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Solid-phase synthesis of serglycin glycopeptides on a new allyl ester linker
Tetrahedron Letters 41 (2000) 6489±6493
Solid-phase synthesis of serglycin glycopeptides on a new allyl ester linker Yuko Nakahara,a Sumie Ando,a Masaki Itakura,b Naomi Kumabe,b Hironobu Hojo,b Yukishige Itoa and Yoshiaki Nakaharaa,b,* a
The Institute of Physical and Chemical Research (RIKEN), Hirosawa 2-1, Wako-shi, Saitama, 351-0198, Japan b Department of Industrial Chemistry, Tokai University, Kitakaname 1117, Hiratsuka-shi, Kanagawa, 259-1292, Japan Received 17 May 2000; revised 22 June 2000; accepted 23 June 2000
Abstract t-Butyl 6-bromo-(E)-4-hexenoate was used as a handle for the solid-phase synthesis of glycopeptides. The handle was ®rst conjugated with Fmoc amino acids to form the allyl esters, which were then attached to the Sieber amide resin via acidic cleavage of the t-butyl esters followed by activation of the liberated carboxylic acids. Solid-phase synthesis was demonstrated for the glycopeptide oligomers modeled after glycosyl Ser-Gly repeating sequence of proteoglycan. # 2000 Elsevier Science Ltd. All rights reserved. Keywords: solid-phase synthesis; glycopeptides; allyl linker; serglycin.
Recently, a large number of syntheses have been performed on a solid support, especially utilizing the improved techniques such as the introduction of the newly designed linkers. Most of them have been directed towards the creation of small molecule libraries by combinatorial chemistry.1 On the other hand, oligopeptides have long been the leaders in the ®eld of solid-phase synthesis, since the ®rst proposal by Merri®eld.2 With respect to the methodologies for peptide synthesis, Boc or Fmoc chemistry is the standard of choice and the oligomers synthesized on the resins are released, in general, under strongly or mildly acidic conditions, depending upon the nature of the speci®c linkers. In contrast, the allyl ester-based HYCRAM and HYCRON linkers, both developed by Kunz, are compatible with Boc and Fmoc strategy and the neutral cleavage condition using Pd(0) catalyst allows detachment of the fully protected peptides from resin. The linkers have successfully been utilized in the synthesis of acid-susceptible glycopeptide.3
* Corresponding author. Fax +81 463 50 2075; e-mail: [email protected] 0040-4039/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. PII: S0040-4039(00)01083-2
6490
In the framework of our project, aiming at synthesis of complex glycopeptides, we now report preparation of a new allyl ester-type linker and the synthesis of proteoglycan core molecules, performed by combination of the linker with the commercial acid-labile Sieber amide resin. This combination appealed to us since either C-protected or C-unprotected glycopeptide block would be obtainable by palladium catalysis and by mildly acidic cleavage (a and b, respectively, in Fig. 1), and further coupling of the liberated peptide blocks would be feasible by condensation in solution. It is to be noted that chemoenzymatic synthesis of glycopeptides utilizing a similar dually-cleavable linker has recently appeared.4
Figure 1.
In order to install an appropriate allyl ester handle, t-butyl 6-bromo-(E)-4-hexenoate 1 was synthesized in 68% yield by reaction of commercially available1,4-dibromo-2-butene and t-butyl acetate in the presence of lithium amides,5 as shown in Scheme 1. Esteri®cation of several Fmoc amino acids with 1 proceeded smoothly via their cesium salts, with the exception of Fmoc proline, while the latter was esteri®ed in 65% yield under the conditions reported by Kunz.3d
Scheme 1.
6491
Serglycin proteoglycans have been named from their structural features, representing an extended sequence of alternating serine and glycine residues.6 The serine residues are heavily glycosylated with clustered chondroitin sulfate or heparin. A number of synthetic eorts have been made on those glycosaminoglycan chains, aiming at the potential biological functions.7 However, there appear to be only a few reports on the synthesis of oligopeptides carrying the proteoglycan-related carbohydrate attachments.8 In order to practice our solid-phase methodology using the allyl ester linker, the serglycin fragments representing the carbohydrate±protein linkage
Scheme 2.
6492
region of proteoglycans were chosen as the target compounds. Treatment of 2 with TFA (tri¯uoroacetic acid) quantitatively aorded the corresponding carboxylic acid 9, which was attached to the Sieber amide resin (0.6 mmol/g) through activation with HBTU (O-benzotriazol1-yl-N,N,N0 ,N0 -tetramethyluronium hexa¯uorophosphate), HOBt (hydroxybenztriazole), and DIEA (diisopropylethylamine) in NMP (N-methylpyrrolidone). All the on-resin reactions were facilitated by use of a vortexing tube-mixer. This loading eciency was estimated to be 99% by ninhydrin monitoring. The resin-bound glycine 10 was then N-deprotected with 20% piperidine in NMP. The known xylosyl serine unit 12 9 (1.5 equiv.) was condensed with the N-deprotected glycin 11 under the conditions with HBTU/HOBt/DIEA in NMP. The resulting dipeptide Xyl-Ser-Gly 15 was then split from the resin by Pd(0)-catalyzed deallylation10 [Pd(Ph3P)4, dimedone, DMSO/ CH2Cl2 quant.], whereas C,N-masked glycopeptide 16 was obtained quantitatively by acidolysis with 2% TFA. The latter was easily deallylated in solution to give the same glycopeptide 15 (quant.). With the dipeptide block 15, further solid-phase synthesis was examined. Removal of the Fmoc group from Xyl-Ser-Gly-resin 13 with 20% piperidine produced 14, which was condensed with 15 under the same conditions as above. The tetrapeptide 18 thus synthesized was detached by Pd-reaction and used for the synthesis of octapeptide. Repeating this procedure, an octaxylosyl hexadecapeptide was eventually synthesized on the solid support. Acidic treatment of the resin 27 followed by preparative HPLC of the released product aorded pure 28 in 79% yield. The yields of the intermediary oligomers 18, 19, 23, and 24 were 96, 100, 99, and 98%, respectively. The structures of them were con®rmed by mass and NMR spectroscopy (Scheme 2).11 In addition, block condensation in solution was also investigated. The N-deprotected tetrapeptide 20 (93%) was prepared in solution from 19, while 25 (86%) was obtained by acidic cleavage of the resin 26. The coupling reaction of tetrapeptides 18 and 20, using HBTU/HOBt/DIEA in NMP, resulted in the formation of 24 (84%), whereas the reaction of 23 and 25 gave 28 (66%). In conclusion, the synthesis of serglycin fragments, up to hexadecapeptide, was accomplished with high eciency by means of Fmoc solid-phase protocol, employing a newly designed allyl linker. The protected glycooligopeptides cleaved from the resin served as the useful building blocks, not only for the solid-phase condensation but also for the coupling in solution. The combination of the allyl linker and the acid-labile Sieber amide resin is promising for the synthesis of acid- and base-labile glycopeptides. Acknowledgements We thank Dr T. Chihara and his sta for elemental analyses, and Ms A. Takahashi for technical assistance.
References 1. For reviews: (a) Hermkens, P. H. H.; Ottenheijm, H. C. J.; Rees, D. Tetrahedron 1996, 52, 4527±4554. (b) Balkenhohl, F.; von dem Bussche-HuÈnnefeld, C.; Lansky, A.; Zechel, C. Angew. Chem., Int. Ed. Engl. 1996, 35, 2288±2337. (c) FruÈchtel, J. S.; Jung, G. Angew. Chem., Int. Ed. Engl. 1996, 35, 17±42. 2. Merri®eld, R. B. J. Am. Chem. Soc. 1963, 85, 2149±2154.
6493 3. (a) Kunz, H.; Dombo, B. Angew. Chem., Int. Ed. Engl. 1988, 27, 711±712. (b) Kosch, W.; MaÈrz, J.; Kunz, H. React. Polym. 1994, 22, 181±194. (c) Seitz, O.; Kunz, H. Angew. Chem., Int. Ed. Engl. 1995, 34, 803±805. (d) Seitz, O.; Kunz, H. J. Org. Chem. 1997, 62, 813±826. 4. Seitz, O.; Wong, C.-H. J. Am. Chem. Soc. 1997, 119, 8766±8776. 5. Dorsch, D.; Kunz, E.; Helmchen, G. Tetrahedron Lett. 1985, 26, 3319±3322. 6. Kresse, H.; Hausser, H.; SchoÈnherr, E. In Proteoglycans; JolleÁs, P., Ed. Small Proteoglycans. BirkhaÈuser: Verlag, Basel, 1994; pp. 73±100. 7. Tamura, J. Trends Glycosci. Glycotechnol. 1994, 6, 29±50. 8. (a) Rio, S.; Beau, J.-M.; Jaquinet, J.-C. Carbohydr. Res. 1991, 219, 7±90. (b) Rio, S.; Beau, J.-M.; Jaquinet, J.-C. Carbohydr. Res. 1993, 244, 295±313. 9. Garg, H. G.; Hasenkamp, T.; Paulsen, H. Carbohydr. Res. 1986, 151, 225±232. 10. Kunz, H.; Unverzagt, C. Angew. Chem., Int. Ed. Engl. 1984, 23, 436±437. 11. Selected physical data are given below. 1: bp. 80 C/3 mm Hg; 1H NMR, 5.75 (m, 2H), 3.93 (d, 2H, J 6.3 Hz), 2.33 (m, 4H), 1.45 (s, 9H). 2: mp. 47±49 C; 7.74 (d, 2H, J 7.6 Hz), 7.59 (brd, 2H, J 7.3 Hz), 7.38±7.29 (m, 4H), 5.83±5.53 (m, 2H), 5.44 (t, 1H, J 5.3 Hz), 4.57 (d, 2H, J 6.3 Hz), 4.39 (d, 2H, J 7.3 Hz), 4.21 (brt, 1 H, J 6.9 Hz), 3.97 (d, 2H, J 5.6 Hz), 2.30 (brs, 4H), 1.43 (s, 9H). 15: 7.76 (d, 2H, J 7.3 Hz), 7.58 (brd, 2H, J 7.3 Hz), 7.42±7.30 (m, 4H), 6.84 (d, 1H, NH), 5.82 (d, 1H, NH), 5.17 (t, 1H, 8.6), 4.95±4.89 (m, 2H), 4.21 (brt, 1H, J 6.8 Hz), 3.69 (m, 1H), 3.39 (m, 1H), 2.04 (s, 3H), 2.02 (s, 6H). 16: 7.77 (d, 2H, J 7.6 Hz), 7.59 (brd, 2H, J 7.6 Hz), 7.42±7.30 (m, 4H), 6.96 (brs, 1H, NH), 5.97 (brs, 1H, NH), 5.84±5.77 (m, 3H), 5.62 (m, 1H), 5.19 (t, 1H, J 8.8 Hz), 4.99±4.89 (m, 2H), 4.60±4.42 (m, 6H), 4.22 (t, 1H, J 6.8 Hz), 3.70 (m, 1H), 3.40 (m, 1H), 2.40±2.32 (m, 4H), 2.05 (s, 3H), 2.03 (s, 6H). 18: 7.76 (d, 2H, J 7.3 Hz), 7.61 (brd, 2H, J 7.6 Hz), 7.41±7.27 (m, 4H), 5.18 (t, 1H, J 8.5 Hz), 5.16 (t, 1H, J 8.5 Hz), 5.00±4.80 (m, 4H), 2.04±2.00 (18H); HR-Fab MS 1045.3322 (M+H). 19: 7.77 (d, 2H, J 7.6 Hz), 7.61 (brd, 2H, J 7.1 Hz), 7.43±7.30 (m, 4H), 5.95 (brs, 1H, NH), 5.85±5.50 (m, 3H), 5.21±5.15 (m, 2H), 5.00±4.88 (m, 4H), 4.22 (t, 1H, J 6.6 Hz), 3.80±3.60 (m, 2H), 3.39±3.33 (m, 2H), 2.38±2.17 (m, 4H), 2.06±2.01 (m, 18H); HR-Fab MS 1156.4221 (M+H). 20: HR-Fab MS 935.3427 (M+H). 23: Fab MS 1849.6 (M+H). 24: Fab MS 11061.2 (M+H). 25: Fab MS 1738.6 (M+H). 28: TOF MS 3591.46 (M+Na).
Solid-phase synthesis of serglycin glycopeptides on a new allyl ester linker Yuko Nakahara,a Sumie Ando,a Masaki Itakura,b Naomi Kumabe,b Hironobu Hojo,b Yukishige Itoa and Yoshiaki Nakaharaa,b,* a
The Institute of Physical and Chemical Research (RIKEN), Hirosawa 2-1, Wako-shi, Saitama, 351-0198, Japan b Department of Industrial Chemistry, Tokai University, Kitakaname 1117, Hiratsuka-shi, Kanagawa, 259-1292, Japan Received 17 May 2000; revised 22 June 2000; accepted 23 June 2000
Abstract t-Butyl 6-bromo-(E)-4-hexenoate was used as a handle for the solid-phase synthesis of glycopeptides. The handle was ®rst conjugated with Fmoc amino acids to form the allyl esters, which were then attached to the Sieber amide resin via acidic cleavage of the t-butyl esters followed by activation of the liberated carboxylic acids. Solid-phase synthesis was demonstrated for the glycopeptide oligomers modeled after glycosyl Ser-Gly repeating sequence of proteoglycan. # 2000 Elsevier Science Ltd. All rights reserved. Keywords: solid-phase synthesis; glycopeptides; allyl linker; serglycin.
Recently, a large number of syntheses have been performed on a solid support, especially utilizing the improved techniques such as the introduction of the newly designed linkers. Most of them have been directed towards the creation of small molecule libraries by combinatorial chemistry.1 On the other hand, oligopeptides have long been the leaders in the ®eld of solid-phase synthesis, since the ®rst proposal by Merri®eld.2 With respect to the methodologies for peptide synthesis, Boc or Fmoc chemistry is the standard of choice and the oligomers synthesized on the resins are released, in general, under strongly or mildly acidic conditions, depending upon the nature of the speci®c linkers. In contrast, the allyl ester-based HYCRAM and HYCRON linkers, both developed by Kunz, are compatible with Boc and Fmoc strategy and the neutral cleavage condition using Pd(0) catalyst allows detachment of the fully protected peptides from resin. The linkers have successfully been utilized in the synthesis of acid-susceptible glycopeptide.3
* Corresponding author. Fax +81 463 50 2075; e-mail: [email protected] 0040-4039/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. PII: S0040-4039(00)01083-2
6490
In the framework of our project, aiming at synthesis of complex glycopeptides, we now report preparation of a new allyl ester-type linker and the synthesis of proteoglycan core molecules, performed by combination of the linker with the commercial acid-labile Sieber amide resin. This combination appealed to us since either C-protected or C-unprotected glycopeptide block would be obtainable by palladium catalysis and by mildly acidic cleavage (a and b, respectively, in Fig. 1), and further coupling of the liberated peptide blocks would be feasible by condensation in solution. It is to be noted that chemoenzymatic synthesis of glycopeptides utilizing a similar dually-cleavable linker has recently appeared.4
Figure 1.
In order to install an appropriate allyl ester handle, t-butyl 6-bromo-(E)-4-hexenoate 1 was synthesized in 68% yield by reaction of commercially available1,4-dibromo-2-butene and t-butyl acetate in the presence of lithium amides,5 as shown in Scheme 1. Esteri®cation of several Fmoc amino acids with 1 proceeded smoothly via their cesium salts, with the exception of Fmoc proline, while the latter was esteri®ed in 65% yield under the conditions reported by Kunz.3d
Scheme 1.
6491
Serglycin proteoglycans have been named from their structural features, representing an extended sequence of alternating serine and glycine residues.6 The serine residues are heavily glycosylated with clustered chondroitin sulfate or heparin. A number of synthetic eorts have been made on those glycosaminoglycan chains, aiming at the potential biological functions.7 However, there appear to be only a few reports on the synthesis of oligopeptides carrying the proteoglycan-related carbohydrate attachments.8 In order to practice our solid-phase methodology using the allyl ester linker, the serglycin fragments representing the carbohydrate±protein linkage
Scheme 2.
6492
region of proteoglycans were chosen as the target compounds. Treatment of 2 with TFA (tri¯uoroacetic acid) quantitatively aorded the corresponding carboxylic acid 9, which was attached to the Sieber amide resin (0.6 mmol/g) through activation with HBTU (O-benzotriazol1-yl-N,N,N0 ,N0 -tetramethyluronium hexa¯uorophosphate), HOBt (hydroxybenztriazole), and DIEA (diisopropylethylamine) in NMP (N-methylpyrrolidone). All the on-resin reactions were facilitated by use of a vortexing tube-mixer. This loading eciency was estimated to be 99% by ninhydrin monitoring. The resin-bound glycine 10 was then N-deprotected with 20% piperidine in NMP. The known xylosyl serine unit 12 9 (1.5 equiv.) was condensed with the N-deprotected glycin 11 under the conditions with HBTU/HOBt/DIEA in NMP. The resulting dipeptide Xyl-Ser-Gly 15 was then split from the resin by Pd(0)-catalyzed deallylation10 [Pd(Ph3P)4, dimedone, DMSO/ CH2Cl2 quant.], whereas C,N-masked glycopeptide 16 was obtained quantitatively by acidolysis with 2% TFA. The latter was easily deallylated in solution to give the same glycopeptide 15 (quant.). With the dipeptide block 15, further solid-phase synthesis was examined. Removal of the Fmoc group from Xyl-Ser-Gly-resin 13 with 20% piperidine produced 14, which was condensed with 15 under the same conditions as above. The tetrapeptide 18 thus synthesized was detached by Pd-reaction and used for the synthesis of octapeptide. Repeating this procedure, an octaxylosyl hexadecapeptide was eventually synthesized on the solid support. Acidic treatment of the resin 27 followed by preparative HPLC of the released product aorded pure 28 in 79% yield. The yields of the intermediary oligomers 18, 19, 23, and 24 were 96, 100, 99, and 98%, respectively. The structures of them were con®rmed by mass and NMR spectroscopy (Scheme 2).11 In addition, block condensation in solution was also investigated. The N-deprotected tetrapeptide 20 (93%) was prepared in solution from 19, while 25 (86%) was obtained by acidic cleavage of the resin 26. The coupling reaction of tetrapeptides 18 and 20, using HBTU/HOBt/DIEA in NMP, resulted in the formation of 24 (84%), whereas the reaction of 23 and 25 gave 28 (66%). In conclusion, the synthesis of serglycin fragments, up to hexadecapeptide, was accomplished with high eciency by means of Fmoc solid-phase protocol, employing a newly designed allyl linker. The protected glycooligopeptides cleaved from the resin served as the useful building blocks, not only for the solid-phase condensation but also for the coupling in solution. The combination of the allyl linker and the acid-labile Sieber amide resin is promising for the synthesis of acid- and base-labile glycopeptides. Acknowledgements We thank Dr T. Chihara and his sta for elemental analyses, and Ms A. Takahashi for technical assistance.
References 1. For reviews: (a) Hermkens, P. H. H.; Ottenheijm, H. C. J.; Rees, D. Tetrahedron 1996, 52, 4527±4554. (b) Balkenhohl, F.; von dem Bussche-HuÈnnefeld, C.; Lansky, A.; Zechel, C. Angew. Chem., Int. Ed. Engl. 1996, 35, 2288±2337. (c) FruÈchtel, J. S.; Jung, G. Angew. Chem., Int. Ed. Engl. 1996, 35, 17±42. 2. Merri®eld, R. B. J. Am. Chem. Soc. 1963, 85, 2149±2154.
6493 3. (a) Kunz, H.; Dombo, B. Angew. Chem., Int. Ed. Engl. 1988, 27, 711±712. (b) Kosch, W.; MaÈrz, J.; Kunz, H. React. Polym. 1994, 22, 181±194. (c) Seitz, O.; Kunz, H. Angew. Chem., Int. Ed. Engl. 1995, 34, 803±805. (d) Seitz, O.; Kunz, H. J. Org. Chem. 1997, 62, 813±826. 4. Seitz, O.; Wong, C.-H. J. Am. Chem. Soc. 1997, 119, 8766±8776. 5. Dorsch, D.; Kunz, E.; Helmchen, G. Tetrahedron Lett. 1985, 26, 3319±3322. 6. Kresse, H.; Hausser, H.; SchoÈnherr, E. In Proteoglycans; JolleÁs, P., Ed. Small Proteoglycans. BirkhaÈuser: Verlag, Basel, 1994; pp. 73±100. 7. Tamura, J. Trends Glycosci. Glycotechnol. 1994, 6, 29±50. 8. (a) Rio, S.; Beau, J.-M.; Jaquinet, J.-C. Carbohydr. Res. 1991, 219, 7±90. (b) Rio, S.; Beau, J.-M.; Jaquinet, J.-C. Carbohydr. Res. 1993, 244, 295±313. 9. Garg, H. G.; Hasenkamp, T.; Paulsen, H. Carbohydr. Res. 1986, 151, 225±232. 10. Kunz, H.; Unverzagt, C. Angew. Chem., Int. Ed. Engl. 1984, 23, 436±437. 11. Selected physical data are given below. 1: bp. 80 C/3 mm Hg; 1H NMR, 5.75 (m, 2H), 3.93 (d, 2H, J 6.3 Hz), 2.33 (m, 4H), 1.45 (s, 9H). 2: mp. 47±49 C; 7.74 (d, 2H, J 7.6 Hz), 7.59 (brd, 2H, J 7.3 Hz), 7.38±7.29 (m, 4H), 5.83±5.53 (m, 2H), 5.44 (t, 1H, J 5.3 Hz), 4.57 (d, 2H, J 6.3 Hz), 4.39 (d, 2H, J 7.3 Hz), 4.21 (brt, 1 H, J 6.9 Hz), 3.97 (d, 2H, J 5.6 Hz), 2.30 (brs, 4H), 1.43 (s, 9H). 15: 7.76 (d, 2H, J 7.3 Hz), 7.58 (brd, 2H, J 7.3 Hz), 7.42±7.30 (m, 4H), 6.84 (d, 1H, NH), 5.82 (d, 1H, NH), 5.17 (t, 1H, 8.6), 4.95±4.89 (m, 2H), 4.21 (brt, 1H, J 6.8 Hz), 3.69 (m, 1H), 3.39 (m, 1H), 2.04 (s, 3H), 2.02 (s, 6H). 16: 7.77 (d, 2H, J 7.6 Hz), 7.59 (brd, 2H, J 7.6 Hz), 7.42±7.30 (m, 4H), 6.96 (brs, 1H, NH), 5.97 (brs, 1H, NH), 5.84±5.77 (m, 3H), 5.62 (m, 1H), 5.19 (t, 1H, J 8.8 Hz), 4.99±4.89 (m, 2H), 4.60±4.42 (m, 6H), 4.22 (t, 1H, J 6.8 Hz), 3.70 (m, 1H), 3.40 (m, 1H), 2.40±2.32 (m, 4H), 2.05 (s, 3H), 2.03 (s, 6H). 18: 7.76 (d, 2H, J 7.3 Hz), 7.61 (brd, 2H, J 7.6 Hz), 7.41±7.27 (m, 4H), 5.18 (t, 1H, J 8.5 Hz), 5.16 (t, 1H, J 8.5 Hz), 5.00±4.80 (m, 4H), 2.04±2.00 (18H); HR-Fab MS 1045.3322 (M+H). 19: 7.77 (d, 2H, J 7.6 Hz), 7.61 (brd, 2H, J 7.1 Hz), 7.43±7.30 (m, 4H), 5.95 (brs, 1H, NH), 5.85±5.50 (m, 3H), 5.21±5.15 (m, 2H), 5.00±4.88 (m, 4H), 4.22 (t, 1H, J 6.6 Hz), 3.80±3.60 (m, 2H), 3.39±3.33 (m, 2H), 2.38±2.17 (m, 4H), 2.06±2.01 (m, 18H); HR-Fab MS 1156.4221 (M+H). 20: HR-Fab MS 935.3427 (M+H). 23: Fab MS 1849.6 (M+H). 24: Fab MS 11061.2 (M+H). 25: Fab MS 1738.6 (M+H). 28: TOF MS 3591.46 (M+Na).