RAPID PEPTIDE SYNTHESIS AT ELEVATED TEMPERATURES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority benefit of U.S. Provisional Patent Application

No. 62/397, 167, filed September 20, 2016. The entire contents of that application are hereby incorporated by reference herein.

FIELD OF THE INVENTION

[0002] The present invention relates to the field of peptide synthesis, and particularly, to methods of rapidly synthesizing peptides at elevated temperatures.

BACKGROUND OF THE INVENTION

[0003] 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.

[0004] Reaction conditions of solid phase peptide synthesis (SPPS) have undergone development and optimization for decades since the development of SPPS by Merrifield (J. Am. Chem. Soc. 85(14): 2149-2154 (1963)). Currently, popular amino acid coupling methods include diisopropyl carbodiimide/l-hydroxybenzotriazole (DIC/HOBt), 2-(lH-benzotriazole-l- yl)-l, l,3,3-tetramethyluronium hexafluorophosphate/diisopropylethylamine (HBTU/DIEA), or 2-(7-aza- 1 H-benzotriazole- 1 -yl)- 1 , 1 ,3,3-tetramethyluronium

hexafluorophosphate/diisopropylethylamine (HATU/DIEA) as coupling reagents/promoters. Fmoc amino group protection is widely used, and 20% piperidine in DMF is used for removal (referred to as deblocking or deprotection) of the Fmoc protecting group at room temperature. However, one drawback to the basic conditions used is potential racemization of the L-amino acids.

[0005] 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. However, 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. As 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.

[0006] 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

recommended to provide sufficient mixing of the Fmoc amino acid solution and the resin.

However, uronium salts such as HBTU, HATU, and HCTU are expensive, especially for the large-scale peptide synthesis needed to produce vaccine epitopes.

[0007] Diisopropyl carbodiimide (DIC) and HOBt ( 1 -hydroxybenzotriazole) 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. However, the longer coupling reaction time needed for good coupling efficacy with DIC/HOBt leads to longer synthesis times and increased expense.

[0008] There is thus a need for a method of synthesizing peptides on a large scale rapidly and inexpensively, while at the same time maintaining good purity, high yield, and low rates of racemization.

BRIEF SUMMARY OF THE INVENTION

[0009] 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.

[0010] Peptides with sensitive residues, such as Cys, Met, and Trp, can be prepared in good yield and purity using the methods of the invention. 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.

[0011] In one embodiment, the invention embraces a method for rapid solid-phase peptide synthesis of a desired peptide sequence on a resin, comprising:

[0012] 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 2 and about 20 minutes at a temperature between about 40 °C and about 80 °C;

[0013] b) contacting the N-terminal-unprotected resin-bound peptide or amino acid with a coupling solution comprising an N-protected amino acid, a carbodiimide activating reagent, and an active ester-forming reagent at a temperature between about 40 °C and about 80 °C, for a time between about 10 minutes to about 60 minutes; and

[0014] c) repeating a) and b) until the desired peptide sequence is formed on the resin.

[0015] In one embodiment, the contacting with a coupling solution can be carried out at a temperature between about 50 °C and about 70 °C.

[0016] In one embodiment, 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;

[0018] a.i) when the scale of synthesis is at or below about 1 mmol of peptide, the contacting with the deblocking solution can be performed for between about 4 minutes to about 10 minutes,

[0019] a.ii) when the scale of synthesis is above about 5 mmol of peptide, the contacting with the deblocking solution can be performed for between about 7 minutes to about 20 minutes, and

[0020] a.iii) when the scale of synthesis is between about 1 mmol of peptide to about 5 mmol of peptide, 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; and

[0021] b) contacting the N-terminal-unprotected resin-bound peptide or amino acid with a coupling solution comprising an N-protected amino acid, a carbodiimide activating reagent, and an active ester-forming reagent at a temperature between about 40 °C and about 80 °C, wherein

[0022] b.i) when the scale of synthesis is at or below about 1 mmol of peptide, the contacting with the coupling solution can be performed for between about 10 minutes to about 30 minutes,

[0023] b.ii) when the scale of synthesis is above about 5 mmol of peptide, the contacting with the coupling solution can be performed for between about 20 minutes to about 60 minutes, and

[0024] b.iii) when the scale of synthesis is between about 1 mmol of peptide to about 5 mmol of peptide, 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; and

[0025] c) repeating a) and b) until the desired peptide sequence is formed on the resin.

[0026] In one embodiment, the contacting with a coupling solution can be carried out at a temperature between about 50 °C and about 70 °C.

[0027] In any of the embodiments of the methods of the invention, 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). In any of the embodiments of the methods, the carbodiimide activating reagent can comprise diisopropylcarbodiimide (DIC). [0028] In any of the embodiments of the methods, 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).

[0029] In any of the embodiments of the methods, the N-protected amino acid and the protected N-terminus can be protected with 9-fluorenylmethoxycarbonyl (Fmoc) groups.

[0030] In any of the embodiments of the methods, 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. In a further embodiment of any of the methods, the resin can comprise a resin selected from the group consisting of 4-methylbenzhydrylamine (MBHA) resin, Wang resin, or aminomethyl (AM) resin.

[0031] In any of the embodiments of the methods, the deblocking solution can comprise a dipolar aprotic solvent, and the coupling solution independently can comprise a dipolar aprotic solvent.

[0032] In any of the embodiments of the methods, 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).

[0033] In any of the embodiments of the methods, 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.

[0034] In any of the embodiments of the methods, 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.

[0035] In any of the embodiments of the methods, the deblocking reagent of the deblocking solution can comprise piperidine.

[0036] In any of the embodiments of the methods, the deblocking solution can comprise about 20% piperidine.

[0037] In any of the embodiments of the methods, 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. [0038] In any of the embodiments of the methods, the washing of the resin can comprise washing the resin with a dipolar aprotic solvent.

[0039] In any of the embodiments of the methods, 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. In any of the embodiments of the methods, 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).

[0040] In any of the embodiments of the methods, 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. In any of the embodiments of the methods, 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. [0041] In any of the embodiments of the methods, 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. In any of the embodiments of the methods, 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%.

[0042] In any of the embodiments of the methods, the peptide synthesis can be performed on a scale to yield about 0.2 mmol to about 0.4 mmol of peptide.

[0043] In any of the embodiments of the methods, 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.

[0044] In any of the embodiments of the methods, the peptide synthesis can be performed without microwave irradiation.

[0045] In any of the embodiments of the methods, the peptide synthesis can be performed without added salts.

[0046] These and other aspects and advantages of the present invention will become apparent from the subsequent detailed description and the appended claims. It is to be understood that one, some, or all of the properties of the various embodiments described herein may be combined to form other embodiments of the present invention where possible.

BRIEF DESCRIPTION OF THE DRAWINGS

[0047] FIG. 1 depicts the purity of various synthesized peptides, versus the temperature of synthesis.

[0048] FIG. 2 depicts the weight gain of the peptide-resins (resin weight after synthesis minus resin weight before synthesis) for the various synthesized peptides. DETAILED DESCRIPTION OF THE INVENTION

[0049] Methods and protocols for rapid heated peptide synthesis are described herein.

I. Definitions and General Matters

[0050] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0051] Unless otherwise stated, all chiral amino acids in naturally occurring peptides and synthetic analogs thereof are in the L-configuration.

[0052] As used herein, "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"). In reference to deblocking of an Fmoc group, the reaction is also referred to as "deFmoc".

[0053] "Crude 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.

[0054] When numerical values are expressed herein using the term "about" or the term

"approximately," it is understood that both the value specified, as well as values reasonably close to the value specified, are included. For example, the description "about 50° C" or

"approximately 50° C" includes both the disclosure of 50° C itself, as well as values close to 50° C. Thus, 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").

[0055] With respect to numerical ranges disclosed in the present description, 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.

[0056] It is understood that reference to 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.

[0057] Some embodiments described herein are recited as "comprising" or "comprises" with respect to their various elements. In alternative embodiments, those elements can be recited with the transitional phrase "consisting essentially of or "consists essentially of as applied to those elements. In further alternative embodiments, those elements can be recited with the transitional phrase "consisting of or "consists of as applied to those elements. Thus, for example, if a composition or method is disclosed herein as comprising A and B, the alternative embodiment for that composition or method of "consisting essentially of A and B" and the alternative embodiment for that composition or method of "consisting of A and B" are also considered to have been disclosed herein. Likewise, embodiments recited as "consisting essentially of or "consisting of with respect to their various elements can also be recited as "comprising" as applied to those elements. Finally, embodiments recited as "consisting essentially of with respect to their various elements can also be recited as "consisting of as applied to those elements, and embodiments recited as "consisting of with respect to their various elements can also be recited as "consisting essentially of as applied to those elements.

[0058] When a device, composition, or system is described as "consisting essentially of the listed elements, the device, composition, or system contains the elements expressly listed, and may contain other elements which do not materially affect the condition being treated (for compositions for treating conditions), or the properties of the described device or system.

However, 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. When a method is described as "consisting essentially of the listed steps, 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.

[0059] As used herein and in the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise.

[0060] Abbreviations:

ACN acetonitrile

AA Amino acid

Boc Tert-butoxycarbonyl

Cl-HOBt 6-chloro- 1 -hydroxybenzotriazole

DCM Dichloromethane

DIC Diisopropylcarbodiimide

DMF Ν,Ν-dimethyl formamide

EDT Ethanedithiol

eq equivalent

Fmoc 9-fluorenylmethyloxycarbonyl

HOBt 1 -hydroxybenzotrizole

HPLC High performance liquid chromatography

hr(s) Hour(s)

He isoleucine

min minutes

mmt 4-methoxytrityl

mtt 4-Methyltrityl

NMP N-methyl -2-pyrrolidone

Pip Piperidine

TFA Trifluoroacetic acid TIS Triisopropylsilane

Trt Trityl

[0061] This disclosure provides several embodiments. It is contemplated that any features from any embodiment can be combined with any features from any other embodiment where possible. In this fashion, hybrid configurations of the disclosed features are within the scope of the present invention.

II. Methods for rapid heated solid-phase peptide synthesis

[0062] The methods of the present invention provide for rapid synthesis of peptides at elevated temperatures, with good resulting yield, purity, and stereochemical purity. Preferably, an automated or semi-automated peptide synthesizer is used to perform the solid-phase peptide synthesis (SPPS).

[0063] Fmoc-based SPPS methods have been described in the art; see, for example, Chan, WC & White, PD, Fmoc solid phase peptide synthesis: A practical approach, Oxford University Press, 2004, which is incorporated herein by reference. Solid-phase peptide synthesis comprises at least the following procedures:

[0064] 1) obtaining a suitable solid support on which to perform the synthesis; typically the solid support used is a polystyrene resin modified with various functional groups, but a variety of solid supports are available;

[0065] 2) attaching the initial N-protected amino acid to the resin or other solid support. As solid-phase peptide synthesis is performed from C-terminus to N-terminus, 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);

[0066] 3) deblocking the initial amino acid to remove the N-protecting group, using a deblocking solution;

[0067] 4) coupling the next amino acid in the sequence to the resin-bound amino acid, using a coupling solution. The amino acid to be coupled is N-protected, to prevent oligomerization when its carboxyl group is activated for coupling to the unprotected amino group of the amino acid on the resin;

[0068] 5) repeating 3) and 4) until the desired peptide sequence has been synthesized.

[0069] Solid-phase peptide synthesis provides a convenient way of isolating the synthetic intermediates, as they are attached to the solid resin. Thus, 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.

[0070] 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.

[0071] A typical protocol for rapid heated peptide synthesis is as follows:

[0072] Temperature used for the synthesis: about 40°C to about 80°C, about 45 °C to about

75°C, about 50°C to about 70°C, or about 55°C to about 65°C,such as about 60°C.

[0073] 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.

[0074] 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. 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.

[0075] 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.

[0076] Washing solvent after coupling: DMF. [0077] Washing after coupling: about 2 to about 3 washes for about 20 to about 40 seconds per each wash.

[0078] Monitoring of coupling completion: 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. In some embodiments of the methods of the invention, 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 (or after double-coupling) is optional, but typically not necessary.

[0079] Deblocking/DeFmoc reagent: about 20% piperidine in DMF.

[0080] 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. Alternatively, 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. Alternatively, 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.

[0081] Washing solvents after deblocking: DMF/DCM. [0082] Washing time after deblocking: about 5 to about 7 washes for about 30 seconds to about 40 seconds per each wash.

[0083] Total cycle time: about 30 minutes to about 35 minutes.

[0084] Synthesis at elevated temperatures enables reduction of coupling time, deblocking time, and washing time. The elevated temperature increases the rate of reaction for coupling and deblocking, and accelerates penetration of solvents and reagents through the resin for further reduction of coupling, deblocking, and washing times. Because of the better accessibility of the solvents to the resin, the number of resin washes after coupling and after deblocking can also be reduced without compromising crude peptide purity, also leading to further reduction in the overall synthesis time. Reducing the number of washes required also reduces needed solvent volume, with a corresponding reduction in the cost of solvent and cost of disposal of used solvent.

Cleavage of peptide-resins and further purification of peptides

[0085] When the desired peptide has been synthesized, 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. As any reactive side chains of the amino acids are also protected during synthesis, 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).

[0086] 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. In some embodiments, the acidic cleavage solution further comprises one or more scavengers, including, but not limited to, ethanedithiol (EDT), triisopropylsilane (TIS), phenol, and thioanisole. Reagents and cleavage conditions known in the art are described, for example, in "Introduction to Cleavage Techniques, Applied Biosystems, Inc.," 1990, pp. 6-12, which is incorporated herein by reference. [0087] In some embodiments, 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.

[0088] 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.

[0089] 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. In some embodiments, the acidic cleavage solution comprises about 2% of EDT by volume. In some embodiments, the acidic cleavage solution comprises about 2% of TIS by volume. In some embodiments, the acidic cleavage solution comprises about 2% of EDT and about 2% of TIS by volume.

[0090] 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. In some embodiments, the acidic cleavage solution comprises about 2% of water by volume. In some embodiments, the acidic cleavage solution comprises about 94% of TFA, 2% of EDT, 2% of TIS and about 2% of water by volume.

[0091] If desired, after cleavage of the peptide from the resin, further purification can be performed. 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.

[0092] 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

[0093] 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. Examples of resins useful in rapid heated peptide synthesis are 4-methylbenzhydrylamine (MB HA) resin, benzhydrylamine (BHA) resin, Wang resin, and aminomethyl (AM) resin. In one embodiment, rapid heated peptide synthesis is performed using 4-methylbenzhydrylamine (MBHA) resin, benzhydrylamine (BHA) resin, Wang resin, or aminomethyl (AM) resin. In one embodiment, rapid heated peptide synthesis is performed using 4-methylbenzhydrylamine (MBHA) resin, Wang resin, or aminomethyl (AM) resin. In one embodiment, 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.

[0094] 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

Millipore, Billerica, Massachusetts, USA).

Temperatures for rapid heated peptide synthesis

[0095] As noted above, elevated temperatures provide for accelerated reaction rates, and better mixing and penetration of solvents and reagents into the resin, which shortens synthesis times and improves overall synthetic efficiency.

[0096] 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. [0097] 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.

[0098] 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

[0099] Extensive testing of reaction conditions has led to optimization of the coupling solutions, activating reagents, and active ester-forming reagents to be used in peptide synthesis. One notable problem during synthesis at elevated temperatures, such as the temperatures used in the current invention, is racemization of the activated amino acid. Indeed, racemization is sometimes observed at room temperature synthesis or synthesis at temperatures below room temperature.

[0100] 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.

[0101] In one embodiment of the invention, the activating reagent comprises a carbodiimide activating reagent. In one embodiment of the invention, 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). In a preferred embodiment, the carbodiimide activating reagent comprises

diisopropylcarbodiimide (DIC).

[0102] In one embodiment of the invention, the active ester-forming reagent used with the activating reagent comprises a benzotriazole or azabenzotnazole reagent. In one embodiment of the invention, 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). In a preferred embodiment, the active ester-forming reagent used with the activating reagent comprises 1-hydroxybenzotriazole (HOBt).

[0103] In a further preferred embodiment, the activating reagent comprises

diisopropylcarbodiimide (DIC), and the active ester-forming reagent used with the activating reagent comprises 1-hydroxybenzotriazole (HOBt).

[0104] In any of the above embodiments, 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.

[0105] 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. [0106] 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.

[0107] 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.

[0108] 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.

[0109] 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.

[0110] 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.

[0111] Typically, transfer of solutions of Fmoc amino acids becomes more difficult as their concentration increases about above 1 M, and thus, the concentration ranges used are preferably at or below 1 M. In a preferred embodiment, 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

[0112] The rapid heated peptide synthesis methods disclosed herein are excellent for synthesis of peptides on a large scale. Using ordinary peptide synthesis methods on a large scale often leads to substandard yields, due to slower diffusion of solvents and reagents and longer reaction times. Rapid heated peptide synthesis overcomes these problems.

[0113] 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.

[0114] 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.

[0115] 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. [0116] 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.

[0117] 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.

[0118] 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.

[0119] 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.

[0120] 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.

[0121] 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. [0122] 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%.

[0123] 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%.

[0124] 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%

[0125] 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%.

[0126] For any synthesis on any scale as described above, whether the yield is given in mg or grams, in mmol, in percent crude purity, or in percent crude yield, 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. For any synthesis on any scale as described above, whether the yield is given in mg or grams, in mmol, in percent crude purity, or in percent crude yield, 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. For any synthesis on any scale as described above, 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 15 residues; or between about 10 residues to about 20 residues, between about 15 residues to about 25 residues, between about 20 residues to about 30 residues, between about 25 residues to about 35 residues, between about 30 residues to about 40 residues, between about 35 residues to about 45 residues, or between about 40 residues to about 50 residues.

[0127] For any synthesis on any scale as described above, whether the yield is given in mg or grams, in mmol, in percent crude purity, or in percent crude yield, 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 about 55,000, about 4,000 to about 50,000, about 4,000 to about 45,000, about 4,000 to about 40,000, about 4,000 to about 35,000, about 4,000 to about 30,000, about 4,000 to about 25,000, about 4,000 to about 20,000, about 4,000 to about 15,000, about 4,000 to about 10,000, about 2,000 to about 4,000, about 2,000 to about 3,000, or about 3,000 to about 5,000.

[0128] 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. In one embodiment, 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.

[0129] For any of the methods described herein, 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%.

Times for washing, deblocking, and coupling; double-coupling

[0130] 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.

[0131] For peptide synthesis performed on a scale above about 5 mmol (for example, about 5 mmol to about 7.5 mmol), 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.

[0132] For peptide synthesis performed on a scale of about 1 mmol to about 5 mmol, either of the above periods— that is, either the shorter coupling periods for up to about 1 mmol, or the longer coupling periods for above 5 mmol— can be used. If speed is of the essence, then the shorter coupling periods should be used. If purity is more important than the shortest synthesis time possible, or if the sequence is known to contain difficult coupling regions that require additional time for completion (for example, coupling of a beta-branched residue to another beta- branched residue, or if the peptide sequence is highly hydrophobic or known to be prone to aggregation, or if prior synthesis of the sequence proceeded with less than optimum yield at a particular coupling), then the longer coupling periods should be used. [0133] If 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.

[0134] Deblocking: 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.

[0135] 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 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. If the deblocking reaction for peptide synthesis performed on a scale of up to about 1 mmol is run as two steps, it 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.

[0136] For peptide synthesis performed on a scale above about 5 mmol (for example, about 5 mmol to about 7.5 mmol), 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 minutes to about 16 minutes, or between about 9 minutes to about 15 minutes. 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.

[0137] For peptide synthesis performed on a scale of above about 5 mmol (for example, about 5 mmol to about 7.5 mmol), 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. If 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.

[0138] For peptide synthesis performed on a scale of about 1 mmol to about 5 mmol, 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.

[0139] 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.

[0140] 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. When 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. Alternatively, 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.

EXAMPLES

[0141] The examples below are intended to be purely exemplary of the invention and should therefore not be considered to limit the invention in any way. The following examples and detailed description are offered by way of illustration and not by way of limitation.

[0142] Reagents used for solid-phase peptide synthesis and subsequent cleavage of peptides from the resin are provided in Table 1 below. Table 1

General synthetic procedure

[0143] 1) Weigh out starting resin. The amount of resin weighed out is dependent on substitution of the resin. Grams of resin = (scale of synthesis in mmol) divided by (substitution of resin in mmol per gram).

[0144] 2) Place resin into reaction vessel(s) (RVs) of synthesizer. Fill solvent bottles and adjust air/nitrogen pressures.

[0145] 3) Set all couplings to single couple or double coupling, with an initial 10 minute swelling function (using DCM/DMF), and a final deblocking function after the peptide is completed. If one or more coupling reactions during the synthesis are expected to be difficult (that is, expected to proceed in lower than average yield, or require additional coupling time), a double coupling can be programmed for those reactions.

[0146] 4) Adjust temperature of heating jacket as appropriate for the synthesis. Allow for it to reach desired temperature before proceeding.

[0147] 5) Record start time and date.

[0148] 6) Run the pre-set programs.

[0149] 7) After the run is over, record end time and date.

[0150] 8) Remove resin from the RVs on the synthesizer, wash with methanol 3-5 times

[0151] 9) Vacuum dry over 24 hours, or air dry in a fume hood. [0152] 10) Record final weight. Net weight gain is (final weight - starting resin weight).

[0153] 11) Cleavage to yield crude peptide.

[0154] 12) Analyze crude peptide purity via Reverse Phase HPLC.

EXAMPLE 1

Synthesis of test peptides at varying temperatures

[0155] The purpose of this experiment was to quantify the differences in resins under different temperature conditions. The variables used for this quantification are (i) peptide purity by RP- HPLC and (ii) weight gain.

[0156] Peptides were synthesized on a CS 136H Automated Synthesizer (CSBio, Menlo Park, California, USA).

[0157] Four of the most common types of resins, AM (Aminomethyl) Resin, CLT (2- Chlorotrityl) Resin, Wang Resin, and MBHA (4-Methylbenzhydrylamine) Resin were used. Peptides varying from 10 amino acids to 38 amino acids long were chosen as test sequences.

[0158] All peptides were synthesized using DIC/HOBt

(diisopropylcarbodiimide/hydroxybenzotriazole) as carbodiimide activating reagent/active ester- forming reagent. Dimethylformamide (DMF) was used as the primary solvent. Each peptide was made at 0.25 mmol scale.

[0159] Heated synthesis at temperatures of 40°C, 60°C, and 80°C was conducted with shorter Deblock and Coupling time: Deblock (7 minutes) and Coupling (40 minutes) as per protocols developed for rapid heated peptide synthesis. Non-heated synthesis (25°C or Room Temperature) was conducted with routine Deblock and Coupling settings: Deblock (30 minutes) and Coupling (2 hours).

[0160] Post synthesis, the resin was extracted from its reaction vessel and washed with methanol (MeOH) 3-5 times and vacuum dried over 24 hours. The amount of starting resin used

(g) was weighed out depending on the substitution of the resin (mmol/g). The weight gain was then calculated by subtracting the starting resin weight from the final dried resin weight.

[0161] A small scale sample of the peptide resin (150-250 beads) was then cleaved using

0.4 mL of Reagent "K" over 2 hours. (Reagent K consists of 94% TFA (trifluoroacetic Acid), 2%

TIS (triisopropylsilane), 2% EDT (ethane dithiol), and 2% deionized water.) The crude sample was then completely dissolved using various ratios of acetonitrile (ACN) and water with 0.1% TFA. The dissolved sample was then used to confirm identity of the peptide via mass spectroscopy. The same sample was used to run RP-HPLC to measure peptide purity.

[0162] In Solid Phase Peptide Synthesis (SPPS), an acceptable synthesis yield is

typically >70%, which can be calculated from actual weight gain divided by the theoretical weight gain. Another important criterion for a good synthesis is the purity of the crude peptide, which is usually > 40%. As long as acceptable synthetic yield and crude purity can be maintained, automated peptide synthesizers can dramatically simplify the laborious bench work of manual SPPS, especially with multiple sequences synthesized on large scales. Rapid heated peptide synthesis performed on CS automated peptide synthesizers (such as the CS 136H

Automated Synthesizer, available from CSBio, Menlo Park, California) can further save time, and frequently gives better synthesis compared to routine SPPS conditions.

[0163] Eight peptide sequences were chosen, using four different resins (MBHA, CLT, Wang, and AM resins), to test rapid peptide synthesis at elevated temperatures. For each resin, one short peptide and one long peptide were synthesized. Table 2 shows the sequences of test peptides synthesized using the methods of the invention.

Table 2

[0164] 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).

[0165] For AM resin and Wang resin, rapid heated synthesis accelerated the

coupling/deblocking reactions and shortened the SPPS time, with a similar synthesis yield and crude peptide purity as non-heated synthesis. [0166] For MBHA resin, rapid heated synthesis shortened SPPS time and provided increased crude peptide purity, but at a lower synthesis yield (10-20% lower)

[0167] For CLT resin, the reaction temperature should be 40°C or lower in order to obtain reasonable weight gain.

[0168] The data shows that rapid heated peptide synthesis can save significant amounts of time, while also resulting in good synthetic yield and purity.

Table 3

25°C

Starting 0.54g 0.33g 0.36g 0.41g 0.50g 0.45g 0.52g 0.43g

Resin

Wt

Ending 1.82g 1.12g 0.46g 1.46g 0.56g 0.84g 1.34g 0.63g

Resin

Wt

Weight 1.28g 0.79g 0.10g 1.05g 0.06g 0.39g 0.82g 0.20g Gain

[0169] The disclosures of all publications, patents, patent applications and published patent applications referred to herein by an identifying citation are hereby incorporated herein by reference in their entirety. Web sites references using "World-Wide-Web" at the beginning of the Uniform Resource Locator (URL) can be accessed by replacing "World-Wide-Web" with "www."

[0170] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is apparent to those skilled in the art that certain changes and modifications will be practiced. Therefore, the description and examples should not be construed as limiting the scope of the invention.