Research Article Received 25 March 2014,
Revised 30 June 2014,
Accepted 16 July 2014
Published online 10 October 2014 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3225
The synthesis of 14C-labeled N-succinimidyl-3maleimidopropionate, a linker molecule for PEGylated biologics Kai Cao,* Brad D. Maxwell, and Samuel J. Bonacorsi Carbon-14 labeled linker molecule, N-succinimidyl-3-maleimidopropionate was prepared for disposition studies of PEGylated biologics. Our new route started with 100 mCi of carbon-14 labeled Potassium cyanide (KCN) to prepare 55 mCi of [1-14C]N-succinimidyl-3-maleimidopropionate, 6 in five steps. This represents a multiple of 5.5× improvement in the yield of the desired labeled product compared with our previous synthesis. Keywords: carbon-14; N-succinimidyl-3-maleimidopropionate (BMPS); linker molecule; PEGylated biologics
Introduction It is widely believed that covalent conjugation of a therapeutic protein with polyethylene glycol (PEG) can extend the pharmacokinetic half-life and hence reduce dosing frequency. The PEG moiety increases the hydrodynamic radius of the protein and shields it from the immune system thus reducing the potential for immunogenicity. A larger hydrodynamic radius may also slow renal excretion, prolong half-life, and shield the protein from proteolytic enzymes.2–5 To fully understand the disposition properties of such biologics and support our ongoing development of PEGylated Adnectins, carbon-14 labeling methods were developed for cross-linking N-succinimidyl-3-maleimidopropionate (BMPS) to a PEG amine (via the reaction of a NHS ester with the primary amine) and adnectins (via the conjugate addition of cysteines within the adnectins with the maleimide).
Experimental 1
13
J. Label Compd. Radiopharm 2014, 57 667–669
[1-14C]3-Benzyloxycarbonylaminopropionitrile, 3 In a 10-mL reaction vial was weighed 2-benzyloxycarbonylaminoethyl14 1-methanesulfonate (795 mg, 2.91 mmol), K CN (129 mg, 100 mCi, 55 mCi/mmol, 1.82 mmol), and potassium iodide (153 mg, 0.91 mmol). To this was added DMSO (2.8 mL). The solution was stirred at room temperature under nitrogen for 18 h. The crude product was purified by preparative HPLC. UV: 260 nm, flow rate: 8 mL/min. Gradient: 0 min, 100% A; 3 min, 100% A; 20 min, 95% B; and 25 min, 100% A. The product retention time was 9.3 min. Pooled HPLC fractions containing the desired product were concentrated under reduced pressure to give 14 [1- C]3-benzyloxycarbonylaminopropionitrile as a white solid 1 2 (351 mg, 92.5 mCi, radiochemical yield 93%). H NMR (300 MHz, [ H6]DMSO) δ 7.25–7.45(m, 5H), 5.04(s, 2H), 3.25(q, J = 6.4 Hz, 2H), 2.64(t, J = 6.4 Hz, 2H). + + MS ESI [M + H] = 205, 207.
[1-14C]N-Benzyloxycarbonyl-β-alanine, 4 14
To [1- C]3-benzyloxycarbonylaminopropionitrile (351 mg, 1.7 mmol, 92.5 mCi) in methanol (4 mL) in a 100-mL flask was added 1 N NaOH (5 mL, 5 mmol) and 30% aqueous hydrogen peroxide (3 mL, 29.4 mmol) at 0 °C with vigorous stirring. The mixture was allowed to warm at room temperature over 1 h and then stirred for 48 h. The volatiles were removed under a gentle stream of nitrogen. About 1 N aqueous HCl
Department of Chemical Synthesis, Radiochemistry Group, Discovery Chemistry, Bristol-Myers Squibb Pharmaceutical Research and Development, P.O. Box 4000, Princeton, NJ 08543, USA *Correspondence to: Kai Cao, Bristol-Myers Squibb, Route 206 and Province Line Road, Department of Chemical Synthesis, Mail-Stop: H14-02, Princeton, NJ 08543, USA. E-mail: [email protected]
Copyright © 2014 John Wiley & Sons, Ltd.
667
H NMR and decoupled C NMR were recorded on a Bruker Avance II 300 MHz spectrometer (Billerica, MA). Mass spectra were recorded on a Thermo Scientific Finnigan LXQ spectrometer (Waltham, MA). Radioactivity determinations were completed with a PerkinElmer Tri-Carb Model 2900TR liquid scintillation counter (Waltham, MA) using standard methods and Ecolite scintillation fluid by MP Biomedicals (Santa Ana, CA). Analytical HPLC analyses were performed on an Agilent 1100 HPLC system (Wilmington, DE), which included a solvent degasser, quaternary pump, autosampler, and diode array detector connected to an IN/US BetaRam flow radioactivity detector with a 0.5-mL detector cell, column: Phenomenex Gemini C18, 5 μm, 4.6 × 150 mm; mobile phase A: 0.1% trifluoroacetic acid in water; and mobile phase B: 0.1% trifluoroacetic acid in acetonitrile. Preparative HPLC was performed on a Varian HPLC system (Wilmington, DE) with two PrepStar 218 pumps (Wilmington, DE) and a ProStar 320 variable UV detector (Wilmington, DE), column: Phenomenex Gemini C18, 5 μm, 10 × 250 mm; mobile phase A: 0.1% trifluoroacetic acid in water; and mobile phase B: 0.1% trifluoroacetic acid in acetonitrile. All reagents were obtained from the Sigma-Aldrich Chemical Company of Milwaukee, WI, USA and were either American Chemical Society (ACS) grade or higher quality. Carbon-14 labeled KCN
was received from ViTrax, Inc of Placentia, CA, USA. Identities of labeled compounds were established by co-analysis with fully characterized unlabeled compounds using HPLC, NMR, and mass spectrometry, when possible. 2-Benzyloxycarbonylaminoethyl-1-methanesulfonate, 2, was prepared following a literature procedure.6
K. Cao et al. (6 mL, 6 mmol) was added to the residue, and the mixture was extracted with ethyl acetate (10 mL × 3). Pooled ethyl acetate extracts were dried over Na2SO4 and filtered, and solvent evaporated under reduced 14 pressure to give [1- C]N-benzyloxycarbonyl-β-alanine (355 mg, 1 83.7 mCi, radiochemical yield 90%) as a white solid. H NMR (300 MHz, 2 [ H6]DMSO) δ 7.20–7.45(m, 5H), 5.01(s, 2H), 3.20(q, J = 6.8 Hz, 2H), 2.38 + + (t, J = 7.1 Hz, 2H). MS ESI [M + H] = 224, 226.
[1-14C]β-Alanine, 5 14
To a 25-mL flask was weighed [1- C]N-benzyloxycarbonyl-β-alanine (355 mg, 1.58 mmol, 83.7 mCi) and 10% Pd/C (17 mg, 0.16 mmol). The flask was equipped with a balloon, degassed, and flushed with nitrogen three times prior to the addition of water and ethanol (5 mL each), followed by the introduction of hydrogen gas. The mixture was stirred at room temperature for 3 h. The reaction mixture was filtered through Celite, and the bed was washed with water (5 mL × 2). The combined filtrates were evaporated under a gentle stream of nitrogen to give 14 138 mg of [1- C]β-alanine (82.1 mCi, radiochemical yield 98%) as a pale 1 2 yellow solid. H NMR (300 MHz, [ H6]DMSO) δ 2.81(t, J = 6.3 Hz, 2H), 2.08 (t, J = 6.5 Hz, 2H).
Scheme 1. Synthesis of carbon-14 labeled-N-succinimidyl-3-maleimidopropionate 14 from K CN.
overall conversion, inconsistency, and difficulty in purifying the desired label product.
[1-14C]3-Maleimidopropionic acid, 6 14
To [1- C]β-alanine (138 mg, 1.52 mmol, 82.1 mCi) was added maleic anhydride (162 mg, 1.65 mmol) and acetic acid (8 mL). The reaction mixture turned cloudy after the first hour. The mixture was stirred at room temperature for 18 h, and then the cloudy suspension was heated to 115 °C for 8 h and became a clear solution. The solvent was evaporated under a gentle stream of nitrogen. The crude product was dissolved in 50% acetonitrile and purified by preparative HPLC. UV: 305 nm, flow rate: 7 mL/min. Gradient: 0 min, 10% B; 15 min, 50% B; 20 min, 95% B; and 25 min, 10% B. The product retention time was 5.9 min. Pooled fractions containing the desired product were 14 concentrated under reduced pressure to give [1- C]3-maleimidopropionic 1 acid (170 mg, 58 mCi, radiochemical yield 70%) as a white solid. H NMR 2 (300 MHz, [ H6]DMSO) δ 7.01(s, 2H), 3.62(t, J = 7.4 Hz, 2H), 2.45(t, 2H, partially buried under DMSO peak).
[1-14C]N-Succinimidyl 3-maleimidopropionate, 7 14
To [1- C]3-maleimidopropionic acid (170 mg, 1.00 mmol) and N-hydroxysuccinimide (114 mg, 1.00 mmol) was added DMF (5 mL). The solution was cooled in an ice water bath under nitrogen. N, N′-diisopropylcarbodiimide (0.31 mL, 2.00 mmol) was added slowly. The ice water bath was removed 5–10 min after the completion of the addition. The solution turned pink in color, and a white precipitate was formed. After stirring at room temperature for 5 h, the reaction flask was placed in an ice bath for 30 min, and the urea was removed via filtration. The solid was washed with DMF (3 mL × 3). Combined filtrates were evaporated under a stream of nitrogen to give the crude product as an off-white solid. The solid was dissolved in acetonitrile (15 mL) and the acetonitrile solution was cooled in an ice water bath for 10 min and filtered. The yellow orange filtrate was evaporated under a stream of nitrogen to give 324 mg of a gray powder as a mixture of 27% diisopropyl urea and 73% desired product (54.5 mCi, radiochemical purity 98%, radiochemical yield: 88%). The product mixture was used as is for subsequent PEGylation and coupling with adnectins without detrimental interference from the diisopropyl urea. 1 2 H NMR (300 MHz, [ H6]DMSO) δ 7.04(s, 2H), 3.74(t, J = 6.9 Hz, 2H), 3.04 (t, J = 6.9 Hz, 2H), 2.79(s, 4H).
Results and discussion
668
Our initial synthesis of a carbon-14 labeled linker molecule started by heating [1,4-14C]maleic acid with β-alanine. The product isolated was then reacted with N-hydroxysuccinimide to produce [14C]BMPS, 1.1 This synthesis suffered from low
www.jlcr.org
Because of the difficulties with the initial approach, an alternative labeling approach was developed. C. A. Townsend and A. Basak reported the synthesis of carbon-13 labeled β-alanine and derivatives.6 Following similar chemistry, we successfully prepared carbon-14 labeled β-alanine from readily available [14C]KCN, see Scheme 1. For the cyanation reaction of benzyloxycarbonylaminoethyl-1-methanesulfonate, 3,6 we discovered that the reaction was completed in 18 h with the addition of 1 eq. of KI, which also gave better yields. Thus 2-benzyloxycarbonylaminoethyl-1methanesulfonate reacted with [14C]KCN in the presence of 1 eq. of KI that gave a 93% conversion to [1-14C]3-benzyloxycarbonylaminopropionitrile, 3, after purification. Subsequent hydrolysis of nitrile 3 with NaOH and H2O2 yielded [1-14C]N-benzyloxycarbonylβ-alanine, 4, in 90% radiochemical yield. Deprotection of 4 with hydrogen gas and 10% Pd/C gave [1-14C]β-alanine, 5, in near quantitative yield. Following the previously reported procedures, [1-14C]3-maleimidopropionic acid 6 was prepared in 70% yield, and [1-14C]N-succinimidyl 3-maleimidopropionate, 7, was prepared in 94% radiochemical yield.1,7,8 The final product is a mixture of 27% diisopropyl urea and 73% of [1-14C]BMPS (determined by 1H NMR). Our earlier attempts of purification using flash chromatography resulted in very low recoveries (
Revised 30 June 2014,
Accepted 16 July 2014
Published online 10 October 2014 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3225
The synthesis of 14C-labeled N-succinimidyl-3maleimidopropionate, a linker molecule for PEGylated biologics Kai Cao,* Brad D. Maxwell, and Samuel J. Bonacorsi Carbon-14 labeled linker molecule, N-succinimidyl-3-maleimidopropionate was prepared for disposition studies of PEGylated biologics. Our new route started with 100 mCi of carbon-14 labeled Potassium cyanide (KCN) to prepare 55 mCi of [1-14C]N-succinimidyl-3-maleimidopropionate, 6 in five steps. This represents a multiple of 5.5× improvement in the yield of the desired labeled product compared with our previous synthesis. Keywords: carbon-14; N-succinimidyl-3-maleimidopropionate (BMPS); linker molecule; PEGylated biologics
Introduction It is widely believed that covalent conjugation of a therapeutic protein with polyethylene glycol (PEG) can extend the pharmacokinetic half-life and hence reduce dosing frequency. The PEG moiety increases the hydrodynamic radius of the protein and shields it from the immune system thus reducing the potential for immunogenicity. A larger hydrodynamic radius may also slow renal excretion, prolong half-life, and shield the protein from proteolytic enzymes.2–5 To fully understand the disposition properties of such biologics and support our ongoing development of PEGylated Adnectins, carbon-14 labeling methods were developed for cross-linking N-succinimidyl-3-maleimidopropionate (BMPS) to a PEG amine (via the reaction of a NHS ester with the primary amine) and adnectins (via the conjugate addition of cysteines within the adnectins with the maleimide).
Experimental 1
13
J. Label Compd. Radiopharm 2014, 57 667–669
[1-14C]3-Benzyloxycarbonylaminopropionitrile, 3 In a 10-mL reaction vial was weighed 2-benzyloxycarbonylaminoethyl14 1-methanesulfonate (795 mg, 2.91 mmol), K CN (129 mg, 100 mCi, 55 mCi/mmol, 1.82 mmol), and potassium iodide (153 mg, 0.91 mmol). To this was added DMSO (2.8 mL). The solution was stirred at room temperature under nitrogen for 18 h. The crude product was purified by preparative HPLC. UV: 260 nm, flow rate: 8 mL/min. Gradient: 0 min, 100% A; 3 min, 100% A; 20 min, 95% B; and 25 min, 100% A. The product retention time was 9.3 min. Pooled HPLC fractions containing the desired product were concentrated under reduced pressure to give 14 [1- C]3-benzyloxycarbonylaminopropionitrile as a white solid 1 2 (351 mg, 92.5 mCi, radiochemical yield 93%). H NMR (300 MHz, [ H6]DMSO) δ 7.25–7.45(m, 5H), 5.04(s, 2H), 3.25(q, J = 6.4 Hz, 2H), 2.64(t, J = 6.4 Hz, 2H). + + MS ESI [M + H] = 205, 207.
[1-14C]N-Benzyloxycarbonyl-β-alanine, 4 14
To [1- C]3-benzyloxycarbonylaminopropionitrile (351 mg, 1.7 mmol, 92.5 mCi) in methanol (4 mL) in a 100-mL flask was added 1 N NaOH (5 mL, 5 mmol) and 30% aqueous hydrogen peroxide (3 mL, 29.4 mmol) at 0 °C with vigorous stirring. The mixture was allowed to warm at room temperature over 1 h and then stirred for 48 h. The volatiles were removed under a gentle stream of nitrogen. About 1 N aqueous HCl
Department of Chemical Synthesis, Radiochemistry Group, Discovery Chemistry, Bristol-Myers Squibb Pharmaceutical Research and Development, P.O. Box 4000, Princeton, NJ 08543, USA *Correspondence to: Kai Cao, Bristol-Myers Squibb, Route 206 and Province Line Road, Department of Chemical Synthesis, Mail-Stop: H14-02, Princeton, NJ 08543, USA. E-mail: [email protected]
Copyright © 2014 John Wiley & Sons, Ltd.
667
H NMR and decoupled C NMR were recorded on a Bruker Avance II 300 MHz spectrometer (Billerica, MA). Mass spectra were recorded on a Thermo Scientific Finnigan LXQ spectrometer (Waltham, MA). Radioactivity determinations were completed with a PerkinElmer Tri-Carb Model 2900TR liquid scintillation counter (Waltham, MA) using standard methods and Ecolite scintillation fluid by MP Biomedicals (Santa Ana, CA). Analytical HPLC analyses were performed on an Agilent 1100 HPLC system (Wilmington, DE), which included a solvent degasser, quaternary pump, autosampler, and diode array detector connected to an IN/US BetaRam flow radioactivity detector with a 0.5-mL detector cell, column: Phenomenex Gemini C18, 5 μm, 4.6 × 150 mm; mobile phase A: 0.1% trifluoroacetic acid in water; and mobile phase B: 0.1% trifluoroacetic acid in acetonitrile. Preparative HPLC was performed on a Varian HPLC system (Wilmington, DE) with two PrepStar 218 pumps (Wilmington, DE) and a ProStar 320 variable UV detector (Wilmington, DE), column: Phenomenex Gemini C18, 5 μm, 10 × 250 mm; mobile phase A: 0.1% trifluoroacetic acid in water; and mobile phase B: 0.1% trifluoroacetic acid in acetonitrile. All reagents were obtained from the Sigma-Aldrich Chemical Company of Milwaukee, WI, USA and were either American Chemical Society (ACS) grade or higher quality. Carbon-14 labeled KCN
was received from ViTrax, Inc of Placentia, CA, USA. Identities of labeled compounds were established by co-analysis with fully characterized unlabeled compounds using HPLC, NMR, and mass spectrometry, when possible. 2-Benzyloxycarbonylaminoethyl-1-methanesulfonate, 2, was prepared following a literature procedure.6
K. Cao et al. (6 mL, 6 mmol) was added to the residue, and the mixture was extracted with ethyl acetate (10 mL × 3). Pooled ethyl acetate extracts were dried over Na2SO4 and filtered, and solvent evaporated under reduced 14 pressure to give [1- C]N-benzyloxycarbonyl-β-alanine (355 mg, 1 83.7 mCi, radiochemical yield 90%) as a white solid. H NMR (300 MHz, 2 [ H6]DMSO) δ 7.20–7.45(m, 5H), 5.01(s, 2H), 3.20(q, J = 6.8 Hz, 2H), 2.38 + + (t, J = 7.1 Hz, 2H). MS ESI [M + H] = 224, 226.
[1-14C]β-Alanine, 5 14
To a 25-mL flask was weighed [1- C]N-benzyloxycarbonyl-β-alanine (355 mg, 1.58 mmol, 83.7 mCi) and 10% Pd/C (17 mg, 0.16 mmol). The flask was equipped with a balloon, degassed, and flushed with nitrogen three times prior to the addition of water and ethanol (5 mL each), followed by the introduction of hydrogen gas. The mixture was stirred at room temperature for 3 h. The reaction mixture was filtered through Celite, and the bed was washed with water (5 mL × 2). The combined filtrates were evaporated under a gentle stream of nitrogen to give 14 138 mg of [1- C]β-alanine (82.1 mCi, radiochemical yield 98%) as a pale 1 2 yellow solid. H NMR (300 MHz, [ H6]DMSO) δ 2.81(t, J = 6.3 Hz, 2H), 2.08 (t, J = 6.5 Hz, 2H).
Scheme 1. Synthesis of carbon-14 labeled-N-succinimidyl-3-maleimidopropionate 14 from K CN.
overall conversion, inconsistency, and difficulty in purifying the desired label product.
[1-14C]3-Maleimidopropionic acid, 6 14
To [1- C]β-alanine (138 mg, 1.52 mmol, 82.1 mCi) was added maleic anhydride (162 mg, 1.65 mmol) and acetic acid (8 mL). The reaction mixture turned cloudy after the first hour. The mixture was stirred at room temperature for 18 h, and then the cloudy suspension was heated to 115 °C for 8 h and became a clear solution. The solvent was evaporated under a gentle stream of nitrogen. The crude product was dissolved in 50% acetonitrile and purified by preparative HPLC. UV: 305 nm, flow rate: 7 mL/min. Gradient: 0 min, 10% B; 15 min, 50% B; 20 min, 95% B; and 25 min, 10% B. The product retention time was 5.9 min. Pooled fractions containing the desired product were 14 concentrated under reduced pressure to give [1- C]3-maleimidopropionic 1 acid (170 mg, 58 mCi, radiochemical yield 70%) as a white solid. H NMR 2 (300 MHz, [ H6]DMSO) δ 7.01(s, 2H), 3.62(t, J = 7.4 Hz, 2H), 2.45(t, 2H, partially buried under DMSO peak).
[1-14C]N-Succinimidyl 3-maleimidopropionate, 7 14
To [1- C]3-maleimidopropionic acid (170 mg, 1.00 mmol) and N-hydroxysuccinimide (114 mg, 1.00 mmol) was added DMF (5 mL). The solution was cooled in an ice water bath under nitrogen. N, N′-diisopropylcarbodiimide (0.31 mL, 2.00 mmol) was added slowly. The ice water bath was removed 5–10 min after the completion of the addition. The solution turned pink in color, and a white precipitate was formed. After stirring at room temperature for 5 h, the reaction flask was placed in an ice bath for 30 min, and the urea was removed via filtration. The solid was washed with DMF (3 mL × 3). Combined filtrates were evaporated under a stream of nitrogen to give the crude product as an off-white solid. The solid was dissolved in acetonitrile (15 mL) and the acetonitrile solution was cooled in an ice water bath for 10 min and filtered. The yellow orange filtrate was evaporated under a stream of nitrogen to give 324 mg of a gray powder as a mixture of 27% diisopropyl urea and 73% desired product (54.5 mCi, radiochemical purity 98%, radiochemical yield: 88%). The product mixture was used as is for subsequent PEGylation and coupling with adnectins without detrimental interference from the diisopropyl urea. 1 2 H NMR (300 MHz, [ H6]DMSO) δ 7.04(s, 2H), 3.74(t, J = 6.9 Hz, 2H), 3.04 (t, J = 6.9 Hz, 2H), 2.79(s, 4H).
Results and discussion
668
Our initial synthesis of a carbon-14 labeled linker molecule started by heating [1,4-14C]maleic acid with β-alanine. The product isolated was then reacted with N-hydroxysuccinimide to produce [14C]BMPS, 1.1 This synthesis suffered from low
www.jlcr.org
Because of the difficulties with the initial approach, an alternative labeling approach was developed. C. A. Townsend and A. Basak reported the synthesis of carbon-13 labeled β-alanine and derivatives.6 Following similar chemistry, we successfully prepared carbon-14 labeled β-alanine from readily available [14C]KCN, see Scheme 1. For the cyanation reaction of benzyloxycarbonylaminoethyl-1-methanesulfonate, 3,6 we discovered that the reaction was completed in 18 h with the addition of 1 eq. of KI, which also gave better yields. Thus 2-benzyloxycarbonylaminoethyl-1methanesulfonate reacted with [14C]KCN in the presence of 1 eq. of KI that gave a 93% conversion to [1-14C]3-benzyloxycarbonylaminopropionitrile, 3, after purification. Subsequent hydrolysis of nitrile 3 with NaOH and H2O2 yielded [1-14C]N-benzyloxycarbonylβ-alanine, 4, in 90% radiochemical yield. Deprotection of 4 with hydrogen gas and 10% Pd/C gave [1-14C]β-alanine, 5, in near quantitative yield. Following the previously reported procedures, [1-14C]3-maleimidopropionic acid 6 was prepared in 70% yield, and [1-14C]N-succinimidyl 3-maleimidopropionate, 7, was prepared in 94% radiochemical yield.1,7,8 The final product is a mixture of 27% diisopropyl urea and 73% of [1-14C]BMPS (determined by 1H NMR). Our earlier attempts of purification using flash chromatography resulted in very low recoveries (
