Use of excess carbodiimide for peptide synthesis at elevated temperatures

Abstract

An improved method of coupling amino acids into peptides or peptidomimetics is disclosed in which the activation and coupling are carried out in the same vessel, in the presence of a carbodiimide in an amount greater than 1 equivalent as compared to the amino acid, in the presence of an activator additive, and at a temperature greater than 30 C.

Claims

1. In a method of coupling initially Fmoc-protected amino acids into peptides or peptidomimetics, the improvement comprising: carrying out activation and coupling in the same vessel; incorporating a carbodiimide in an amount between 1.5 and 4 equivalents as compared to the amino acid to be activated, wherein the amino acid is initially Fmoc-protected; in the presence of an activator additive; and at a temperature greater than 70 C.

2. A method according to claim 1 in which the activated amino acid is coupled to at least one other amino acid that is linked to a solid phase resin.

3. A method according to claim 1 limited to a total coupling time less than 10 minutes.

4. A method according to claim 1 limited to a total coupling time less than 15 minutes.

5. A method according to claim 1 wherein the activator additive is present in an amount between 1 and 1.5 equivalents compared to the amino acid to be activated.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a flow diagram of Carbodiimide Based Activation.

(2) FIG. 2 is a flow diagram of Onium Salt Based Activation.

DETAILED DESCRIPTION

(3) An improved carbodiimide coupling method at elevated temperatures is presented that avoids the use of a strong base while simultaneously increasing coupling efficiency. This method is based on the unexpected improvement in coupling from the use of increased amounts of carbodiimide (greater than 1 equivalent) relative to the amino acid. It was found that this combination uniquely improved coupling efficiency through more rapid formation of the o-acylisourea intermediate, avoidance of expected N-acylurea peptide formation and/or capping of the terminal amino group, and reduction in epimerization compared to the use of base with carbodiimide coupling. The efficiency of this method was verified by synthesizing three difficult peptide sequences under various conditions as shown in Tables 1-3.

(4) The invention also applies to the synthesis of peptidomimetics; i.e., small protein-like chains designed to mimic a peptide. Because the coupling reactions described herein are for the most part identical as between peptides and peptidomimetics, the invention will be described in terms of peptides. The skilled person will, of course, understand this completely.

(5) In a similar manner, the skilled person will understand that the term amino acid is used herein in its broadest sense to refer to the organic compounds that contain both amine and carboxylic acid functional groups usually along with a side chain. The skilled person is well aware of and familiar with the 22 amino acids that are naturally incorporated into polypeptides and are referred to as proteinogenic or natural amino acids. Again, because the basic coupling reactions are not limited to these particular molecules, the skilled personal will recognize that the advantages of the invention also apply to non-proteinogenic amino acids (aka non-natural amino acids) of which 40 have been added into proteins using established synthetic steps. The results described herein use the well-established single letter designations for the amino acids in the synthesized peptides.

(6) The basics of solid phase peptide chemistry have been well-established starting with the pioneering work of Merrifield. (R. B. Merrifield (1963) Solid Phase Peptide Synthesis I, The Synthesis of a Tetrapeptide, J. Am. Chem. Soc. 85 (14), 2149-2154). The frequently used Fmoc (9-fluorenylmethyloxycarbonyl) protecting-group approach is well described in references that are easily available to the skilled person. (e.g., Chan and White, Fmoc solid phase peptide synthesis, a practical approach, Oxford University Press (2000)).

(7) The LIBERTY BLUE instrument referred to in the experiments is available from CEM Corporation of Matthews N.C. Relevant US patents dealing with the subject of solid phase peptide synthesis at elevated temperatures and using microwave irradiation include, but are not necessarily limited to, the following: U.S. Pat. Nos. 7,393,920; 7,550,560; 7,563,865; 7,939,628; 7,902,488; 7,582,728; 8,153,761; 8,058,393; 8,426,560; 8,846,862; 9,211,522. The contents of these are incorporated entirely herein by reference.

(8) TABLE-US-00001 TABLE 1 Synthesis of Thymosin with Various Amounts of Carbodiimide Temp Coupling DIEA % Purity Entry ( C.) Time Activation (Equivalents) (UPLC-MS) 1 90 2 DIC/Oxyma 0 63 (1:1) 2 90 2 DIC/Oxyma 0.1 70 (1:1) 3 90 2 DIC/Oxyma 0 75 (1:1)

(9) Experiment Conditions:

(10) Peptide Sequence (Thymosin)=(SEQ ID NO. 1) SDAAVDTSSEITTKDLKEKKEVVEEAEN-NH2

(11) Synthesis Scale=0.1 mmol

(12) Resin=Rink Amide MBHA Polystyrene Resin (0.38 mmol/g)

(13) Instrument=Liberty Blue Microwave Peptide Synthesizer (CEM Corp., Matthews, N.C.)

(14) Deprotection=3 mL of a 10% (w/v) piperazine in EtOH:NMP (1:9)

(15) Microwave Deprotection Method=1 min at 90 C.

(16) Washing=Post-Deprotection (2 mL, 2 mL, 3 mLDMF); Post-Coupling=None

(17) Coupling=5-fold excess of AA/DIC/Oxyma in 4 mL solution

(18) Cleavage=5 mL of TFA/TIS/H2O/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.)

(19) 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, 2.1100 mm)

(20) TABLE-US-00002 TABLE 2 Synthesis of GRP with Various Amounts of Carbodiimide Temp Coupling DIEA % Puriiy Entry ( C.) Time Activation (Equivalents) (UPLC-MS) 1 90 2 DIC/Oxyma 0 62 (1:1) 3 90 2 DIC/Oxyma 0 74 (2:1)

(21) Experiment Conditions:

(22) Peptide Sequence (GRP)=(SEQ ID NO. 2) VPLPAGGGTVLTKMYPRGNHWAVGHLM-NH2

(23) Synthesis Scale=0.1 mmol

(24) Resin=Rink Amide MBHA Polystyrene Resin (0.35 mmol/g)

(25) Instrument=Liberty Blue Microwave Peptide Synthesizer (CEM Corp., Matthews, N.C.)

(26) Deprotection=3 mL of a 10% (w/v) piperazine in EtOH:NMP (1:9)

(27) Microwave Deprotection Method=1 min at 90 C.

(28) Washing=Post-Deprotection (2 mL, 2 mL, 3 mLDMF); Post-Coupling=None

(29) Coupling=5-fold excess of AA/DIC/Oxyma in 4 mL solution

(30) Cleavage=5 mL of TFA/TIS/H2O/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.)

(31) 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, 2.1100 mm).

(32) TABLE-US-00003 TABLE 3 Synthesis of Ubiquitin with Various Amounts of Carbodiimide Temp Coupling DIEA % Purity Entry ( C.) Time Activation (Equivalents) (UPLC-MS) 1 90 2 DIC/Oxyma 0 >68 (1:1) 3 90 2 DIC/Oxyma 0 >73 (2:1)

(33) Experiment Conditions:

(34) Peptide Sequence (Ubiquitin)=(SEQ ID NO. 3) MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG-NH2

(35) Synthesis Scale=0.1 mmol

(36) Resin=Fmoc-PAL-PEG-PS resin (0.20 mmol/g)

(37) Instrument=Liberty Blue Microwave Peptide Synthesizer (CEM Corp., Matthews, N.C.)

(38) Deprotection=4 mL of a 10% (w/v) piperazine in EtOH:NMP (1:9)+0.1 M HOBt

(39) Microwave Deprotection Method=1 min at 90 C.

(40) Washing=Post-Deprotection (44 mL DMF); Post-Coupling=None

(41) Coupling=5-fold excess of AA/DIC/Oxyma in 4 mL solution

(42) Cleavage=5 mL of TFA/TIS/H2O/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.)

(43) 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, 2.1100 mm)

(44) As shown in Table 1, a significant increase in purity was observed by increasing the carbodiimide excess relative to the amino acid (75% vs. 63%). Similar improvements were observed from other peptides synthesized as shown in Tables 2 and 3. Together, these results show that not only was coupling efficiency increased, but also that potential capping from the carbodiimide was avoided. This is presumably from the increased kinetics of acylation relative the capping which thereby avoids this potential side reaction. Additionally, the increased temperature of the coupling reaction is also helpful for ensuring the urea formed from the carbodiimide is fully soluble and removed during subsequent draining and washing steps. Therefore, elevated temperatures provide protection against potential solubility issues from larger amounts of urea generated through the use of greater than 1 equivalent of carbodiimide.

(45) The epimerization of each amino acid was then investigated through hydrolysis, subsequent derivatization, and analysis by gas chromatography (CAT GmbH). We observed extremely low levels of epimerization using the excess carbodiimide method. This is presumably because the coupling reaction is completed the fastest (short lifetime for activated species) and no external base is present. Therefore, this method offers advantages over any previous method described for coupling at elevated temperatures to date.

(46) TABLE-US-00004 TABLE 4 Epimerization Analysis of the Synthesis of Thymosin with 2-Equivalents of Carbodiimide Alanine 0.14% D-Enantiomer Valine 0.10% D-Enantiomer Threonine >99.7% L-Threonine <0.10% D-Threonine <0.10% L-allo Threonine <0.10% D-allo Threonine Isoleucine >99.7% L-Isoleucine <0.10% D-Isoleucine <0.10% L-allo-Isoleucine <0.10% D-allo-Isoleucine Leucine 0.12% D-Enantiomer Serine 0.11% D-Enantiomer Aspartic acid 0.10% D-Enantiomer Glutamic acid 0.22% D-Enantiomer Lysine <0.10% D-Enantiomer

(47) The use of bases during the coupling process is not ideal as they can lead to undesirable side reactions. Collins et al. showed how cysteine epimerization was minimal at 90 C. under a carbodiimide based coupling method without the presence of any base. (J. Collins et al., High-Efficiency Solid Phase Peptide Synthesis (HE-SPPS), Org. Lett., vol. 16, pp. 940-943, 2014). Palasek et al. had shown how significant cysteine epimerization can occur under onium salt activation methods with the presence of DIEA and NMM present at 2 equivalents. (S. Palasek, Z. Cox et al., Limiting racemization and aspartimide formation in microwave-enhanced Fmoc solid phase peptide synthesis, J. Pept. Sci., vol. 13, pp. 143-148, 2007). It is also known that the Fmoc protecting group is slowly labile to DIEA. This can be increased at higher temperatures and leads to undesirable insertion sequences which can be difficult to separate.

(48) TABLE-US-00005 TABLE 5 Comparison of Carbodiimide and Onium Salt Activation Strategies for Peptide Coupling at Elevated Temperature NEW STANDARD ONIUM SALTS METHOD CARBODIIMIDE [Aminium] [Phosphonium] DIC/Oxyma DIC/Oxyma HBTU/DIEA PyBOP/DIEA Feature (>1:1) (1:1) (0.9:2) (1:2) Coupling FASTEST FAST LONGER - LONGER - Time Temperature Temperature Required limited limited Synthesis HIGHEST HIGH MODERATE MODERATE Purity Pre- NO NO NO* NO activation (w/slight required deficit) Stability of BEST BEST LIMITED LIMITED activated ester formed Epimerization OK OK BAD BAD of Cysteine derivatives -lactam OK OK BAD BAD formation of Arginine Stability of YES LIMITED YES YES hyper-acid sensitive resins Stability of GOOD GOOD LESS LESS activator STABLE STABLE reagents in solution

(49) TABLE-US-00006 TABLE 6 Comparison of Carbodiimide Activation Strategies for Peptide Coupling at Elevated Temperature NEW NEW STANDARD METHOD METHOD CARBODIIMIDE DIC/Oxyma DIC/Oxyma/DIEA DIC/Oxyma Feature (1:1) (1:1:0.1) (1:1) Formation of FASTEST SLIGHTLY OK O-acylisourea REDUCED Synthesis HIGHEST HIGHER HIGH Purity Pre-activation NO NO NO required Stability of BEST GOOD BEST activated ester formed Epimerization of BEST OK BEST Cysteine derivatives -lactam BEST OK BEST formation of Arginine Stability of OK YES NO hyper-acid sensitive resins Stability of GOOD GOOD GOOD activator reagents in solution

(50) In the 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.