WO2013115813A1 - Synthèse de peptide en phase solide, soluble dans l'eau - Google Patents

Synthèse de peptide en phase solide, soluble dans l'eau Download PDF

Info

Publication number
WO2013115813A1
WO2013115813A1 PCT/US2012/023461 US2012023461W WO2013115813A1 WO 2013115813 A1 WO2013115813 A1 WO 2013115813A1 US 2012023461 W US2012023461 W US 2012023461W WO 2013115813 A1 WO2013115813 A1 WO 2013115813A1
Authority
WO
WIPO (PCT)
Prior art keywords
alcohol
water
group
washing
protected
Prior art date
Application number
PCT/US2012/023461
Other languages
English (en)
Inventor
Jonathan M. Collins
Original Assignee
Cem Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Cem Corporation filed Critical Cem Corporation
Priority to PCT/US2012/023461 priority Critical patent/WO2013115813A1/fr
Publication of WO2013115813A1 publication Critical patent/WO2013115813A1/fr

Links

Classifications

    • 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
    • C07K1/061—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 protecting groups
    • C07K1/063—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 protecting groups for alpha-amino functions
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present invention relates to solid phase peptide synthesis (SPPS) and to a method of carrying out SPPS reactions in aqueous solutions.
  • SPPS solid phase peptide synthesis
  • Peptides are linked chains of amino acids which in turn are the basic building blocks for most living organisms. Peptides are also the precursors of proteins; i.e., long complex chains of amino acids. Peptides and proteins are fundamental to human and animal life, and they drive, affect, or control a wide variety of natural processes. As a result, the study of peptides and proteins and the capability to synthesize peptides and proteins are of significant interest in the biological sciences and medicine.
  • Solid phase peptide synthesis is a technique in which an initial amino acid is linked to a solid particle and then additional amino acids are added to the first acid to form the peptide chain. Because the chain is attached to a particle, it can be washed and otherwise treated with additional solvents or rinses while being maintained in a discrete vessel and handled (at least to some extent) as a solid. SPPS thus allows solution phase chemistry to be carried out in a manner that has some of the convenience of handling solids.
  • DCM dimethyl formamide
  • NMP n-methylpyrrolidone
  • DMSO dimethyl sulfoxide
  • DCM dichloromethane
  • DCM is typically mixed with DMF or NMP because the N-alpha protecting groups Fmoc (e.g., fluorenylmethyloxycarbonyl chloride) and Boc (e.g., tert- butoxycarbonyl) frequently used in SPPS are typically hydrophobic and insoluble in water.
  • Fmoc and Boc e.g., tert-butoxycarbonyl
  • DBF dibenzofulvene
  • the DBF can, however, also react with the free amine group effectively capping the end of the peptide chain.
  • Some deprotection employ a short initial deprotection step to flush most of the DBF out of the reaction vessel and then use a second longer deprotection with fresh piperidine solution to reduce this potential side reaction. This approach may be unnecessary, however, because a typical 20% deprotection solution has a large excess of piperidine versus potential DBF. For example, a synthesis at 0.1 mmol scale using a 7 ml_ solution of a 20% piperidine in DMF would have a ratio of piperidine to total potential DBF of approximately 710:1 .
  • an aqueous based—i.e., water-soluble— scheme for peptide synthesis, and particularly SPPS, represents a worthwhile ongoing technological goal.
  • Galanis Organic Letters, Vol. 1 1 , No. 20, pp. 4488-4491 (2009)
  • Galanis Organic Letters, Vol. 1 1 , No. 20, pp. 4488-4491 (2009)
  • a conventional Boc protecting group in the presence of specific resins, linkers, activating agents and a zwitterion detergent to produce a single demonstrative Leu-Enkephalin peptide.
  • Sps has a solubility comparable to that of Esc, but synthesizing Esc appears to be more complicated and expensive. Additionally, a different synthesis scheme must be used for cysteine (Cys) and methionine (Met) in order to avoid oxidation of their sulfur groups.
  • Cys cysteine
  • Metal methionine
  • a larger number of aromatic rings in a protecting group molecule can enhance the UV absorption for conventional monitoring purposes. The additional rings, however, also minimize or eliminate water solubility.
  • a reaction product is drawn after the deprotection step and measured under UV absorption.
  • Fmoc will absorb characteristic UV frequencies (e.g., 300 nanometers) in amount proportional to its concentration and thus the amount of detected Fmoc will provide an indication of the extent to which deprotection has proceeded
  • Pms, Esc, and Sps have the advantage of some water solubility, but Pms and Esc cannot be tracked in conventional UV monitoring in the same manner as conventional Fmoc. Sps can be monitored by UV, but its difficult and costly synthesis tends to discourage its use. As a result, the increased water solubility of these compounds is less helpful in an overall sense.
  • compositions tend to produce low purity peptides at each step thus limiting overall peptide length.
  • amino acids used in SPPS include side chain protecting groups (e.g., trityl "Trt” and t-butyl “tBu”) which are hydrophobic.
  • side chain protecting groups e.g., trityl "Trt” and t-butyl “tBu”
  • the activated species tend to get hydrolyzed in water and will no longer react with the growing peptide chain.
  • the invention is an improvement in solid phase peptide synthesis that includes deprotecting an amino acid and then washing the deprotected acid in a solvent selected from the group consisting of water, alcohol, and mixtures of water and alcohol.
  • the invention includes the steps of deprotecting an amino group in its protected form that is protected with a protecting group containing an alpha, beta ( ⁇ , ⁇ ) unsaturated sulfone and then washing the deprotected acid in a solvent selected from the group consisting of water, alcohol, and mixtures of water and alcohol.
  • the amino acid is protected with a protecting group that acts as a Michael Reaction acceptor in the presence of a Michael Reaction donor
  • the protecting group is selected from the group consisting of Bsmoc, Nsmoc, Bspoc and Mspoc; and with Bsmoc being typical.
  • the invention is a solid phase peptide synthesis method that includes the improvement of deprotecting an amino acid that is soluble in aqueous environments in its protected form, and then washing the deprotected acid in a solvent selected from the group consisting of water, alcohol, and mixtures of water and alcohol [0030]
  • the invention is a solid phase peptide synthesis method that includes the improvement of deprotecting a Bsmoc-protected amino acid, and then washing the deprotected acid in a solvent selected from the group consisting of water, alcohol, and mixtures of water and alcohol.
  • the invention is a solid phase peptide synthesis method that includes the steps of coupling a protected acid to a resin-based peptide or a resin-based amino acid; and washing the coupled composition in a solvent selected from the group consisting of water, alcohol, and mixtures of water and alcohol.
  • the invention is a composition that includes a solid phase resin, an amino acid protected with a protecting group containing an ⁇ , ⁇ -unsaturated sulfone, a solvent selected from the group consisting of water, alcohol, and mixtures of water and alcohol, a base for deprotecting the protected amino acid, and the adduct formed by the reaction between the deprotecting base and the ⁇ , ⁇ -unsaturated sulfone protecting group.
  • the invention is a composition that includes a solid phase resin, a solvent selected from the group consisting of water, alcohol, and mixtures of water and alcohol, an unactivated ⁇ , ⁇ -unsaturated sulfone based protected amino acid, and activated portions of the ⁇ , ⁇ -unsaturated sulfone based protected amino acid.
  • the invention is a process for accelerating the solid phase synthesis of peptides by carrying out one or more of the steps of deprotecting an amino group in its protected form that is protected with a protecting group containing an ⁇ , ⁇ - unsaturated sulfone and linked to solid phase resin particles by admixing the protected linked acid with a deprotecting solution while irradiating the admixed acid and solution with microwaves; activating a second amino acid by adding the second acid and an activating solution; coupling the second amino acid to the first acid while irradiating the composition with microwaves; and successively deprotecting, activating, and coupling a plurality of amino acids into a peptide.
  • Figure 1 illustrates a conventional Boc deprotection.
  • Figure 2 illustrates a conventional Fmoc deprotection.
  • Figure 3 illustrates the alternative pathways for Fmoc being scavenged by a base and as reacting with a non-protected amino acid to cap a peptide chain.
  • Figure 4 illustrates an ⁇ , ⁇ -unsaturated sulfone protecting an amino acid.
  • Figure 5 illustrates a Bsmoc deprotection according to the present invention.
  • Figure 6 is the output plot of an HPLC chromatograph separation of the products of a peptide synthesis incorporating aspects of the present invention.
  • Figure 7 is a mass spectrum of a relevant fraction from the HPLC chromatograph represented by Figure 6.
  • Figure 8 is the output plot of an HPLC chromatograph separation of the products of another peptide synthesis incorporating aspects of the present invention.
  • Figure 9 is a mass spectrum of a relevant fraction from the HPLC chromatograph represented by Figure 8.
  • Figure 10 is the output plot of an HPLC chromatograph separation of the products of another peptide synthesis incorporating aspects of the present invention.
  • Figure 1 1 is a mass spectrum of a relevant fraction from the HPLC
  • Figure 12 is the output plot of an HPLC chromatograph separation of the products of another peptide synthesis incorporating aspects of the present invention.
  • Figure 13 is a mass spectrum of a relevant fraction from the HPLC
  • the invention is a solid phase peptide synthesis method in which the improvement comprises using one or more amino acids that are protected with a protecting group includes an ⁇ , ⁇ -unsaturated sulfone.
  • a protecting group acts as a Michael Reaction acceptor in the presence of a Michael Reaction donor.
  • the washing steps are carried out in a solvent selected from the group consisting of water, alcohol, and mixtures of water and alcohol.
  • an organic solvent system is (i.e., continues to be) advantageous for the deprotection and coupling steps, but that significant advantages can be obtained by carrying out the washing steps in an aqueous environment, including solvent systems that include water, or polar alcohols, or mixtures of water and alcohols.
  • solvent systems that include water, or polar alcohols, or mixtures of water and alcohols.
  • aqueous solvent systems water, alcohol, or water and alcohol
  • the invention represents the recognition (in part) that the washing steps can be carried out in water, or a polar alcohol, or an alcohol and water solvent system, if the N alpha protecting group incorporates an alpha, beta unsaturated sulfone system.
  • the invention represents the recognition that effective washing requires that the solid phase resin swell adequately in water (or the water-alcohol solvent system) to effectively ensure complete washing.
  • PEG polyethylene glycol
  • the protecting group is selected from the group consisting of Bsmoc, Nsmoc, Bspoc and Mspoc; and with Bsmoc being typical.
  • a Michael Addition reaction is the nucleophilic addition of a nucleophile to an alpha, beta unsaturated carbonyl compound.
  • the nucleophile is the Michael Donor (e.g., piperidine) and the alpha, beta unsaturated carbonyl compound is the Michael Acceptor (e.g. an alkene).
  • the amino acid protecting group has a Michael acceptor site that includes an alpha, beta-unsatu rated sulfone.
  • the use of the alpha, beta unsaturated sulfone system for the protecting group offers at least two advantages in an aqueous-based washing step.
  • the sulfone functional group is extremely soluble in water. This in turn greatly increases the solubility of amino acids that use this class of protecting groups.
  • the alpha, beta unsaturated sulfone compositions have a higher base lability than do Fmoc protected amino acids.
  • the alpha, beta unsaturated sulfone protecting groups can be removed by a more reactive Michael addition mechanism (as opposed to the beta-elimination mechanism used to remove Fmoc).
  • This higher lability permits a correspondingly lower amount (concentration) of the base to be utilized for the deprotection step.
  • the moderated base (i) reduces or effectively eliminates the undesired aspartimide formation that tends to occur during either the next deprotection step or a subsequent washing step when small amounts of the base remain behind, and (ii) minimizes or eliminates undesired formation of hydroxide ion (OH " ) in any of the aqueous based steps or environments.
  • soluble in water in its protected form means that the composition has the degree of solubility necessary for the desired reaction to proceed in an aqueous solvent system.
  • soluble does not imply unlimited solubility in any or all amounts.
  • Bsmoc refers to 1 ,1 -dioxobenzo[b]thiphene-2- ylmethyloxycarbonyl. Bsmoc is also referred to by the "common name"
  • Bsmoc is typically represented by the following formula:
  • SPPS synthesis is set forth by Carpino et al in the Journal or Organic Chemistry, 1999, 64 (12) at pages 4324-4338.
  • Nsmoc 1 ,1 -dioxonaptho[1 ,2- b]thiophene-2-methyloxycarbonyl; "a-Nsmoc"
  • Nsmoc derivatives of all 20 standard amino acids have been successfully made and used in SPPS.
  • the Nsmoc group shows similar advantages to the Bsmoc group, but appears somewhat more expensive to produce because of its additional six member carbon ring.
  • the Nsmoc group is also predicted to result in a lower acylation rate than the Bsmoc group, but comparable to the Fmoc group because of their similar size.
  • two other Nsmoc isomers can be produced; i.e., with the second aromatic ring in a different position with respect to the S0 2 group.
  • ⁇ , ⁇ -unsaturated sulfone protecting groups that can function as the Michael acceptor include 2-tert-butylsulfonyl-2-propenoxycarbonyl (Bspoc) and 2- methylsulfonyl-3-phenyl-1 -prop-2-enyloxycarbonyl (Mspoc); see, e.g., Carpino et al., The 2-methylsulfonyl-3-phenyl-1 -prop-2-enyloxycarbonyl (Mspoc) Amino Protecting Group, J. Org. Chem. 1999, 64, 8399-8401 .
  • washing solvent as between and among water, alcohol, and water-alcohol mixtures (as well as the water:alcohol ratio of any given mixture) will depend to some greater or lesser extent upon the amino acids desired for the target peptide, or the base selected for deprotection, or a combination of these factors.
  • the straightforward nature of the invention enables the skilled person to make the selection on a case-by-case basis and without undue experimentation.
  • the method can also include irradiating the acid and the solvent with microwaves during the deprotection step.
  • irradiating the acid and the solvent with microwaves during the deprotection step.
  • a detailed description of an instrument suitable for microwave irradiation is the SPPS context is set forth in commonly-owned U.S. Patent No. 7,393,920 (and in a number or related patents and published applications).
  • the protected amino acid is one of the essential amino acids that remains sufficiently water-soluble (in both its activated and protected forms) when protected with the relevant protecting group to wash with the water (or water- alcohol) solvent system of the invention; e.g. an amino acid protected with Bsmoc.
  • water is used as a solvent and a base that is soluble in water is used in an amount and to the extent necessary to deprotect the acid.
  • a favorable base will be one for which the base and its deprotection adduct can be successfully removed (washed) in the aqueous-based solvent system of the present invention.
  • the method can comprise repeating the steps of deprotecting, washing, coupling, and washing for a second protected acid. Thereafter, the steps can be repeated to add a third protected amino acid, and thereafter a successive plurality of protected amino acids to produce a desired peptide.
  • the invention is a method of solid phase peptide synthesis in which the improvement includes the steps of deprotecting an amino group in its protected form that is protected with a protecting group containing a Michael acceptor site composed of an ⁇ , ⁇ -unsaturated sulfone, and then washing the deprotected acid in a solvent selected from the group consisting of water, alcohol, and mixtures of water and alcohol.
  • a solvent selected from the group consisting of water, alcohol, and mixtures of water and alcohol.
  • the advantages of the water or alcohol or mixture solvent system can be used for the washing step independently of whether or not the solvent system is used for the deprotection step.
  • the acid is protected with a protecting group selected from the group consisting Bsmoc, Nsmoc, Bspoc and Mspoc, with a Bsmoc-protected amino acid being most typical.
  • the washing step can be carried out in the presence of microwave irradiation on an as-needed or as-desired basis.
  • the alcohol again can be selected from the group consisting of methanol, ethanol, 1 -propanol, 2-propanol, n- butanol, isobutanol, sec-butanol, and tert-butanol.
  • the invention is a method of solid phase peptide synthesis comprising deprotecting an amino group in its protected form that is protected with a protecting group containing a Michael acceptor site composed of an ⁇ , ⁇ -unsaturated sulfone, coupling the deprotected acid to a resin-based peptide or a resin-based amino acid, and then washing the coupled composition in a solvent selected from the group consisting of water, alcohol, and mixtures of water and alcohol.
  • a solvent selected from the group consisting of water, alcohol, and mixtures of water and alcohol.
  • Bsmoc, Nsmoc, Bspoc and Mspoc protected amino acids are again exemplary.
  • the step of washing the coupled composition can likewise be enhanced in some circumstances by the use of microwave irradiation.
  • the alcohols used for the water- alcohol mixture solvent system can be those mentioned previously and the bases used to deprotect the protected amino acids can be those bases named previously.
  • the invention is a solid phase peptide synthesis method that includes the following steps: deprotecting an amino group in its protected form that is protected with a protecting group containing a Michael acceptor site composed of an ⁇ , ⁇ - unsaturated sulfone; washing the deprotected acid in a solvent selected from the group consisting of water, alcohol, and mixtures of water and alcohol; coupling the deprotected acid to a resin-based peptide or a resin-based amino acid; and washing the coupled composition in a solvent selected from the group consisting of water, alcohol, and mixtures of water and alcohol.
  • Bsmoc, Nsmoc, Bspoc and Mspoc protected amino acids are again exemplary.
  • microwaves can be applied during the deprotection step or the coupling step, including the steps of coupling single acids together or the step of coupling a sequential acid to a resin-based peptide or a resin based amino acid.
  • appropriate alcohols can include methanol, ethanol, 1 -propanol, 2-propanol, n-butanol, isobutanol, sec-butanol, and tert-butanol.
  • any appropriate base can be used to deprotect the relevant amino acids, but in exemplary embodiments, including Bsmoc-protected acids, weaker bases (e.g., piperazine, morpholine) can help minimize hydroxide (OH " ) concentration in an aqueous environment (and thus minimize racemization). Also, a single alcohol wash step prior to coupling appears to likewise help minimize hydrolysis of the activated amino acid being added by minimizing water present during the coupling step (i.e., activated amino acids are known to be susceptible to hydrolysis in water).
  • weaker bases e.g., piperazine, morpholine
  • OH " hydroxide
  • a single alcohol wash step prior to coupling appears to likewise help minimize hydrolysis of the activated amino acid being added by minimizing water present during the coupling step (i.e., activated amino acids are known to be susceptible to hydrolysis in water).
  • the deprotecting, coupling and washing steps can be repeated to add a second amino acid that is likewise initially protected with Bsmoc to the first amino acid.
  • the steps can be repeated for a third and thereafter successive plurality of Bsmoc-protected acids to form a peptide chain.
  • the method can further include the step of cleaving the peptide chain from the solid phase resin, and microwave radiation can be applied to enhance the cleaving step.
  • the invention is a composition.
  • the composition comprises a mixture of a solid phase resin and a solution.
  • the solution comprises an amino acid and an amino acid protecting group, both dissolved in the same solvent.
  • the protecting group includes an ⁇ , ⁇ -unsaturated sulfone, which in exemplary embodiments acts as a Michael Reaction acceptor in the presence of a Michael Reaction donor.
  • the solvent is selected from the group consisting of water, alcohol, and mixtures of water and alcohol.
  • the composition further comprises a base that is soluble in the solvent system.
  • the base is soluble in water alone.
  • Water soluble bases appropriate for the composition include mild alkyl hydroxide bases, sodium hydroxide, lithium hydroxide, sodium carbonate, piperidine, 4-(Amino methyl)piperidine and piperazine.
  • Bsmoc (or Nsmoc, Bspoc or Mspoc) and an amino acid are dissolved in the same solvent.
  • the alcohol in the composition can in exemplary embodiments be selected from the group consisting of methanol, ethanol, 1 -propanol, 2-propanol, n-butanol, isobutanol, sec-butanol, and tert-butanol.
  • the solid phase resin includes polyethylene glycol, either as the resin itself or as ethylene-oxide spacer groups between a polystyrene resin and its functional groups.
  • the invention represents the recognition that effective washing requires that the solid phase resin swell adequately in water (or the water-alcohol solvent system) to effectively ensure complete washing.
  • PEG polyethylene glycol
  • 1 % cross-linked polystyrene resins will demonstrate a swelling factor (original size to solvated size) of at least 3 in some organic solvents and greater than 5 in other solvents.
  • polystyrene resins do not swell in water (i.e., swelling factor equal 1 .0) and have a swelling factor of less than two in methanol.
  • polyethylene glycol resins tend to show greater swelling in all solvents (polar and nonpolar) then do polystyrene resins. According to publications of Sigma Aldrich (St. Louis Missouri USA), certain PEG resins will demonstrate swelling factors greater than 10 in water. In the context of the present invention, the H-Rink Amide-ChemMatrix® resin from PCAS BioMatrix Inc. of Quebec Canada is particularly favorable.
  • PEG "spacer" resins offer improved performance as compared to conventional polystyrene resins.
  • a spacer resin includes a polystyrene matrix, but also includes several (typically between 5 and 10) ethylene oxide units between the polystyrene matrix and the reactive sites.
  • the spacer groups help modify the hydrophobic properties of the polystyrene backbone and the spacer resins demonstrate a higher mobility which in turn increases the rate of reaction.
  • the aqueous solvent system can be enhanced by the presence of a detergent to help render a protected acid soluble in the aqueous-based solvent system.
  • a detergent to help render a protected acid soluble in the aqueous-based solvent system.
  • soluble is used herein in its usual sense; i.e., the desired or necessary amount of protected acid will completely dissolve in the solvent system.
  • solubility is a relative term that can also be quantified based on the amount of a particular material that will dissolve in a particular solvent.
  • the respective compositions are considered soluble if they will dissolve in water in the amounts typically required to successfully carry out solid phase peptide synthesis.
  • Detergents are water soluble molecules classified according to their hydrophilic or hydrophobic character (or the degree of each) and their ionic groups. These characteristics establish the behavior of the detergent with respect to the protecting groups, the peptide chain, and individual amino acids.
  • a detergent has a hydrophobic tail that associates to form micelles, or that aggregates, or interacts with other molecules (lipids, proteins). In solution, detergents help keep molecules in solution by dissociating aggregates, and unfolding larger molecules
  • Typical detergents that are helpful include nonionic detergents, cationic detergents, anionic detergents, and zwitterionic detergents. Particular detergents that are useful include octyl phenyl ethylene oxide; sodium lauryl sulfate; and sodium dodecyl sulfate.
  • the method can include activating the deprotected acid with an activator.
  • an activator Any activator that carries out the appropriate advantages (i.e. making the oxygen a better leaving group) and that otherwise is consistent with the overall SPPS reaction is appropriate.
  • Representative activating agents include carbodiimides and triazoles.
  • HBTU 0-Benzotriazolyl-N,N,N',N'-tetramethyluronium hexafluorophospate
  • TBTU 2-(1 H-Benzotriazole-1 -yl)-1 ,1 ,3,3-Tetramethyluronium Tetrafluoro Borate
  • Boc-histidine(tosyl) Boc-histidine(tosyl)
  • the invention is a process for accelerating the solid phase synthesis of peptides by carrying out one or more of the steps of deprotecting the alpha amino group of an amino acid in its protected form that is protected with a protecting group containing an ⁇ , ⁇ -unsaturated sulfone and linked to solid phase resin particles by admixing the protected linked acid with a deprotecting solution while irradiating the admixed acid and solution with microwaves.
  • the method includes activating a second amino acid and then coupling the second amino acid to the first amino acid while irradiating the composition with microwaves. Thereafter the method includes
  • the amino acid is protected with Bsmoc, Nsmoc, Bspoc or Mspoc.
  • An instrument suitable for use in the method is described in detail in commonly assigned US Patent No. 7,393,920. The same description is set forth in other commonly assigned U.S. patents resulting from divisional and continuing applications and has also been published in Europe, for example at EP 1 491 552 and EP 1 923 396. These descriptions provide the skilled person with the information helpful to practicing the method.
  • the method can comprise cyclically repeating the steps of deprotecting, activating, and coupling for three or more amino acids in succession to thereby synthesize a desired peptide.
  • any of the methods described herein typically comprises cleaving the linked peptide from the solid phase resin by admixing the linked peptide with the cleaving composition.
  • cleavage is carried out while irradiating the composition with microwaves.
  • the cleaving compositions and protocol are to some extent dictated by the amino acids in the peptide chain and in some cases by the side protecting groups that those amino acids may carry.
  • an acid is used to carry out the cleaving step.
  • the acid should carry out the necessary cleavage without adversely affecting or interfering with the desired peptide and any desired groups (e.g., side chain protecting groups) that are attached to the amino acids in the peptide.
  • Trifluoroacetic acid and hydrofluoric acid are common cleaving agents, but are often mixed with small proportions of complementary compositions such as water, phenol and ethanedithiol (EDT).
  • Trifluoromethane sulfonic acid TFMSA or
  • TMSOTf trimethylsilyltrifluoromethanesulfonate
  • Cleavage is typically carried out in the presence of scavenger compositions (e.g., water, phenol, EDT) which protect the peptide from undesired side reactions during and after the cleaving step.
  • scavenger compositions e.g., water, phenol, EDT
  • the scavengers are generally selected based upon the protecting groups that are present. Thus, the selection is to some extent customized by the skilled person, who can select the appropriate scavengers without undue experimentation.
  • Bsmoc can be synthesized using several reaction pathways.
  • Bsmoc is formed from commercially available 1 -benzothiophene through hydroxymethylation followed by peracid oxidation.
  • the starting material 1 - benzothiophene is readily available at modest pricing.
  • the protecting group e.g., Bsmoc
  • a Michael Addition reaction is the nucleophilic addition of a nucleophile to an electrophilic ⁇ , ⁇ unsaturated compound.
  • the nucleophile is the Michael Donor (e.g., piperazine) and the ⁇ , ⁇ unsaturated compound is the Michael Acceptor (e.g. an alkene).
  • the protecting groups developed by Carpino contain a Michael Acceptor group.
  • the Michael Acceptor group for these compounds is an activated alkene group.
  • a Michael Donor typically a base such as piperidine or piperazine initiates the reaction and forms a Michael Adduct with the protecting group. Formation of the Michael Adduct leads to an intramolecular rearrangement that cleaves the protecting group from the amino acid.
  • the Michael addition process eliminates DBF; thus DBF never raises an issue.
  • the amount of base can be selected as needed for cleavage rather than in order to provide a large excess for scavenging DBF.
  • Michael addition is more reactive than the beta elimination steps of an Fmoc deprotection. This also provides the opportunity to use a weaker base (pKa) or a lower concentration of a stronger base.
  • pKa weaker base
  • a smaller amount of base is preferred because it will tend to form less hydroxide ion (OH " ), minimize base catalyzed side reactions during deprotection, lower reagent costs, and reduce waste toxicity
  • the ⁇ , ⁇ group that is deblocked through a Michael addition is more base-labile than are conventional beta-elimination groups such as Fmoc. This in turn reduces or eliminates side reactions and is likewise advantageous for the aqueous solvent system because it also minimizes or eliminates the production of hydroxide ion.
  • the deprotection also serves as the scavenging action so that no reactive intermediate is present to react with the free amine group.
  • the Bsmoc group is also more reactive to attack by secondary amines than the Fmoc group. These factors likewise lower the strength or concentration of the base needed in the Bsmoc deprotection reaction.
  • Bsmoc As compared to Fmoc, the structure of Bsmoc appears more soluble in water based upon its heterocyclic 5-membered ring that has an S0 2 group present. Bsmoc appears to be more soluble because it contains only one additional six-membered carbon ring. A comparison between an Fmoc and Bsmoc compound has been observed in rapid solution phase synthesis. In this type of synthesis, TAEA (tris(2-aminoethyl)amine) is used for deprotection and its adduct with Bsmoc is soluble in water, while its adduct with Fmoc is not.
  • TAEA tris(2-aminoethyl)amine
  • the potential water soluble methods for the Bsmoc reagent can be performed with or without assistance of microwave energy.
  • the sulfone-containing protecting groups described herein present opportunities for monitoring after completion of either or both of the deprotection and coupling reactions.
  • the single S0 2 group in these compounds is unique to most, and in many calls all, of the other reagents used during the step-wise assembly of the peptide.
  • This S0 2 group can be monitored by infrared radiation (IR) to determine the quantitative amounts of Bsmoc (or Nsmoc, Bspoc or Mspoc) present at the end of each reaction.
  • IR infrared radiation
  • Evidence of the S0 2 group can be used to determine an incomplete removal of Bsmoc at the end of the deprotection. This is advantageous to the UV approach in that it does not require performing the reaction twice to make a comparison.
  • the coupling reaction can be monitored by IR absorption in two possible ways.
  • the first method is to determine the IR absorption immediately after addition of the amino acid and activator reagents. This provides a baseline for total Bsmoc (Nsmoc, Bspoc, Mspoc) in the reaction vessel at the user defined excess.
  • the IR absorption is then again determined and compared to the initial value (addition of pure solvent in identical volume to amino acid activator solution may be necessary for comparison).
  • a 100% complete coupling reaction should yield an IR absorption ratio that is proportional to the excess used. This approach is advantageous because it only requires the coupling reaction to be performed one time.
  • a second approach could make a comparison of the IR absorption after two subsequent coupling reactions in a manner identical to that currently used by UV for monitoring the Fmoc deprotection step.
  • the acids generally carry side chain protecting groups and such sidechain protected acids can be used in accordance with the present invention.
  • Common (typical) amino acid side chain protecting groups include the following examples. Trityl- (Trt) is typically used on Cysteine (Cys), Histidine (His), Asparagine (Asn), and Glutamine (Gin).
  • tert-Butyl (tBu) is typically used on Aspartic Acid (Asp), Glutamic Acid (Glu), Serine (Ser), Threonine (Thr), and Tyrosine (Tyr).
  • tert- butoxycarbonyl (Boc) is typically used on Lysine (Boc), and Tryptophan (Trp).
  • 2,2,4,6,7- Pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) is typically used on Arginine (Arg).
  • Dmcp Dimethylcyclopropyl
  • All of these side chain protecting groups are non-polar.
  • the Pbf group contains some polarity with the sulfonyl group, but is still largely non-polar.
  • the deprotection can be carried out using amino acids protected with the Michael addition acceptor compounds, including, but not limited to Bsmoc, Nsmoc, Bspoc and Mspoc. Any one or more (or all) of the deprotection, washing, activation, coupling or cleaving steps can be carried out in water or in a water-alcohol system, with or without a detergent. Any one or more (or all) of these steps can likewise be enhanced by applying microwave irradiation.
  • Example 1 (65-74 ACP) was synthesized using conditions otherwise identical to Example 1 , but with Bsmoc protected acids instead of Fmoc protected acids.
  • the side chain protecting groups on the amino acids were otherwise conventional, and the following amino acid derivatives were used
  • Figure 1 illustrates successful synthesis, but with less than desirable purity, particularly based upon the presence of significant aspartimide related side products. These side products are presumed to result from the presence of hydroxide ion (OH-) which in turn is generated by the reaction between piperidine and water.
  • OH- hydroxide ion
  • Figures 3 and 4 further demonstrate some of the results.
  • Figure 3 is another HPLC plot and Figure 4 is the mass spectrum of the relevant fraction taken from the HPLC. The results show that the purity of the product was higher than Example 2, presumably because the weaker base piperazine reduces (as compared to piperidine) the formation of the hydroxide ion.
  • Table 4 and Figures 5 and 6 illustrate that the peptide was successfully synthesized at high purity using this approach.
  • the purity was as high as any observed with conventional organic solvents (e.g., DMF, N-methylpyrrolidone) for the washing steps.
  • organic solvents e.g., DMF, N-methylpyrrolidone
  • the isopropyl alcohol wash prior to the coupling reaction appears to be helpful in reducing hydrolysis of the activated ester thus leading to higher coupling efficiency.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne un procédé de synthèse de peptide en phase solide. Le procédé comprend les étapes éliminant la protection d'un groupe amino sous sa forme protégée qui est protégée par un groupe protecteur qui comprend une sulfone α,β-insaturée ; le lavage de l'acide déprotégé dans un solvant choisi dans le groupe constitué par l'eau, l'alcool et les mélanges d'eau et d'alcool ; le couplage de l'acide déprotégé à un peptide à base de résine ou un acide aminé à base de résine ; le lavage de la composition couplée dans un solvant choisi dans le groupe constitué par l'eau, l'alcool et les mélanges d'eau et d'alcool.
PCT/US2012/023461 2012-02-01 2012-02-01 Synthèse de peptide en phase solide, soluble dans l'eau WO2013115813A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/US2012/023461 WO2013115813A1 (fr) 2012-02-01 2012-02-01 Synthèse de peptide en phase solide, soluble dans l'eau

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US2012/023461 WO2013115813A1 (fr) 2012-02-01 2012-02-01 Synthèse de peptide en phase solide, soluble dans l'eau

Publications (1)

Publication Number Publication Date
WO2013115813A1 true WO2013115813A1 (fr) 2013-08-08

Family

ID=45569770

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2012/023461 WO2013115813A1 (fr) 2012-02-01 2012-02-01 Synthèse de peptide en phase solide, soluble dans l'eau

Country Status (1)

Country Link
WO (1) WO2013115813A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016050764A1 (fr) * 2014-09-29 2016-04-07 Technische Universität Darmstadt Procédé de synthèse de peptides et appareil de mise en œuvre d'un procédé de synthèse en phase solide de peptides
WO2019101939A1 (fr) 2017-11-24 2019-05-31 Sulfotools Gmbh Procédé de préparation de peptides
WO2019101940A1 (fr) 2017-11-24 2019-05-31 Sulfotools Gmbh Procédé de préparation de peptides

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0401817A2 (fr) * 1989-06-09 1990-12-12 Research Corporation Technologies, Inc. Réactifs pour synthèse rapide de peptides
EP1491552A2 (fr) 2003-06-23 2004-12-29 CEM Corporation Procédé et appareil pour la synthèse des peptides assistée par micro-ondes
EP1923396A2 (fr) 2006-10-24 2008-05-21 CEM Corporation Synthèse de peptides assistée par micro-ondes
WO2012024224A1 (fr) * 2010-08-16 2012-02-23 Cem Corporation Synthèse sur phase solide de peptides hydrosolubles

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0401817A2 (fr) * 1989-06-09 1990-12-12 Research Corporation Technologies, Inc. Réactifs pour synthèse rapide de peptides
EP1491552A2 (fr) 2003-06-23 2004-12-29 CEM Corporation Procédé et appareil pour la synthèse des peptides assistée par micro-ondes
US7393920B2 (en) 2003-06-23 2008-07-01 Cem Corporation Microwave-assisted peptide synthesis
EP1923396A2 (fr) 2006-10-24 2008-05-21 CEM Corporation Synthèse de peptides assistée par micro-ondes
WO2012024224A1 (fr) * 2010-08-16 2012-02-23 Cem Corporation Synthèse sur phase solide de peptides hydrosolubles

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
CARPINO A L ET AL: "New family of base- and nucleophile-sensitive amino-protecting groups. A Michael.based debloking process. Practical utilization of the 1,1-dioxobenzo[b]thiophene-2-ylmethyloxycarbony (Bsmoc) group", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, ACS PUBLICATIONS, US, vol. 119, 1 January 1997 (1997-01-01), pages 9915 - 9916, XP002426192, ISSN: 0002-7863, DOI: 10.1021/JA9713690 *
CARPINO ET AL., JOURNAL OR ORGANIC CHEMISTRY, vol. 64, no. 12, 1999, pages 4324 - 4338
CARPINO ET AL.: "The 2-methylsulfonyl-3-phenyl-1-proP-2-enyloxycarbonyl (Mspoc) Amino Protecting Group", J. ORG. CHEM., vol. 64, 1999, pages 8399 - 8401, XP055008325, DOI: doi:10.1021/jo990541a
CARPINO ET AL: "The 1,1-Dioxobenzo[b]thiophene-2-ylmethyloxycarbonyl ( Bsmoc ) amino-protecting group", JOURNAL OF ORGANIC CHEMISTRY, ACS, US, vol. 64, no. 12, 1 January 1999 (1999-01-01), pages 4324 - 4338, XP002203970, ISSN: 0022-3263, DOI: 10.1021/JO982140L *
GALANIS, ORGANIC LETTERS, vol. 11, no. 20, 2009, pages 4488 - 4491
HOJO ET AL., CHEM. PHARM. BULL., vol. 52, 2004, pages 422 - 427
HOJO K ET AL: "2-(4-Sulfophenylsulfonyl)ethoxycarbonyl group: a new water-soluble N-protecting group and its application to solid phase peptide synthesis in water", TETRAHEDRON LETTERS, ELSEVIER, AMSTERDAM, NL, vol. 45, no. 50, 6 December 2004 (2004-12-06), pages 9293 - 9295, XP004641320, ISSN: 0040-4039, DOI: 10.1016/J.TETLET.2004.10.095 *
HOJO, K.; MAEDA, M.; KAWASAKI, K., TETRAHEDRON LETT., vol. 45, 2004, pages 9293
R. B. MERRIFIELD: "Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 85, no. 14, 1963, pages 2149, XP002257754, DOI: doi:10.1021/ja00897a025
SHAHNAZ GHASSEMI: "Supported Nucleophiles for Rapid Removal of Bsmoc-Amino Protecting Group Utilizing Microwave Heating", THE 231ST ACS NATIONAL MEETING, ATLANTA, GA, MARCH 26-30, 2006, 28 March 2006 (2006-03-28), XP055008351 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016050764A1 (fr) * 2014-09-29 2016-04-07 Technische Universität Darmstadt Procédé de synthèse de peptides et appareil de mise en œuvre d'un procédé de synthèse en phase solide de peptides
US11466050B2 (en) 2014-09-29 2022-10-11 Sulfotools Gmbh Method for peptide synthesis and apparatus for carrying out a method for solid phase synthesis of peptides
WO2019101939A1 (fr) 2017-11-24 2019-05-31 Sulfotools Gmbh Procédé de préparation de peptides
WO2019101940A1 (fr) 2017-11-24 2019-05-31 Sulfotools Gmbh Procédé de préparation de peptides
US11319340B2 (en) 2017-11-24 2022-05-03 Sulfotools Gmbh Method for preparing peptides
US11976094B2 (en) 2017-11-24 2024-05-07 Sulfotools Gmbh Method for preparing peptides

Similar Documents

Publication Publication Date Title
US20120041173A1 (en) Water soluble solid phase peptide synthesis
Ralhan et al. Piperazine and DBU: a safer alternative for rapid and efficient Fmoc deprotection in solid phase peptide synthesis
Sheppeck II et al. A convenient and scaleable procedure for removing the Fmoc group in solution
Isidro‐Llobet et al. p‐Nitrobenzyloxycarbonyl (pNZ) as a Temporary Nα‐Protecting Group in Orthogonal Solid‐Phase Peptide Synthesis–Avoiding Diketopiperazine and Aspartimide Formation
Pires et al. Some mechanistic aspects on Fmoc solid phase peptide synthesis
Chan et al. Basic procedures
AU2017204172B2 (en) Improved coupling method for peptide synthesis at elevated temperatures
Chen et al. A mild removal of Fmoc group using sodium azide
WO2013115813A1 (fr) Synthèse de peptide en phase solide, soluble dans l'eau
RU2727200C2 (ru) Способ пептидного синтеза и устройство для осуществления способа твердофазного пептидного синтеза
US20120157563A1 (en) Water soluble solid phase peptide synthesis
Hayashi et al. Simultaneous and traceless ligation of peptide fragments on DNA scaffold
WO2019101939A1 (fr) Procédé de préparation de peptides
Marine et al. Azide‐rich peptides via an on‐resin diazotransfer reaction
DK3044204T3 (en) PHOTOLABLE LINKS FOR PHASE PHYSICAL SYNTHESIS OF HYDRAZIDES AND PYRANOPYRAZOLES
KR20240073850A (ko) 수성 고체상 펩티드 합성
US6583318B2 (en) Method for synthesis of α-sulfonamido amide, carboxylic acid and hydroxamic acid derivatives
Ede et al. Synthesis of hydroxamic acids using SynPhase™ crowns: development of the hydroxylamine trityl linker
Pedersen et al. Peptide release, side-chain deprotection, work-up, and isolation
WO2019101940A1 (fr) Procédé de préparation de peptides
Mergler et al. SASRIN™, a Versatile Tool in Peptide Synthesis and Solid-Phase Organic Chemistry
US20150011778A1 (en) Use of trifluoroacetamide for n-terminal protection
Kumar et al. Photocleavable 2-nitrobenzylamino anchored polystyrene-butanediol dimethacrylate supports for the synthesis of protected peptides, peptide amides and peptide N-alkyl amides
Mora A catch-and-release purification method to simplify the synthesis of cysteine-rich peptides
Blanc Synthesis on solid phase of a bicyclic octapeptide amatoxin

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 12703218

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 12703218

Country of ref document: EP

Kind code of ref document: A1