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
  • C07K1/04—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
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
  • B01J2219/00277—Apparatus
  • B01J2219/00351—Means for dispensing and evacuation of reagents
  • B01J2219/00418—Means for dispensing and evacuation of reagents using pressure
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
  • B01J2219/00583—Features relative to the processes being carried out
  • B01J2219/0059—Sequential processes
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
  • B01J2219/00583—Features relative to the processes being carried out
  • B01J2219/00596—Solid-phase processes
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
  • B01J2219/00718—Type of compounds synthesised
  • B01J2219/0072—Organic compounds
  • B01J2219/00725—Peptides
• • C—CHEMISTRY; METALLURGY
  • C40—COMBINATORIAL TECHNOLOGY
  • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
  • C40B40/00—Libraries per se, e.g. arrays, mixtures
  • C40B40/04—Libraries containing only organic compounds
  • C40B40/10—Libraries containing peptides or polypeptides, or derivatives thereof
• • C—CHEMISTRY; METALLURGY
  • C40—COMBINATORIAL TECHNOLOGY
  • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
  • C40B60/00—Apparatus specially adapted for use in combinatorial chemistry or with libraries
  • C40B60/14—Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries

Definitions

• This invention relates to the synthesis of peptides, polypeptides and proteins. More particularly, the invention relates to an improvement in peptide, polypeptide and protein synthesis wherein the peptide, polypeptide or protein is "grown" on an insoluble support or carrier by a series of stepwise coupling reactions.
• the insoluble support is provided a reactive substituent group as by chloromethylation, carboxylation, hydroxymethylation, etc. and a protected amino acid coupled thereto either via the amino or carboxyl group.
• the peptide is synthesized in a stepwise manner on the insoluble support.
• the peptide may be assembled either from the amine or the carboxyl terminus, in practice the latter is preferred, mainly because of lower extents of racemization when N ⁇ -urethane-protected, carboxyl activated amino acids are used for coupling reactions.
• the insoluble support and its appended peptide chain are neutralized if necessary and washed before the addition of the next amino acid residue.
• the polypeptide is removed from the solid support by use of a suitable cleaving reagent, and any necessary deprotections completed. The final peptide is then subjected to extensive purification.
• the reactive sites on the insoluble supports employed in the synthesis are located on and within the support at varying degrees or depths of accessibility. Because of shrinkage and swelling of the resin during a normal synthetic procedure, the number of "inaccessible sites” may change throughout the procedure. This means that a reactive site which is inaccessible during one particular cycle may in practice become accessible during a subsequent cycle.
• Yet another object of the invention is to provide an automatable method wherein long chain polypeptides of greater than 20 amino acids can be prepared on a large scale, with little, if any, side reactions.
• a further object of the invention is to provide a method wherein coupling reactions are complete at every stage of the synthesis regardless of the sequence or structure of the peptide so that the polypeptide or protein product of desired chain length or size requires minimum purification or in many instances no purification after cleavage from the support.
• the flow reactor is pressurized to at least 100 psi or 0.00145 dyne/cm 2 , up to 1,000 psi or 0-0145 dyne/cm 2 although pressures of up to 10,000 psi or 0.145 dyne/cm 2 or more may be used.
• the flow rate of the reactants and reagents employed will fall in the range of 4 to 50 ml per minute or more depending on the size of the reactor used.
• the first amino acid passed through the reactor packed with the insoluble support containing substituent groups reactive with said amino acid is a protected amino acid.
• a protected amino acid is coupled to said support by condensation reaction with said substituent groups, said coupled first amino acid is deprotected by passing a deprotecting agent through the flow reactor, a second similar or dissimilar protected amino acid is passed through said reactor and coupled to said first coupled amino acid, said coupled second amino acid is deprotected and the process repeated until the desired polypeptide is obtained, the improvement being that during said synthesis the reactants and reagents are passed through the reactor in a continuous flow and the reaction pressure in said reactor is at the defined elevated pressures in order to obtain rapid reactions and quantitative yields of the desired products.
• reaction rates achieved under the pressurized flow system of the invention compared to conventional solid-phase polypeptide coupling reactions are unexpectedly rapid. For example, coupling reactions which typically take, hours by the conventional procedures are completed in minutes. Such rapid rates of reaction result in the complete elimination of undesirable side reactions that occur because of the tendency of activated amino acids to decompose or rearrange during the long reaction times required for conventional synthetic procedures.
• a pressurized flow reactor may be used to increase rates and efficiencies of reactions in sequencing of polypeptides, proteins and nucleic acids.
• the use of a pressurized flow reactor in accordance with the teachings of the invention results in increased rates of reaction and also drives the reaction to completion, the both of which combine to result in rapid synthesis of pure materials in complicated, repetitive, sequential synthetic procedures.
• polypeptides as used in the specification and the appended claims is meant to include peptides and proteins.
• an insoluble solid support or matrix advantageously in bead form such as any of the conventional solid-phase polymeric substrates conventionally employed for the synthesis of polypeptides can be utilized.
• polymeric resins are crosslinked polystyrene resins, crosslinked polyacrylamides, glass beads, clays, celite, crosslinked dextran, and similar insoluble solid supports which either naturally contain reactive sites for coupling with the amino acid components or which can be provided with such reactive sites.
• Insoluble supports particularly preferred are derivatized, crosslinked polystyrene resins, such as chloromethylated or hydroxymethylated, crosslinked polystyrene resins, benzhydrylamine resins and the like.
• the crosslinked polystyrenes are normally copolymers of styrene and a crosslinking agent preferably formed by way of a pearl or bead polymerization process using an aqueous suspension system.
• Preferred crosslinking agents for preparation of the crosslinked polystyrene resins include divinyl compounds such as para-divinylbenzene, meta-divinylbenzene, divinylcyclohexane, butadiene, and the like.
• resin supports containing higher than normal levels of crosslinking e.g.
• the supporting resin containing the C-terminal amino acid of the proposed peptide chain must be prepared. This can be accomplished in the same reactor and under the pressurized flow conditions of the present invention.
• hydroxymethylated resins may be esterified in the flow reactor by esterification with a protected amino acid mixed or symmetrical anhydride, catalyzed by 4-dimethylaminopyridine.
• the attachment of the first i.e.
• C-terminal amino acid may be prepared for instance by esterifying a suitably protected amino acid with the reactive site or substituent group on the insoluble support such as chloromethy1ated or hydroxymethylated crosslinked polystyrene resins.
• the esterification reaction is accomplished directly with the chloromethylated resin or via suitable activation of the protected amino acid in the case of the hydroxymethylated resin.
• protecting groups for terminal reactive amino groups which have been employed in peptide synthesis.
• the protecting groups of choice in prior art syntheses have been the benzyloxycarbonyl and especially the t-butyl-oxycarbonyl groups but, because of the extremely strong acidic conditions required for their removal, more labile groups are preferred for high performance peptide synthesis.
• protecting groups and method of attachment of the first amino acid to the resin support are recommended.
• sidechain protecting groups, as well as the linkage between the resin and the growing peptide chain be completely stable to the various conditions used throughout the synthesis.
• these protecting groups and the linkage to the resin should be sufficiently labile so that final deprotection of the peptide and its cleavage from the support may be accomplished under the mildest possible conditions without decomposition of sensitive amino acids or destruction of the peptide.
• Conventional solid-phase techniques use N ⁇ -t-butyloxycarbonyl protection in conjunction with benzyl sidechain protection and attachment to the resin.
• t-Butyloxycarbonyl group removal requires relatively strongly acidic conditions (e.g. 50% trifluoroacetic acid:dichloromethane), while final deprotection and cleavage from the resin requires even more strongly acidic conditions (e.g. anhydrous hydrogen fluoride or hydrogen bromide/acetic acid), which can lead to extensive decomposition of the final product. Furthermore, the lack of complete selectivity during N ⁇ -deprotection can lead to partial cleavage of the growing peptide chain and consequent reduction in yield, as well as the possibility of contamination by truncated sequences.
• relatively strongly acidic conditions e.g. 50% trifluoroacetic acid:dichloromethane
• final deprotection and cleavage from the resin requires even more strongly acidic conditions (e.g. anhydrous hydrogen fluoride or hydrogen bromide/acetic acid), which can lead to extensive decomposition of the final product.
• anhydrous hydrogen fluoride or hydrogen bromide/acetic acid e.g
• Termination by trifluoroacetylation of the amine terminus during deprotection with trifluoroacetic acid has also been shown to be a serious side reaction in this synthetic scheme.
• t-butyloxycarbonyl and benzyloxycarbonyl protecting groups can.be used in the present invention, it is preferred that N ⁇ -protecting groups which are removed under the mildest possible conditions be used.
• Such groups are biphenylisopropyloxycarbonyl, adamantylisopropyloxycarbonyl and nitrophenylsulfenyl groups which are removable under mild, acidic conditions, or the 9-fluorenyImetHyIoxycarbonyl or trifluoroacetyl groups which are cleaved under mild, basic conditions.
• resins of the p-alkoxybenzyl alcohol type as [proposed by Wang, J. Amer. Chem. Soc. 95, 1328 (1973)], which are cleaved under relatively mild, acidic conditions (e.g. 50:50 trifluoroacetic acid: dichloromethane) are superior.
• a preferred combination for the invention utilizes N ⁇ -fluorenylmethyloxycarbonyl in conjunction with t-butyl sidechain protection and the p-alkoxybenzyl alcohol resin. This results in complete selectivity, with N ⁇ -deprotection under basic conditions and final cleavage and sidechain deprotection under acidic conditions. Use of this combination also reduces the danger of cleavage of the growing peptide chain from the resin during the synthesis and minimizes decomposition during final deprotection and cleavage of the product. Furthermore, coupling cycles are shortened substantially since no neutralization is required after the deprotection step.
• the resulting product is commonly analyzed using standard procedures such as spectrophotometric or quantitative amino acid analysis to determine the amino acid content for the purpose of calculating the amounts of subsequent amino acid reactants and deprotecting agents to be used in the synthesis.
• the thus-prepared C-terminal amino acid-containing, support is packed into a suitable continuous flow reactor.
• the reactor may take any desired shape or form so long as it is capable of withstanding the elevated pressures under which the synthesis of the present invention may be conducted.
• the preferred reactors are column reactors having an inlet and an outlet so as to maximize the contact time between solvent or reactants and the growing peptide chains on the resin, thus maximizing the efficiency of the process.
• the remaining synthesis to form the desired polypeptide sequence is carried out as follows. Before coupling of the second amino acid residue can take place, the first residue already on the support must be deprotected. Deprotection of the first amino acid residue on the resin as well as on each of the subsequently coupled amino acid residues can be carried out by pumping through the reactor an appropriate deprotecting agent.
• the deprotecting agents employed for this purpose are well known to those of ordinary skill in the peptide synthesis art and the particular deprotecting agent employed in any given instance will depend of course upon the protecting group on the amino acid/resin.
• the protecting group is t-butyloxycarbonyl, trifluoroacetic acid, methanesulfonic acid or hydrochloric acid in a suitable solvent such as dioxane or dichloromethane may be used.
• the protecting group is biphenylisopropyloxycarbonyl, mild acidic solvolysis (for example with 1% trifluoroacetic acid in dichloromethane) is the preferred method of deprotection.
• the N ⁇ -protecting group is 9-fluorenylmethyloxycarbonyl
• the preferred deprotecting agent is piperidine in dimethyformamide (DMF).
• concentrations of the particular deprotecting agent in the solvent will vary depending again upon the particular protecting agent employed but will ordinarily range from about 5 to 50% by volume.
• a sufficient volume of deprotecting agent is pumped through the reactor over a period of time sufficient to effect complete removal of the protecting groups. Frequently, multiple reactor volumes (for example 2-10 column volumes over a period of 5-30 minutes) of the solution containing the deprotecting agent are passed through the reactor to ensure complete removal of the protecting group.
• the resin is washed with a suitable solvent, normally the solvent in which the deprotecting agent was dissolved, in order to remove excess deprotecting agent.
• the peptide on the resin must be neutralized by washing with an appropriate base such as triethylamine in a solvent such as dichloromethane. Any excess triethylamine and triethylammonium chloride, or trifluoroacetate, formed may be removed by repeated washings with a suitable solvent such as dichloromethane or dimethylformamide. The free ⁇ -amino group, thus prepared, is now ready for coupling with the next protected amino acid.
• an appropriate base such as triethylamine in a solvent such as dichloromethane. Any excess triethylamine and triethylammonium chloride, or trifluoroacetate, formed may be removed by repeated washings with a suitable solvent such as dichloromethane or dimethylformamide.
• N ⁇ -protected amino acid is first activated, that is, converted into a reactive form, for instance, by converting the amino acid into an active ester or anhydride or by activation with dicychlohexylcarbodiimide, carbonyldiimidazole or other activating agents.
• the most preferred activated amino acid derivatives are the mixed anhydride, symmetrical anhydride or active ester derivatives (such as the p-nitrophenyl esters catalyzed by 1-hydroxybenzotriazole). These activated amino acids are preferred because of the short coupling times, quantitative yields and minimization or complete elimination of side reactions they provide.
• a solution of the activated protected second amino is then passed into and through the reactor packed with the support now containing an unprotected C-terminal amino acid.
• an excess of the activated, protected amino acid per equivalent of the first amino acid on the resin is employed although the excess required may be limited by recycling solutions through the column.
• the common practice is to pass more than one reactor volume of the activated, protected amino acid through the reactor to ensure complete reaction. It should be understood that any of the conventional methods of activating amino acids for the purpose of coupling with another amino acid may be applied to this method. These procedures should be well-known to anyone skilled in the art of peptide chemistry.
• the attached protected amino acid is then deprotected, neutralized (if necessary) and washed as described above before coupling of the next amino acid derivative is effected. This procedure is repeated until the desired sequence of amino acids has been assembled on the insoluble support.
• Resins of the p-alkoxybenzyl alcohol type are preferred because of the relatively mild cleavage conditions (for example 50% trifluoroacetic acid: dichloromethane) which are required. These mild cleavage conditions minimize or eliminate completely the destruction of sensitive amino acids during the cleavage process.
• the entire series of coupling reactions in the abovedescribed methods, from the second amino acid, (and if desired the first amino acid) to the last is conducted in a pressurized reactor, preferably under a reactor pressure of at least 40 psi or 0.00058 dyne/cm up to 1,000 psi or 0.0145 dyne/cm 2 , although pressures of up to 10,000 psi or 0.145 dyne/cm 2 or more may be used.
• Reactor pressures of this level can be generated by use of commercial pressurizing equipment and methods.
• any of the commercially available reciprocating pumps capable of generating the required pressures and flow rates can be used and the reactants, reagents and wash solvents pumped directly into and through the reactor.
• the reactants, reagents and wash solvents may be pumped through the reactor by means of pressurization with an inert gas such as nitrogen and the pressure in the reactor regulated by controlling the volume of inert gas released to transfer the reactants, reagents and wash materials into and through the reactor.
• the scale of the peptide syntheses in the pressurized flow reactor may vary widely, being limited solely by the capacity of the pressurized pun-ping means utilized. For example, reactors in the range 5-100 ml in volume may be used for the production of up to tens of grams of peptides, although larger reactors (up to 1000 ml or more) may be used if necessary.
• the completed peptide sequence can be removed from the insoluble support by any of the standard methods as, for instance, by cleavage with anhydrous hydrogen fluoride, transesterification, acidolysis, aminolysis, etc.
• This cleavage is most conveniently accomplished by extruding the peptide-support from the reactor and treating it with the cleavage reagent at atmospheric pressure.
• the cleavage may also be carried out in the reactor and under the elevated reactor pressures of the invention provided the reactor is resistant to the cleavage reagent.
• the resulting peptide is found to be remarkably homogeneous and to require no or minimal purification. Because of the very low contamination of by-products overall yields are found to be surprisingly high and whatever purification is necessary can be carried out with relative ease. Such purifications, if required, are preferably carried out by partition chromatography, ion exchange chromatography, gel permeation chromatography, countercurrent distribution and the like.
• Illustrations of peptides, polypeptides and proteins which can be obtained by the method of the invention are enkephalins, angiotensin, oxytocin, vasopressin, leuteinizing hormone releasing hormone, somatostatin, gastrin, insulin, glucagon, ribonuclease, endorphins, etc.
• Example 1 Synthesis of L-Leucyl-L-alanyl-glycyl-L-valine using 9-Fluorenylmethyloxycarbonyl (Fmoc)- protected Amino Acid Mixed Anhydrides p-Benzyloxybenzyl alcohol polystyrene resin (2% crosslinked) was esterified with Fmoc-L-valine by the method of Wang [J. Amer. Chem. Soc. 95, 1328 (1973)] to give a substitution level of 0.6 moles/gram as determined by the spectrophotometric method of Meienhofer et al. [Int. J. Pept. Prot. Res. 13, 35 (1979)].
• the resin (1.2 g) was swollen in dry DMF, packed into a stainless steel column reactor (1.0 ⁇ 5 cm) and treated with 10% (w.v) peperidine in D-MF at a flow-rate of 5 ml/minute for 10 minutes.
• the column reactor was washed to neutral pH with DIM-F at a flow-rate of approximately 20 ml/minute over a 5 minute period before treating with a solution of Fmoc-glycine mixed anhydride [prepared by treating a solution of Fmocglycine (3.5 mmoles) and N-methylmorpholine (3.5 mmoles) in DMF (40 ml) with isobutyl chloroformate (3.2 moles)] over a 10 minute period.
• Example 2 Synthesis of L-Leucyl-L-alanyl-glycyl-L-valine using Fmoc-protected Amino Acid Symmetrical Anhydrides
• the symmetrical anhydrides were prepared by treating the appropriate Fmoc-amino acid (2.4 mmoles) with dicyclohexylcarbodiimide (1.2 mmoles) in DMF (12 ml). The solution was filtered to remove dicyclohexylurea prior to use. Couplings were allowed to proceed for 10 minutes. Deprotection, cleavage and isolation of the product as described under Example I gave 0.242 g of the tetrapeptide (99%) yield) which was homogeneous ( 99% pure) by chromatographic analysis.
• Example 3 Synthesis of L-Leucyl-L-alanyl-glycyl-L-valine using Fmoc-protected Amino Acid p-Nitrophenyl Esters The synthesis described under Example 2 was repeated using p-nitrophenyl esters catalyzed by 1-hydroxybenzotriazole for activation. Couplings were allowed to proceed for 15 minutes using solutions of the appropriate Fmoc-amino acid p-nitrophenyl esters (1.2 mmoles, 2.3 equivalents) and 1-hydroxybenzotriazole (1.2 mmoles) in DMF (12 ml).
• Example 4 Synthesis of L-Leucyl-L-alanyl-glycyl-L- valine using Biphenylisopropyloxycarbonyl (Bpoc) - protected Amino Acid Mixed Anhydrides p-Benzyloxybenzyl alcohol polystyrene resin (2% crosslinked) was esterified with Bpoc-L-valine by the method of Wang, to give a substitution level, as determined by nitrogen analysis, of 0.49 mmoles/gram.
• This resin (1.2 g) was swollen in dichloromethane and packed into a stainless steel column reactor (1.0 ⁇ 5 cm). Cleavage of the Bpoc-group was effected by passing a 1% solution of trifluoroacetic acid in dichloromethane through the column for 10 minutes at a flow-rate of 5 ml/minute. The column was subsequently washed with dichloromethane (10 ml/min., 5 minutes), neutralized with 5% diisopropylethylamine in dichloromethane (10 ml/min., 5 minutes) and finally washed to neutrality with dichloromethane (10 ml/min., 5 minutes).
• Bpoc-glycine mixed anhydride (0.03 M, 80 ml) (generated as described under Example 1) was passed through the column over a 15 minute period. The resin was washed with dichloromethane (10 ml/min., 10 minutes) before repeating the total cycle using Bpoc alanine and Boc-leucine mixed anhydrides. The entire synthesis was carried out under 200 psi or 0.0029 dyne/ cm 2 nitrogen pressure. The tetrapeptide was isolated as described under Example 1 (0.273 g, 99% yield) and shown to be homogeneous (>99% pure) by chromatographic analysis.
• Example 5 Synthesis of L-Prolyl-L-prolyl-L-valine using Fmoc-protected Amino Acid Mixed Anhydrides
• the synthesis was carried out in dichloromethane as solvent, except for Fmoccleavage cycles which utilized 10% piperidine in DMF.
• the tripeptide was cleaved and deprotected as described in Example 1 and shown to contain 2% isobutyldxycarbonyl-L-prolyl-L-valine (the "wrong-opening" product) by reverse phase high performance liquid chromatography.
• Example 6 Synthesis of Methionine -Enkephalin (L-Tyrosylglycyl-glycyl-L-phenylalanyl-L-methionine) using Fmoc-Amino Acid Mixed Anhydrides
• the resin was treated sequentially with the mixed anhydrides (16 mmoles/100 ml; 10 minutes) of Fmoc-glycine, Fmoc-O-t-butyl-L-serine, Fmoc-L-alanine, Fmoc-S-t-butyl-L-aspartic acid, Fmoc-glycine (twice), Fmoc-L-alanine, and finally Boc-L-tryptophan.
• the synthesis was carried out under 1000 psi or 0.0145 dyne/cm 2 nitrogen pressure.
• the peptide-resin was removed from the reactor and cleaved and deprotected by suspending in trifluoroacetic acid:ethanedithiol: dichloromethane (90:10:20) for 1.75 hours under nitrogen at room temperature. After filtration and evaporation of the solvent the residue was washed with ether to give delta-sleep inducing peptide (1.70 g, 100%). Analysis by reverse phase high performance liquid chromatography showed the peptide to be>95% pure, with only trace contamination by peptide impurities.

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• 1982
  • 1982-03-09 WO PCT/US1982/000307 patent/WO1982003077A1/fr not_active Application Discontinuation
  • 1982-03-09 EP EP19820901235 patent/EP0073828A4/fr not_active Withdrawn
  • 1982-03-09 ZA ZA821537A patent/ZA821537B/xx unknown

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