COUPLING METHOD FOR PEPTIDE SYNTHESIS AT ELEVATED TEMPERATURES

Abstract

An improved method for coupling amino acids into peptides or peptidomimetics is disclosed that includes the steps of combining an amino acid, a carbodiimide, an activator additive, and a base at less than 1 equivalent compared to the amino acid to be activated; and carrying out the activation and coupling at a temperature greater than 30° C.

Claims

1. In a method for coupling amino acids into peptides or peptidomimetics, the improvement comprising: combining an amino acid, a carbodiimide, an activator additive, and a base, with the base in an amount of less than 1 equivalent compared to the amino acid to be activated; and carrying out the activation and coupling at a temperature greater than 30° C.

2. A method according to claim 1 carried out within a total coupling time less than 10 minutes.

3. A method according to claim 1 carried out within a total coupling time less than 4 minutes.

4. A method according to claim 1 carried out within a total coupling time less than 2 minutes.

5. A solid phase peptide synthesis method according to claim 1.

6. A method according to claim 5 wherein the base is selected from the group consisting of DIEA, NMM, TMP, TEA and combinations thereof.

7. A method according to claim 5 wherein the amount of base is no more than 0.2 equivalent based on the amount of amino acid present.

8. A method according to claim 5 wherein the amount of base is no more than 0.1 equivalent based on the amount of amino acid present.

9. A method according to claim 5 wherein the carbodiimide is selected from the group consisting of DCC, DIC, EDC, and mixtures thereof.

10. A method according to claim 9 wherein the activator additive is selected from the group consisting of HOBt, HOAt, 6-Cl-HOBt, Oxyma, NHS and mixtures thereof.

11. A method according to claim 5 wherein the activation and coupling are carried out at temperature of between about 30° C. and 110° C.

12. A method according to claim 5 wherein the activation and coupling are carried out at temperature of at least about 60° C.

13. A method according to claim 5 wherein the activation and coupling are carried out at temperature of at least about 75° C.

14. A method according to claim 5 wherein the activation and coupling are carried out at temperature of at least about 90° C.

15. A method according to claim 1 wherein the amino acid is selected from the group consisting of essential amino acids, conditionally essential amino acids and dispensable amino acids.

16. In a method for coupling carboxylic acids and amines, the improvement comprising: combining a carboxylic acid, an amine, a carbodiimide, an activator additive, and a base, with the base in an amount of less than 1 equivalent compared to the amine; and carrying out activation and coupling at a temperature greater than 30° C.

17. A method according to claim 16 carried out within a total coupling time less than 10 minutes.

18. A method according to claim 16 carried out within a total coupling time less than 4 minutes.

19. A method according to claim 16 carried out within a total coupling time less than 2 minutes.

20. A solid phase peptide synthesis method according to claim 16.

21. A method according to claim 20 wherein the base is selected from the group consisting of DIEA, NMM, TMP, TEA and combinations thereof.

22. A method according to claim 20 wherein the amount of base is no more than 0.2 equivalent based on the amount of amine present.

23. A method according to claim 20 wherein the amount of base is no more than 0.1 equivalent based on the amount of amine acid present.

24. A method according to claim 20 wherein the carbodiimide is selected from the group consisting of DCC, DIC, EDC, and mixtures thereof.

25. A method according to claim 24 wherein the activator additive is selected from the group consisting of HOBt, HOAt, 6-Cl-HOBt, Oxyma, NHS and mixtures thereof.

26. A method according to claim 20 wherein the activation and coupling are carried out at temperature of between about 30° C. and 110° C.

27. A method according to claim 20 wherein the activation and coupling are carried out at temperature of at least about 60° C.

28. A method according to claim 20 wherein the activation and coupling are carried out at temperature of at least about 75° C.

29. A method according to claim 20 wherein the activation and coupling are carried out at temperature of at least about 90° C.

30. In a solid phase method for coupling amino acids into peptides or peptidomimetics, the improvement comprising: combining a hyper-acid sensitive linker connecting a peptide and a resin, an amino acid, a carbodiimide, an activator additive, and a base; and carrying out the activation and coupling at a temperature greater than 30° C.

31. A method according to claim 30 in which the base is present in an amount of less than 1 equivalent compared to the amino acid to be activated.

32. A method according to claim 31 in which the base is present in an amount of 0.2 equivalent compared to the amino acid to be activated.

33. A method according to claim 31 in which the base is present in an amount of 0.1 equivalent compared to the amino acid to be activated.

34. A method according to claim 30 in which the linker is selected from the group consisting of 2 chlorotrityl and Trityl.

35. A method according to claim 30 in which the carbodiimide is selected from the group consisting of DCC, DIC, EDC, and mixtures thereof.

36. A method according to claim 30 in which the activator additive is selected from the group consisting of HOBt, HOAt, 6-Cl-HOBt, Oxyma, NHS and mixtures thereof.

37. A method according to claim 30 in which the activation and coupling are carried out at a temperature of between about 90° C. and 110° C.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The foregoing and other objects and advantages of the invention and the manner in which the same are accomplished will become clearer based on the followed detailed description taken in conjunction with the accompanying drawings.

[0041] FIG. 1 is an illustration of the well understood possible pathways for carbodiimide based activation.

[0042] FIG. 2 is a diagram of the well understood reaction pathways for onium salt based activation.

[0043] FIG. 3 is a UPLC (ultra performance liquid chromatography) chromatogram from a Thymosin synthesis carried out at 90° C. following an onium salt activation in the presence of 2 equivalents of base.

[0044] FIG. 4 is a UPLC chromatogram from a Thymosin synthesis carried out at 90° C. in the presence of Oxyma, but without any base.

[0045] FIG. 5 is a mass spectrum from a Thymosin synthesis carried out at 90° C. in the presence of Oxyma, but without any base.

[0046] FIG. 6 is a UPLC chromatography gram from a Thymosin synthesis carried out at 90° C. in the presence of Oxyma and one equivalent of base.

[0047] FIG. 7 is a UPLC chromatogram from a Thymosin synthesis carried out at 90° C. with Oxyma and in the presence of 0.1 equivalents of base.

[0048] FIG. 8 is a UPLC chromatogram from a Thymosin synthesis carried out at 100 C with Oxyma and in the presence of 0.1 equivalent of base.

[0049] FIG. 9 is a mass spectrum from a Thymosin synthesis carried out at 100° C. with Oxyma and in the presence of 0.1 equivalent of base.

[0050] FIG. 10 is a plot comparing peptide purity to the amount of base included for a coupling reaction carried out at 90° C.

[0051] FIG. 11 illustrates one racemization pathway for cysteine in solid phase peptide synthesis.

[0052] FIG. 12 illustrates a potential lactam formation reaction in SPPS that is specific to arginine.

[0053] FIG. 13 is an HPLC (high pressure liquid chromatography) chromatogram of ABRF 1992 peptide carried out at 90° C. using HBTU and two equivalents of base.

[0054] FIG. 14 is a UPLC chromatogram of ABRF 1992 peptide carried out at 90° C. using DIC and Oxyma, but without any base.

[0055] FIG. 15 is the mass spectrum of the same reaction as FIG. 14.

[0056] FIG. 16 is a UPLC chromatogram of the same reaction as FIG. 14, but using 0.1 equivalent of base.

DETAILED DESCRIPTION

[0057] In a broad sense, the invention incorporates a base in a manner that improves SPPS carried out at elevated temperature. Potentially, many different types of bases could be used for this process. As used herein, phrases such as “1 equivalent of base” or “0.1 equivalent of base” will always refer to the amount of base present as compared to the amount of amino acid present, unless some other meaning is clearly indicated in context. In addition to the bases set forth in the accompanying examples, Applicants believe that trimethylamine (“TEA”) will be useful in the same or similar circumstances.

[0058] Additionally, those of skill in this art will recognize that the invention and its advantages can be expressed in terms of the reaction of a carboxylic acid and an amine.

[0059] A limitation of onium salt based methods is that they require at least 1 equivalent of base compared to the amino acid and activator to complete activation. This is because a carboxylate anion must be generated on each amino acid to be activated so that it can perform a nucleophilic attack on the onium salt activator as shown in FIG. 2. We verified the need for the base by synthesizing a known difficult 28 mer peptide (Thymosin) with various amounts of base (DIEA) as shown in Table 1. FIG. 3 is the UPLC chromatogram for the experiment that used HBTU for activation and 2 equivalents of DIEA as the base (Entry 5). FIG. 3 shows many (undesired) fragments and an overall lack of purity.

TABLE-US-00001 TABLE 1 Synthesis of Thymosin with Onium Salt Activation at Various Base Equivalents Temp Coupling DIEA % Purity Entry (° C.) Time Activation (Equivalents) (UPLC-MS) 1 75 5 HBTU/DIEA 2 44 2 75 5 HCTU/DIEA 2 39 3 75 5 HATU/DIEA 2 35 4 90 2 HCTU/DIEA 2 56 5 90 2 HBTU/DIEA 2 57 6 90 2 HBTU/DIEA 1 39 7 90 2 HBTU/DIEA 0.5 14 8 90 2 HBTU/DIEA 0.1 0

[0060] Experiment Conditions: [0061] Peptide Sequence (Thymosin)=SDAAVDTSSEITTKDLKEKKEVVEEAEN-NH.sub.2 [0062] Synthesis Scale=0.1 mmol [0063] Resin=Rink Amide MBHA Polystyrene Resin (0.38 mmol/g) [0064] Instrument=Liberty Blue Microwave Peptide Synthesizer (CEM Corp., Matthews, N.C.) [0065] Deprotection=3 mL of a 10% (w/v) piperazine in EtOH:NMP (1:9) [0066] Microwave Deprotection Method=1 min at 90° C. [0067] Washing=Post-Deprotection (2 mL, 2 mL, 3 mL-DMF); Post-Coupling=None [0068] Coupling=5-fold excess of AA/HBTU/DIEA (1:0.9:variable) in 4 mL solution [0069] Cleavage=5 mL of TFA/TIS/H.sub.2O/DODt (92.5:2.5:2.5:2.5) for 30 min at 38° C. in an Accent MW cleavage system (CEM Corp., Matthews, N.C.) [0070] Analysis=Peptides were analyzed on a Waters UPLC ACQUITY H-Class with 3100 Single Quad MS using acetonitrile/water with 0.1% TFA as the solvent system on C18 Column (1.7 mm, 2.1×100 mm)

[0071] In contrast, this same peptide (Thymosin) could be synthesized at higher purity (63%; Table 2, Entry 11) without the presence of any base using carbodiimide based activation (DIC) and the common activator additive Oxyma.

[0072] We then investigated the addition of bases during the entire activation and subsequent acylation step at 1 to 2 equivalents compared to the amino acid to be activated. These approaches resulted in either a decrease or similar purity compared to the control experiment without base.

[0073] Beyermann et al and Carpino (1999) have suggested that the purity could be somewhat increased by adding the base after activation is complete, and described how formation of the O-acylisourea can be hindered by the presence of a strong base under room temperature conditions. Adding the base after activation, however, increases the complexity of potential automation and is also difficult to perform without slowing down the overall coupling process and increasing the manipulative steps required (which can increase the complexity of any corresponding automation step). Additionally, at the elevated temperatures used in these experiments, we did not observe a significant benefit from the presence of TMP during the entire coupling process. In comparison, Carpino (1999) used TMP under room temperature conditions to offer improvements for carbodiimide based coupling processes.

TABLE-US-00002 TABLE 2 Synthesis of Thymosin with Carbodimide Activation and zero or at least 1 Equivalents of Base Temp Coupling Base % Purity Entry (° C.) Time Additive (Equivalents) (UPLC-MS) 1 60 5 HOBt None 38 2 60 5 Oxyma None 52 3 90 2 Oxyma None 63 4 90 4 Oxyma None 67 5 90 2 Oxyma DIEA—(1) 59 6 90 2 Oxyma DIEA—(2) 55 7 90 2 Oxyma NMM—(1) 64 8 90 2 Oxyma NMM—(2) 49 9 90 2 Oxyrna TMP—(1) 63 10 100 2 Oxyma None 61 11 110 2 Oxyma None 63

[0074] Experiment Conditions: [0075] Peptide Sequence (Thymosin)=SDAAVDTSSEITTKDLKEKKEVVEEAEN-NH.sub.2 [0076] Synthesis Scale=0.1 mmol [0077] Resin=Rink Amide MBHA Polystyrene Resin (0.38 mmol/g) [0078] Instrument=Liberty Blue Microwave Peptide Synthesizer (CEM Corp., Matthews, N.C.) [0079] Deprotection=3 mL of a 10% (w/v) piperazine in EtOH:NMP (1:9) [0080] Microwave Deprotection Method=1 min at 90° C. [0081] Washing=Post-Deprotection (2 mL, 2 mL, 3 mL-DMF); Post-Coupling=None [0082] Coupling=5-fold excess of AA/DIC/Additive (1:1:1) in 4 mL solution [0083] Cleavage=5 mL of TFA/TIS/H.sub.2O/DODt (92.5:2.5:2.5:2.5) for 30 min at 38° C. in an Accent MW cleavage system (CEM Corp., Matthews, N.C.) [0084] Analysis=Peptides were analyzed on a Waters UPLC ACQUITY H-Class with 3100 Single Quad MS using acetonitrile/water with 0.1% TFA as the solvent system on C18 Column (1.7 mm, 2.1×100 mm)

[0085] FIGS. 4 and 6 are respective chromatograms from Table 2 and FIG. 5 is a mass spectrum corresponding to the experiment of FIG. 4. These demonstrate generally similar performance as between no base (63% in FIG. 4) and 1 equivalent of base (59% in FIG. 6) for carbodiimide based coupling at elevated temperatures.

[0086] In favorable comparison to prior art efforts, the use of the invention raised the purity up to 73% by using only small amounts of base (DIEA), and specifically much less than 1 equivalent as compared to the amount of amino acid and carbodiimide activator. Using the invention, the presence of a base at a low excess does not significantly hinder O-acylisourea formation at elevated temperature and simultaneously improves the subsequent acylation step.

[0087] Table 3 categorizes some of these results at elevated temperatures and using several different amounts of base, but all at less than one equivalent.

TABLE-US-00003 TABLE 3 Synthesis of Thymosin with Carbodiimide Activation and less than 1 Base Equivalent Coupling Coupling Base % Purity Entry Temp (° C.) Time (Equivalents) (UPLC-MS) 1 90 2 DIEA—0.05 61 2 90 2 DIEA—0.1  70 3 90 2 DIEA—0.4  70 4 90 2 DIEA—0.8  67 5 100 2 DIEA—0.1  73 6 110 2 DIEA—0.1  73

[0088] Experiment Conditions: [0089] Peptide Sequence (Thymosin)=SDAAVDTSSEITTKDLKEKKEVVEEAEN-NH.sub.2 [0090] Synthesis Scale=0.1 mmol [0091] Resin=Rink Amide MBHA Polystyrene Resin (0.38 mmol/g) [0092] Instrument=Liberty Blue Microwave Peptide Synthesizer (CEM Corp., Matthews, N.C.) [0093] Deprotection=3 mL of a 10% (w/v) piperazine in EtOH:NMP (1:9) [0094] Microwave Deprotection Method=1 min at 90° C. [0095] Washing=Post-Deprotection (2 mL, 2 mL, 3 mL-DMF); Post-Coupling=None [0096] Coupling=5-fold excess of AA/DIC/Oxyma (1:1:1) in 4 mL solution [0097] Cleavage=5 mL of TFA/TIS/H.sub.2O/DODt (92.5:2.5:2.5:2.5) for 30 min at 38° C. in an Accent MW cleavage system (CEM Corp., Matthews, N.C.) [0098] Analysis=Peptides were analyzed on a Waters UPLC ACQUITY H-Class with 3100 Single Quad MS using acetonitrile/water with 0.1% TFA as the solvent system on C18 Column (1.7 mm, 2.1×100 mm)

[0099] FIGS. 7, 8 and 9 are UPLC chromatograms and a mass spectrum from the Table 3 results and demonstrate the improvements of the invention for the synthesis of Thymosin at various temperatures. FIG. 7 shows 70% purity using 0.1 equivalent of base at 90° C.; FIG. 8 shows 73% purity using 0.1 equivalent of base at 100° C.; and FIG. 9 is the mass spectrum of the experiment of FIG. 8.

[0100] The ability to achieve these improvements in synthesis with the presence of only a very small amount of base is surprising and uniquely valuable. Because larger amounts of base have been shown to drive the acylation reaction to completion under room temperature conditions, the small amounts of base used in the invention are counter-intuitive. In fact, Carpino (1999) demonstrated that a difficult acylation reaction improved as the amount of base (DIEA) present was increased up to 4 equivalents during the room temperature synthesis of a difficult peptide.

[0101] Although the inventors do not wish to be bound by any theory, it appears that the results from the present invention may result from the effect of elevated temperature on the stability of an activated amino acid derivative. To evaluate this possibility we carried out a set of experiments using a pre-activated representative Fmoc amino acid at various elevated temperature conditions. Immediately after pre-activating for a designed time interval, the activated amino acid derivative was cooled to room temperature and then added at 1 equivalent to a 4 mer peptide on a resin under similar conditions in all cases. This demonstrated the amount of activated amino acid that survived the activation process at high temperatures to in turn demonstrate the relative stability.

[0102] As shown in Table 4, at elevated temperatures there exists a negative correlation between the stability of the activated amino acid derivative and the amount of base present. This shows that higher amounts of base present in the coupling reaction lead to faster destruction of the activated amino acid species thereby reducing subsequent acylation efficiency. It should be noted that the acylation process regenerates the acidic activator additive. Therefore, the activated amino acid species should have a somewhat longer lifetime in an in-situ activation process where the acidic additive is generated simultaneously with activation, and thus would partially offset the presence of base.

TABLE-US-00004 TABLE 4 Stability of activated amino acid esters in the presence of base from various pre-activation conditions Pre-activation Acti- Acti- % (2 min/90° C.) Base H.sub.2O vation vation Coupled 1.0 equiv. (Equiv- Added Temp Time (UPLC- Entry active ester alents) (mL) (° C.) (min) MS) 1 Amino Acid/ DIEA None 90 2 1 PyAOP (1:1) (2) 2 Amino Acid/ DIEA None 90 2 40 PyAOP (1:1) (1) 3 Amino Acid/DIC/ None None 90 2 90 Oxyma (1:1:1) 4 Amino Acid/DIC/ None 0.5 mL 90 2 36 Oxyma (1:1:1) 5 Amino Acid/DIC/ None None 110 2 48 Oxyma (1:1:1) 6 Amino Acid/DIC/ DIEA None 90 2 22 Oxyma (1:1:1) (1) 7 Amino Acid/DIC/ DIEA 0.5 mL 90 2 5 Oxyma (1:1:1) (1) 8 Amino Acid/DIC/ DIEA None 90 2 44 Oxyma (1:1:1) (0.1) 9 Amino Acid/DIC/ DIEA 0.5 mL 90 2 38 Oxyma (1:1:1) (0.1) 10 Amino Acid/DIC/ NMM None 90 2 24 Oxyma (1:1:1) (2) 11 Amino Acid/DIC/ TMP None 90 2 47 Oxyma (1:1:1) (1)

[0103] Experiment Conditions: [0104] Peptide Sequence=DYING-NH.sub.2 [0105] Synthesis Scale=0.1 mmol [0106] Resin=Rink Amide MBHA Polystyrene Resin (0.38 mmol/g) [0107] Instrument=Liberty Blue Microwave Peptide Synthesizer (CEM Corp., Matthews, N.C.) [0108] Deprotection=3 mL of a 10% (w/v) piperazine in EtOH:NMP (1:9) [0109] Microwave Deprotection Method=1 min at 90° C. [0110] Washing=Post-Deprotection (2 mL, 2 mL, 3 mL-DMF); Post-Coupling=None [0111] Coupling (for all amino acids except Fmoc-Asp(OtBu)-OH=5-fold excess of AA/DIC/Oxyma (1:1:1) in 4 mL solution [0112] Coupling (Fmoc-Asp(OtBu)-OH)=The amino acid was pre-activated as described in Table 4 and cooled to room temperature before coupling. Subsequent coupling was performed for 2 min at 90° C. [0113] Cleavage=5 mL of TFA/TIS/H.sub.2O/DODt (92.5:2.5:2.5:2.5) for 30 min at 38° C. in an Accent MW cleavage system (CEM Corp., Matthews, N.C.) [0114] Analysis=Peptides were analyzed on a Waters UPLC ACQUITY H-Class with 3100 Single Quad MS using acetonitrile/water with 0.1% TFA as the solvent system on C18 Column (1.7 mm, 2.1×100 mm)

Water

[0115] Tofteng et al [A. Tofteng, S. Pedersen, D. Staerk and K. Jensen, “Effect of Residual Water and Microwave Heating on the Half-Life of the Reagents and Reactive Intermediates in Peptide Synthesis,” Chemistry, vol. 18, pp. 9024-9031, 2012] recently examined the influence of water on the stability of activated amino acids in water. The authors noted a correlation between the stability of certain coupling reagents and activated esters based on varying amounts of water (from 50-18,000 ppm) in DMF. Additionally, the authors compared the efficiency of DIC/Oxyma/DIEA (1:1:2) versus DIC/Oxyma (1:1) and observed no significant difference in the synthesis purity of a 10 mer peptide.

[0116] We found that even in the presence of large amounts of water (e.g., more than 300,000 ppm) an activated ester was still 36% intact after 2 min at 90° C. (Table 4, entry 4) versus 90% intact without additional water added (Table 4, entry 3). This difference suggested that the ester has higher stability in the presence of more than 300,000 ppm of water than in the absence of any additional water, but in the presence of 1 equivalent of DIEA (Table 4, entry 6). The presence of more than 300,000 ppm and 1 equivalent of DIEA (Table 4, entry 7) displayed only a 5% survival rate.

[0117] An onium salt based coupling provided further indication of the instability of an activated ester at elevated temperature in the presence of a base. An ester generated from the onium salt PyAOP was only 1% intact after 2 min at 90° C. in the presence of 2 equivalents of base (Table 4, entry 1). The stability could be increased to 40% (Table 4, entry 2) by reducing the base equivalents to 1. This method (no added water) was ineffective, however, in synthesizing the difficult Thymosin sequence (Table 1, entry 6).

[0118] Without being bound by theory, these results indicate that the presence of a base during the coupling process is a primary factor affecting stability of an activated amino acid species at elevated temperature. The pH is strongly affected by a strong non-nucleophilic base (such as DIEA, or trimethylamine; “TEA”) which can accelerate destruction of an activated amino acid species. Specifically, the non-nucleophilic nature of a tertiary amine can attract protons from any water present, which in turn generates hydroxide (OH.sup.−1) ions. These hydroxide ions rapidly (and undesirably) hydrolyze the activated esters, thus quenching the desired coupling. Secondarily, the non-nucleophilic base can catalyze attack from amines present in the solvent (e.g., dimethylamine). Additionally, the hindrance of the base may also affect its destructive impact on the activated species. For example, the base NMM is significantly weaker than DIEA, but is less hindered. These factors together appear to cause NMM to reduce the stability of an activated species more than expected based upon its basicity alone.

[0119] It has previously been noted that both basicity and steric hindrance properties of a base may play a role in its ability to cause epimerization in peptide synthesis (L. Carpino and A. El-Faham, “Effect of Teriary Bases on O-Benzotriazolyuronium Salt-Induced Peptide Segment Coupling,” J. Org. Chem., vol. 59, pp. 695-698, 1994). Therefore, an important feature of the present invention is that it identifies a key variable in a carbodiimide coupling process affecting stability of activated amino acid species at elevated temperatures, and that it provides an improved method which uniquely improves synthesis quality with only a minimal amount of base present. Minimizing the amount of the base thereby limits generation of other nucleophiles which could otherwise quickly react with and destroy an electrophilic activated species at elevated temperatures.

[0120] To further investigate the role of base during the coupling reaction a known very difficult coupling reaction was explored; specifically, coupling an Fmoc-Aib-OH residue onto another Aib residue, a reaction previously explored by Tofteng, supra. We were able to reproduce Tofteng's results and achieve a 92% purity with a 20 min coupling at 75° C. As an improvement, however, we were able to nearly match this result in only 6 minutes at 90° C. in the presence of 0.1 equivalent of DIEA. The presence of 0.1 equivalents of DIEA was superior to both 0 and 1.0 equivalents of DIEA at each coupling time tested; e.g., Table 5 and FIG. 10 (in which the base is the main variable; all of the reactions having been carried out at either 90° C. or 100° C.). These results show that less than 1 equivalent of a base is uniquely suited for elevated temperature coupling because it provide an optimal balance between the stability of the activated amino acid and a basic environment for accelerating acylation.

TABLE-US-00005 TABLE 5 Coupling Fmoc-Aib-OH onto Aib-Ile-Asp(OtBu)-Tyr(tBu)-Ile-Asn(Trt)-Gly-NH.sub.2 under various conditions Coupling Coupling Base % Purity Entry Temp (° C.) Time (min) (Equivalents) (UPLC-MS) 1 75 20 None 92 2 90 2 None 30 3 90 2 DIEA—(0.1) 53 4 90 2 DIEA—(1.0) 24 5 90 4 None 65 6 90 4 DIEA—(0.1) 68 7 90 4 DIEA—(1.0) 62 8 90 6 None 73 9 90 6 DIEA—(0.1) 76 10 90 6 DIEA—(1.0) 72 11 100 6 DIEA—(0.1) 89 12 100 10 None 86 13 100 10 DIEA—(0.1) 93 14 100 10 DIEA—(1.0) 85

[0121] Experiment Conditions: [0122] Peptide Sequence=Fmoc-Aib-Aib-IDYING-NH.sub.2 [0123] Synthesis Scale=0.1 mmol [0124] Resin=Rink Amide MBHA Polystyrene Resin (0.38 mmol/g) [0125] Instrument=Liberty Blue Microwave Peptide Synthesizer (CEM Corp., Matthews, N.C.) [0126] Deprotection=3 mL of a 10% (w/v) piperazine in EtOH:NMP (1:9) [0127] Microwave Deprotection Method=1 min at 90° C. [0128] Washing=Post-Deprotection (2 mL, 2 mL, 3 mL-DMF); Post-Coupling=None [0129] Coupling=5-fold excess of AA/DIC/Oxyma (1:1:1) in 4 mL solution [0130] Cleavage=5 mL of TFA/TIS/H.sub.2O/DODt (92.5:2.5:2.5:2.5) for 30 min at 38° C. in an Accent MW cleavage system (CEM Corp., Matthews, N.C.) [0131] Analysis=Peptides were analyzed on a Waters UPLC ACQUITY H-Class with 3100 Single Quad MS using acetonitrile/water with 0.1% TFA as the solvent system on C18 Column (1.7 mm, 2.1×100 mm)

Cysteine

[0132] It is well documented that conversion of an amino acid to an activated ester increases the acidity of the alpha (α)-carbon's proton. Cysteine derivatives are particularly susceptible to epimerization due to the electron withdrawing effect of the side chain sulfur atom as shown in FIG. 11. Significant epimerization of cysteine has been observed under elevated temperature coupling conditions using onium salt activation strategies. Replacing DIEA or NMM with the more hindered base TMP has been shown to reduce epimerization levels for cysteine during HBTU coupling (Palasek). TMP appears less effective, however, for difficult couplings and is not recommended as a standard replacement for DIEA. Reducing coupling temperature to 50° C. or less has reduced, but not eliminated, cysteine epimerization. The lower temperature is not ideal, however, because a lower coupling temperature can result in incomplete coupling and longer reaction time. Recently, Collins et al (J. Collins, K. Porter, S. Singh and G. Vanier, “High-Efficiency Solid Phase Peptide Synthesis (HE-SPPS),” Org. Lett., vol. 16, pp. 940-943, 2014) it showed that the use of a carbodiimide based activation method without the presence of any base (DIC/Oxyma) minimized cysteine epimerization even at coupling temperatures as high as 90° C. The inventors have discovered, however, that a small amount of base can be added to this same process without significantly increasing cysteine epimerization (Table 6, entries 2 and 6). This was tested on the same peptide sequence previously studied by Palasek and Collins et al (J. Collins, K. Porter, S. Singh and G. Vanier, “High-Efficiency Solid Phase Peptide Synthesis (HE-SPPS),” Org. Lett., vol. 16, pp. 940-943, 2014) which contains a cysteine coupling and is susceptible to epimerization. Table 6 summarizes these results.

TABLE-US-00006 TABLE 6 Cysteine Epimerization during ABC 20mer synthesis under various carbodiimide coupling conditions Coupling Coupling Base % Purity Entry Temp (° C.) Time (Equivalents) (UPLC-MS) % D-Cys 1 90 2 None 72 0.69 2 90 2 DIEA—(0.1) 78 1.46 3 90 4 DIEA—(0.1) 74 Not measured 4 90 2 DIEA—(1.0) 51 3.91 5 90 2 NMM—(2.0) 68 8.65 6 100 2 DIEA—(0.1) 76 0.85

[0133] Experiment Conditions: [0134] Peptide Sequence (ABC 20mer)=VYWTSPFMKLIHEQCNRADG-NH.sub.2 [0135] Synthesis Scale=0.1 mmol [0136] Resin=Rink Amide MBHA Polystyrene Resin (0.38 mmol/g) [0137] Instrument=Liberty Blue Microwave Peptide Synthesizer (CEM Corp., Matthews, N.C.) [0138] Deprotection=3 mL of a 10% (w/v) Piperazine in EtOH:NMP (1:9) [0139] Microwave Deprotection Method=1 min at 90° C. [0140] Washing=Post-Deprotection (2 mL, 2 mL, 3 mL-DMF); Post-Coupling=None [0141] Coupling=5-fold excess of AA/DIC/Oxyma (1:1:1) in 4 mL solution [0142] Cleavage=5 mL of TFA/TIS/H.sub.2O/DODt (92.5:2.5:2.5:2.5) for 30 min at 38° C. in an Accent MW cleavage system (CEM Corp., Matthews, N.C.) [0143] Analysis=Peptides were analyzed on a Waters UPLC ACQUITY H-Class with 3100 Single Quad MS using acetonitrile/water with 0.1% TFA as the solvent system on C18 Column (1.7 mm, 2.1×100 mm) [0144] Epimerization Analysis=GC-MS after hydrolysis/derivatization w/deuterium labeling (C.A.T. GmbH)

[0145] Arginine

[0146] It is well known that during the coupling reaction the nucleophilic side chain of arginine is susceptible to forming a δ-lactam [M. Cezari and L. Juliano, “Studies on lactam formation during coupling procedures of N alpha z-N omega-protected arginine derivatives,” J Pept. Res., vol. 9, pp. 88-91, 1996]. Activating the carboxylic acid promotes attack by the highly basic δ-guanidino group (pKa=12.5) as shown in FIG. 12. This irreversible reaction converts an activated arginine derivative into an inactive species by ejecting the activator. This intramolecular side reaction increases at elevated temperatures leading to significant arginine deletion (P. White, J. Collins and Z. Cox, “Comparative study of conventional and microwave assisted synthesis,” in 19th American Peptide Symposium, San Diego, Calif., 2005). As a potential alternative, arginine can be coupled at room temperature for a long initial period (e.g., about 30 minutes) followed by a shorter time at a higher temperature; J. Collins, “Microwave-Enhanced Synthesis of Peptides, Proteins, and Peptidomimetics,” in Microwaves in Organic Synthesis 3rd Ed., Weinheim, Germany, Wiley-VCH Verlag & Co. KGaA, 2013, pp. 897-960. This method is disadvantageously slow, however, and requires twice as much arginine because the coupling must be repeated.

[0147] In contrast, the invention provides previously undocumented advantages of carbodiimide coupling methods at high temperatures for arginine coupling. In particular, arginine can be coupled at very high temperatures (up to 90° C.) without significant δ-lactam formation using standard carbodiimide coupling chemistry. This appears to be due to the more acidic coupling environment of standard carbodiimide coupling methods which reduce the propensity of nucleophilic attack by the nucleophilic arginine side chain. A similar effect has been observed with a cyclization reaction of an ornithine derivative in the presence of base with both DIC/HOBt/DIEA (1:1:1) and PyBOP/DIEA activation systems (T. Lescrinier, R. Busson, H. Winter, C. Hendrix, G. Janssen, C. Pannecouque, J. Rozenski, A. Aerschot and P. Herdewijn, “a-Amino acids derived from ornithine as building blocks for peptide synthesis,” J. Pept. Res., vol. 49, pp. 183-189, 1997). The inventors noted that eliminating the base from the activation method was beneficial in eliminating the intramolecular side reaction. As a particular advantage, adding only a small amount of base still allowed arginine to be coupled at 90° C. without significant δ-lactam formation. Because the amount of base added was minimal, the overall pH was lower than in standard onium salt coupling methods. The less basic conditions allowed the resulting coupling behavior to mimic standard carbodiimide coupling chemistry in regards to δ-lactam formation, while simultaneously providing the other benefits of this coupling method.

TABLE-US-00007 TABLE 7 Synthesis of ABRF 1992 peptide with known δ-Lactam Formation side reaction Coupling Coupling % Purity Activation Temp Time Base (UPLC- Entry Method (° C.) (min) (Equivalents) MS) 1 HBTU/DIEA 90 2 DIEA—(2)   37 (0.9:2) 2 DIC/Oxyma (1:1) 90 2 None 87 3 DIC/Oxyma (1:1) 90 2 DIEA—(0.1) 82 4 DIC/Oxyma (1:1) 100 2 DIEA—(0.1) 78

[0148] Experiment Conditions: [0149] Peptide Sequence (ABRF 1992)=GVRGDKGNPGWPGAPY [0150] Synthesis Scale=0.1 mmol [0151] Resin=Fmoc-Tyr(tBu)-Wang Resin (0.64 mmol/g) [0152] Instrument=Liberty Blue Microwave Peptide Synthesizer (CEM Corp., Matthews, N.C.) [0153] Deprotection=3 mL of a 10% (w/v) piperazine in EtOH:NMP (1:9) [0154] Microwave Deprotection Method=1 min at 90° C. [0155] Washing=Post-Deprotection (2 mL, 2 mL, 3 mL-DMF); Post-Coupling=None [0156] Coupling=5-fold excess of amino acid in 4 mL solution [0157] Cleavage=5 mL of TFA/TIS/H.sub.2O/DODt (92.5:2.5:2.5:2.5) for 30 min at 38° C. in an Accent MW cleavage system (CEM Corp., Matthews, N.C.) [0158] Analysis (entry 1)=Peptide was analyzed on a Waters Atlantis C18 column (2.1×150 mm) at 214 nm with a gradient of 5-70% MeCN (0.1% formic acid), 0-20 min. Mass analysis was performed using an LCQ Advantage ion trap mass spectrometer with electrospray ionization (Thermo Electron). [0159] Analysis (entry 2-4)=Peptides were analyzed on a Waters UPLC ACQUITY H-Class with 3100 Single Quad MS using acetonitrile/water with 0.1% TFA as the solvent system on C18 Column (1.7 mm, 2.1×100 mm)

[0160] FIGS. 13-16 reflect data from the experiments listed in Table 7. In particular, FIG. 13 illustrates the relatively poor results at elevated temperatures using 2 equivalents of base, while FIG. 16 illustrates the much better results at the same temperature using 0.1 equivalent of base.

[0161] The invention's modification to carbodiimide based activation raises the pH to avoid undesirable features of carbodiimide based coupling such as premature cleavage of hyper-acid sensitive linkers at elevated temperature. By only adding a small amount of base, however, the unique properties of a carbodiimide based coupling are maintained (long lifetime of activated ester, minimal epimerization of cysteine derivatives, and avoidance of δ-lactam formation of arginine derivatives). This is because the overall pH of the coupling reaction is kept closer to 7—which is ideal for avoiding both basic and acidic catalyzed side reactions—while simultaneously raising the pH somewhat, which increases the rate of acylation.

[0162] Hyper-Acid Sensitive Linkers

[0163] Hyper-acid sensitive linkers such as 2-chlorotrityl and Trityl in SPPS have the capacity to overcome key side reactions and to generate fully protected peptide fragments useful in peptide condensation reactions. Nevertheless, premature cleavage of these linker bonds is a concern at higher temperatures due to their increased lability. Common activators used in SPPS (HOBt, HOAt, 6-Cl-HOBt, Oxyma) are acidic and can act like common cleavage acids (e.g., acetic acid) and cleave the peptide-resin bond prematurely; R. E.-F. A. a. A. F. Subirós-Funosas, “Use of Oxyma as pH modulatory agent to be used in the prevention of base-driven side reactions and its effect on 2-chlorotrityl chloride resin,” Pept. Sci., vol. 98, pp. 89-97, 2012. Higher temperatures tend to increase the premature cleavage from acidic activator additives.

[0164] Standard carbodiimide coupling chemistry at elevated temperatures up to 60° C. has successfully avoided premature cleavage. Friligou et al (I. Friligou, E. Papadimitriou, D. Gatos, J. Matsoukas and T. Tselios, “Microwave-assisted solid-phase peptide synthesis of the 60-110 domain of human pleiotrophin on 2-chlorotrityl resin,” Amino Acids, vol. 40, pp. 1431-1440, 2011) described a successful synthesis of a 51 mer peptide with DIC/HOBt (1:1) activation for 5 min at 60° C. maximum temperature. The desired product was obtained in 30 hours at 60% crude purity and 51% crude yield. Accordingly, limiting the temperature to 60° C. or less appears to avoid premature coupling when using hyper-acid sensitive resins; J. Collins, “Microwave-Enhanced Synthesis of Peptides, Proteins, and Peptidomimetics,” in Microwaves in Organic Synthesis 3rd Ed., Weinheim, Germany, Wiley-VCH Verlag & Co. KGaA, 2013, pp. 897-960.

[0165] Limiting the coupling temperature to 60° C., however, has two main disadvantages. First, the 60° C. temperature may not provide enough energy to complete difficult couplings. Second, coupling at lower temperatures requires longer reaction times thereby significantly increasing the total synthesis time. As an example, the method of Friligou et al resulted in low purity when synthesizing the difficult Thymosin peptide (Table 2, entry 1-2). Synthesizing this same peptide using a coupling temperature of 90° C., however, resulted in a significantly higher crude purity and a reduced synthesis time. Therefore, a method that allows for higher temperatures at higher yields using hyper-acid sensitive linkers would be of significant value.

[0166] The inventors have discovered that adding small amounts of base significantly enhances the yield of the well-known .sup.65-74ACP peptide when synthesized on a 2-chlorotrityl linker at 90° C. (Table 8). The addition of 0.1 equivalents of DIEA increased the yield 134% for DIC/HOBt and 176% for DIC/Oxyma activation.

TABLE-US-00008 TABLE 8 Improved Yield for the 2-chlorotrityl linker with the addition of base to Carbodiimide Based Couplings at Elevated Temperature Coupling Coupling Base % Purity Activation Temp Time (Equiv- (UPLC- Entry Method (° C.) (min) alents) MS) Yield 1 DIC/HOBt (1:1) 60 5 None 89 91 2 DIC/HOBt (1:1) 60 5 DIEA- 89 92 (0.1) 3 DIC/HOBt (1:1) 90 2 None 87 29 4 DIC/HOBt (1:1) 90 2 DIEA- 92 68 (0.1) 5 DIC/Oxyma 90 2 None 86 17 (1:1) 6 DIC/Oxyma 90 2 DIEA- 91 47 (1:1) (0.1) 7 DIC/Oxyma 90 2 DIEA- 91 44 (1:1) (0.8)

[0167] Experiment Conditions: [0168] Peptide Sequence (.sup.65-74ACP)=VQAAIDYING [0169] Synthesis Scale=0.1 mmol [0170] Resin=Fmoc-Gly-2-Chlorotrityl-Resin (0.68 mmol/g) [0171] Instrument=Liberty Blue Microwave Peptide Synthesizer (CEM Corp., Matthews, N.C.) [0172] Deprotection=3 mL of a 10% (w/v) piperazine in EtOH:NMP (1:9) [0173] Microwave Deprotection Method=1 min at 90° C. [0174] Washing=Post-Deprotection (2 mL, 2 mL, 3 mL-DMF); Post-Coupling=None [0175] Coupling=5-fold excess of amino acid in 4 mL solution [0176] Cleavage=5 mL of TFA/TIS/H.sub.2O/DODt (92.5:2.5:2.5:2.5) for 30 min at 38° C. in an Accent MW cleavage system (CEM Corp., Matthews, N.C.) [0177] Analysis=Peptides were analyzed on a Waters UPLC ACQUITY H-Class with 3100 Single Quad MS using acetonitrile/water with 0.1% TFA as the solvent system on C18 Column (1.7 mm, 2.1×100 mm)

[0178] The inventors have also discovered that adding small amounts of base significantly enhances the yield of the well-known .sup.65-74ACP peptide when synthesized on a Trityl linker at 90° C. (Table 9). Adding 0.1 equivalents of DIEA resulted in complete stability with both DIC/HOBt and DIC/Oxyma activation. This represents a 35% yield increase for DIC/HOBt and a 153% yield increase for DIC/Oxyma. In general, the Trityl linker appears somewhat more stable than the 2-chlorotrityl linker under these conditions at elevated temperatures.

TABLE-US-00009 TABLE 9 Improved Yield for the Trityl linker with the addition of base to Carbodiimide Based Couplings at Elevated Temperature Coupling Coupling Base % Purity Activation Temp Time (Equiv- (UPLC- Entry Method (° C.) (min) alents) MS) Yield 1 HBTU/DIEA Room 30 DIEA- 94 98 Temper- (2.0) ature 2 DIC/HOBt (1:1) 90 2 None 93 72 3 DIC/HOBt (1:1) 90 2 DIEA- 95 97 (0.1) 4 DIC/Oxyma 90 2 None 90 38 (1:1) 5 DIC/Oxyma 90 2 DIEA- 95 96 (1:1) (0.1)

[0179] Experiment Conditions: [0180] Peptide Sequence (.sup.65-74ACP)=VQAAIDYING [0181] Synthesis Scale=0.1 mmol [0182] Resin=Fmoc-Gly-NovaSyn-TGT-Resin (0.19 mmol/g) [0183] Instrument=Liberty Blue Microwave Peptide Synthesizer (CEM Corp., Matthews, N.C.) [0184] Deprotection=3 mL of a 10% (w/v) piperazine in EtOH:NMP (1:9) [0185] Microwave Deprotection Method (entry 1)=5 min+10 min at room temperature [0186] Microwave Deprotection Method (entries 2-6)=1 min at 90° C. [0187] Washing (entry 1)=Post-Deprotection (5×5 mL-DMF); Post-Coupling=(5×5 mL-DMF) [0188] Washing (entries 2-6)=Post-Deprotection (2 mL, 2 mL, 3 mL-DMF); Post-Coupling=None [0189] Coupling=5-fold excess of amino acid in 4 mL solution [0190] Cleavage=5 mL of TFA/TIS/H.sub.2O/DODt (92.5:2.5:2.5:2.5) for 30 min at 38° C. in an Accent MW cleavage system (CEM Corp., Matthews, N.C.) [0191] Analysis=Peptides were analyzed on a Waters UPLC ACQUITY H-Class with 3100 Single Quad MS using acetonitrile/water with 0.1% TFA as the solvent system on C18 Column (1.7 mm, 2.1×100 mm)

[0192] Tables 10 and 11 summarize the comparative advantages of the invention.

TABLE-US-00010 TABLE 10 Comparison of Carbodiimide and Onium Salt Activation Strategies for Peptide Coupling at Elevated Temperature NEW METHOD STANDARD ONIUM SALTS DIC/ CAR- [Aminium] Oxyma/ BODIIMIDE HBTU/ [Phosphonium] DIEA DIC/Oxyma DIEA PyBOP/DIEA Feature (1:1:0.1) (1:1) (0.9:2) (1:2) Coupling Time FASTEST FAST LONGER— LONGER— Required Temper- Temper- ature ature limited limited Synthesis HIGHEST HIGH MOD- MOD- Purity ERATE ERATE Pre-activation NO NO NO* NO required (w/slight deficit) Stability of GOOD BEST LIMITED LIMITED activated ester formed Epimerization OK OK BAD BAD of Cysteine derivatives σ-lactam OK OK BAD BAD formation of Arginine Stability of YES NO YES YES hyper-acid sensitive resins Stability of GOOD GOOD LESS LESS activator STABLE STABLE reagents in solution

TABLE-US-00011 TABLE 11 Comparison of Carbodiimide Activation Strategies for Peptide Coupling at Elevated Temperature NEW METHOD CARBODIIMIDE DIC/ STANDARD w/Full Base Oxyma/ CARBODIIMIDE Equivalent DIEA DIC/Oxyma DIC/Oxyma/DIEA Feature (1:1:0.1) (1:1) (1:1:1) or (1:1:2) Coupling Time FASTEST FAST LONGER— Required Temperature Limited Synthesis Purity HIGHEST HIGH HIGH/ MODERATE Pre-activation NO NO PREFERABLE— required (except cysteine and arginine) Stability of GOOD BEST LOW activated ester formed Epimerization of OK OK BAD Cysteine derivatives σ-lactam formation OK OK BAD of Arginine Stability of hyper- YES NO YES acid sensitive resins Stability of GOOD GOOD GOOD activator reagents in solution

[0193] In the drawings and specification there has been set forth a preferred embodiment of the invention, and although specific terms have been employed, they are used in a generic and descriptive sense only and not for purposes of limitation, the scope of the invention being defined in the claims.