used in concentrations of about 0.1 M to about 0.5 M in solution, such as about 0.2 M to about 0.4 M in solution. Scale, yield, and purity of rapid heated peptide synthesis
  • the methods of the current invention can be used to perform peptide synthesis on an amount of resin, or to yield an amount of peptide, of at least about 0.2 mmol, at least about 0.3 mmol, at least about 0.4 mmol, at least about 0.5 mmol, at least about 0.6 mmol, at least about 0.7 mmol, at least about 0.8 mmol, at least about 0.9 mmol, at least about 1 mmol, at least about 1.25 mmol, at least about 1.5 mmol, at least about 2 mmol, at least about 3 mmol, at least about 4 mmol, at least about 5 mmol, at least about 6 mmol, at least about 7 mmol, at least about 7.5 mmol, at least about 8 mmol, at least about 9 mmol, or at least about 10 mmol of peptide.
  • the methods of the current invention can be used to perform peptide synthesis on an amount of resin, or to yield an amount of peptide, of up to about 0.2 mmol, up to about 0.3 mmol, up to about 0.4 mmol, up to about 0.5 mmol, up to about 0.6 mmol, up to about 0.7 mmol, up to about 0.8 mmol, up to about 0.9 mmol, up to about 1 mmol, up to about 1.25 mmol, up to about 1.5 mmol, up to about 2 mmol, up to about 3 mmol, up to about 4 mmol, up to about 5 mmol, up to about 6 mmol, up to about 7 mmol, up to about 7.5 mmol, up to about 8 mmol, up to about 9 mmol, or up to about 10 mmol of peptide.
  • the methods of the current invention can be used to perform peptide synthesis on an amount of resin, or to yield an amount of peptide, of about 0.1 mmol to about 1 mmol, about 0.1 mmol to about 2 mmol, about 0.1 mmol to about 3 mmol, about 0.1 mmol to about 4 mmol, about 0.1 mmol to about 5 mmol, about 0.1 mmol to about 0.8 mmol, about 0.1 mmol to about 0.6 mmol, or about 0.1 mmol to about 0.4 mmol of peptide.
  • the methods of the current invention can be used to perform peptide synthesis on an amount of resin, or to yield an amount of peptide, of about 0.2 mmol to about 1 mmol, about 0.2 mmol to about 1 mmol, about 0.2 mmol to about 2 mmol, about 0.2 mmol to about 3 mmol, about 0.2 mmol to about 4 mmol, about 0.2 mmol to about 5 mmol, about 0.2 mmol to about 0.8 mmol, about 0.2 mmol to about 0.6 mmol, or about 0.2 mmol to about 0.4 mmol of peptide.
  • the methods of the current invention can be used to perform peptide synthesis on an amount of resin, or to yield an amount of peptide, of about 0.2 mmol to about 0.4 mmol of peptide.
  • the methods of the current invention can be used to perform peptide synthesis on an amount of resin, or to yield an amount of peptide, of about 1 mmol to about 5 mmol, about 2 mmol to about 5 mmol, about 3 mmol to about 5 mmol, about 4 mmol to about 5 mmol, about 5 mmol to about 7.5 mmol, about 5 mmol to about 7 mmol, about 6 mmol to about 7 mmol, about 2.5 mmol to about 7.5 mmol, or about 4 to about 6 mmol of peptide.
  • the methods of the current invention can be used to perform peptide synthesis on an amount of resin, or to yield an amount of peptide, of about 1 mmol to about 2 mmol of peptide.
  • the methods of the current invention can be used to perform peptide synthesis on a scale to yield at least about 50 mg, at least about 75 mg, at least about 100 mg, at least about 200 mg, at least about 500 mg, at least about 750 mg, at least about 1 g, at least about 2 g, at least about 3 g, at least about 4 g, at least about 5 g, at least about 7.5 g, at least about 10 g, at least about 12.5 g, at least about 15 g, or at least about 20 g of peptide.
  • the methods of the current invention can be used to perform peptide synthesis on a scale to yield about 50 mg to about 20 g, about 75 mg to about 20 g, about 100 mg to about 20 g, about 200 mg to about 20 g, about 500 mg to about 20 g, about 750 mg to about 20 g, about 1 g to about 20 g, about 2 g to about 20 g, about 3 g to about 20 g, about 4 g to about 20 g, about 5 g to about 20 g, about 7.5 g to about 20 g, about 10 g to about 20 g, about 12.5 g to about 20 g, or about 15 g to about 20 g of peptide.
  • the methods of the current invention can be used to perform peptide synthesis on a scale to yield about 50 mg to about 10 g, about 75 mg to about 10 g, about 100 mg to about 10 g, about 200 mg to about 10 g, about 500 mg to about 10 g, about 750 mg to about 10 g, about 1 g to about 10 g, about 2 g to about 10 g, about 3 g to about 10 g, about 4 g to about 10 g, about 5 g to about 10 g, or about 7.5 g to about 10 g of peptide.
  • the methods of the current invention can be used to perform peptide synthesis on a scale to yield about 50 mg to about 5 g, about 75 mg to about 5 g, about 100 mg to about 5 g, about 200 mg to about 5 g, about 500 mg to about 5 g, about 750 mg to about 5 g, about 1 g to about 5 g, about 2 g to about 5 g, about 3 g to about 5 g, or about 4 g to about 5 g of peptide.
  • the methods of the current invention can be used to perform peptide synthesis on a scale to yield at least about 50 mg to about lg, at least about 50 mg to about 750 mg, at least about 50 mg to about 500 mg, at least about 50 mg to about 250 mg, or at least about 50 mg to about 100 mg of peptide.
  • the methods of the current invention can be used to perform peptide synthesis at a crude peptide purity of at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%.
  • the methods of the current invention can be used to perform peptide synthesis at a crude peptide purity of about 30% to about 90%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, about 80% to about 90%, about 30% to about 90%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, about 30% to about 50%, about 30% to about 40%, about 40% to about 90%, about 40% to about 80%, about 40% to about 70%, about 40% to about 60%, or about 40% to about 50%.
  • the methods of the current invention can be used to perform peptide synthesis at a crude peptide purity of about (0.95) N x 100%, about (0.98) N x 100%, or about (0.99) N x 100%, where N is the number of couplings performed in the synthesis (typically, the number of residues in the peptide minus one).
  • the methods of the current invention can be used to perform peptide synthesis at a crude peptide purity of about (0.95) N x 100% to about (0.98) N x 100%, or about (0.95) N x 100% to about (0.99) N x 100%
  • the methods of the current invention can be used to perform peptide synthesis at a crude peptide yield of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%, where yield is measured with respect to the molar amount of protected amino acid-resin (or peptide-resin, if synthesis begins with a pre-formed peptide attached to the resin) used at the beginning of synthesis.
  • the methods of the current invention can be used to perform peptide synthesis at a crude peptide yield of about 30% to about 90%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, about 30% to about 50%, about 40% to about 90%, about 40% to about 80%, about 40% to about 70%, about 25% to about 75%, about 25% to about 50%, about 20% to about 80%, or about 20% to about 70%.
  • the peptide yield can be for a peptide of at least about 10 residues, at least about 15 residues, at least about 20 residues, at least about 25 residues, at least about 30 residues, at least about 35 residues, at least about 40 residues, at least about 45 residues, at least about 50 residues, at least about 60 residues, at least about 70 residues, at least about 75 residues, at least about 80 residues, at least about 90 residues, or at least about 100 residues.
  • the peptide yield can be for a peptide of about 10 residues in length or less, about 15 residues in length or less, about 20 residues in length or less, about 25 residues in length or less, about 30 residues in length or less, about 35 residues in length or less, about 40 residues in length or less, about 45 residues in length or less, about 50 residues in length or less, about 60 residues in length or less, about 70 residues in length or less, about 75 residues in length or less, about 80 residues in length or less, about 90 residues in length or less, or about 100 residues in length or less.
  • the crude peptide yield can be for a peptide of between about 10 residues to about 100 residues, between about 10 residues to about 90 residues, between about 10 residues to about 80 residues, between about 10 residues to about 75 residues, between about 10 residues to about 70 residues, between about 10 residues to about 60 residues, between about 10 residues to about 50 residues, between about 15 residues to about 50 residues, between about 20 residues to about 50 residues, between about 25 residues to about 50 residues, between about 30 residues to about 50 residues, between about 35 residues to about 50 residues, between about 40 residues to about 50 residues, or between about 45 residues to about 50 residues; or between about 10 residues to about 45 residues, between about 10 residues to about 40 residues, between about 10 residues to about 35 residues, between about 10 residues to about 30 residues, between about 10 residues to about 25 residues, between about 10 residues to about 20 residues, between about 10 residues to about 90 residues, between about 10 residues to
  • the peptide yield can be for a peptide having a molecular weight in Daltons of about 1,000 to about 55,000, about 1 ,000 to about 50,000, about 1,000 to about 45,000, about 1 ,000 to about 40,000, about 1,000 to about 35,000, about 1,000 to about 30,000, about 1 ,000 to about 25,000, about 1,000 to about 20,000, about 1,000 to about 15,000, about 1,000 to about 10,000, about 1,000 to about 5,000, about 1,000 to about 2,500, about 1,000 to about 2,000, about 2,000 to about 55,000, about 2,000 to about 50,000, about 2,000 to about 45,000, about 2,000 to about 40,000, about 2,000 to about 35,000, about 2,000 to about 30,000, about 2,000 to about 25,000, about 2,000 to about 20,000, about 2,000 to about 15,000, about 2,000 to about 10,000, about 2,000 to about 5,000, about 4,000 to
  • a typical estimate for the number of residues in a peptide is (molecular weight of peptide) divided by (110); see Lehninger Principles of Biochemistry, Fifth Edition, David L. Nelson, Albert L. Lehninger, Michael M. Cox, W.H. Freeman & Co. New York: 2008, at page 84.
  • a peptide with 100 amino acid residues may typically range from a molecular weight of about 9,000 to about 15,000; a peptide with 50 residues may typically range from a molecular weight of about 4,000 to about 8,000.
  • the methods of the invention are used for synthesis of peptides having a molecular weight of about 20,000 Daltons or less. In one embodiment, the methods of the invention are used for synthesis of peptides of length about 100 residues or less.
  • the maximum racemization resulting from the method (that is, from the coupling step of the method, where the N-terminal-unprotected resin- bound peptide or amino acid is contacted with a coupling solution) at any position in the sequence can be less than about 1%, less than about 0.5%, less than about 0.2%, less than about 0.1%, less than about 0.05%, or less than about 0.01%, such as between about 0.05% to about 1%, between about 0.05% to about 0.5%, between about 0.05% to about 0.2%, between about 0.05% to about 0.1%, between about 0.01% to about 1%, between about 0.01% to about 0.5%, between about 0.01% to about 0.2%, between about 0.01% to about 0.1%, or between about 0.01% to about 0.05%.
  • Coupling For peptide synthesis performed on a scale of up to about 1 mmol (for example, about 0.1 mmol to about 1 mmol), the coupling reaction is typically run for between about 10 minutes to about 30 minutes, about 10 minutes to about 25 minutes, about 10 minutes to about 20 minutes, about 10 minutes to about 15 minutes, about 15 minutes to about 30 minutes, about 15 minutes to about 25 minutes, about 15 minutes to about 20 minutes, about 20 minutes to about 30 minutes, about 20 minutes to about 25 minutes, about 25 minutes to about 30 minutes, or about 15 minutes to about 25 minutes.
  • a preferred range of coupling time for peptide synthesis performed on a scale of up to about 1 mmol is about 15 minutes to about 25 minutes.
  • a preferred coupling time for peptide synthesis performed on a scale of up to about 1 mmol is about 20 minutes.
  • the coupling reaction is typically run for between about 15 minutes to about 60 minutes, about 15 minutes to about 50 minutes, about 15 minutes to about 45 minutes, about 15 minutes to about 40 minutes, about 15 minutes to about 30 minutes, about 20 minutes to about 60 minutes, about 20 minutes to about 50 minutes, about 20 minutes to about 45 minutes, about 20 minutes to about 40 minutes, about 20 minutes to about 30 minutes, about 25 minutes to about 60 minutes, about 25 minutes to about 50 minutes, about 25 minutes to about 45 minutes, about 25 minutes to about 40 minutes, about 25 minutes to about 30 minutes, about 30 minutes to about 60 minutes, about 30 minutes to about 50 minutes, about 30 minutes to about 45 minutes, about 30 minutes to about 40 minutes, or about 20 minutes to about 30 minutes.
  • a preferred range of coupling time for peptide synthesis performed on a scale above about 5 mmol is about 20 minutes to about 60 minutes, such as about 30 minutes to about 50 minutes.
  • a preferred coupling time for peptide synthesis performed on a scale above about 5 mmol is about 40 minutes.
  • a coupling reaction is incomplete, such as less than about 99% complete, less than about 98% complete, less than about 97% complete, or less than about 95% complete, a second coupling ("double-coupling") can be performed. The amount of completion of the coupling reaction can be determined using the Kaiser ninhydrin test.
  • the deblocking reaction For peptide synthesis performed on a scale of up to about 1 mmol (for example, about 0.1 mmol to about 1 mmol), the deblocking reaction, if run as a single step, can be performed for about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, or about 10 minutes, such as between about 2 minutes to about 10 minutes, between about 3 minutes to about 10 minutes, between about 4 minutes to about 10 minutes, between about 5 minutes to about 10 minutes, between about 6 minutes to about 10 minutes, between about 7 minutes to about 10 minutes, between about 8 minutes to about 10 minutes, between about 9 minutes to about 10 minutes, between about 2 minutes to about 8 minutes, between about 3 minutes to about 8 minutes, between about 4 minutes to about 8 minutes, between about 4 minutes to about 7 minutes, between about 4 minutes to about 6 minutes, between about 4 minutes to about 5 minutes, between about 5 minutes to about 9 minutes, or between about 6 minutes to about 8 minutes.
  • a preferred range for the length of the deblocking reaction for peptide synthesis performed on a scale of up to about 1 mmol, when run as a single step, is about 5 minutes to about 10 minutes.
  • a preferred time for the length of the deblocking reaction for peptide synthesis performed on a scale of up to about 1 mmol, when run as a single step, is about 7 minutes.
  • the deblocking reaction can also be run as two steps, where the resin is contacted with a first portion of deblocking solution for the first step; the first portion of the deblocking solution is drained; and then the resin is contacted with a second portion of deblocking solution for the second step.
  • the deblocking reaction for peptide synthesis performed on a scale of up to about 1 mmol can be performed for about 1 minute to about 3 minutes for the first step and between about 3 minutes to about 7 minutes for the second step; or about 1 minute to about 3 minutes for the first step and between about 4 minutes to about 6 minutes for the second step.
  • Preferred ranges for the length of the deblocking reaction for peptide synthesis performed on a scale of up to about 1 mmol, when run as two steps are about 2 minutes to about 5 minutes for the first step and about 2 minutes to about 10 minutes for the second step, such as about 2 minutes for the first step and about 5 minutes for the second step.
  • the deblocking reaction if run as a single step, can be performed for about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 1 1 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, or about 20 minutes, such as between about 4 minutes to about 20 minutes, between about 4 minutes to about 15 minutes, between about 4 minutes to about 10 minutes, between about 5 minutes to about 20 minutes, between about 7 minutes to about 20 minutes, between about 8 minutes to about 20 minutes, between about 10 minutes to about 20 minutes, between about 5 minutes to about 15 minutes, between about 6 minutes to about 15 minutes, between about 7 minutes to about 15 minutes, between about 8 minutes to about 15 minutes, between about 9 minutes to about 15 minutes, between about 10 minutes to about 15 minutes, between about 6 minutes to about 18 minutes, between about 7 minutes to about 17 minutes, between about 8
  • a preferred range for the length of the deblocking reaction for peptide synthesis performed on a scale of above about 5 mmol, when run as a single step, is about 7 minutes to about 16 minutes.
  • a preferred time for the length of the deblocking reaction for peptide synthesis performed on a scale of above about 5 mmol, when run as a single step, is about 12 minutes.
  • the deblocking reaction can also be run as two steps, where the resin is contacted with a first portion of deblocking solution for the first step; the first portion of the deblocking solution is drained; and then the resin is contacted with a second portion of deblocking solution for the second step.
  • the deblocking reaction for peptide synthesis performed on a scale of above about 5 mmol is run as two steps, it can be performed for about 2 minutes to about 6 minutes for the first step and between about 5 minutes to about 15 minutes for the second step; or about 3 minutes to about 5 minutes for the first step and between about 7.5 minutes to about 12.5 minutes for the second step.
  • Preferred ranges for the length of the deblocking reaction for peptide synthesis performed on a scale of above about 5 mmol, when run as two steps are about 3 minutes to about 5 minutes for the first step and about 7.5 minutes to about 12.5 minutes for the second step, such as about 4 minutes for the first step and about 10 minutes for the second step.
  • any of the above deblocking periods— that is, either the shorter one-step or two-step deblocking periods for up to about 1 mmol, or the longer one-step or two-step deblocking periods for above 5 mmol— can be used. If speed is of the essence, then the shorter deblocking periods should be used. If purity is more important than the shortest synthesis time possible, or if the sequence is known to contain difficult regions that require additional time for deblocking (for example, if the peptide sequence is highly hydrophobic or known to be prone to aggregation), then the longer
  • deblocking periods should be used.
  • Washing Washes of the resin can be done after coupling (that is, after removal of the coupling solution from the resin), typically for about 20 seconds to 1 minute or about 20 seconds to 40 seconds. Typically, about 1, 2, 3, or 4 washes are done after removal of the coupling reagents, such as about 2 or 3 washes of about 20 to 40 seconds each.
  • Washes of the resin can be done after deblocking (that is, after removal of the deblocking solution from the resin), typically for about 20 seconds to 1 minute, about 30 seconds to 1 minute, about 20 seconds to 40 seconds, or about 30 seconds to 40 seconds. Typically, about 3, 4, 5, 6, 7, 8, or 9 washes are done after removal of the deblocking solution, such as about 5 to 7 washes of about 30 to 40 seconds each.
  • the deblocking reaction is run as two steps, the resin can be washed after the first step of the deblocking reaction, followed by the second step of the deblocking reaction, followed by additional washes.
  • the first and second steps of the deblocking reaction can be run without intervening washes, and the resin is washed after the second step of the deblocking reaction.
  • AM Aminomethyl
  • CLT (2- Chlorotrityl) Resin
  • Wang Resin Wang Resin
  • MBHA 4-Methylbenzhydrylamine
  • Reagent K consists of 94% TFA (trifluoroacetic Acid), 2%
  • TIS triisopropylsilane
  • EDT ethane dithiol
  • deionized water 2% deionized water.
  • ACN acetonitrile
  • SPPS Solid Phase Peptide Synthesis
  • Automated Synthesizer available from CSBio, Menlo Park, California) can further save time, and frequently gives better synthesis compared to routine SPPS conditions.
  • Table 3 shows the results of rapid heated peptide synthesis at 40°C, 60°C, and 80°C for the test sequences of Table 2, along with a comparative experiment done at room temperature (25°C).
  • reaction temperature should be 40°C or lower in order to obtain reasonable weight gain.

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Abstract

L'invention concerne des procédés de synthèse rapide de peptides en phase solide (SPPS) à des températures élevées. Les procédés de l'invention réduisent le temps nécessaire à la synthèse peptidique de manière significative, tout en fournissant un peptide brut avec un bon rendement, une pureté et une pureté stéréochimique.
PCT/US2017/052099 2016-09-20 2017-09-18 Synthèse rapide de peptides à températures élevées WO2018057470A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5233044A (en) * 1989-06-08 1993-08-03 Millipore Corporation Active esters for solid phase peptide synthesis
US6028172A (en) * 1997-02-11 2000-02-22 Mallinckrodt Inc. Reactor and method for solid phase peptide synthesis
US20040063918A1 (en) * 2002-07-16 2004-04-01 Young Travis G. Methods and materials for the synthesis of modified peptides

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5233044A (en) * 1989-06-08 1993-08-03 Millipore Corporation Active esters for solid phase peptide synthesis
US6028172A (en) * 1997-02-11 2000-02-22 Mallinckrodt Inc. Reactor and method for solid phase peptide synthesis
US20040063918A1 (en) * 2002-07-16 2004-04-01 Young Travis G. Methods and materials for the synthesis of modified peptides

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