Multicyclic peptides and methods for their preparation
11192918 · 2021-12-07
Assignee
Inventors
- Gaston Julia Johannes Richelle (Amsterdam, NL)
- Dieuwertje Emma Streefkerk (Amsterdam, NL)
- Jan Herman van Maarseveen (Amsterdam, NL)
- Peter Timmerman (Amsterdam, NL)
Cpc classification
G01N2500/04
PHYSICS
C07K7/56
CHEMISTRY; METALLURGY
C07K7/64
CHEMISTRY; METALLURGY
C07K1/047
CHEMISTRY; METALLURGY
G01N33/54353
PHYSICS
C07K1/1077
CHEMISTRY; METALLURGY
International classification
C07K1/107
CHEMISTRY; METALLURGY
G01N33/543
PHYSICS
C07K7/56
CHEMISTRY; METALLURGY
C40B30/04
CHEMISTRY; METALLURGY
C07K7/64
CHEMISTRY; METALLURGY
Abstract
The invention relates to methods for preparing a compound comprising a peptide attached to a molecular scaffold whereby multiple peptide loops are formed, to compounds that can be obtained with such methods and uses thereof.
Claims
1. A method for preparing a compound comprising a peptide attached to a molecular scaffold, the method comprising: 1) performing a thiolate nucleophilic substitution reaction between a peptide and a molecular scaffold to form two or three thioether linkages between said peptide and said molecular scaffold; and 2) performing a subsequent reaction between said peptide and said molecular scaffold selected from the group consisting of an oxime-ligation reaction, an alkyne-azide cycloaddition, a thiol-ene reaction, a hydrazone ligation reaction, a Diels Alder reaction, a tetrazine ligation reaction, a disulfide bridge formation and a ring-closing metathesis reaction to form two or three further linkages between said peptide and said molecular scaffold, thereby forming three to six peptide loops resulting from the formation of two or three thioether linkages between said peptide and said molecular scaffold in step 1 and two or three further linkages between said peptide and said molecular scaffold in step 2; whereby: said peptide and said molecular scaffold comprise two or three reactive groups capable of participating in said thiolate nucleophilic substitution reaction and two or three reactive groups capable of participating in said reaction in step 2) prior to performing said reactions, and said molecular scaffold comprises an aromatic or heteroaromatic cyclic moiety, a 6-membered cycloalkyl or a 6-membered cycloalkylene and possesses twofold or threefold symmetry.
2. The method according to claim 1 wherein said reaction in step 2) is selected from the group consisting of an oxime-ligation reaction, an alkyne-azide cycloaddition and a thiol-ene reaction.
3. The method according to claim 1 wherein said molecular scaffold prior to performing said reactions in steps 1) and 2) comprises: two reactive groups capable of participating in said thiolate nucleophilic substitution reaction and two reactive groups capable of participating in said reaction in step 2), or three reactive groups capable of participating in said thiolate nucleophilic substitution reaction and three reactive groups capable of participating in said reaction in step 2).
4. The method according to claim 1 wherein said peptide, prior to performing said reactions in steps 1) and 2), comprises two or three thiol groups and said molecular scaffold, prior to performing said reactions in steps 1) and 2), comprises two or three halides attached to activated methylene groups.
5. The method according to claim 1 wherein said peptide is a linear peptide prior to performing said reactions in steps 1) and 2).
6. The method according to claim 1 further comprising introducing one or more linkages in said peptide.
7. The method according to claim 1 wherein said scaffold, prior to performing said reactions in steps 1) and 2), comprises a free rotatable bond located between a part of the scaffold that comprises two or three reactive groups capable of participating in said thiolate nucleophilic substitution reaction and a part of the scaffold that comprises said two or three reactive groups capable of participating in said reaction in step 2).
8. The method according to claim 1 wherein: said peptide and said molecular scaffold, prior to performing said reactions in steps 1) and 2), comprise two reactive groups capable of participating in said thiolate nucleophilic substitution reaction and two reactive groups capable of participating in said reaction in step 2), said molecular scaffold possesses C2 symmetry, and said molecular scaffold, prior to performing said reactions in steps 1) and 2), comprises a free rotatable bond located between a part of the scaffold that comprises said two reactive groups capable of participating in the thiolate nucleophilic substitution reaction and a part of the scaffold that comprises said two reactive groups capable of participating in the reaction in step 2).
9. A compound comprising a peptide attached to a molecular scaffold, wherein: i. said peptide is attached to said molecular scaffold by four to six linkages; ii. said molecular scaffold comprises an aromatic or heteroaromatic cyclic moiety or a 6-membered cycloalkyl or cycloalkylene and possesses twofold or threefold symmetry; iii. said compound comprises three to six peptide loops formed as a result of attachment of said peptide to said molecular scaffold by said four to six linkages; iv. two or three of said linkages are thioether linkages; and v. two or three of said linkages result from a reaction selected from the group consisting of an oxime-ligation reaction, an alkyne-azide cycloaddition, a thiol-ene reaction, a hydrazone ligation reaction, a Diels Alder reaction, a tetrazine ligation reaction, a disulfide bridge formation and a ring-closing metathesis reaction.
10. The compound according to claim 9 wherein said compound is essentially in one or two regioisomeric forms.
11. The compound according to claim 9 wherein said peptide comprises an intra-peptide linkage.
12. The compound according to claim 9 wherein a part of said molecular scaffold comprising said two or three thioether linkages and a part of said molecular scaffold comprising said two or three linkages resulting from a reaction selected from the group consisting of an oxime-ligation reaction, an alkyne-azide cycloaddition, a thiol ene reaction, a hydrazone ligation reaction, a Diels Alder reaction, a tetrazine ligation reaction, a disulfide bridge formation and a ring-closing metathesis reaction are separated by a singly bonded pair of atoms other than hydrogen atoms.
13. The compound according to claim 9, wherein said compound comprises a genetic package displaying said peptide and comprising a nucleic acid encoding said peptide.
14. A library comprising a plurality of compounds according to claim 9.
15. A method for identifying a compound capable of binding to a target of interest, comprising contacting a library of compounds according to claim 14 with the target of interest, determining binding of said compounds to said target and selecting a compound that binds to said target.
16. The method according to claim 15, wherein said target of interest is a receptor, a ligand, an antibody, a cytokine, or a hormone.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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EXAMPLES
List of Abbreviations
(31) TABLE-US-00001 Boc tert-Butyloxycarbonyl Fmoc- 9-Fluorenylmethyl N-succinimidyl OSu carbonate Cbz Carboxybenzyl HATU (1-[Bis(dimethylamino) methylene]-1H-1,2,3-triazolo[4,5- b]pyridinium 3-oxid hexafluorophosphate) CBZ- N-(Benzyloxycarbonyloxy) OSu succinimide CLiPS Chemical Linkage of Peptides onto Scaffolds CuAAC copper-catalyzed alkyne- IBX 2-Iodoxybenzoic acid azide cycloaddition MeCN Acetonitrile DBE 1,2-Dibromoethane NBS N-Bromo succinimide DBU 1,8-Diazabicycloundec-7- NMP N-Methyl-2-pyrrolidone ene DIAD Diisopropyl pAcF Para-acetyl Phenylalanine azodicarboxylate DIPEA N,N-Diisopropylethylamine P.E. Petroleum Ether 40-60 DMAP 4-Dimethylaminopyridine Pd/C Pd on activated carbon DMF N,N-Dimethylformamide Phth Phthalimide DMSO Dimethylsulfoxide THF Tetrahydrofuran ESI Electron Spray Ionization THP Tetrahydropyran EtOAc Ethyl acetate THPTA Tris- hydroxypropyltriazolylmethylamine Fmoc Fluorenylmethyloxycarbonyl TLC Thin Layer Chromatography TMS Trimethylsilyl
Example 1. Coupling of Peptide and Scaffold Via Thiolate Nucleophilic Substitution Reaction and Oxime Ligation
(32) Amino Acids
(33) Example of the Synthesis of a Phthalimide Protected Amino-Oxy Containing Amino Acid
(34) ##STR00006##
(35) In a flame dried flask, under N.sub.2 flow, Fmoc-Asp-OtBu (100 g, 2.41 mmol) was dissolved in 15 ml freshly distilled THF. After cooling the mixture on ice, BH.sub.3.SMe.sub.2 (484 μL, 5.10 mmol, 2.1 equiv) was dropwise added. The mixture was warmed to rt. and stirred overnight, after which TLC showed full conversion of the starting material. The reaction mixture was quenched with saturated NH.sub.4Cl solution and extracted with EtOAc. The collected organic phases were washed with brine and dried over Na.sub.2SO.sub.4. The volatiles were removed under reduced pressure, yielding a colorless oil. Flash column chromatography (2:2:0.5—CH.sub.2Cl.sub.2:P.E.:EtOAc) yielded the homoserine-derived product as a colorless oil (833 mg, 2.10 mmol, 86%). .sup.1H NMR (400 MHz, Chloroform-d) δ 7.79 (d, J=7.5 Hz, 2H), 7.62 (d, J=7.4 Hz, 2H), 7.43 (t, J=7.5 Hz, 2H), 7.35 (t, J=7.3 Hz, 2H), 5.64 (d, J=7.4 Hz, 1H), 4.55-4.36 (m, 3H), 4.25 (t, J=6.9 Hz, 1H), 3.77-3.67 (m, 1H), 3.62 (td, J=11.8, 11.2, 3.2 Hz, 1H), 3.03 (br. s, 1H), 2.19 (ddt, J=14.4, 9.9, 4.7 Hz, 1H), 1.64 (ddd, J=13.7, 7.9, 3.1 Hz, 1H), 1.50 (s, 9H). .sup.13C NMR (126 MHz, CDCl3) δ 171.69, 156.92, 143.79, 143.59, 141.29, 127.73, 127.07, 125.06, 125.00, 120.00, 119.97, 82.54, 67.14, 58.30, 51.52, 47.17, 36.05, 27.97.
(36) In a flame dried flask, under N.sub.2 flow, the purified Fmoc-protected homoserine (750 mg, 1.89 mmol) was dissolved in 11 ml anhydrous THF. The solution was cooled on an ice bath, and PPh.sub.3 (544 mg, 2.08 mmol, 1.1 equiv), N-hydroxyphthalimide (338 mg, 2.08 mmol, 1.1 equiv) were added. DIAD (408 μL, 2.08 mmol, 1.1 equiv) was added dropwise, and the mixture was warmed to rt. After stirring for 5 hours, the volatiles were removed under reduced pressure. The thick orange oil was immobilized on silica, after which flash column chromatography (3:2:0.5—P.E.:CH.sub.2Cl.sub.2:EtOAc) yielded the phthalimide-protected homoserine as an off-white solid (793 mg, 1.47 mmol, 78%). .sup.1H NMR (500 MHz, Chloroform-d) δ 7.88 (dd, J=5.0, 3.1 Hz, 3H), 7.83-7.72 (m, 4H), 7.70 (t, J=7.3 Hz, 2H), 7.40 (t, J=7.4 Hz, 2H), 7.36-7.27 (m, 2H), 6.33 (d, J=8.1 Hz, 1H), 4.55 (q, J=5.7 Hz, 1H), 4.49-4.39 (m, 2H), 4.35 (t, J=5.7 Hz, 2H), 4.28 (t, J=7.2 Hz, 1H), 2.36 (d, J=5.6 Hz, 2H), 1.51 (s, 9H).
(37) .sup.13C NMR (126 MHz, CDCl.sub.3) δ 171.70, 156.97, 143.76, 143.55, 141.29, 127.74, 127.07, 125.06, 124.98, 120.01, 119.98, 82.61, 67.15, 58.24, 51.39, 47.15, 36.17, 27.96.
(38) The phthalimide derivative (100 mg, 0.18 mmol) was dissolved in 700 μL freshly distilled CH.sub.2Cl.sub.2. HCOOH (1.4 mL) was added and the reaction mixture was stirred overnight at rt. The volatiles were removed under reduced pressure, to yield the tBu-deprotected Fmoc-homoserine derivative (84 mg, 0.17 mmol, 95%) as a colorless foam. .sup.1H NMR (400 MHz, Chloroform-d) δ 7.88 (dd, J=5.3, 3.1 Hz, 2H), 7.79 (dd, J=5.4, 3.1 Hz, 2H), 7.75 (d, J=7.5 Hz, 2H), 7.69 (t, J=6.6 Hz, 2H), 7.39 (t, J=7.5 Hz, 2H), 7.35-7.29 (m, 2H), 6.33 (d, J=8.3 Hz, 1H), 4.57-4.48 (m, 1H), 4.40 (dd, J=7.4, 2.9 Hz, 2H), 4.34 (t, J=6.0 Hz, 2H), 4.27 (t, J=7.2 Hz, 1H), 2.35 (q, J=5.9 Hz, 2H). MS (ESI) [M+H].sup.+ calc 486.48. found 486.7.
(39) Example of the Synthesis of a Boc-Protected Amino-Oxy Containing Amino Acid
(40) ##STR00007##
(41) In a flame-dried flask, under Ar-pressure, Fmoc-Asp(OtBu)-OH (43.106 g, 106.12 mmol, 1 equiv) was suspended in 400 ml of anhydrous MeOH. Cs.sub.2CO.sub.3 (17.288 g, 53.06 mmol, 0.5 equiv) was added and the mixture immediately becomes a colorless solution, which was subsequently stirred for 45 min. The volatiles were removed under reduced pressure, yielding a while solid. The residue is dissolved in 500 ml anhydrous MeCN and benzyl bromide (37.86 ml, 318.36 mmol, 3 equiv) was added. The mixture was stirred for 3 hours at rt. during which a white precipitate forms. The volatiles were removed and the remaining solid was washed with water and EtOH twice, yielding the desired Fmoc-Asp(OtBu)-OBn as a white solid, in quantitative yield. .sup.1H-NMR (400 MHz, Chloroform-d) δ 7.78 (d, J=7.5 Hz, 2H), 7.62 (d, J=6.9 Hz, 2H), 7.42 (t, J=7.4 Hz, 2H), 7.33 (m, J=15.3, 7.5 Hz, 7H), 5.93 (d, J=8.5 Hz, 1H), 5.23 (dd, J=33.0, 12.4 Hz, 1H), 4.70 (dt, J=8.4, 4.1 Hz, 1H), 4.51-4.40 (m, 1H), 4.40-4.30 (m, 1H), 4.26 (t, J=7.1 Hz, 1H), 2.92 (ddd, J=70.8, 17.0, 4.4 Hz, 2H), 1.45 (s, 9H). .sup.13C NMR (101 MHz, CDCl3) δ 170.70, 169.85, 155.89, 143.81, 143.59, 141.14, 135.15, 128.47, 128.31, 128.13, 127.60, 126.97, 125.08, 125.03, 119.87, 81.74, 67.34, 67.17, 50.55, 46.98, 37.62, 27.89.
(42) Fmoc-Asp(OtBu)-OBn (1.604 g, 3.197 mmol) was dissolved in 15 ml of freshly distilled CH.sub.2Cl.sub.2. 15 ml HCOOH is added to the solution and the mixture is stirred overnight at rt, after which TLC showed full conversion of the starting material. The volatiles were removed under reduced pressure and the remnants of HCOOH were removed by co-evaporation with CH.sub.2Cl.sub.2, yielding Fmoc-Asp(OH)—OBn as a white solid (1.310 g, 2.94 mmol, 92%). .sup.1H NMR (400 MHz, Chloroform-d) δ 11.15 (s, 1H), 7.80 (d, J=7.5 Hz, 2H), 7.66 (d, J=7.4 Hz, 2H), 7.44 (t, J=7.4 Hz, 2H), 7.36 (d, J=7.4 Hz, 7H), 6.16 (d, J=8.5 Hz, 1H), 5.26 (s, 2H), 4.83 (dt, J=8.6, 4.4 Hz, 1H), 4.50 (dd, J=10.4, 7.4 Hz, 1H), 4.46-4.38 (m, 1H), 4.26 (t, J=7.1 Hz, 1H), 3.18 (dd, J=17.4, 4.6 Hz, 1H), 3.01 (dd, J=17.4, 4.2 Hz, 1H).sup.13C NMR (101 MHz, CDCl.sub.3) δ 175.36, 170.34, 156.04, 143.53, 143.39, 141.02, 134.84, 128.34, 128.20, 127.94, 127.51, 126.87, 124.90, 119.76, 67.44, 67.21, 50.18, 46.79, 36.12.
(43) In a flame-dried flask, under N.sub.2 flow, Fmoc-Asp(OH)—OBn (11.11 g, 25 mmol) was dissolved in 175 ml of freshly distilled THF. The reaction mixture is cooled to 0° C., after which BH.sub.3.SMe.sub.2 (11.85 ml, 125 mmol, 5 equiv) is added dropwise over 1 hour. The mixture is stirred on ice for 2 h, and subsequently warmed to rt and stirred overnight, after which TLC showed full consumption of the starting material. The mixture was carefully quenched with sat. NH.sub.4Cl solution and extracted with EtOAc (3×). The organic phase was washed with 1M KHSO.sub.4, brine and dried over Na.sub.2SO.sub.4. The volatiles were removed under reduced pressure, after which Fmoc-homoSer-OBn crystallizes as a white solid (10.74 g, 24.86 mmol, 99%). .sup.1H NMR (400 MHz, Chloroform-d) δ 7.79 (d, J=7.4 Hz, 2H), 7.62 (d, J=7.3 Hz, 2H), 7.43 (t, J=7.4 Hz, 3H), 7.40-7.29 (m, 8H), 5.76 (d, J=7.7 Hz, 1H), 5.29-5.11 (m, 2H), 4.68-4.57 (m, 1H), 4.55-4.39 (m, 2H), 4.24 (t, J=6.6 Hz, 1H), 3.79-3.67 (m, 1H), 3.61 (t, J=9.8 Hz, 1H), 2.21 (ddt, J=14.4, 9.4, 4.4 Hz, 1H), 1.75 (ddd, J=14.1, 9.4, 4.4 Hz, 1H).
(44) In a flame-dried flask, under N.sub.2, Fmoc-homoSer-OBn (7.01 g, 16.21 mmol) was dissolved in 125 ml of anhydrous THF. Subsequently Boc.sub.2NOH (3.97 g, 17.02 mmol, 1.05 equiv) and PPh.sub.3 (4.46 g, 17.02 mmol, 1.05 equiv) were added, and the flask was cooled on an ice bath. DIAD (4.29 ml, 17.02 mmol, 1.05 equiv) was added dropwise via a syringe pump (4.4 ml/h). The mixture was warmed to rt, and stirred overnight. The volatiles were removed under reduced pressure, after which the mixture was immobilized in silica. Column chromatography (6:2:1—P.E.: CH.sub.2Cl.sub.2:EtOAc) provided the product as a white solid (7.514 g, 11.60 mmol, 72%). .sup.1H NMR (500 MHz, Chloroform-d) δ 7.77 (d, J=7.5 Hz, 2H), 7.67 (d, J=7.3 Hz, 2H), 7.40 (t, J=7.4 Hz, 2H), 7.37-7.33 (m, 2H), 7.33-7.24 (m, 5H), 6.63 (d, J=8.6 Hz, 1H), 5.20 (s, 2H), 4.65 (dt, J=10.3, 5.5 Hz, 1H), 4.42 (dd, J=10.2, 7.5 Hz, 1H), 4.38-4.29 (m, 1H), 4.24 (t, J=7.2 Hz, 1H), 4.16-4.07 (m, 1H), 4.03-3.93 (m, 1H), 2.29 (q, J=10.2, 6.0 Hz, 2H), 2.23 (dd, J=12.8, 8.1 Hz, 1H), 1.55 (s, 18H). .sup.13C NMR (126 MHz, CDCl.sub.3) δ 171.63, 156.42, 150.34, 144.08, 143.95, 141.28, 135.52, 128.54, 128.31, 128.17, 127.66, 127.65, 127.07, 127.06, 125.32, 125.31, 119.92, 84.41, 72.54, 67.18, 67.14, 52.04, 47.21, 29.64, 28.10. HR-MS FD m/z [M.sup.+] calcd for C.sub.36H.sub.42N.sub.2O.sub.9: 646.2890. found 646.2866.
(45) Fmoc-homoserine(ONBoc.sub.2)-Benzyl-ester (11.32 g, 17.51 mmol) was dissolved in 150 ml of CH.sub.2Cl.sub.2. TFA (2.14 ml, 27.94 mmol, 1.6 equiv). It was stirred overnight, after which NMR showed incomplete conversion. 0.9 ml (11.75 mmol, 0.67 equiv) of TFA was added and the reaction mixture was again stirred overnight. The volatiles were removed, after which the mono-Boc compound was purified via column chromatography (6:4:1—P.E.: CH.sub.2Cl.sub.2:EtOAc) yielding a white solid 5.17 g (9.22 mmol, 53%). .sup.1H NMR (500 MHz, Chloroform-d) δ 7.79 (d, J=7.6 Hz, 2H), 7.69 (d, J=7.5 Hz, 2H), 7.42 (t, J=7.5 Hz, 3H), 7.40-7.27 (m, 9H), 6.57 (s, 1H), 5.22 (s, 2H), 4.64 (q, J=6.5 Hz, 1H), 4.42 (tt, J=17.9, 8.9 Hz, 2H), 4.26 (t, J=7.4 Hz, 1H), 4.01 (ddd, J=11.4, 7.4, 4.3 Hz, 1H), 3.93 (dt, J=10.5, 5.3 Hz, 1H), 2.20 (tq, J=15.4, 9.8, 9.2 Hz, 2H), 1.52 (s, 9H). .sup.13C NMR (126 MHz, CDCl.sub.3) δ 171.96, 157.13, 156.32, 143.98, 143.79, 141.21, 135.33, 128.49, 128.30, 128.19, 127.59, 127.00, 125.21, 119.85, 82.05, 72.89, 67.16, 67.05, 51.81, 47.11, 29.92, 28.14. IR (cm.sup.−1) 3285, 3065, 2977, 2933, 1702, 1529, 1477, 1449, 1391, 1367, 1337, 1248, 1214, 1159, 1104, 1080, 1057, 1003, 909, 853, 757, 737. HR-MS FD m/z [M+] calcd for C.sub.31H.sub.34N.sub.2O7: 546.2336. found 546.2366. mp 43° C.
(46) In a flame-dried flask, Fmoc-homoserine(ONHBoc)-Benzyl-ester (5.17 g, 9.22 mmol) was dissolved in 200 ml EtOH. The flask was degassed and Pd/C (10 wt % loading, 256 mg) was added. The flask was evacuated and purged with 112 three times and the reaction mixture was stirred under H.sub.2 pressure (balloon) for 4 h at rt. TLC showed full conversion of the starting material and the reaction flask was purged with N.sub.2. The mixture was filtered over Celite and eluted with EtOH. The volatiles were evaporated under reduced pressure, yielding the desired Fmoc-homoserine(ONHBoc)-OH amino acid as a white solid (4.20 g, 9.21 mmol, 99%). .sup.1H NMR (400 MHz, Chloroform-d) δ 7.78 (d, J=7.5 Hz, 2H), 7.67 (d, J=7.3 Hz, 2H), 7.54 (s, 1H), 7.41 (t, J=7.4 Hz, 2H), 7.32 (t, J=7.4 Hz, 2H), 6.66 (s, 1H), 4.63 (q, J=6.1 Hz, 1H), 4.41 (p, J=10.3 Hz, 3H), 4.26 (t, J=7.2 Hz, 2H), 4.11-3.93 (m, 3H), 2.20 (d, J=4.5 Hz, 2H), 1.52 (s, 9H).
(47) Example of the Synthesis of a Ketone-Containing Amino Acid: Synthesis of Para-Acetyl Phenylalanine
(48) ##STR00008##
(49) H-Phe-OH (33.073 g, 200 mmol) was added to a flask equipped with a reflux condenser, and suspended in 170 ml EtOH. Ac.sub.2O (52 ml, 540 mmol, 2.7 equiv) was added and the solution was stirred at reflux overnight. The volatiles (acetic acid remnants) were removed under reduced pressure, yielding a yellowish sticky oil. This oil was redissolved in 170 ml of EtOH and concentrated HCl (4 ml, cat) was added. The mixture was heated to reflux and stirred overnight. The volatiles were removed under reduced pressure. The yellow oil was redissolved in EtOAc and washed with a 1M KHSO.sub.4 solution, sat. NaHCO.sub.3 solution and brine, and subsequently dried over Na.sub.2SO.sub.4. The volatiles were removed under reduced pressure, yielding Ac-Phe-OEt as an off-white solid (38.89 g, 165.31 mmol, 82%). .sup.1H NMR (500 MHz, Chloroform-d) δ 7.28 (dt, J=15.6, 7.0 Hz, 3H), 7.13 (d, J=6.9 Hz, 2H), 6.16 (d, J=7.2 Hz, 1H), 4.87 (q, J=6.0 Hz, 1H), 4.17 (q, J=7.1 Hz, 2H), 3.12 (tt, J=13.8, 7.0 Hz, 2H), 1.99 (s, 3H), 1.25 (t, J=7.1 Hz, 3H). .sup.13C NMR (126 MHz, CDCl3) δ 171.69, 169.61, 135.95, 129.26, 128.45, 127.00, 61.43, 53.16, 37.90, 23.04, 14.06. IR (cm.sup.−1) 3312, 3025, 3002, 2973, 2932, 2908, 1946, 1728, 1641, 1530, 1493, 1480, 1444, 1398, 1374, 1345, 1319, 1259, 1220, 1198, 1156, 1129, 1075, 1033, 1021, 966, 929, 909, 867, 824, 813, 764, 745. mp (° C.) 67.
(50) To a flame-dried flask, under N.sub.2 flow and at 0° C., AlCl.sub.3 (12.40 g, 93.00 mmol, 5.5 equiv) was added, followed by the dropwise addition of AcCl (7.2 ml, 101.27 mmol, 6.0 equiv). To the chunky suspension, a solution of Ac-Phe-OEt (4.00 g, 17.00 mmol, 1 equiv) in 18 ml of CH.sub.2Cl.sub.2 was added dropwise. The dark orange solution was stirred for 30 min on ice, then the mixture was warmed to rt and stirred overnight. The mixture was crashed onto ice with 10% 1M HCl solution. The product was extracted with CH.sub.2Cl.sub.2, and the organic phase was washed twice with a sat. NaHCO.sub.3 solution and water. After drying over Na.sub.2SO.sub.4, the volatiles were removed under reduced pressure to yield a dark brown oil. Flash column chromatography (1:2—P.E.:EtOAc) provided the acylated product as a yellowish oil, which crystallizes upon standing (4.26 g, 15.36 mmol, 90%). .sup.1H NMR (400 MHz, Chloroform-d) δ 7.84 (d, J=8.2 Hz, 2H), 7.19 (d, J=8.2 Hz, 2H), 6.17 (d, J=7.5 Hz, 1H), 4.86 (dd, J=19.7, 7.7 Hz, 1H), 4.14 (q, J=6.2 Hz, 2H), 3.19 (dd, J=13.8, 6.1 Hz, 1H), 3.10 (dd, J=13.8, 5.9 Hz, 1H), 2.54 (s, 3H), 1.95 (s, 3H), 1.21 (t, J=7.1 Hz, 3H). .sup.13C NMR (101 MHz, CDCl3) δ 197.50, 171.19, 169.48, 141.59, 135.78, 129.39, 128.32, 61.51, 52.79, 37.74, 26.38, 22.90, 13.94.
(51) To a flask, equipped with a reflux condenser, the acylated product (7.00 g, 25.24 mmol) was added. A 9M HCl solution was added (100 ml, excess) and the slight orange mixture was heated to 90° C. and stirred for 6 h. The mixture was cooled to rt, yielding a precipitate. This precipitate was filtered and washed with acetone and Et.sub.2O to yield the fully deprotected H-p-AcPhe-OH product as fine, slightly yellow needles (3.769 mg, 15.41 mmol, 61%). The remaining solution was evaporated to dryness, yielding a yellow solid, which was washed with acetone and Et.sub.2O. Filtration yielded the second batch of the product as a pale yellow solid (2.316 g, 9.47 mmol, 37%). .sup.1H NMR (500 MHz, Deuterium Oxide) δ 7.89 (d, J=8.1 Hz, 2H), 7.36 (d, J=8.1 Hz, 2H), 4.28-4.20 (m, 1H), 3.26 (ddd, J=58.6, 14.5, 6.7 Hz, 2H), 2.55 (s, 3H). .sup.13C NMR (126 MHz, D2O) δ 203.57, 171.59, 140.59, 135.99, 129.81, 129.29, 54.09, 35.75, 26.31.
(52) The free H-p-AcPhe-OH (1.00 g, 4.09 mmol) was dissolved in 11 ml of 1,4-dioxane, after which 15 ml of an aqueous saturated NaHCO.sub.3 solution was added, and the solution was subsequently cooled to 0° C. A solution of Fmoc-OSu (1.45 g, 4.29 mmol, 1.05 equiv) in 10 ml of acetone was added in a dropwise fashion. The flask was warmed to rt, and the solution was stirred overnight. The volatiles were removed under reduced pressure and the remaining solution was diluted with EtOAc. The organic phase was washed with an 1M KHSO.sub.4 solution (8 times) followed by brine. After drying over Na.sub.2SO.sub.4, the volatiles were vaporized under reduced pressure, yielding Fmoc-pAcF—OH as an off-white solid (1.72 g, 3.99 mmol, 97%). .sup.1H NMR (500 MHz, Methanol-d.sub.4) δ 7.89 (d, J=8.1 Hz, 2H), 7.78 (d, J=7.5 Hz, 2H), 7.57 (dd, J=11.1, 7.7 Hz, 2H), 7.38 (t, J=6.4 Hz, 4H), 7.28 (q, J=7.0 Hz, 2H), 7.20 (d, J=7.3 Hz, 1H), 4.50 (dd, J=9.6, 4.6 Hz, 1H), 4.30 (dd, J=10.5, 7.2 Hz, 1H), 4.22 (dd, J=10.4, 7.1 Hz, 1H), 4.12 (t, J=6.9 Hz, 1H), 3.31 (d, J=4.5 Hz, 1H), 3.03 (dd, J=13.7, 9.8 Hz, 1H), 2.52 (s, 3H). .sup.13C NMR (126 MHz, MeOD) δ 198.79, 173.70, 156.85, 143.75, 143.52, 141.09, 135.40, 129.28, 128.16, 127.31, 126.68, 124.83, 124.74, 119.44, 66.49, 55.01, 46.89, 37.15, 25.20. LC-MS (ESI), tr 7.46, ([M+H].sup.+ calc. 429.47. found 429.8).
(53) Peptide Synthesis
(54) Peptides were synthesized on solid phase using a 4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxy (RinkAmide) resin (Bachem, Germany), on a Prelude (Protein Technologies Inc., USA) synthesizer. All Fmoc-amino acids were purchased from Biosolve (Netherlands), Bachem (Germany) or Fluorochem Ltd. (UK), with side-chain functionalities protected according to the Fmoc-protocol ((N-tBoc (KW), OtBu (DESTY), N-Trt (HNQ), S-Trt (C) or N-Pbf (R) groups). Canonical amino acids were coupled with a 4-fold excess of HATU: amino acid: DIPEA (1:1:2) in NMP, with a 15 min activation time using double coupling. Unnatural amino acids were coupled with 2-fold excess of HATU: amino acid: DIPEA (1:1:2) in NMP, with a 60 min activation time using double coupling. Fmoc deprotection was performed using a 20% piperidine solution in NMP. Acylation of the N-terminus of the peptide was performed by reacting the resin with NMP:Ac.sub.2O:DIPEA (10:1:0.1 v/v/v) for 30 min at rt. The acylated peptide was cleaved from the resin cleavage and coincides with removal of protective groups. The cleavage cocktail (60 ml/mmol resin) which consist of 80 v % TFA, 7.5 wt % phenol, 5 v % thioanisole, 2.5 v % tri-isopropyl silane, 5 v % MilliQ water, and after 2 h stirring at rt, the resin was filtered off and the crude peptide is precipitated with ice-cold ether:pentane—1:1. The pellet is dissolved in MeCN:H.sub.2O (1:1) and lyophilized. Preparative HPLC is performed to further purify the peptide.
(55) Examples of Synthesized Peptides
(56) TABLE-US-00002 Sequence MW calc. MW found (ESI) Ac-CE (pAcF)A(pAcF)KC-NH.sub.2 973.15 972.5 Ac-CEK(pAcF)AS(pAcF)KDC-NH.sub.2 1303.49 1302.6 Ac-CES(pAcF)AK(pAcF)KAC-NH.sub.2 1259.48 1258.9 Ac-CERKF(pAcF)SGAV(pAcF)KLYSC-NH.sub.2 2010.68 1005.22 [M + 2H].sup.2+ Ac-(pAcF)ERKFCSGAVCKLYS(pAcF)-NH.sub.2 2010.68 1005.82 [M + 2H].sup.2+ Ac-CEQFhS(ONH.sub.2)AKFhS(ONH.sub.2)LKNC-NH.sub.2 1604.18 1604.21 Ac-CEWFhS(ONH.sub.2)SIKhS(ONH.sub.2)LKGC-NH.sub.2 1587.18 1588.60 Ac-CERKFhS(ONH.sub.2)SGAVhS(ONH.sub.2)KLYSC-NH.sub.2 1864.35 933.97 [M + 2H].sup.2+ Ac-hS(ONH.sub.2)EQFCAKFCLKNhS(ONH.sub.2)-NH.sub.2 1604.18 1604.35 Ac-hS(ONH.sub.2)ERKFCSGAVCKLYShS(ONH.sub.2)-NH.sub.2 1864.50 932.99 [M + 2H].sup.2+ Ac-CEQSK(Aoa)AKFK(Aoa)YKNC-NH.sub.2 1766.19 883.19 [M + 2H].sup.2+ Ac-CERKFK(Aoa)SGAVK(Aoa)KLYSC-NH.sub.2 2034.70 1017.81 [M + 2H].sup.2+ Ac-K(Aoa)EQFCAKFCLKNK(Aoa)-NH.sub.2 1774.38 887.64 [M + 2H].sup.2+ Ac-K(Aoa)ERKFCSGAVCKLYSK(Aoa)-NH.sub.2 2034.70 1017.36 [M + 2H].sup.2+
T4 Scaffolds
(57) ##STR00009##
(58) To a flame dried flask, under N.sub.2 flow, benzylamine (5 ml, 45.77 mmol) was added, followed by bromoacetaldehyde dietethyl acetal (16 ml, 106.36 mmol, 2.3 equiv) and NEt.sub.3 (18 ml, 129.05 mmol, 2.8 mmol). The yellowish mixture was stirred at 100° C. for 18 h. The mixture was diluted with EtOAc and washed with H.sub.2O and a saturated solution of NaHCO.sub.3 and brine, and subsequently dried over Na.sub.2SO.sub.4. The volatiles were removed under reduced pressure to yield an orange oil. Flash column chromatography (9:1—P.E.:EtOAc) yields the benzyl-protected amine as a pale yellow oil in 31% yield (6.08 g, 17.91 mmol). .sup.1H NMR (300 MHz, Chloroform-d) δ 7.30 (ddt, J=21.7, 13.9, 7.0 Hz, 5H), 4.58 (t, J=5.2 Hz, 2H), 3.81 (s, 2H), 3.65 (dq, J=9.2, 7.1 Hz, 4H), 3.51 (dq, J=9.3, 7.0 Hz, 4H), 2.76 (d, J=5.2 Hz, 4H), 1.20 (t, J=7.1 Hz, 12H). .sup.13C NMR (101 MHz, CDCl.sub.3) δ 139.75, 128.68, 127.86, 126.59, 102.25, 61.75, 59.97, 57.21, 15.17. IR (cm.sup.−1) 3027, 2973, 2928, 2876, 1602, 1494, 1452, 1372, 1345, 1267, 1114, 1056, 1023, 916, 849, 816, 739, 698. HR-MS (FD) 339.23924, (calc. 339.23828)
(59) The benzyl-protected amine (5.008 g, 14.75 mmol) was dissolved in 150 ml EtOH, and the solution was degassed and flushed with N.sub.2. Pd/C (10% loading, 256 mg) was added and hydrogen pressure was applied (H.sub.2 filled balloon) after evacuation/saturation (3×). The mixture was stirred for 4 h at rt, after TLC indicated full conversion of the starting material. The solution was filtered over a Na.sub.2SO.sub.4/Celite pad and eluted with EtOH. The volatiles were removed under reduced pressure, yielding the acetal protected amine as a pale yellow oil (3.480 g, 13.98 mmol, 94%). .sup.1H NMR (400 MHz, Chloroform-d) δ 4.44 (t, J=5.6 Hz, 2H), 3.60-3.50 (m, 4H), 3.44-3.34 (m, 4H), 2.59 (d, J=5.6 Hz, 4H), 1.06 (t, J=7.1 Hz, 12H). .sup.13C NMR (101 MHz, CDCl.sub.3) δ 101.70, 61.98, 51.62, 14.99. IR (cm.sup.−1) 2974, 2876, 1455, 1372, 1346, 1282, 1223, 1118, 1056, 1021, 944, 854, 813, 603, 504. HR-MS (FD) 249.19077 (calc. 249.19401)
(60) In a flame-dried flask, under N.sub.2 flow, 1,2,4,5-tetrakis(bromomethyl)benzene (2.50 g, 5.55 mmol, 3 equiv) was dissolved in 200 ml of freshly distilled MeCN and DIPEA (650 μL, 6.77 mmol, 1.2 equiv) was added. The secondary amine product (461 mg, 1.85 mmol, 1 equiv) was dissolved in 30 ml MeCN, and added dropwise to the durene solution. After stirring overnight, the reaction mixture was concentrated, and immobilized on silica, after which the scaffold was obtained via flash column chromatography (2:1—CH.sub.2Cl.sub.2: EtOAc to 15% MeOH in CH.sub.2Cl.sub.2), as a bright orange waxy solid (468 mg, 0.87 mmol, 47%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.58 (s, 2H), 5.06 (s, 4H), 4.87 (s, 4H), 3.80 (d, J=4.8 Hz, 4H), 3.66-3.53 (m, 4H), 3.44-3.36 (m, 4H), 3.16 (dt, J=7.4, 3.7 Hz, 2H), 1.32-1.22 (m, 12H). LC-MS (ESI) tr 6.76 min, [M].sup.+ calc. 538.34. found 538.2.
(61) ##STR00010##
(62) In a flame-dried flask, under N.sub.2 flow, tert-butyl 3,5-bis(bromomethyl)benzoate (500 mg, 1.37 mmol) was dissolved in 27 ml anhydrous DMF. KPhth (1015 mg, 5.48 mmol, 4 equiv) was added next, and the mixture was heated to 125° and stirred overnight. The suspension was cooled to rt and the mixture was evaporated to dryness. The mixture was dissolved in CH.sub.2Cl.sub.2 and washed with water, 1M KHSO.sub.4, saturated aqueous NaHCO.sub.3 and water. After drying over Na.sub.2SO.sub.4 the volatiles were removed under reduced pressure. The phthalimide remnants were removed via flash column chromatography (3:1—P.E:EtOAc to remove first spot, then increased to 4:1—EtOAc:P.E.), yielding the phthalimide product as a white solid (533 mg, 1.07 mmol, 78%). .sup.1H NMR (300 MHz, Chloroform-d) δ 7.90 (d, J=1.4 Hz, 2H), 7.85 (dd, J=5.4, 3.1 Hz, 4H), 7.72 (dd, J=5.4, 3.1 Hz, 4H), 7.64 (s, 1H), 4.87 (s, 4H), 1.56 (s, 9H). .sup.13C NMR (75 MHz, CDCl.sub.3) δ 167.88, 164.97, 137.03, 134.06, 133.01, 132.31, 132.02, 128.57, 123.46, 81.35, 41.10, 28.11. IR (cm.sup.−1) 2975, 2938, 1769, 1706, 1607, 1554, 1536, 1466, 1427, 1391, 1367, 1342, 1310, 1257, 1231, 1155, 1122, 1099, 1086, 973, 957, 918, 896, 845, 794, 774, 726, 710, 695. HR-MS FD m/z [M.sup.+] calcd for C.sub.29H.sub.24N.sub.2O.sub.6: 496.1634. found 496.1634. mp 212° C.
(63) The phthalimide compound (468 mg, 0.94 mmol) was dissolved in 5 ml freshly distilled CH.sub.2Cl.sub.2 and HCOOH was added (10 ml, excess). The mixture was stirred overnight at rt, during which a precipitate forms. The solids were filtered off, and dried. The liberated acid was obtained as an off-white powder (398 mg, 0.91 mmol, 96%), which was used without further purification. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 13.10 (s, 1H), 7.88 (q, J=4.4 Hz, 8H), 7.76 (s, 2H), 7.53 (s, 1H), 4.82 (s, 4H). .sup.13C NMR (126 MHz, CDCl.sub.3) δ 167.61, 166.70, 137.65, 134.61, 131.43, 130.93, 127.27, 123.26, 40.44. IR (cm.sup.−1) 3064, 1771, 1704, 1604, 1466, 1428, 1393, 1359, 1343, 1311, 1261, 1240, 1190, 1170, 1112, 1102, 1086, 1071, 980, 960, 914, 885, 834, 792, 773, 746, 725, 710. mp 228° C. (sublimates), 351° C. melts. HRMS FD m/z [M.sup.+] calcd for C.sub.25H.sub.16N.sub.2O.sub.6: 440.1008. found 440.1004.
(64) In a flame-dried flask, under N.sub.2 flow, the Phth-acid (1000 mg, 2.27 mmol), was suspended in 15 ml of anhydrous DMF. HATU (949 mg, 2.497 mmol, 1.1 equiv) and DIPEA (1 ml, 5.74 mmol, 2.5 equiv) were added, yielding a clear solution. The mixture was stirred for 30 min, after which the acetal protected amine (594 mg, 2.38 mmol, 1.05 equiv) was added. The mixture was stirred overnight, after which it was diluted with EtOAc and washed with H.sub.2O and brine. After drying over Na.sub.2SO.sub.4, filtration and evaporation of the volatiles, the phthalimide-amide is obtained as a pale brown solid, which is used without further purification. (1540 mg, 2.27 mmol, quant). .sup.1H NMR (500 MHz, Chloroform-d) δ 7.82 (dd, J=5.3, 3.1 Hz, 4H), 7.70 (dd, J=5.4, 3.0 Hz, 4H), 7.54 (s, 1H), 7.33 (s, 2H), 4.82 (s, 4H), 4.77 (t, J=4.8 Hz, 1H), 4.33 (t, J=4.7 Hz, 1H), 3.80-3.68 (m, 2H), 3.63 (d, J=4.9 Hz, 2H), 3.57 (dt, J=15.5, 6.7 Hz, 3H), 3.44 (dt, J=14.8, 7.0 Hz, 3H), 3.38 (d, J=4.8 Hz, 2H), 3.25 (p, J=7.1 Hz, 2H), 1.20 (t, J=6.6 Hz, 8H), 1.02 (t, J=6.8 Hz, 6H). .sup.13C NMR (126 MHz, CDCl.sub.3) δ 171.76, 167.76, 137.71, 137.21, 134.03, 132.05, 129.72, 126.25, 123.39, 101.49, 100.98, 63.56, 63.26, 52.70, 49.26, 41.12, 15.42, 15.20. IR cm.sup.−1 2974, 2929, 2877, 1765, 1702, 1635, 1605, 1468, 1445, 1421, 1394, 1348, 1329, 1313, 1260, 1233, 1173, 1119, 1103, 1054, 1016, 958, 960, 938, 914, 886, 846, 799, 733, 712 HR-MS FD m/z [M.sup.+] calcd for C.sub.37H.sub.41N.sub.3O.sub.9: 671.2843. found 671.2842 mp 161-164° C.
(65) The phthalimide amide (1530 mg, 2.27 mmol) was suspended in 20 ml of Toluene/EtOH (1:2). Hydrazine hydrate (51% solution in water, 1.42 ml, 22.77 mmol, 10 equiv) was added and the mixture was stirred at reflux for 2 hours, during which a thick precipitate has formed. The mixture was cooled to rt after which the solids were filtered off, and washed with CH.sub.2Cl.sub.2. The volatiles were removed under reduced pressure, yielding the crude diamine in quantitative yield, which was used without further purification. .sup.1H NMR (400 MHz, DMSO-d6) δ 7.33 (s, 1H), 7.14 (s, 2H), 5.76 (s, 2H), 4.73 (s, 1H), 4.54 (s, 1H), 3.73 (s, 4H), 3.71-3.63 (m, 2H), 3.62-3.55 (m, 3H), 3.55-3.40 (m, 5H), 3.35 (d, J=12.5 Hz, 4H), 1.16 (t, J=6.3 Hz, 8H), 1.08-0.95 (m, 6H).
(66) The crude diamine (contains water, 1190 mg, est. 2.27 equiv) was dissolved in 55 ml CH.sub.2Cl.sub.2 with 5 ml EtOH added. Then NaHCO.sub.3(1334 mg, 15.89 mmol, 7 equiv) and Bromoacetic acid N-hydroxysuccinimide ester (1768 mg, 7.49 mmol, 3.3 equiv) were added and the mixture was stirred for 1 hour, after which TLC analysis showed full conversion. The reaction mixture was diluted with water and extracted with CH.sub.2Cl.sub.2. The organic phase was subsequently washed with water, brine and a sat. solution of NaHCO.sub.3. After drying over Na.sub.2SO.sub.4, filtration and evaporation of the volatiles under reduced pressure, the crude product was obtained, which was purified by column chromatography (7:1—EtOAc:P.E.), yielding the desired scaffold as a fluffy white solid (950 mg, 1.54 mmol, 64% overall). .sup.1H NMR (500 MHz, Chloroform-d) δ 7.20 (t, J=5.9 Hz, 2H), 7.18 (s, 2H), 7.13 (s, 1H), 4.79 (t, J=4.9 Hz, 1H), 4.46 (t, J=4.8 Hz, 1H), 4.36 (d, J=5.9 Hz, 4H), 3.88 (s, 4H), 3.75 (p, J=7.2 Hz, 2H), 3.66 (d, J=4.9 Hz, 2H), 3.58 (dq, J=14.1, 6.9 Hz, 5H), 3.43 (d, J=4.8 Hz, 2H), 3.37 (dt, J=15.5, 7.2 Hz, 2H), 1.22 (t, J=6.9 Hz, 6H), 1.13 (t, J=6.9 Hz, 6H). .sup.13C NMR (126 MHz, CDCl.sub.3) δ 172.11, 165.89, 138.56, 137.35, 127.32, 125.09, 101.14, 100.80, 63.33, 52.68, 48.78, 43.40, 28.86, 15.28. IR (cm.sup.−1) 3262, 3067, 2971, 2873, 1678, 1665, 1599, 1480, 1424, 1373, 1341, 1297, 1266, 1248, 1227, 1209, 1177, 1120, 1063, 1027, 933, 905, 858, 830, 799, 763, 723, 708. HR-MS FD m/z [M.sup.+] calcd for C.sub.25H.sub.39Br.sub.2N.sub.3O.sub.7: 651.1155. found 651.1136. mp 74° C.
(67) ##STR00011##
(68) The synthesis of Bis-Boc hydroxylamine. To a flame-dried flask, under N.sub.2 flow, N-Benzylhydroxylamine hydrochloride (3.794 g, 23.77 mmol) was suspended in 30 ml of freshly distilled MeCN. NEt.sub.3 (3.65 ml, 26.15 mmol, 1.1 equiv) was added and the mixture was stirred for 2 h at rt. The solids were filtered off, and washed with 30 ml of MeCN. The remaining liquid was cooled to 0° C. and a solution of Boc.sub.2O (5.931 g, 27.19 mmol, 1.14 equiv) in 30 ml of MeCN was added dropwise. The solution was subsequently warmed to rt and stirred overnight. A second portion of Boc.sub.2O (8.300 g, 38.03 mmol, 1.6 equiv) in 30 ml of MeCN was added, together with DMAP (290 mg, 2.38 mmol, 0.1 equiv). The solution was warmed to 40° C. and stirred for 6 h, after which it was cooled to rt. The volatiles were removed under reduced pressure, yielding a white waxy solid. The residue was dissolved in EtOAc and washed with a 1M sodium phosphate buffer (pH 7), followed by brine and the organic phase was dried over Na.sub.2SO.sub.4. The volatiles were removed via rotary evaporation, yielding the N,N-bis-Boc protected N-Benzylhydroxylamine as a colorless oil, which solidified to a waxy solid upon standing (7.424 g, 22.96 mmol, 96%).
(69) All of the previously obtained product was dissolved in 35 ml of EtOH. The solvent was deoxygenated and flushed with N.sub.2. Pd/C (5 wt % loading, 436 mg, 5 mol %) was added and 112 pressure was added via a balloon. After three cycles of evacuation/saturation, the mixture was stirred at rt for 16 h, when TLC showed no starting material. The reaction mixture was filtered over a Celite pad and was eluted with EtOH. The volatiles were removed via rotary evaporation, and the resulting oil was redissolved in EtOAc. It was washed with a 1M NaOH solution trice, after which the aqueous phase was carefully neutralized to pH 7 with a 1M KHSO.sub.4 solution. Then, the product was extracted with EtOAc, and the collected organic phases were washed with brine and dried over Na.sub.2SO.sub.4. The volatiles were removed under reduced pressure, yielding a sticky oil, which crystallized upon standing, yielding bis-Boc hydroxylamine as a white crystalline solid (4.586 g, 19.66 mmol, 85%). Analytical data are in accordance with those reported in literature (Jayasekara, P. S.; Jacobson. 2014).
(70) To a flask, diethanolamine (4.8 ml, 50 mmol) was added and dissolved in 170 ml of dioxane and 100 ml of sat. NaHCO.sub.3 solution (aq). Then, a solution of Cbz-OSu (13.09 g, 52.5 mmol, 1.05 equiv) in 125 ml of acetone was added to the mixture in a dropwise fashion. The mixture was stirred overnight at rt, after which the volatiles were removed under reduced pressure. The resulting slurry was redissolved in EtOAc and washed alternatingly with water and a 1M KHSO.sub.4 solution (aq) (6×) and brine. After drying over Na.sub.2SO.sub.4, the volatiles were removed under reduced pressure, yielding the Cbz-protected diethanolamine product colorless oil (10.065 g, 42.06 mmol, 84%). .sup.1H NMR (400 MHz, Chloroform-d) δ 7.43-7.31 (m, 5H), 5.15 (s, 2H), 3.85 (s, 3H), 3.78 (s, 2H), 3.50 (p, J=4.6 Hz, 4H). .sup.13C NMR (75 MHz, CDCl.sub.3) δ 156.24, 135.93, 128.04, 127.59, 127.30, 66.81, 60.82, 60.43, 51.78, 51.20. IR (cm.sup.−1) 3368, 3064, 3032, 3942, 2879, 1671, 1496, 1473, 1455, 1415, 1363, 1262, 1217, 1130, 1046, 989, 906, 858, 768, 734, 696.
(71) In a flame-dried flask, under N.sub.2 flow, Cbz-protected diethanolamine (10.0 g, 41.79 mmol) was dissolved in 230 ml of freshly distilled THF. PPh.sub.3 (23.02 g, 87.76 mmol, 2.1 equiv) and Boc.sub.2N—OH (20.47 g, 87.76 mmol, 2.1 equiv) were added, and the solution was cooled to 0° C. DIAD (17.3 ml, 87.76 mmol, 2.1 equiv) was added dropwise via a syringe pump (5 ml/h). The mixture was warmed to rt and stirred overnight. The volatiles were removed under reduced pressure, providing a yellow oil. Flash column chromatography (3:2:0.5—P.E.:CH.sub.2Cl.sub.2:EtOAc) yielded the protected amino-oxy compound as a colorless oil (21.39 g, 31.94 mmol, 76%). .sup.1H NMR (400 MHz, Chloroform-d) δ 7.39-7.30 (m, 5H), 5.14 (s, 2H), 4.10 (t, J=5.0 Hz, 2H), 4.04 (t, J=5.5 Hz, 2H), 3.67 (q, J=5.2 Hz, 4H), 1.53 (s, 18H), 1.51 (s, 18H). .sup.13C NMR (101 MHz, CDCl3) δ 155.73, 149.88, 136.43, 128.49, 128.03, 127.93, 83.72, 83.68, 75.00, 74.90, 67.21, 47.26, 46.68, 28.01. IR (cm.sup.−1) 2979, 2936, 1792, 1751, 1703, 1475, 1457, 1412, 1393, 1368, 1344, 1271, 1247, 1140, 1109, 1092, 1038, 1004, 912, 890, 848, 794, 768, 751, 735, 697.
(72) The protected amino-oxy compound (840 mg, 1.2 mmol, 1 equiv) was dissolved in 20 ml of CH.sub.2Cl.sub.2. TFA (368 μL, 4.8 mmol, 4 equiv) was added dropwise, after which the mixture was stirred for 16 h at rt. TLC and NMR analysis showed the mono-deprotection. The volatiles were removed under reduced pressure. To remove the Cbz-group, the oily residue was redissolved in 25 ml of EtOH. The solution was evacuated, and purged with N.sub.2. Pd/C (10 wt %, 75 mg) was added. H.sub.2 pressure was applied via a balloon. The solution was thrice evacuated and saturated with H.sub.2. The solution was stirred for 3 h at rt, after which it was filtered over a Celite pad. After elution with EtOH the volatiles were removed under reduced pressure, yielding an opaque oil. Further purification via flash column chromatography (EtOAc:EtOH—5:1) yielded the deprotected amine 1 as a very sticky foam (363 mg, 1.08 mmol, 90%). .sup.1H NMR (400 MHz, Chloroform-d) δ 4.26-4.13 (m, 4H), 3.40-3.18 (m, 4H), 1.50 (s, 18H). .sup.13C NMR (126 MHz, CDCl.sub.3) δ 158.30, 82.98, 71.54, 45.93, 28.17. IR (cm.sup.−1) 3198, 2981, 2938, 1672, 1446, 1395, 1369, 1287, 1253, 1201, 1161, 1112, 1051, 1011, 925, 837, 799, 774, 721.
(73) ##STR00012##
(74) In a flame-dried flask, under N.sub.2-flow, 1,2,4,5-tetrakis(bromomethyl)benzene (1.350 g, 3 mmol, 3 equiv) was dissolved in 175 ml of freshly distilled MeCN. DIPEA (348 μL, 2 mmol, 2 equiv) was added and the mixture was stirred until all solids had dissolved. A solution of the deprotected amine 1 (335 mg, 1 mmol, 1 equiv) in 20 ml of MeCN was added to the durene solution in dropwise fashion over the course of an hour, and the mixture was stirred overnight. Full consumption of the starting material was shown via LC-MS analysis. Flash column chromatography (EtOAc, then 5:1 up to 2:1 EtOAc:EtOH) yielded T4N-1 as a yellow foam (400 mg, 0.64 mmol, 64%). .sup.1H NMR (400 MHz, CH.sub.3CN+D.sub.2O) δ 7.47 (s, 2H), 5.05 (s, 4H), 4.74 (s, 4H), 4.18 (s, 4H), 3.95-3.88 (m, 411), 1.43 (s, 18H). .sup.13C NMR (101 MHz, CH.sub.3CN+D.sub.2O) δ 157.78, 138.90, 134.88, 126.62, 82.51, 70.58, 69.38, 60.80, 30.16, 28.10. LC-MS [M].sup.+ calc. 624.39. found 624.4.
(75) ##STR00013##
(76) In a flame-dried flask, under N.sub.2 flow, 3,5-dimethyl benzoic acid (2.00 g, 13.31 mmol) was suspended in 1.6 ml of toluene. Thionyl chloride (2 ml, 27.6 mmol, 2.06 equiv) was added and the mixture was warmed to a gentle reflux and stirred for 4 hours. The volatiles were removed under reduced pressure, after which the oily residue was diluted with 4 ml of freshly distilled CH.sub.2Cl.sub.2. t-BuOH (2.05 ml, 21.31 mmol, 1.6 equiv) was added followed by pyridine (1.13 ml, 13.98 mmol, 1.05 equiv). The mixture was stirred for 12 hours, after which the solids are removed by filtration and washed with CH.sub.2Cl.sub.2. The organic phase was washed with 4M HCl, water, 2M NaOH and water. After drying over K.sub.2CO.sub.3, the volatiles are removed, yielding the OtBu-ester as a colorless oil (2.48 g, 12.03 mmol, 90%). .sup.1H NMR (400 MHz, Chloroform-d) δ 7.63 (s, 2H), 7.17 (s, 1H), 2.38 (s, 6H), 1.62 (s, 9H). .sup.13C NMR (101 MHz, CDCl.sub.3) δ 166.12, 137.75, 133.99, 127.09, 80.70, 28.18, 21.13.
(77) In a flame-dried flask, under N.sub.2 flow, the OtBu-ester (13.95 g, 67.64 mol) was dissolved in freshly distilled CH.sub.2Cl.sub.2 (250 ml). NBS (25.28 g, 142.04 mmol, 2.1 equiv) was added and the mixture was degassed and flushed with N.sub.2. The flask was irradiated with a commercially available halogen construction lamp (500 W), heating the mixture to a gentle reflux. The mixture was stirred for 1.5 hours, after which .sup.1H-NMR showed the reaction completed*. The mixture was diluted with CH.sub.2Cl.sub.2 and washed with water. After drying over Na.sub.2SO.sub.4, the volatiles were removed under reduced pressure, yielding a colorless oil. The mixture was crystallized from hexane, providing the brominated product as a white solid (7.56 g, 20.77 mmol, 31%). A second crystallization yielded another 1.71 g (4.70 mmol, 7%). *The mixture contains both doubly-brominated product (<15%), as well as <15% incompletely brominated starting material. .sup.1H NMR (400 MHz, Chloroform-d) δ 7.94 (s, 2H), 7.60 (s, 1H), 4.51 (s, 4H), 1.61 (s, .sup.13C NMR (101 MHz, CDCl.sub.3) δ 164.31, 138.54, 133.28, 133.10, 129.69, 81.56, 31.94, 28.01. IR (cm.sup.−1) 3013, 2982, 2969, 2932, 1790, 1714, 1604, 1472, 1449, 1390, 1369, 1319, 1236, 1213, 1154, 1110, 1060, 999, 973, 953, 918, 893, 846, 794, 771, 753, 734, 692. mp 52° C.
(78) The brominated product (5.00 g, 13.73 mmol) was dissolved in freshly distilled CH.sub.2Cl.sub.2 (50 ml). HCOOH was added and the solution was stirred overnight at rt, after which .sup.1H-NMR showed completion of the reaction. The volatiles were removed under reduced pressure and co-evaporation with CH.sub.2Cl.sub.2 (3×) yielded the acid product as a white solid (3.98 g, 12.92 mmol, 94%) which was used without further purification. .sup.1H NMR (400 MHz, Chloroform-d) δ 8.10 (s, 2H), 7.71 (s, 1H), 4.55 (s, 4H). .sup.13C NMR (101 MHz, CDCl.sub.3) δ 170.03, 139.08, 134.64, 130.45, 130.31, 31.53. IR (cm.sup.−1) 2971, 2821, 2710, 2604, 2539, 1686, 1603, 1460, 1437, 1420, 1308, 1278, 1248, 1211, 1162, 1111, 1056, 998, 938, 927, 904, 855, 771, 728, 691, 662. mp 123° C. (sublimates), 142° C. (melts).
(79) To a flame-dried flask, under N.sub.2 flow, the acid product (2.00 g, 6.49 mmol) was added and suspended in SOCl.sub.2 (9 ml, excess). The mixture was warmed to reflux and the orange solution was stirred for 4 hours. Then, the temperature was lowered to 50° C., and the mixture was stirred overnight. Remnants of SOCl.sub.2 were removed under reduced pressure, and were co-evaporated with toluene twice. The resulting orange oil was dissolved in 50 ml of freshly distilled CH.sub.2Cl.sub.2 and DMAP (50 mg, 6 mol %) was added. A solution of amine 1 (1.94 g, 5.78 mmol, 0.9 equiv) and DIPEA (1.13 ml, 6.49 mmol, 1 equiv) in 10 ml of CH.sub.2Cl.sub.2 was added dropwise to the acid chloride solution. After 2 h, the reaction shows complete conversion. The mixture is transferred to a separator funnel and washed with a 1M KHSO.sub.4 solution (aq) and brine, end dried over Na.sub.2SO.sub.4. The volatiles were removed under reduced pressure, providing an orange oil. Flash column chromatography (3:1-2:1-4:3-pentane:EtOAc) yields the product as a yellowish sticky solid. Via .sup.1H-NMR, .sup.13C-NMR and LC-MS, it was determined that the product contains not 2 bromides, but halogen exchange had taken place, and some fraction of the product contained Cl instead of Br. Based on NMR data, this accounts for 30% of the mass. The follow-up chemistry is not subject to adverse effects. Therefore the molecular weight of the compound is now 612.02 instead of 625.36, yielding the T4N-2 scaffold in 21% yield (763 mg, 1.24 mmol). .sup.1H NMR (400 MHz, Chloroform-d) δ 8.37 (s, 1H), 7.95 (s, 1H), 7.38 (s, 1H), 7.34 (s, 1H), 4.50 (s, 1H), 4.39 (s, 3H), 4.01 (s, 2H), 3.87 (s, 2H), 3.79 (s, 2H), 3.49 (s, 2H), 1.39 (d, J=7.0 Hz, 18H). .sup.13C NMR (75 MHz, CDCl.sub.3) δ 171.60, 156.97, 156.71, 138.90, 138.57, 137.23, 137.18, 130.45, 129.98, 127.42, 126.90, 81.68, 81.38, 74.31, 73.53, 48.09, 45.10, 43.97, 32.06, 28.23. (underlined are the peaks belonging to the Chloride substituent). LC-MS shows 2 peaks, 1) tr 8.11, (minor) where 1×Cl, 1×Br, [M+H].sup.+ calc. 581.91. found 581.91, as well as m/z 604.2 (Na.sup.+), m/z 482.0 (—Boc+H.sup.+) and m/z 382.2 (−2Boc+2H.sup.+). 2) tr 8.18 (major) where 2× Br, [M+H].sup.+ calc. 626.38. found 625.8, as well as m/z 648.1 (Na.sup.+), m/z 525.9 (—Boc+H.sup.+) and m/z 426.1 (−2Boc+2H.sup.+).
(80) ##STR00014##
(81) In a flame-dried flask, under N.sub.2 flow, tert-butyl 3,5-bis(bromomethyl)benzoate (500 mg, 1.37 mmol) was dissolved in 27 ml anhydrous DMF. KPhth (1015 mg, 5.48 mmol, 4 equiv) was added next, and the mixture was heated to 125° and stirred overnight. The suspension was cooled to rt and the mixture was evaporated to dryness. The mixture was dissolved in CH.sub.2Cl.sub.2 and washed with water, 1M KHSO.sub.4, saturated aqueous NaHCO.sub.3 and water. After drying over Na.sub.2SO.sub.4 the volatiles were removed under reduced pressure. The phthalimide remnants were removed via flash column chromatography (3:1—P.E:EtOAc to remove first spot, then increased to 4:1—EtOAc:P.E.), yielding the phthalimide product as a white solid (533 mg, 1.07 mmol, 78%). .sup.1H NMR (300 MHz, Chloroform-d) δ 7.90 (d, J=1.4 Hz, 2H), 7.85 (dd, J=5.4, 3.1 Hz, 4H), 7.72 (dd, J=5.4, 3.1 Hz, 4H), 7.64 (s, 1H), 4.87 (s, 4H), 1.56 (s, 9H). .sup.13C NMR (75 MHz, CDCl.sub.3) δ 167.88, 164.97, 137.03, 134.06, 133.01, 132.31, 132.02, 128.57, 123.46, 81.35, 41.10, 28.11. IR (cm.sup.−1) 2975, 2938, 1769, 1706, 1607, 1554, 1536, 1466, 1427, 1391, 1367, 1342, 1310, 1257, 1231, 1155, 1122, 1099, 1086, 973, 957, 918, 896, 845, 794, 774, 726, 710, 695. mp 212° C.
(82) The phthalimide compound (468 mg, 0.94 mmol) was dissolved in 5 ml freshly distilled CH.sub.2Cl.sub.2 and HCOOH was added (10 ml, excess). The mixture was stirred overnight at rt, during which a precipitate forms. The solids were filtered off, and dried. The liberated acid was obtained as an off-white powder (398 mg, 0.91 mmol, 96%), which was used without further purification. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 13.10 (s, 1H), 7.88 (q, J=4.4 Hz, 8H), 7.76 (s, 2H), 7.53 (s, 1H), 4.82 (s, 4H). .sup.13C NMR (126 MHz, CDCl.sub.3) δ 167.61, 166.70, 137.65, 134.61, 131.43, 130.93, 127.27, 123.26, 40.44.
(83) The crude acid (298 mg, 0.68 mmol) was suspended in 3 ml of DMF. HATU (283 mg, 0.74 mmol, 1.1 equiv) was added, followed by DIPEA (235 μL, 1.35 mmol, 2 equiv). The mixture was stirred for 15 min, after which amine 1 (283 mg, 0.74 mmol, 1.1 equiv) was dissolved in 4 ml DMF, and added dropwise to the reaction mixture. After 4 h, full conversion is observed via LC-MS analysis. The mixture was concentrated under reduced pressure, and the residue was dissolved in EtOAc and washed with water (5×) followed by brine. After drying over Na.sub.2SO.sub.4, the volatiles were removed under reduced pressure, and the product was purified via flash column chromatography (1:1—P.E.:EtOAc) yielding the amide as a white foam (375 mg, 0.50 mmol, 73%). .sup.1H NMR (300 MHz, Chloroform-d) δ 8.42 (s, 1H), 8.08 (s, 1H), 7.68 (dd, J=5.3, 3.1 Hz, 4H), 7.58 (dd, J=5.4, 3.1 Hz, 4H), 7.43 (s, 1H), 7.28 (s, 2H), 4.71 (s, 4H), 3.97 (s, 2H), 3.85 (s, 2H), 3.75 (s, 2H), 3.42 (s, 2H), 1.33 (s, 18H). .sup.13C NMR (75 MHz, CDCl.sub.3) δ 171.87, 167.75, 156.88, 156.63, 137.24, 137.06, 134.10, 131.77, 129.64, 126.22, 123.33, 81.39, 81.07, 74.27, 73.41, 47.96, 43.87, 40.98, 28.17. IR (cm.sup.−1) 3266, 2976, 2934, 1770, 1707, 1624, 1468, 1426, 1391, 1366, 1344, 1248, 1161, 1108, 1050, 1012, 955, 728, 711. mp 78° C.
(84) The amide compound (1866 mg, 2.36 mmol) was added to a flask, and dissolved in EtOH:Toluene—2:1 (31 ml). Hydrazine hydrate (50% solution in water, 1.6 ml, 23.6 mml, 10 equiv) was added and the mixture was heated to reflux, where a solid starts to precipitate after 30 min. The mixture was stirred at reflux overnight, after which the yellow suspension was cooled to rt. The solids were filtered off and washed with CH.sub.2Cl.sub.2 (3×). The volatiles were removed under reduced pressure, yielding the bis-amine as a white solid (1084 mg, 2.18 mmol, 92%), which was used in the next reaction step without further purification. .sup.1H NMR (500 MHz, Chloroform-d) δ 8.51 (s, 1H), 8.21 (s, 1H), 7.33 (s, 2H), 7.31 (s, 1H), 4.11 (s, 2H), 4.00 (s, 2H), 3.92 (s, 4H), 3.87 (s, 2H), 3.59 (s, 2H), 1.49 (s, 18H). .sup.13C NMR (126 MHz, CDCl.sub.3) δ 173.28, 157.34, 156.60, 143.71, 136.76, 127.11, 124.05, 81.65, 81.48, 74.01, 73.35, 48.03, 45.97, 43.39, 38.62, 28.26.
(85) The bis-amine (688 mg, 1.38 mmol) was dissolved in 36 ml of freshly distilled CH.sub.2Cl.sub.2, and 7 ml of EtOH was added to dissolve the amine fully. NaHCO.sub.3(s) (381 mg, 4.53 mmol, 3.3 equiv) was added. Bromoacetic acid N-hydroxy succinimide ester (1071 mg, 4.54 mmol, 3.3 equiv) was added and the reaction mixture was stirred at rt for 30 min, after which LC-MS analysis showed full conversion. The volatiles were removed under reduced pressure, after which the residue was dissolved in CH.sub.2Cl.sub.2, and washed with 1M KHSO.sub.4, water, brine and dried over Na.sub.2SO.sub.4, and the solvent was again removed. Flash column chromatography (7:1—EtOAc:P.E.) yielded the T4N-3 scaffold as a white foam (635 ngm 0.86 mmol, 62%). .sup.1H NMR (500 MHz, Chloroform-d) δ 8.30 (d, J=15.0 Hz, 2H), 7.62 (t, J=5.9 Hz, 2H), 7.21 (s, 2H), 7.16 (s, 1H), 4.32 (d, J=5.9 Hz, 4H), 4.02 (s, 2H), 3.92 (s, 2H), 3.84 (s, 4H), 3.77 (s, 2H), 3.46 (s, 2H), 1.44 (s, 18H). .sup.13C NMR (126 MHz, CDCl.sub.3, rt) δ 172.47, 166.58, 157.14, 156.76, 138.92, 136.62, 128.38, 127.39, 125.56, 124.58, 81.80, 81.66, 73.71, 73.61, 73.20, 72.99, 48.22, 44.09, 43.72, 43.35, 42.97, 29.64, 29.50, 28.86, 28.74, 28.23, 27.85, 26.96.
(86) T6 Scaffolds
(87) ##STR00015##
(88) Benzene-1,3,5-triyltrimethanol (400 mg, 2.38 mmol) was suspended in 8 ml of tBuOH, and IBX (4.00 g, 14.27 mmol, 6 equiv) was added. The mixture was heated to reflux and stirred for 5 h. The suspension was cooled to rt and the solids were filtered off, and subsequently washed with CH.sub.2Cl.sub.2. The clear liquid was evaporated to dryness, yielding the tri-aldehyde as white powder (385 mg, 2.38 mmol, quant). .sup.1H NMR (500 MHz, Chloroform-d) δ 10.21 (s, 3H), 8.64 (s, 3H). .sup.13C NMR (126 MHz, CDCl3) δ 189.78, 137.80, 134.76.
(89) In a flame-dried flask, under N.sub.2, the tri-aldehyde (162 mg, 1.00 mmol) was dissolved in 6 ml of 1:1 CHCl.sub.3:MeOH. Then 2,2-diethoxyethanamine (653 μL, 4.5 mmol, 4.5 equiv) was added in a dropwise fashion. The mixture was stirred for 1 h at rt. after which the volatiles were removed under reduced pressure to yield the imine as an orange oil (499 mg, 0.98 mmol, 98%). The residue (458 mg, 0.90 mmol) was dissolved in 4 ml of MeOH and cooled to 0° C. NaBH.sub.4 (204 mg, 5.42 mmol, 6 equiv) was added and the mixture was stirred for 1 hour, after which TLC showed full completion. The reaction mixture was quenched with sat. NH.sub.4Cl solution and extracted with CH.sub.2Cl.sub.2. The combined organic phases were washed with sat. NaHCO.sub.3 solution and brine, and subsequently dried over Na.sub.2SO.sub.4. The volatiles were removed under reduced pressure to yield the tri-amine as a pale yellow oil (457 mg, 0.88 mmol, 98%), which was used as the crude, without further purification. .sup.1H NMR .sup.1H NMR (500 MHz, Chloroform-d) δ 7.12 (s, 3H), 4.58 (t, J=5.5 Hz, 3H), 3.74 (s, 6H), 3.63 (dt, J=14.2, 7.0 Hz, 6H), 3.53-3.42 (m, 6H), 2.70 (d, J=5.5 Hz, 6H), 1.15 (t, J=7.1 Hz, 15H). .sup.13C NMR .sup.13C NMR (126 MHz, CDCl.sub.3) 140.22, 126.42, 101.91, 62.10, 53.58, 51.46, 15.18.
(90) The tri-amine (457 mg, 0.89 mmol, 1 equiv) was dissolved in 7.5 ml of CH.sub.2Cl.sub.2, after which 13 ml of a sat. NaHCO.sub.3 solution was added. The mixture was cooled to 0° C. and a solution of bromoacetyl bromide (227 μL, 2.70 mmol, 4.5 equiv) in 6 ml of CH.sub.2Cl.sub.2 was added in a dropwise fashion. The biphasic solution was warmed to rt and stirred for 2 h, after which TLC showed full conversion. The product was extracted with CH.sub.2Cl.sub.2 and the organic phase was washed with sat. NaHCO.sub.3 solution and dried over Na.sub.2SO.sub.4. The volatiles were removed under reduced pressure, yielding an orange oil. Flash column chromatography (1:1—P.E.:EtOAc) provided the T6C-2 scaffold as a pale orange oil (318 mg, 0.36 mmol, 41%). .sup.1H NMR (500 MHz, Chloroform-d) δ 7.06-6.81 (m, 3H), 4.69 (d, J=6.3 Hz, 3H), 4.60 (d, J=8.2 Hz, 4H), 4.44 (dt, J=18.1, 4.5 Hz, 2H), 4.07 (t. J=11.0 Hz, 4H), 3.78 (s, 2H), 3.75-3.62 (m, 6H), 3.59-3.48 (m, 2H), 3.44 (dt, J=15.3, 7.4 Hz, 4H), 3.36 (s, 6H), 1.20-1.12 (m, 18H). .sup.13C NMR (126 MHz, CDCl3) δ 168.38, 168.29, 168.20, 167.41, 167.38, 138.89, 138.42, 138.31, 138.05, 137.89, 137.56, 126.16, 126.01, 124.34, 123.57, 100.83, 100.79, 100.73, 64.11, 64.03, 63.85, 63.81, 53.12, 53.09, 51.01, 50.96, 49.74, 49.65, 49.55, 49.38, 49.31, 49.21, 27.03, 26.74, 26.46, 26.30, 26.14, 25.92, 15.34, 15.25.
(91) ##STR00016##
(92) To a flame-dried flask, under Ar pressure (balloon), iodobenzene (28 ml, 250 mmol) was added, followed by TFA (100 ml) and CHCl.sub.3 (240 ml). Oxone (116 g, 377 mmol, 1.5 equiv) was added under vigorous stirring. Five minutes after the addition of all the oxone, the mixture was put on ice for 30 min, due to heat evolution. The ice bath was removed and the mixture was stirred for 2 days at rt. The solids were suspended in CHCl.sub.3 and filtered. The collected filtrate was evaporated to dryness under reduced pressure, yielding the bis-trifluoroacetoxy iodobenzene as an off-white solid (89.3 g, 208 mmol, 83%). .sup.1H NMR (300 MHz, Chloroform-d) δ 8.20 (d, J=7.8 Hz, 2H), 7.74 (t, J=7.4 Hz, 1H), 7.62 (t, J=7.8 Hz, 2H).
(93) In a flame-dried flask, under N.sub.2 flow, iodine (102 g, 40 mmol, 6 equiv) was dissolved in 120 ml of CCl.sub.4. Mesitylene was added, followed by bis-trifluoroacetoxy-iodobenzene (50 g, 116.3 mmol, 1.7 equiv). The bright purple solution was stirred overnight, during which a white-ish cake was formed on the side. The solids were filtered off, resulting in a yellowish solid, containing I.sub.2 crystals. The cake was washed with acetone until the cake no longer gives off an orange color. The tris-iodinated product was isolated as an off-white solid (25.74 g, 51.70 mmol, 76%). .sup.1H NMR (400 MHz, Chloroform-d) δ 3.01 (s, 9H).
(94) A solution was made of Ac.sub.2O (470 ml), AcOH (235 ml) and H.sub.2SO.sub.4 (47 ml), to which 1,3,5-triiodo-2,4,6-trimethylbenzene was added (25 g, 50.2 mmol), yielding a milky pink suspension. KMnO.sub.4 (31.82 g, 301.3 mmol, 4.1 equiv) was added in small portions over 3 hours, due to the heat evolution after every step. The yellow suspension was stirred over the weekend. The solution was concentrated, after which water was added. The aqueous phase was extracted with CH.sub.2Cl.sub.2 (6×), and the collected organic phases were washed with brine, and dried over Na.sub.2SO.sub.4. After concentration, the product was precipitated from acetone. The precipitate was filtered, washed with acetone and dried under reduced pressure, yielding the tri-acetate compound as a fine, off-white powder (14.78 g, 22.0 mmol, 44%). .sup.1H NMR (400 MHz, Chloroform-d) δ 5.72 (s, 6H), 2.13 (s, 9H).
(95) Synthesis of tert-butyl (prop-2-yn-1-yloxy)carbamate: To a flame-dried flask, under N.sub.2 flow, propargyl bromide (80% wt. solution in toluene, 10 ml, 89.76 mmol), was dissolved in 266 ml of freshly distilled MeCN and the solution was cooled to 0° C. Tert-butyl hydroxycarbamate (13.75 g, 116.7 mmol, 1.15 equiv) was added, followed by DBU (17.45 ml, 116.7 mmol, 1.3 equiv). The mixture was stirred for 30 min at 0° C., after which the mixture was warmed to rt and stirred for another hour. The volatiles were removed under reduced pressure and the residue was suspended in CH.sub.2Cl.sub.2. A saturated solution of NaHCO.sub.3 was added and the organic phase was washed twice, followed by brine. After drying over Na.sub.2SO.sub.4, the residue was concentrated. Flash column purification (10:1—P.E.:EtOAc) yielded the acetylene-product as a colorless oil (1.28 g, 59.65 mmol, 66%). .sup.1H NMR (400 MHz, Chloroform-d) δ 7.36 (s, 1H), 4.50 (d, J=2.3 Hz, 2H), 2.52 (t, J=2.3 Hz, 1H), 1.51 (s, 9H).
(96) To a flame-dried high pressure tube, under Ar flow, the tri-acetoxy compound (100 mg, 0.149 mmol), PdCl.sub.2(PPh.sub.3).sub.2 (7.0 mg, 8.9 μmol 6 mol %) and Cud (1.3 mg, 6.6 μmol, 4.5 mol %) were added and suspended in 1.2 ml of NEt.sub.3. To the yellow suspension, tert-butyl (prop-2-yn-1-yloxy)carbamate (112 mg, 0.54 mmol, 3.6 equiv) was added. The Lube was sealed and heated to 50° C. and the mixture was stirred overnight. The resulting brown suspension was filtered over Celite and eluted with CH.sub.2Cl.sub.2. The volatiles were removed under reduced pressure. Flash column purification (2:1 to 1:1—P.E.:EtOAc) yielded the tri-alkyne product as a yellowish foam (75 mg, 0.094 mmol, 62%). .sup.1H NMR (500 MHz, Chloroform-d) δ 7.99 (s, 3H), 5.50 (s, 6H), 4.74 (s, 6H), 2.12 (s, 9H), 1.51 (s, 27H). .sup.13C NMR (126 MHz, CDCl3) δ 171.25, 156.43, 139.06, 126.23, 95.14, 81.95, 80.92, 64.14, 63.18, 28.15, 20.76.
(97) In a flame-dried flask under N.sub.2 flow, the tri alkyne (2.19 g, 2.73 mmol, 1 equiv) was dissolved in 100 ml EtOH. The solution was degassed and purged with N.sub.2. NEt.sub.3 was added (5% v/v, 5.2 ml,) and the solution was degassed and purged with N.sub.2. Pd/C (10% wt. loading, 500 mg, 20 mol %) was added and the solution was degassed. H.sub.2 pressure was applied (balloon) and the reaction vessel was degassed and purged with H.sub.2 (3×) to saturate the flask with H.sub.2. The mixture was stirred overnight under H.sub.2 pressure, after which TLC analysis and .sup.1H-NMR showed full consumption of the starting material. The reaction mixture was filtered over Celite and eluted with CH.sub.2Cl.sub.2. The volatiles were removed under reduced pressure. Flash column chromatography (2:1 to 1:1—P.E.:EtOAc) yielded tri-alkene the product as a yellow foam (1.86 g, 2.298 mmol, 84%). .sup.1H NMR (400 MHz, Chloroform-d) δ 7.76 (d, J=13.0 Hz, 3H), 6.59 (dt, J=33.3, 14.4 Hz, 3H), 6.05 (dt, J=11.8, 6.6 Hz, 3H), 4.90 (d, J=56.2 Hz, 6H), 4.02 (s, 6H), 1.93 (s, 9H), 1.35 (s, 27H). .sup.13C NMR (101 MHz, CDCl.sub.3) δ 170.34, 156.65, 139.58, 139.49 (broad peak), 131.16, 130.79, 129.59, 129.38, 81.34, 72.54, 62.19, 27.94, 20.50.
(98) The tri-alkene product (163 mg, 0.201 mmol) was dissolved in 9 ml EtOH. K.sub.2CO.sub.3 (5 mg, cat.) was added and the opaque solution was stirred overnight. The volatiles were removed under reduced pressure. The residue was dissolved in CH.sub.2Cl.sub.2 and washed with brine. The aqueous phase was twice more extracted with CH.sub.2Cl.sub.2 and dried over Na.sub.2SO.sub.4. The volatiles were removed under reduced pressure, yielding the alkene alcohol a pale yellow foam. .sup.1H NMR (300 MHz, Chloroform-d) δ 7.71 (s, 2H), 7.03-6.86 (m, 3H), 6.30-6.08 (m, 3H), 4.54 (s, 6H), 4.25 (d, J=8.4 Hz, 6H), 2.80 (t, J=6.5 Hz, 2H), 1.45 (s, 27H).
(99) The alkene alcohol residue was dissolved in 7 ml EtOH. The solution was degassed and purged with N.sub.2. Pd/C (10 wt % loading, 32 mg, 10%) was added and the solution was degassed. H.sub.2 pressure was applied (balloon) and the reaction vessel was degassed and purged with H.sub.2 (3×) to saturate the flask with H.sub.2. The mixture was stirred overnight under H.sub.2 pressure, after which TLC analysis and .sup.1H-NMR showed full conversion of the starting material. The mixture was filtered over Celite and eluted with EtOH. The volatiles were removed under reduced pressure, yielding the desired alkane product as a colorless foam (131 mg, 0.190 mmol, 94%). .sup.1H NMR (500 MHz, Chloroform-d) δ 8.14 (s, 3H), 4.65 (s, 6H), 3.92 (s, 6H), 3.49 (br s, 3H), 3.02-2.85 (m, 6H), 1.82 (s, 6H), 1.50 (s, 27H). .sup.13C NMR (126 MHz, CDCl.sub.3) δ 157.23, 141.79, 135.33, 81.51, 76.29, 58.40, 30.76, 28.29, 26.08. (IR cm.sup.−1) 3433 (br), 3263 (br), 2973, 2926, 1710, 1477, 1455, 1392, 1366, 1276, 1250, 1162, 1110, 1003, 754. mp (° C.) 68
(100) In a flame-dried flask, under Ar flow, the alkane product (50 mg, 72.6 μmol) was dissolved in 2.5 ml freshly distilled CH.sub.2Cl.sub.2 and cooled to 0° C. Pyridine (26 μl, 0.33 mmol, 4.5 equiv) was added, followed by dropwise addition of PBr.sub.3 (24 μL, 0.25 mmol, 3.5 equiv). After 1 h on ice, the solution was warmed to rt. and becomes opaque. LC-MS analysis showed full conversion to the desired product. The reaction mixture was diluted with EtOAc and quenched with NaHCO.sub.3. After neutralization with KHSO.sub.4, the product was extracted with EtOAc (3×). The collected organic phases were washed with brine and dried over Na.sub.2SO.sub.4. After filtration, the volatiles were removed under reduced pressure, yielding T6N-1 as a colorless foam (20 mg, 22.8 μmot 31%). .sup.1H NMR (500 MHz, Chloroform-d) δ 7.22 (s, 3H), 4.60 (s, 6H), 4.03 (t, J=5.8 Hz, 6H), 3.01 (t, J=9.5, 8.2 Hz, 9H), 1.99 (d, J=12.9 Hz, 6H), 1.51 (s, 27H). .sup.13C NMR (126 MHz, CDCl.sub.3) δ 157.08, 142.83, 133.61, 81.90, 76.26, 29.60, 28.75, 28.25, 26.48. IR (cm.sup.−1) 3275, 2925, 2854, 1712, 1476, 1454, 1366, 1248, 1161, 1107, 773, 517. mp (° C.) 56-57
(101) ##STR00017##
(102) To a flame-dried flask, under N.sub.2, mesitylene (0.7 ml, 5 mmol) was added, followed by acetic acid (2.6 ml) and HBr in AcOH (33% wt solution) (3.5 ml). Then para-formaldehyde (570 mg, 18.8 mmol, 3.7 equiv) was added. The solution was stirred for at 95° C. After 3 h solids started to develop. The mixture was stirred for another 9 h, after which het mixture was cooled to rt. After crashing the mixture onto ice, the solids were filtered and dried. The solid was recrystallized from CH.sub.2Cl.sub.2: P.E. to yield the desired bromomethylated product as white needles (1.99 g, 5 mmol, 99%). .sup.1H NMR (500 MHz, Chloroform-d) δ 4.61 (s, 6H), 2.50 (s, 9H). .sup.13C NMR (126 MHz, CDCl.sub.3) δ 137.84, 133.18, 29.93, 15.38.
(103) In a flame-dried flask, under N.sub.2, tris(bromomethyl)mesitylene (5.012 g, 12.53 mmol) was suspended in 200 ml of anhydrous DMF. Potassium Phthalimide (KPhth, 14.00 g, 75.58 mmol, 6 equiv) was added and the slurry was vigorously stirred for 18 h at 125° C. The white solid was filtered off, and washed twice with DMF, water and acetone. The white amorphous solid was dried on high vacuum, yielding the phthalimide derivative in 78% (5.846 g, 9.78 mmol). .sup.1H NMR (400 MHz, Chloroform-d) δ 7.80 (dd, J=5.4, 3.0 Hz, 6H), 7.70 (dd, J=5.4, 3.1 Hz, 6H), 4.97 (s, 6H), 2.52 (s, 9H).
(104) In a flame-dried flask, under N.sub.2 flow, the phthalimide derivative (2.50 g, 4.18 mmol) was suspended in 30 ml of DBE. Br.sub.2 (711 μL, 13.80 mmol, 33 equiv) was added and the mixture was heated to reflux, while irradiated with a commercially available 500 W halogen lamp. After two hours, the solution had become light orange, after which .sup.1H-NMR showed the reaction had not reached full conversion. A second portion of Br.sub.2 (400 μL, 7.76 mmol, 1.86 equiv) was added and reflux and irradiation was continued for two more hours, after which .sup.1H-NMR showed full conversion. The reaction mixture was quenched with a 0.5M Na.sub.2S.sub.2O.sub.3 solution, and extracted with CH.sub.2Cl.sub.2. A sticky yellow solid was obtained after removal of the volatiles under reduced pressure. Flash column chromatography (3:1 to 1:1—P.E.:EtOAc) provided the brominated product as a white solid (3.09 g, 3.71, 88%). Analytical data is in concurrence with those reported in literature (Roelens et al. 2009).
(105) In a flame-dried flask, under N.sub.2 flow, the brominated compound (2.50 g, 3 mmol), was suspended in 42 ml of freshly distilled MeCN, and the mixture was cooled to 0° C. N-Boc hydroxylamine (1.80 g, 13.5 mmol, 4.5 equiv), was added after which DBU (1.80 ml, 12 mmol, 4 equiv) was added dropwise. The suspension turned to a clear yellowish solution after 10 min. The mixture was warmed to rt and stirred overnight. The volatiles were removed under reduced pressure. The residue was dissolved in CH.sub.2Cl.sub.2 and washed with a sat. NaHCO.sub.3 solution and brine followed by drying over Na.sub.2SO.sub.4. After filtration and removal of the volatiles under reduced pressure, the crude product was obtained as a bright yellow foam. Flash column purification (2.5:1—P.E.:EtOAc) yielded the amino-oxy functionalized product as a bright yellow solid (1.43 g, 1.44 mmol, 48%). .sup.1H NMR (500 MHz, Chloroform-d) δ 7.80 (dd, J=5.4, 3.0 Hz, 6H), 7.70 (dd, J=5.4, 3.1 Hz, 6H), 7.26 (s, 3H), 5.45 (s, 6H), 5.39 (s, 6H), 1.42 (s, 27H). .sup.13C NMR (126 MHz, CDCl.sub.3) δ 168.25, 156.16, 139.09, 135.69, 133.95, 131.95, 123.20, 81.60, 72.47, 36.81, 28.15.
(106) The amino-oxy functionalized product (200 mg, 0.202 mmol) was suspended in 3.5 ml of EtOH:toluene (2:1), after which hydrazine-hydrate (50% sol. in water, μL, mmol, equiv) was added. The suspension was heated to reflux. When the mixture has reached 70° C., it becomes a colorless solution. After stirring for 20 minutes at reflux, a white solid starts to precipitate. Reflux was continued for 12 hours, after which the solids were filtered off and the residue was evaporated to dryness, yielding the tris-amine as an off-white solid (118 mg, 0.198 mmol, 98%), which was used without further purification in the next step. .sup.1H NMR (500 MHz, Chloroform-d) δ 5.16 (s, 6H), 4.12 (s, 6H), 1.53 (s, 27H). .sup.13C NMR (126 MHz, CDCl.sub.3) δ 157.25, 145.71, 131.96, 81.51, 72.04, 39.78, 28.33.
(107) The tris-amine was suspended in 7 ml CHCl.sub.3 and the suspension was cooled to 0° C. 7 ml of a sat. solution NaHCO.sub.3 is added. Bromoacetyl bromide (70 μL, 0.786 mmol, 4 equiv) was added dropwise and the solution was warmed to rt and stirred for 3 hours. LC-MS confirmed the absence of s.m. and the product was extracted with CH.sub.2Cl.sub.2. The organic phase was washed with brine and dried over Na.sub.2SO.sub.4 and the volatiles were removed under reduced pressure. The product was purified via flash column chromatography (1:1—P.E.:EtOAc), yielding the scaffold T6N-2 as a colorless solid (120 mg, 0.125 mmol, 63%). .sup.1H NMR (500 MHz, Chloroform-d) δ 8.81 (s, 3H), 8.13 (s, 3H), 5.31 (s, 6H), 4.79 (d, J=6.2 Hz, 6H), 3.85 (s, 6H), 1.47 (s, 27H). .sup.13C NMR (126 MHz, CDCl3) δ 166.73, 157.56, 141.69, 133.99, 82.53, 71.98, 37.72, 28.98, 28.24. MS (EST) calc. 964.5. found 964.8.
(108) Peptide Cyclization Experiments
(109) For Ketone in the Peptide, Amino-Oxy in the Scaffold
(110) CLiPS Reaction
(111) For a typical CLIPS experiment, the peptide is dissolved in a 3:2 DMF:MilliQ solution (max. 0.25 mM). Then the scaffold is added (0.9 equiv), as a solution in MeCN or MeCN:H.sub.2O (depending on the scaffold). The resulting peptide solution is adjusted to pH 7.8-8, by addition of an NH.sub.4HCO.sub.3 solution in water. The progress of the reaction is monitored via reversed phase LC-MS or UPLC-MS. Once the reaction is complete (typically 15-30 min), deprotection of the amino-oxy protecting group can commence.
(112) Liberation of the Amino-Oxy Grout) and Oxime Ligation
(113) To the CLiPS reaction mixture, an equal volume of 6M HCl (aq) is added, to initiate Boc-deprotection. The reaction mixture is stirred typically for 1 hour, to ensure full liberation of all amino-oxy moieties, which can be confirmed via reversed phase LC-MS or UPLC-MS. It is important to note that oxime ligation already occurs during these conditions, but full conversion to the twice oximed peptide construct is not reached. The pH of the reaction mixture is adjusted to approximately pH ˜4, using a 5.5M NaOH (aq) solution (typically 95 vol % of the added HCl solution). The reaction mixture is then left for 1 to 16 hours, until full conversion is reached, as judged by reversed phase LC-MS or UPLC-MS.
(114) Examples of Peptide Cyclization Reactions
Example A: T4N-3 Cyclization with a Small Peptide
(115) Peptide Ac—CE(pAcF)A(pAcF)KC—NH.sub.2 (1.10 mg, 1.13 μmol) was dissolved in 1 ml 3:2 DMF:H.sub.2O (1.13 mM). Scaffold T4N-3 (6.10 mg in 500 μl MeCN stock, used 65 μl, 0.8 equiv) was added followed by 50 μl of a 1M NH.sub.4HCO.sub.3 solution, to reach pH 8.5.
(116) The reaction mixture is acidified using 1 ml of 6M HCl solution, for Boc-deprotection. The reaction mixture is stirred at rt for 1 h, after which the pH is corrected to pH4 using 810 μl of a 5.5M NaOH (aq) solution. The reaction mixture was stirred overnight at rt.
Example B: T4N-3 Cyclization with a Larger Peptide
(117) Peptide Ac—CEK(pAcF)AS(pAcF)KDC—NH.sub.2 (1.30 mg, 0.99 μInca) was dissolved in 1 ml 3:2 DMF:H.sub.2O (1.13 mM). Scaffold T4N-3 (6.10 mg in 500 μl MeCN stock, used 57 μl, 0.7 equiv) was added followed by 50 μl of a 1M NH.sub.4HCO.sub.3 solution, to reach pH 8.5.
(118) The reaction mixture is acidified using 1 ml of 6M HCl solution, for Boc-deprotection. The reaction mixture is stirred at rt for 1 h, after which the pH is corrected to pH4 using 790 μl of a 5.5M NaOH (aq) solution. The reaction mixture was stirred overnight at rt.
(119) General Procedure for T4C-3 Cyclization Experiments with a Peptide Containing an Amino-Oxy Residue:
(120) The peptide is dissolved in 1:1 DMSO:H.sub.2O to a final concentration of 50 mM, then scaffold is added (0.63 equiv, from stock solution). The reaction mixture is basified to pH >8 by adding 1M NH.sub.4HCO.sub.3. General scale is 0.3 mg of peptide, which calls for 30 μL of 1M NH.sub.4HCO.sub.3-solution. After UPLC shows the CLIPS reaction has reached full conversion (generally within 30 min.), the reaction is acidified using 15% TFA solution. An equal volume to the base is added (generally 30 uL). The solution is left overnight, yielding the oxime product in quantitative yield.
Example C: T4C-3 Cyclization with Medium-Sized Peptide
(121) Peptide Ac—CEQF hS(ONH.sub.2)AKF hS(ONH.sub.2)LKNC—NH.sub.2 (0.22 mg) is dissolved in 274 μL DMSO:H.sub.2O (1:1). Scaffold T4C-3 is added (2.31 mg in 200 μL MeCN, 4.27 μL was added). Then 30 μL of a 1M solution NH.sub.4HCO.sub.3 is added, after which CLIPS takes place within 20 min.
Example D: T4C-3 Cyclization with Medium-Sized Peptide
(122) Peptide Ac—CERKFK(Aoa)SGAVK(Aoa)KLYSC—NH.sub.2 (0.26 mg) is dissolved in 254 μL DMSO:H.sub.2O (1:1). Scaffold T4C-3 is added (0.93 mg in 100 μL MeCN, 5.66 μL was added). Then 30 μL of a 1M solution NH.sub.4HCO.sub.3 is added, after which CLIPS takes place within 20 min.
Example E: T4C-3 Cyclization with Medium-Sized Peptide—Aminooxy at Termini
(123) Peptide Ac—K(Aoa)EQFCAKFCLKNK(Aoa)-NH.sub.2 (0.41 mg) is dissolved in 460 μL DMSO:H.sub.2O (1:1). Scaffold T4C-3 is added (0.78 mg in 100 μL MeCN, 12.19 μL was added). Then 40 μL of a 1M solution NH.sub.4HCO.sub.3 is added, after which CLIPS takes place within 20 min.
Example E: T4C-3 Cyclization with Medium-Sized Peptide—Aminooxy at Termini
(124) Peptide Ac—K(Aoa)ERKFCSGAVCKLYSK(Aoa)-NH.sub.2 (0.23 mg) is dissolved in 226 μL DMSO:H.sub.2O (1:1). Scaffold T4C-3 is added (0.93 mg in 100 μL MeCN, 5.00 μL was added). Then 30 μL of a 1M solution NH.sub.4HCO.sub.3 is added, after which CLIPS takes place within 20 min.
Example 2. Coupling of Peptide and Scaffold Via Thiolate Nucleophilic Substitution Reaction and Alkyne-Azide Cycloaddition
(125) 1. Synthesis Routes for T4(−≡).sub.2-Scaffolds
(126) The synthesis routes for different T4(−≡).sub.2-scaffolds is shown in scheme 1.
(127) ##STR00018## ##STR00019## ##STR00020##
2. Experimental Procedures and Spectroscopic Data of Compounds
General Section
(128) Unless stated otherwise, reactions were performed without special precautions like drying or N.sub.2/Argon atmosphere. Dried CH.sub.2Cl.sub.2 and CH.sub.3CN were obtained by distilling these solvents with CaH.sub.2 as drying agent. Dried THF was obtained by distillation with sodium. All dried solvents were stored under N.sub.2 atmosphere. Dry DMF and DMSO on 4 Å molecular sieves were obtained from Sigma-Aldrich and stored under N.sub.2 atmosphere. Reagents were purchased with the highest purity (usually >98%) from Sigma Aldrich and Fluorochem and used as received. Reactions were monitored with thin layer chromatography (TLC) carried out on 0.25 mm E. Merck silica gel plates (60E-254). SilaFlash® P60 (particle size 40-63 μm) was used for silica column chromatography. NMR spectra were recorded on Bruker DRX-300, 400 and 500 MHz instruments and calibrated on residual undeuterated solvent signals as internal standard. The .sup.1H-NMR multiplicities were abbreviated as followed: s=singlet, d=doublet, t=triplet, q=quartet, quint=quintet, m=multiplet. High resolution mass spectra (HRMS) were recorded on an AccuTOF GC v 4 g, JMS-T100GCV Mass spectrometer (JEOL, Japan). FD/FI probe equipped with FD Emitter, Carbotec or Linden (Germany), FD 10 μm. Current rate 51.2 mA/min over 1.2 min machine using field desorption (FD) as ionization method. Melting points were recorded on a Wagner & Munz Polytherm A melting point apparatus and are uncorrected. IR spectra were recorded on a Bruker Alpha FTIR machine.
(129) T4 Scaffolds
(130) Scaffold T4(−≡).sub.2-1
(131) ##STR00021##
(132) To a stirred solution of 1,2,4,5-tetrakis(bromomethyl)benzene (8.13 mmol, 3.85 g, 3 equiv) and DIPEA (5.42 mmol, 0.9 mL, 2 equiv) in dry CH.sub.3CN (550 mL) was added dropwise dipropargylamine (2.709 mmol, 263 mg, 1 equiv) in dry CH.sub.3CN (30 mL). After consumption of the amine (2 h), the solvent was evaporated in vacuo, Et.sub.2O (100 mL) was added and the mixture was stirred for 30 min. The precipitate was isolated and filtered over a silica plug (100% CH.sub.2Cl.sub.2 to CH.sub.2Cl.sub.2/MeOH 9:1). The solvents were evaporated and the product was lyophilized to obtain the scaffold T4(−≡).sub.2-1 (quantitative, containing DIPEA salt) as a grey powder. The scaffold was used as such. .sup.1H NMR (400 MHz, D.sub.2O/CD.sub.3CN 9:1) δ 7.74 (s, 4H), 5.28 (s, 4H), 4.97 (s, 4H), 4.76 (d, 4H), 3.56 (t, 2H). .sup.13C NMR (400 MHz, D.sub.2O/CD.sub.3CN 9:1) S 140.9, 136.0, 129.0, 85.8, 73.7, 68.9, 54.7, 45.5, 32.9. IR ν 2978, 2932, 2663, 2617, 2121, 1426, 1391, 1182, 1135 cm.sup.−1. HRMS (EI.sup.+) m/z calculated for C.sub.16H.sub.16Br.sub.2N, 379.9649. found 379.9676.
(133) Scaffold T4(−≡).sub.2-2
(134) Diiodoarene 1
(135) ##STR00022##
(136) Durene (114 mmol, 15.3 g, 1 equiv.) and [bis(trifluoroacetoxy)iodo]benzene (145 mmol, 62.4 g, 1.3 equiv.) were added and the mixture was stirred at rt overnight. The solvent was evaporated and 0.1 M NaOH-solution (100 mL) was added. The product was extracted with CH.sub.2Cl.sub.2 (3×75 mL), followed by washing of the organic layer with H.sub.2O (1×100 mL) and brine (1×100 mL). The organic layer was dried over MgSO.sub.4, filtered and concentrated in vacuo. The crude product was triturated in MeOH and 1 was collected via vacuum filtration (74%). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 2.63 (s, 12H). .sup.13C NMR (400 MHz, CDCl.sub.3) δ 138.0, 112.4, 30.0. IR ν 1396, 1160, 971, 673 cm.sup.−1. HRMS (EI.sup.+) m/z calculated for C.sub.10H.sub.12I.sub.2 385.9028. found 385.8989. Spectral data in agreement with reported data (Zhdankin et al, 2017).
(137) TMS-Acetylene 2
(138) ##STR00023##
(139) To a solution of 1 (3.02 g, 7.82 mmol, 1 equiv), Pd(PPh.sub.3).sub.2Cl.sub.2 (0.380 g, 0.541 mmol, 0.07 equiv) and CuI (0.101 g, 0.530 mmol, 0.07 equiv) in Et.sub.2NH was added ethynyltrimethylsilane (2.45 mL, 17.3 mmol, 2.2 equiv) and the reaction was stirred at RT overnight. The solvent was evaporated followed by extraction with CH.sub.2Cl.sub.2 (3×100 mL) and washing of the organic layer with H.sub.2O (2×200 mL) and brine (250 mL). The combined organic layers were dried over MgSO.sub.4 and concentrated in vacuo. The product was purified via column chromatography (PE) leading to 2 as off-white crystals (82%). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 2.38 (s, 12H), 0.27 (s, 18H). .sup.13C NMR (400 MHz, CDCl.sub.3) δ 136.0, 123.4, 104.2, 103.2, 18.5, 0.27. IR ν 2957, 2138, 1270, 1073, 863 cm.sup.−1. HRMS (EI.sup.+) m/z calculated for C.sub.20H.sub.30Si.sub.2 326.1886. found 326.1870.
(140) Tetrakis(Bromomethyl)Arene 3
(141) ##STR00024##
(142) 2 (2.30 g, 7.06 mmol, 1 equiv) was dissolved in CCl.sub.4 (50 mL). NBS (6.1 g, 34.3, 5 equiv) and dibenzoylperoxide (452 mg, 1.88 mmol, 0.3 equiv) were added and the mixture was refluxed overnight. The solvents were evaporated and the crude product was purified with column chromatography (100% PE to PE/EtOAc 9:1). Subsequent recrystallization from PE gave 3 as a white powder in 10% yield. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 4.83 (s, 4H), 0.34 (s, 9H). .sup.13C NMR (400 MHz, CDCl.sub.3) δ 139.6, 125.6, 109.8, 98.5, 27.8, 0.24. IR ν 2957, 2154, 1420, 1283, 1247, 1200, 957, 837 cm.sup.−1. HRMS (FD.sup.+) m/z calculated for C.sub.20H.sub.26Br.sub.4Si.sub.2 641.8266. found 641.8285.
(143) Scaffold T4(−≡).sub.2-2
(144) ##STR00025##
(145) Compound 3 (300 mg, 0.480 mmol, 1 equiv) was suspended in dry MeOH (24 mL) and cooled to 0° C. K.sub.2CO.sub.3 (13.0 mg, 0.104 mmol, 0.2 equiv) was added and the mixture was stirred for 30 min. The solvent was removed in vacuo, and CH.sub.2Cl.sub.2 was added followed by washings with H.sub.2O (2×15 mL) and brine (20 mL). The organic layer was dried over MgSO.sub.4, filtered and concentrated. The crude product (0.482 mmol, 240 mg, 3 equiv.) and DIPEA (0.321 mmol, 56 μL, 2 equiv.) were dissolved in dry CH.sub.2Cl.sub.2/CH.sub.3CN (3/1:40 mL) under argon atmosphere. The mixture was heated slightly with a heat gun to ensure complete solvation. Piperidine (0.161 mmol, 16 μL, 1 equiv.) in CH.sub.3CN (2 mL) was added dropwise at a rate of 0.1 mL/h. The reaction mixture was stirred overnight, followed by concentration of the mixture. Column chromatography was carried out to isolate the product T4(−≡).sub.2-2 as a colorless oil. The product was then lyophilized to obtain a white solid (71%). .sup.1H NMR (400 mHz, CD.sub.3OD) δ 5.09 (s, 4H), 4.92 (s, 4H), 4.59 (s, 2H), 3.67-3.70 (t, 4H), 2.00-2.05 (q, 4H), 1.76-1.81 (q, 4H). .sup.13C NMR (300 MHz, CD.sub.3OD) δ 141.88, 137.66, 120.52, 92.61, 77.49, 69.36, 63.55, 27.03, 22.43, 21.92. IR ν 943.54, 1033.55, 1061.91, 1078.57, 1114.23, 1160.01, 1172.68, 1209.72, 1270.77, 1323.98, 1444.45, 1719.71, 2871.49, 3175.00, 3251.47, 3360.79. HRMS (ESI.sup.+) m/z calculated for C.sub.19H.sub.20Br.sub.2N.sup.+ 419.9962. found 421.9943. According to LCMS, an amount of Br—Cl exchange was observed which could have happened after the washings with brine in the previous step. However, this did not cause a problem in the follow-up CLIPS reactions.
(146) Scaffold T4(−≡).sub.2-3
(147) Tert-Butyl Ester 4
(148) ##STR00026##
(149) 3,5-dimethylbenzoic acid (10.0 gram, 66.6 mmol, 1 equiv) was dissolved in toluene (8 mL) under nitrogen atmosphere. SOCl.sub.2 (10.0 mL, 137 mmol, 2.06 equiv) was added and the mixture was refluxed for 2.5 h. The temperature was decreased to RT and the mixture was stirred overnight. After evaporation of the solvents, CH.sub.2Cl.sub.2 (20 mL) was added followed by tert-butanol (8.01 gram, 108 mmol, 1.6 equiv) and pyridine (5.53 g, 69.9 mmol, 1.05 equiv) and the mixture was stirred overnight. The mixture was filtered and the mother layer was concentrated in vacuo. The crude product was purified via column filtration (EtOAc) yielding ester 4 as a colorless oil (51%). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.61 (s, 2H), 7.15 (s, 1H), 2.35 (s, 6H), 1.60 (s, 9H). .sup.13C NMR (400 MHz, CDCl.sub.3) δ 166.2, 137.9, 134.1, 132.0, 127.2, 80.8, 28.3, 21.3. IR ν 3006, 1711, 1315, 1231, 1159 cm.sup.−1. HRMS (EI.sup.+) m/z calculated for C.sub.13H.sub.18O.sub.2 206.1307. found 206.1297.
(150) Bis(Bromomethyl)-Tert-Butyl Ester 5
(151) ##STR00027##
(152) Compound 4 (6.79 g, 32.9 mmol, 1 equiv) was dissolved in CH.sub.2Cl.sub.2 (130 mL) under inert atmosphere. N-bromosuccinimide (12.2 g, 68.5 mmol, 2.1 equiv) was added and the mixture was irradiated with a lamp (hν). The lamp was removed after 1 h and the mixture was stirred overnight. H.sub.2O (100 mL) was added to the mixture and the layers were separated. The organic layer was washed with H.sub.2O (3×100 mL) and brine (1×200 mL). The organic layer was dried over MgSO.sub.4, filtered and concentrated in vacuo resulting in a colorless oil. The purified product was obtained via recrystallization from hexanes yielding 5 as a white solid in 42% yield. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.92 (s, 2H), 7.59 (s, 1H), 4.50 (s, 4H), 1.60 (s, 9H). .sup.13C NMR (400 MHz, CDCl.sub.3) δ 164.7, 138.6, 133.6, 131.3, 130.0, 81.9, 32.2, 28.3. IR ν 2977, 1713, 1241, 1160 cm.sup.−1. HRMS (EI.sup.+) m/z calculated for C.sub.13H.sub.16Br.sub.2O.sub.2 361.9517. found 361.9503.
(153) Benzoic Acid 6
(154) ##STR00028##
(155) Compound 5 (4.90 g, 13.5 mmol, 1 equiv) was dissolved in dry CH.sub.2Cl.sub.2 (under inert atmosphere. Formic acid (54 mL of a 0.25M solution) was added and the mixture was stirred overnight. Solvents were evaporated yielding product 6 as a white powder in a yield of 97% with no need for further purification. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.08 (s, 2H), 7.69 (s, 1H), 4.52 (s, 4H). .sup.13C NMR (400 MHz, CDCl.sub.3) δ 170.8, 139.3, 135.0, 130.7, 130.6, 31.8. IR ν 2972, 1694, 1254 cm.sup.−1. HRMS (EI.sup.+) m/z calculated for C.sub.9H.sub.8Br.sub.2O.sub.2 305.8891. found 305.8889.
(156) Scaffold T4(−≡).sub.2-3
(157) ##STR00029##
(158) Compound 6 (3.92 g, 12.7 mmol, 1 equiv) was dissolved in SOCl.sub.2 (15 mL) and refluxed overnight at room temperature. After evaporation of the volatiles, the acyl chloride was dissolved in dry CH.sub.2Cl.sub.2 (130 mL). DMAP (37 mg, 0.30 mmol, 0.02 equiv) was added to the mixture, followed by dropwise addition of dipropargylamine (1.42 mL, 1.23 mmol, 1.05 equiv) in CH.sub.2Cl.sub.2 (13 mL) at 0° C. The temperature was increased to room temperature, and after completion of the reaction H.sub.2O (100 mL) and CH.sub.2Cl.sub.2 (50 mL) were added. The layers were separated and the organic layer was washed with H.sub.2O (2×200 mL) and brine (1×250 mL). The organic layer was dried over MgSO.sub.4, filtered and concentrated in vamp. Column purification of the crude product yielded the desired scaffold T4(−≡).sub.2-3 as a white powder in a yield of 48%. .sup.1H NMR (400 MHz, CDCl.sub.3, measured at −50° C.) δ 7.55-7.51 (m, 3H), 4.47 (s, 4H), 4.46 (d, 2H, part of AB), 4.15 (d, 2H, part of AB), 2.44 (t, 1H, part of AB), 2.31 (t, 1H, part of AB). .sup.13C NMR (400 MHz, CDCl.sub.3, measured at −50° C.) δ 169.6, 139.0, 135.4, 131.8, 127.9, 74.0, 72.8, 38.5 (rotamer signal A), 33.9 (rotamer signal B), 32.2. IR ν 3277, 1644, 1599, 1452, 1219 cm.sup.−1. HRMS (EI.sup.+) m/z calculated for C.sub.15H.sub.13Br.sub.2NO 380.9364. found 380.9345.
(159) Scaffold T4(−≡).sub.2-4
(160) Bisalcohol 7
(161) ##STR00030##
(162) 5-bromoisophtalic acid (25.0 gram, 0.102 mol, 1 equiv) was acid (25.0 gram, 0.102 mol, 1 equiv) was dissolved in dry THF (500 mL) at 0° C. under argon atmosphere. 10M Borane dimethylsulfide complex (50 mL, 0.50 mmol, 5 equiv) was added and the mixture was stirred at room temperature overnight. H.sub.2O (1000 mL) was added carefully to the mixture followed by addition of EtOAc (1000 mL). After separation of the layers, the organic layer was washed with H.sub.2O (3×750 mL) and brine (1000 mL). The organic layer was dried over MgSO.sub.4, filtered and concentrated in vacuo yielding alcohol 7 as a white powder in 85% yield with no need for further purification. .sup.1H NMR (400 MHz, DMSO) δ 7.36 (s, 2H), 7.25 (s, 1H), 5.30 (t, 2H), 4.48 (d, 4H). .sup.13C NMR (400 MHz, DMSO) δ 145.2, 127.1, 123.1, 121.3, 62.1. IR ν 3210, 2851, 1602, 1419 cm.sup.−1. HRMS (FD.sup.+) m/z calculated for C.sub.8H.sub.9BrO.sub.2 215.9786, found 215.9798. Spectral data in agreement with reported data (Wytko and Weiss, 1994).
(163) THP-Ether 8
(164) ##STR00031##
(165) 7 (4.18 g, 19.2 mmol, 1 equiv) was dissolved in dry THF (9 mL) under nitrogen atmosphere. 3,4-Dihydro-2H-pyran (5.3 mL, 58 mmol, 3 equiv) and PPTS (417 mg, 1.66 mmol, 0.09 equiv) were added and the mixture was stirred 48 h. H.sub.2O (50 mL) was added and the product was extracted with EtOAc (3×50 mL). The combined organic layers were washed with brine (1×50 mL) and dried over MgSO.sub.4. After filtration and concentration in vacuo, the product was filtered over a plug of silica leading to 8 as a colorless oil in a yield of 90%. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.44 (s, 2H), 7.25 (s, 1H), 4.75 (d 2H, part of AB), 4.70 (t, 2H), 4.46 (d, 2H, part of AB), 3.92-3.87 (m, 2H), 3.58-3.52 (m, 2H), 1.87-1.52 (m, 12H). .sup.13C NMR (400 MHz, CDCl.sub.3) δ 140.8, 129.8, 125.5, 122.7, 98.1, 68.1, 62.3, 30.6, 25.6, 19.4. IR ν 2940, 2869, 1574, 1386, 1119, 1024 cm.sup.−1. HRMS (FD.sup.+) m/z calculated for C.sub.18H.sub.25BrO.sub.4 384.0936. found 384.0950. Spectral data in agreement with reported data..sup.[1]
(166) Boronic Ester 9
(167) ##STR00032##
(168) 8 (10.4 g, 27.0 mmol, 1 equiv), B.sub.2Pin.sub.2 (8.41 g, 32.8 mmol, 1.2 equiv), Pd(dba).sub.2 (158 mg, 0.275 mmol, 0.01 equiv), DPEPhos (146 mg, 0.271 mmol, 0.01 equiv) and sodium acetate (4.50 g, 55 mmol, 2 equiv) were combined in dry toluene (35 mL) under nitrogen atmosphere. The mixture was heated to reflux and stirred over weekend. H.sub.2O (50 mL) was added, layers were separated and the organic layer was washed with H.sub.2O (3×50 mL) and brine (1×250 mL). Drying with MgSO.sub.4, filtration and concentration followed by purification via column chromatography (PE/EtOAc: 9:1 to 6:1) yielded compound 9 as a colorless oil in a yield of 86%. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.71 (s, 2H), 7.52 (s, 1H), 4.79 (d, 2H, part of AB), 4.71 (t, 2H), 4.50 (d, 2H, part of AB), 3.95-3.90 (m, 2H), 3.56-3.53 (m, 2H), 1.90-1.50 (m, 12H), 1.34 (s, 12H). .sup.13C NMR (400 MHz, CDCl.sub.3) δ 137.9, 133.8, 130.7, 98.0, 84.0, 69.0, 62.2, 30.7, 25.6, 25.0, 19.5. IR ν 2975, 2870, 1606, 1372, 1344, 1122, 1034 cm.sup.−1. HRMS (FD.sup.+) m/z calculated for C.sub.24H.sub.37BO.sub.6 431.2719. found 431.2655.
(169) THP-Biaryl 10
(170) ##STR00033##
(171) 9 (7.36 g, 17.0 mmol, 1.05 equiv) and 7 (3.52 g, 16.2 mmol, 6, 17.0 mmol, 1.05 equiv) and 7 (3.52 g, 16.2 mmol, 1 equiv) were added in dioxane/H.sub.2O (2:1, 85 mL) under nitrogen atmosphere. Potassium carbonate (6.70 g, 48.6 mmol, 3 equiv) and Pd(dppf)Cl.sub.2 (1.21 g, 1.64 mmol, 0.1 equiv) were added and the mixture was stirred overnight at 60° C. The mixture was poured in H.sub.2O (500 mL) and the mixture was extracted with EtOAc (3×400 mL). The combined organic layers were washed with brine (1×1000 mL) and dried over MgSO.sub.4, filtered and concentrated in vacuo. Purification via column chromatography (PE/EtOAc 1:1 to 1:3) yielded 10 as a colorless oil in a yield of 86%. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.48 (s, 2H), 7.46 (s, 2H), 7.35 (s, 1H), 7.28 (s, 1H), 4.81 (d, 2H, part of AB), 4.73 (t, 2H), 4.65 (s, 4H), 4.53 (d, 2H, part of AB), 3.96-3.91 (m, 2H), 3.59-3.53 (m, 2H), 3.11 (br s, 2H), 1.90-1.52 (m, 12H). .sup.13C NMR (400 MHz, CDCl.sub.3) δ 141.9, 141.4, 141.2, 138.9, 126.6, 126.1, 124.9, 124.5, 98.0, 68.9, 65.0, 62.4, 30.6, 25.5, 19.5. IR ν 3395, 2940, 2868, 1601, 1119, 1025 cm.sup.−1. HRMS (FD.sup.+) m/z calculated for C.sub.26H.sub.34O.sub.6 442.2355. found 442.2347.
(172) Propargylic Ether 11
(173) ##STR00034##
(174) 10 (3.42 g, 7.75 mmol, 1 equiv) was added to a suspension of NaH (691 mg, 17.3 mmol, 2.2 equiv) in THF (45 mL) at 0° C. After stirring for 1 h, propargyl bromide (1.3 mL, 17.3 mmol, 2.2 equiv) was added dropwise and the mixture was stirred overnight at rt. H.sub.2O (500 mL) was added and the product was extracted with EtOAc (4×300 mL). The combined organic layers were washed with brine (1000 mL), dried over MgSO.sub.4 and concentrated in vacuo. Purification by column chromatography (PE/EtOAc 4:1) furnished 11 as a colorless oil (70%). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.54 (s, 2H), 7.52 (s, 2H), 7.37 (s, 1H), 7.34 (s, 1H), 4.85 (d, 2H, part of AB), 4.74 (t, 2H), 4.68 (s, 4H), 4.57 (d, 2H, part of AB), 4.22 (d, 4H), 3.97-3.92 (m, 2H), 3.59-3.52 (m, 2H), 2.49 (t, 2H), 1.92-1.50 (m, 12H). .sup.13C NMR (400 MHz, CDCl.sub.3) δ 141.7, 141.1, 139.1, 138.2, 126.8, 126.7, 126.6, 126.1, 98.0, 79.7, 74.9, 71.5, 68.9, 62.4, 57.4, 30.7, 25.6, 19.5. IR ν 3285, 2941, 2868, 1601, 1385, 1118, 1078, 1034 cm.sup.−1. HRMS (FD.sup.+) m/z calculated for C.sub.32H.sub.38O.sub.6 518.2668. found 518.2675.
(175) Bisalcohol 12
(176) ##STR00035##
(177) 11 (2.78 g, 5.37 mmol, 1 equiv) was dissolved in EtOH (30 mL) and PPTS (4.02 g, 15.8 mmol, 2.9 equiv) was added. The mixture was stirred for 2 h at 55° C., followed by addition of 1420 (50 mL) and extraction with EtOAc (3×100 mL). The combined organic layers were dried over MgSO.sub.4 and concentrated in vacuo. Column chromatography (PE/EtOAC 2:1 to 1:2) afforded the 12 in 98% as a colorless oil. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.47 (s, 2H), 7.41 (s, 2H), 7.30 (s, 1H), 7.23 (s, 1H), 4.61 (s, 4H), 4.57 (s, 4H), 4.20 (d, 4H), 3.72 (s, 2H), 2.52 (t, 2H). .sup.13C NMR (400 MHz, CDCl.sub.3) δ 141.8, 141.2, 140.7, 138.1, 126.7, 126.3, 124.7, 124.5, 79.6, 75.1, 71.4, 64.6, 57.4. IR ν 3287, 2860, 1601, 1445, 1350, 1254, 1162, 1071 cm.sup.−1. HRMS (FD.sup.+) m/z calculated for C.sub.22H.sub.22O.sub.4 350.1518. found 350.1515.
(178) Scaffold T4(−≡).sub.2-4
(179) ##STR00036##
(180) Compound 12 (861 mg, 2.46 mmol, 1 equiv) and Et.sub.3N (860 μL, 6.15 mmol, 2.5 equiv) was dissolved in dry THF (25 mL) at 0° C. followed by the dropwise addition of MsCl (761 μL, 9.84 mmol, 4 equiv). The mixture was stirred at RT overnight and subsequently quenched with H.sub.2O (100 mL) for 1 h. The product was extracted with EtOAc (3×150 mL) and washed with sat. NaBr-solution (300 mL). The organic layer was dried over MgSO.sub.4 and concentrated in vacuo. The resulting colorless oil was dissolved in dry THF (20 mL), LiBr (855 mg, 9.84 mmol, 4 equiv) was added and the mixture was stirred for 2 h. H.sub.2O (100 mL) was added followed by extraction with EtOAc (3×200 mL). The combined organic layers were washed with sat. NaBr-solution (500 mL), dried over MgSO.sub.4 and concentrated in vacuo. Purification by column chromatography (PE/EtOAc 5:1 to 3:1) yielded scaffold T4(−≡).sub.2-4 as a white powder (80%). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.55 (s, 2H), 7.52 (s, 2H), 7.41 (s, 1H), 7.37 (s, 1H), 4.68 (s, 4H), 4.53 (s, 4H), 4.24 (d, 4H), 2.50 (t, 2H). .sup.13C NMR (400 MHz, CDCl.sub.3) δ 142.1, 140.5, 139.1, 138.5, 128.7, 128.1, 127.1, 126.4, 79.6, 75.0, 71.4, 57.6, 32.9, 29.8. IR ν 3289, 2924, 2853, 1601, 1448, 1352, 1214, 1082 cm.sup.−1. HRMS (EI.sup.+) m/z calculated for C.sub.22H.sub.20Br.sub.2O.sub.2 473.9830. found 473.9840.
(181) Scaffold T6(E).sub.3-1
(182) Phthalimide 13
(183) ##STR00037##
(184) 1,3,5-tris(bromomethyl)mesitylene (19.8 mmol, 7.88 g, 1 equiv.) was dissolved in anhydrous DMF (300 mL) under inert atmosphere. Phthalimide potassium salt (120 mmol, 22.2 g, 6 equiv.) was added and the mixture was refluxed overnight. After cooling down to room temperature, the mixture was filtered and successively washed with DMF (2×50 mL), H.sub.2O (2×100 mL) and acetone (100 mL). After drying of the residue in vacuo, product 13 was isolated as a white solid (16.8 mmol, 85%). Spectral data was in agreement with reported data (Roelens et al. 2009).
(185) Bromide 14
(186) ##STR00038##
(187) Phthalimide 13 (18.1 mmol, 10.8 g, 1 equiv.) was suspended in 1,2-dibromoethane (120 mL) and bromine (60.2 mmol, 3.1 mL, 3.3 equiv.) was added. The mixture was stirred and irradiated at 120° C. for 25 min and afterwards additionally irradiated without heating for 2.5 h. The excess of bromine was quenched by adding an aqueous solution of thiosulfate ( . . . M) and the product was extracted with dibromoethane (2×50 mL). The combined organic layers were washed with saturated NaHCO.sub.3-solution (100 mL), brine (150 mL) and dried over MgSO.sub.4, filtered and concentrated in vacuo. Purification by column chromatography (CH.sub.2Cl.sub.2) yielded 14 as a yellow solid (9.11 mmol, 51%). Spectral data was in agreement with reported data (Roelens et al. 2009).
(188) Propargylic Ether 15
(189) ##STR00039##
(190) 14 (8.45 mmol, 7.05 g, 1 equiv, prepared according to literature procedure) was suspended in DMSO (85 mL) and silver triflate (28.6 mmol, 7.34 g, 3.4 equiv.) was added. The mixture was stirred vigorously under darkness for 1 h followed by addition of DIPEA (42.3 mmol, 7.4 mL, 5 equiv.) and stirring for 1 h. Water (50 mL) was added and the mixture was filtered, followed by extraction with CH.sub.2Cl.sub.2 (4×100 mL). The combined organic layers were washed with 2M HCl (2×75 mL) and water (100 mL) and dried over MgSO.sub.4, filtered and concentrated. The crude yellow solid (7.40 mmol, 88%) was dissolved in dry CH.sub.2Cl.sub.2 (170 mL) under inert atmosphere. In situ generated trimethyl(2-propyn-1-yloxy)silane (3.5 equiv.) and triethylsilane (22.6 mmol, 3.4 mL, 3.4 equiv.) were added and the mixture was cooled to −60° C. TMSOTf (3.42 mmol, 620 μL, 0.5 equiv.) was added and the mixture was stirred overnight at rt. After dilution with DCM (200 mL), the mixture was washed with H.sub.2O (200 mL), brine (250 mL) and dried over MgSO.sub.4. Filtration, concentration and purification by column chromatography (CH.sub.2Cl.sub.2/EtOAc 95:5) yielded 15 as a white solid (3.987 mmol, 59%). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.79-7.65 (dq, 12H), 5.08 (s, 6H), 4.96 (s, 6H), 3.97 (d, 6H), 2.22 (t, 3H). .sup.13C NMR (400 MHz, CDCl.sub.3) δ 168.17, 137.76, 132.25, 133.90, 132.23, 123.34, 79.47, 74.69, 66.40, 57.80, 37.00. IR ν 1770, 1391, 1058, 712 cm.sup.−1. HRMS (FD.sup.+) m/z calculated for C.sub.45H.sub.33N.sub.3O.sub.9 759.2217. found 759.2191.
(191) Scaffold T6(−≡).sub.3-1
(192) ##STR00040##
(193) To a suspension of propargylic ether 15 (0.341 mmol, 259 mg, 1 equiv.) in EtOH/toluene (3:1, 3.5 mL) under inert atmosphere was added methylhydrazine (2.09 mmol, 110 μL, 6.1 equiv.). The mixture was stirred overnight at 90° C. After completion, the mixture was poured into a 40% KOH-solution (25 mL) and extracted with CH.sub.2Cl.sub.2 (3×25 mL). The combined organic layers were dried over MgSO4, filtered and concentrated. The crude white solid (0.252 mmol, 74%) was used in the follow-up reaction without further purification. Bromoacetylbromide (23.0 mmol, 2 mL, 17 equiv.) was added in dry CH.sub.2Cl.sub.2 (15 mL) and the mixture was cooled to 0° C. Triamine X (1.35 mmol, 497 mg, 1 equiv.) in CH.sub.2Cl.sub.2 (15 mL) was added dropwise to the mixture. After completion, the mixture was quenched with saturated NaHCO.sub.3-solution (25 mL) and extracted with CH.sub.2Cl.sub.2 (3×30 mL). The combined organic layers were washed with H.sub.2O (50 mL), brine (35 mL) and dried over MgSO.sub.4. Concentration and purification with column chromatography (PE/EtOAc 2:1 to 5:1) yielded the desired T6(−≡).sub.3-1 scaffold (0.739 mmol, 55%) as a white powder. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.16 (t, 3H), 4.82 (s, 6H), 4.72 (d, 6H), 4.36 (d, 6H), 3.86 (s, 6H), 2.58 (t, 3H). .sup.13C NMR (400 MHz, CDCl.sub.3) δ 164.85, 139.08, 136.91, 78.80, 76.22, 65.91, 58.51, 38.37, 29.09. IR ν 3277, 1641, 1537, 1070 cm.sup.−1. HRMS (FD.sup.+) m/z calculated for C.sub.27H.sub.30Br.sub.3, N.sub.3O.sub.6 731.9791. found 731.9744.
(194) 3. Solid-Phase Peptide Synthesis (SPPS)
(195) General Section:
(196) Amino acids are indicated by single-letter codes; peptides are acetylated at the N-terminus and amidated at the C-terminus. Unnatural amino acid azidohomoalanine is abbreviated as [Aha].
(197) General Procedure for Fmoc-Synthesis of Peptides:
(198) Peptides were synthesized on solid-phase using a 4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxy (RinkAmide) resin (BACHEM, Germany) on a Prelude (Protein Technologies Incl., USA), Symfony (Protein Technologies Inc., USA), Syro(I) (MultiSyntech, Germany) synthesizer. All Fmoc-amino acids were purchased from Biosolve (Netherlands) or Bachem GmbH (Germany) with appropriate side-chain functionalities protected as N-t-Boc (amino acids KW), O-t-Bu (DESTY), N-Trt (HNQ), S-Trt (C) or N-Pbf (R) groups. All solvents used in peptide synthesis (piperidine, trifluoroacetic acid, NMP and DMF were bought from Biosolve (Netherlands) in peptide grade quality. Fmoc-azidohomoalanine-OH (Fmoc-Aha-OH) was synthesized according to a literature procedure described by Spring et al. in 2011. Amino acids were dissolved in DMF (200 mM) and used as such. Piperidine was used as a 20% stock solution in NMP, HATU as an 0.4M stock solution in DMF, and DIPEA as a 2M stock solution in NMP. Standard amino acids, including [Aha] were coupled via a single-coupling protocol (5-fold excess of HATU/amino acid and 10-fold excess of DIPEA) with a reaction time of 1 hour. In case of difficult amino acid couplings, e.g. R, K and C, the double-coupling protocol (10-fold excess of HATU/amino acid and 20-fold excess of DIPEA) with a reaction time of 2×15 min was used. Acetylation (Ac) of the N-terminus of the peptide was performed by reacting the resin with NMP/Ac.sub.2O/DIPEA (10:1:0.1) for 30 minutes at room temperature. The acetylated peptide was cleaved from the resin by reaction with a cocktail of TFA/MiliQ/thioanisole/DODT/TIS (90:5:2.5:5:2.5) for 2 hours at room temperature. Precipitation of the peptide with Et.sub.2O/pentane (1:1) followed by lyophilization of the precipitated peptide afforded the crude peptide. Purification of the crude peptide was performed by reversed-phase HPLC (mobile phase consists of gradient mixture of eluent-A(milliQ-H.sub.2O containing 0.05% TFA) and eluent-B (ACN containing 0.05% TFA).
(199) 4. CLIPS/CuAAC Ligation-Cyclization
(200) Cyclizations with T4 Scaffolds
(201) General Information
(202) Ligation-cyclization reactions were measured on a UPLC-ESMS system (3 min, 5-80% B, Acquity UPLC Peptide BEH C18 Column, 130 Å, 1.7 μm, 2.1×50 mm with UV detection (λ=215 nm) and positive ion current for MS analysis, unless stated otherwise. Linear peptides are described with a number (#) followed by in subscript the loop length (y), e.g. #.sub.333 (for a certain peptide with 3×3×3 peptide loops). Monocyclic CLIPS-peptides are described with covalent attachment to the T4(−≡).sub.2-@ (where @ is the scaffold number which is 1,2,3 or 4), e.g. [#.sub.333-T4(−≡).sub.2-@]. Tricyclic CLIPS/CuAAC peptides are described with a Roman Number instead of an Arabic number, e.g. the product of tricyclization of peptide 1.sub.333 with scaffold T4(−≡).sub.2-4 will be described as [I.sub.333-T4(−≡).sub.2-4].
(203) ##STR00041##
General Procedures for the One-Pot CLIPS/CuAAC Ligation-Cyclization
(204) Linear peptide (1.0 equiv) was dissolved in DMF/H.sub.2O (2:1, 0.5 mM) and 0.8 equiv of a 10 mM stock solution of T4(−≡).sub.2-scaffold (in DMF) was added to the mixture. The pH of the solution was adjusted to 8 by adding NH.sub.4HCO.sub.3-solution (1M) in order to start the CLIPS reaction. After complete consumption of the linear peptide, a pre-incubated cocktail of CuSO.sub.4 (2 equiv), THPTA-ligand (2 equiv) and sodium ascorbate (10 equiv) in H.sub.2O was added to the reaction mixture. After completion, 0.1M EDTA-solution (5 equiv) was added to the mixture to quench the reaction, followed by immediate reversed phase HPLC purification or lyophilization.
(205) CuAAC-Reaction Completion Check: Staudinger Reduction
(206) To check whether CuAAC reaction was completed, to a 70 uL UPLC sample was added 20 uL 1M TCEP. The mixture was incubated for 24 h and analyzed with UPLC. Both CLIPS/CuAAC and CuAAC/CLIPS were carried out. When CuAAC was carried out first, the free thiols of cysteine residues were prone to oxidation along with severe coordination towards copper(I) leading to formation of S—S oxidized peptide. Furthermore, CLIPS reactions can be performed under micromolar concentrations thereby limiting the oligomerization of the peptide-scaffold constructs. For these reasons it is recommended to always start with CLIPS prior to CuAAC.
(207) Full Experimental Procedure for CLIPS/CLICK Cyclization of Peptide Ac—CQWG[Aha]KSR[Aha]FIIC—NH.sub.2 on Scaffold T4(−≡).sub.2-3
(208) ##STR00042##
(209) CLIPS: Peptide Ac—CQWG[Aha]KSR[Aha]FIIC—NH.sub.2 (1.00 mg, 0.612 μmol, 1 equiv.)
(210) was dissolved in DMF/H.sub.2O (2:1, 1.2 mL) to give a 0.5 mM solution. Then, scaffold T4(−≡).sub.2-3 (1.71 mg, 4.45 μmol) was dissolved in DMF (445 μL) to give a 10 mM solution and 49 μL (0.8 equiv.) of this solution was added to the peptide solution. Subsequently, 10 μL of an NH.sub.4HCO.sub.3-solution (1M) was added to reach pH=8 in order to start the reaction. Complete consumption of the peptide and formation of the monocyclic peptide was confirmed by UPLC/UV in combination with ESI-MS analysis (
(211) CLICK: A 10 mM stock solution of copper(II) sulfate pentahydrate in H.sub.2O was prepared by dissolving copper(II)sulfate pentahydrate (5.0 mg, 20 μmol) in 500 μL H.sub.2O. A 10 mM stock solution of THPTA ligand in H.sub.2O was prepared by dissolving THPTA (5.0 mg, 12 μmol) in 1.2 mL H.sub.2O. Both compounds were added in twofold molar excess with respect to the peptide. 122 μL of a 10 mM copper(II)sulfate stock solution and 122 μL of a 10 mM THPTA stock solution were combined in a separated vial, followed by the addition of 5 equiv. of sodium ascorbate (1.2 mg, 6.06 μmol). Eventual equivalents compared to peptide are 2/2/10 for copper/ligand/ascorbate, respectively). Subsequently, the Cu(I)/THPTA/ascorbate mix was added to the peptide solution, followed by direct analysis with UPLC/UV and ESI-MS. Therefore, a 50 μL sample of the reaction mixture was mixed with 20 μL of a 0.1 M EDTA-solution (pH=7.8) in order to complex all added Cu(I) ions and thus quench the CLICK-reaction. After 1 minute, the reaction was essentially complete according to UPLC-MS (
(212) Full Experimental Procedure for CLIPS/CuAAC Cyclization of Peptide Ac—CQWG[Aha]KAS[Aha]FSEC—NH.sub.2 on Scaffold T4(−≡).sub.2-3
(213) ##STR00043##
(214) CLIPS: peptide Ac—CQWG[Aha]KAS[Aha]FSEC—NH.sub.2 (5.30 mg, 3.44 μmol, 1 equiv) was dissolved in DMF/H.sub.2O (2:1, 6.9 mL) to give a 0.5 mM solution. Then, scaffold T4(—≡).sub.2-3 (0.275 μL, 2.75 μmol, 0.8 equiv from a 10 mM solution in DMF) was added. Subsequently, 20 μL of an NH.sub.4HCO.sub.3-solution (200 mM) was added to reach pH=8 in order to start the reaction.
(215) CuAAC: a pre-incubated mix of CuSO.sub.4/THPTA/Asc (2:2:10 equiv compared to linear peptide) in H.sub.2O was added to the CLIPS-mixture in order to start the CuAAC-reactions.
(216)
(217) After completion, the reaction was quenched by adding 0.1M EDTA-solution and directly purified on RP-HPLC yielding the tricyclic peptide in 18% yield (
(218) Full Experimental Procedure for CLIPS/CuAAC Cyclization of Peptide Ac—CQWG[Aha]KAS[Aha]FSEC—NH.sub.2 on Scaffold T4(−≡).sub.2-4
(219) ##STR00044##
(220) CLIPS: peptide Ac—CQWG[Aha]KAS[Aha]FSEC—NH.sub.2(9.64 mg, 6.26 μmol, 1 equiv) was dissolved in DMF/H.sub.2O (2:1, 12.6 mL) to give a 0.5 mM solution. Then, scaffold T4(—≡).sub.2-4 (0.501 μL, 5.01 μmol, 0.8 equiv from a 10 mM solution in DMF) was added. Subsequently, 504 of an NH.sub.4HCO.sub.3-solution (200 mM) was added to reach pH=8 in order to start the reaction.
(221) CuAAC: a pre-incubated mix of CuSO.sub.4/THPTA/Asc (2:2:10 equiv compared to linear peptide) in H.sub.2O was added to the CLIPS-mixture in order to start the CuAAC-reactions.
(222) After completion, the reaction was quenched by adding 0.1M EDTA-solution and directly purified on RP-HPLC yielding the tricyclic peptide in 28% yield (
(223) Further CLIPS/CuAAC Reactions
(224) CLIPS/CuAAC cyclizations were carried out with 19 different peptides and scaffolds T4(−≡).sub.2-1, T4(−≡).sub.2-42, T4(−≡).sub.2-3 and T4(−≡).sub.2-4 as described above in the general procedures for the one-pot CLIPS/CuAAC ligation-cyclization described above and in analogy with the full experimental procedures for CLIPS/CLICK cyclizations described above. The results (including retention times, MW.sub.calc/found for linear peptides, monocyclic CLIPS-peptides and tricyclic CLIPS/CuAAC-peptides) of all reactions are shown in table 1.
(225) TABLE-US-00003 TABLE 1 Rt given in min, MW given in Da, N-termini were acetylated, C-terminal amide, positive ion mode (For T4(-≡).sub.2-1 scaffold reactions, the mass-Br.sup.- is reported), samples were measured on a UPLC-ESMS system (3 min, 5-80%B, Acquity UPLC Peptide BEH C18 Column, 130ÅA, 1.7 gm, 2.1x50 mm with UV detection (λ = 215 nm) and positive ion current for MS analysis. R.sub.t T4(-≡).sub.2-1 R.sub.t T4(-≡).sub.2-1 R.sub.t T4(-≡).sub.2-2 R.sub.t T4(-≡).sub.2-2 R.sub.t linear CLIPS CLIPS/CuAAC CLIPS CLIPS/CuAAC Peptide Code Sequence [MW.sub.calc/found] [MW.sub.calc/found] [MW.sub.calc/found] [MW.sub.calc/found] [MW.sub.calc/found] 1 1.sub.333 CQWG[Aha]KAS[Aha]FSEC 1.16 1.08 0.85 n.d. n.d. [1538.7/1538.5] [1758.0/1757.5] [1758.0/1758.1] and 0.86 [1758.0/1758.1] 2 2.sub.333 CNSN[Aha]SKE[Aha]TWNC 0.87 n.d. n.d. n.d. n.d. [1578.7/1579.1] 3 3.sub.333 CQYR[Aha]KIL[Aha]KGRC 0.85 n.d. n.d. n.d. n.d. [1661.0/1661.2] 4 4.sub.333 CAIP[Aha]RYR[Aha]NVTC 1.03 n.d. n.d. n.d. n.d. [1588.8/1589.6] 5 5.sub.333 CTHW[Aha]QEK[Aha]SGNC 0.85 n.d. n.d. n.d. n.d. [1585.7/1586.5] 6 6.sub.333 CHPY[Aha]RQV[Aha]TVDC 0.92 n.d. n.d. n.d. n.d. [1613.8/1613.4] 7 7.sub.333 CDHV[Aha]KFY[Aha]RHDC 0.85 n.d. n.d. n.d. n.d. [1715.9/1716.7] 8 8.sub.333 CNEG[Aha]SHN[Aha]GIKC 0.72 n.d. n.d. n.d. n.d. [1454.6/1454.2] 9 9.sub.333 CQLQ[Aha]GSY[Aha]RFIC 1.40 n.d. n.d. n.d. n.d. [1610.8/1611.2] 10 10.sub.222 CQW[Aha]KA[Aha]FSC 1.45 1.26 0.84/0.91 n.d. n.d. [1265.4/1265.7] [1484.7/1484.1] [1484.7/1484.9] R.sub.t T4(-≡).sub.2-3 R.sub.t T4(-≡).sub.2-3 R.sub.t T4(-≡).sub.2-4 R.sub.t T4(-≡).sub.2-4 CLIPS CLIPS/CuAAC CLIPS CLIPS/CuAAC Peptide Code Sequence [MW.sub.calc/found] [MW.sub.calc/found] [MW.sub.calc/found] [MW.sub.calc/found] 1 1.sub.333 CQWG[Aha]KAS[Aha]FSEC 1.34 0.94 1.63 1.21 [1759.9/1759.4] [1759.9/1759.7] [1853.1/1852.7] [1853.1/1852.8] 2 2.sub.333 CNSN[Aha]SKE[Aha]TWNC 1.10 0.82 1.42 1.08 [1800.0/1800.7] [1800.0/1800.3] [1893.1/1894.6] [1893.1/1893.4] 3 3.sub.333 CQYR[Aha]KIL[Aha]KGRC 0.99 0.79 1.20 0.88 [1882.3/1882.7] [1882.3/1882.6] [1975.4/1975.6] [1975.4/1975.8] 4 4.sub.333 CAIP[Aha]RYR[Aha]NVTC 1.21 0.93 1.49 1.07 [1810.1/1810.4] [1810.1/1809.7] [1903.2/1903.5] [1903.2/1903.7] 5 5.sub.333 CTHW[Aha]QEK[Aha]SGNC 1.03 0.75 1.32 0.99 [1807.0/1807.3] [1807.0/1806.7] [1900.1/1901.3] [1900.1/1899.9] 6 6.sub.333 CHPY[Aha]RQV[Aha]TVDC n.d. n.d. 1.43 1.04 [1928.2/1927.9] [1928.2/1928.1] 7 7.sub.333 CDHV[Aha]KFY[Aha]RHDC n.d. n.d. 1.23 0.94 [2030.3/2030.7] [2030.3/2030.8] 8 8.sub.333 CNEG[Aha]SHN[Aha]GIKC n.d. n.d. 1.30 0.94 [1769.0/1769.3] [1769.0/1769.6] 9 9.sub.333 CQLQ[Aha]GSY[Aha]RFIC n.d. n.d. 1.85 1.37 [1925.2/1925.5] [1925.2/1925.9] 10 10.sub.222 CQW[Aha]KA[Aha]FSC 1.60 1.00 1.86 1.24 [1486.7/1486.4] [1486.7/1485.9] [1579.8/1580.2] [1579.8/1579.4] and 1.03 [1486.7/1485.5] R.sub.t T4(-≡).sub.2-1 R.sub.t T4(-≡).sub.2-1 R.sub.t T4(-≡).sub.2-2 R.sub.t T4(-≡).sub.2-2 R.sub.t linear CLIPS CLIPS/CuAAC CLIPS CLIPS/CuAAC Peptide Code Sequence [MW.sub.calc/found] [MW.sub.calc/found] [MW.sub.calc/found] [MW.sub.calc/found] [MW.sub.calc/found] 11 11.sub.222 CES[Aha]FA[Aha]KKC 1.11 1.17 0.94/0.96 0.91 — [1208.4/1208.6] [1427.7/1427.1] [1427.7/1426.0] +1467.7/1467.6+ 12 12.sub.222 ACGS[Aha]FE[Aha]KNCG 0.96 1.20 0.82/0.94 0.95 — [1308.4/1308.7] [1527.7/1527.3] [1527.7/1526.4] +1567.8/1567.4+ 13 13.sub.222 NACEE[Aha]FK[Aha]KSC 0.97 1.15 — 0.89 — [1451.6/1451.5] [1670.9/1670.2] [1711.0/1710.9] 14 14.sub.111 CQ[Aha]K[Aha]FC 0.92 1.05 0.68/0.70 n.d. n.d. [921.1/921.7] [1140.4/1409.3] [1140.4/1139.3] 15 15.sub.111 CE[Aha]F[Aha]KC 1.11 1.14 0.63 (br) n.d. n.d. [922.1/921.9] [1141.4/1141.5] [1141.4/1142.1] 16 16.sub.444 CQWGA[Aha]KASE[Aha]FSEKC 1.10 1.00 0.79 n.d. n.d. [1867.1/1867.3] [2086.4/2086.4] [2086.4/2087.2] and 0.81 [2086.4/2086.0] 17 17.sub.555 CQWGAS[Aha]KASEV[Aha]FSEKGC 1.10 n.d. n.d. n.d. n.d. [2110.3/2110.0] 18 18.sub.333 [Aha]QWGCKASCFSE[Aha] 1.14 1.12 0.84 n.d. n.d. [1538.7/1538.5] [1758.0/1758.4] [1758.0/1758.2] and 0.87 [1758.0/1757.7] 19 19.sub.111 [Aha]QCKCF[Aha] 0.99 0.96 0.641 n.d. n.d. [921.1/921.8] [1140.4/1140.5] [1140.4/1140.1] and 0.68 [1140.4/1140.3] R.sub.t T4(-≡).sub.2-3 R.sub.t T4(-≡).sub.2-3 R.sub.t T4(-≡).sub.2-4 R.sub.t T4(-≡).sub.2-4 CLIPS CLIPS/CuAAC CLIPS CLIPS/CuAAC Peptide Code Sequence [MW.sub.calc/found] [MW.sub.calc/found] [MW.sub.calc/found] [MW.sub.calc/found] 11 11.sub.222 CES[Aha]FA[Aha]KKC 1.12 0.68 1.45 1.09 [1429.6/1429.9] [1429.6/1476.9] [1522.8/1522.6] [1522.8/1522.5] and 0.72 [1429.6/1429.3] 12 12.sub.222 ACGS[Aha]FE[Aha]KNCG 1.16 0.71 (br) 1.48 1.07 [1529.7/1529.6] [1529.7/1529.6] [1622.8/1622.9] [1622.8/1623.0] 13 13.sub.222 NACEE[Aha]FK[Aha]KSC 1.15 0.64 1.48 0.99 [1672.9/1672.7] [1672.9/1672.2] [1766.0/1765.6] [1766.0/1766.2] and 0.72 [1672.9/1672.4] 14 14.sub.111 CQ[Aha]K[Aha]FC 1.35 0.79 1.68 1.12 [1142.3/1141.4] [1142.3/1142.7] [1235.5/1235.8] [1235.5/1235.0] and 0.82 and 1.18 [1142.3/1142.7] [1235.5/1235.3] 15 15.sub.111 CE[Aha]F[Aha]KC 1.47 0.83 (br) 1.81 1.04 [1143.3/1143.2] [1143.3/1143.3] [1236.4/126.3] [1236.4/1236.8] and 1.10 [1236.4/1237.2] 16 16.sub.444 CQWGA[Aha]KASE[Aha]FSEKC 1.22 0.91 1.46 1.08 [2088.3/2088.2] [2088.3/2088.2] [2181.4/2181.1] [2181.4/2180.7] 17 17.sub.555 CQWGAS[Aha]KASEV[Aha]FSEKGC 1.21 0.92 1.45 1.09 [2331.6/2330.8] [2331.6/2330.9] [2424.7/2425.9] [2424.7/2424.5] 18 18.sub.333 [Aha]QWGCKASCFSE[Aha] 1.38 1.00 1.66 1.20 [1759.9/1759.4] [1759.9/1759.8] [1853.1/1851.1] [1853.1/1853.0] 19 19.sub.111 [Aha]QCKCF[Aha] 1.37 0.82 1.76 1.21 [1142.3/1142.1] [1142.3/1142.7] [1235.5/1235.6] [1235.5/1235.9]
General Procedure for T4 Cyclizations of Monocycles to Generate Tetracyclic Peptides:
(226) ##STR00045##
(227) Monocyclic peptides can be created via various methods for head-to-tail cyclizations (e.g. Schmidt et al. 2017 and Timmerman et al. 2009)
(228) CLIPS: monocyclic peptide 20.sub.4444 (0.52, 0.233 μmol, 1 equiv) was dissolved in 480 μL DMF/H.sub.2O (2:1) to give a 0.5 mM solution. Then, scaffold T4(−≡).sub.2-4 (19 μL, 0.186 mol, 0.8 equiv from a 10 mM stock solution in DMF) was added. Subsequently, 40 μL of an aqueous NH.sub.4HCO.sub.3-solution (200 mM) was added to reach pH=8 in order to start the reaction.
(229) CuAAC: a pre-incubated mix of CuSO.sub.4/THPTA/Asc (2:2:10 equiv compared to linear peptide) in H.sub.2O was added to the CLIPS-mixture in order to start the CuAAC-reactions.
(230) Two head-to-tail cyclized peptides were further cyclized using different T4 scaffolds to yield tetracyclic peptides. The UPLC-MS chromatogram of head-to-tail cyclized peptides, bicyclic CLIPS peptide and tetracyclic CLIPS/CuAAC peptides are shown in
(231) TABLE-US-00004 ILCQWGA[Aha]KASE[Aha]FSKVCPK: 20.sub.4444 + T4(-≡).sub.2-3 (FIG. 22), ILCQWGA[Aha]KASE[Aha]FSKVCPK: 20.sub.4444 + T4(-≡).sub.2-4 (FIG. 23), ILKCQKGAT[Aha]KASEK[Aha]NHSKVCPK 21.sub.5555 + T4 (-≡).sub.2-3 (FIG. 24), and ILKCQKGAT[Aha]KASEK[Aha]NHSKVCPK 21.sub.5555 + T4(-≡).sub.2-4 (FIG. 25).
Cyclizations with T6 Scaffolds
General Procedure T6 Cyclizations for Generation of Pentacyclic Peptides
(232) CLIPS: Linear peptide 22.sub.11111 (0.72 mg, 0.533 mol, 1 equiv) was dissolved in DMF/H.sub.2O (1:1) to give a 0.5 mM solution. Then, scaffold T6(−≡).sub.3.1 (43 μL, 0.426 μmol, 0.8 equiv from a 10 mM stock solution in DMF) was added. Subsequently, 40 μL of an aqueous NH.sub.4HCO.sub.3-solution (200 mM) was added to reach pH=8 in order to start the reaction.
(233) CuAAC: a pre-incubated mix of CuSO.sub.4/THPTA/Asc (2:2:10 equiv compared to linear peptide) in H.sub.2O was added to the CLIPS-mixture in order to start the CuAAC-reactions.
(234) peptides were cyclized using different T4 scaffolds to yield tetracyclic peptides. The UPLC-MS chromatogram of head-to-tail cyclized peptides, bicyclic CLIPS peptide and tetracyclic CLIPS/CuAAC peptides are shown in
(235) TABLE-US-00005 Ac-CQ[Aha]KCF[Aha]ACK[Aha]-NH.sub.2: 22.sub.11111 + T6(-≡).sub.3-1 (FIG. 26), Ac-CQW[Aha]KACFS[Aha]ATCKN[Aha]-NH.sub.2: 23.sub.22222 + T6-(≡).sub.3-1 (FIG. 27), H-CQWGA[Aha]KASECFSEK[Aha]ATKGCGNKG[Aha]-NH.sub.2: 24.sub.44444 + T6-(≡).sub.3-1 (FIG. 28), and H-CQWGAS[Aha]KASEVCFSEKG[Aha]ATKGKCGNKGE[Aha]-NH.sub.2: 25.sub.55555 + T6-(≡).sub.3-1 (FIG. 29).
Example 3. Identification of Biologically Active Tetracycle
(236) A peptide with sequence R[Aha]FRLPCRQLRCFRLP[Aha]RQL-OcamL (wherein OcamL is the recognition site of omnilase-1 enzyme) was enzymatically head-to-tail cyclized using omniligase-1 as described in Schmidt et al. 2017. This peptide was subsequently coupled to two different T4 scaffolds, T4(−≡).sub.2-3 and T4(−≡).sub.2-4, yielding tetracyclic peptides.
(237) Biological activity against coagulation factor XIIA, expressed as half maximal inhibitory concentration (IC50), was determined by a residual fluorescence assay as described in Baeriswyl et al 2015, Middendorp et al. 2017 and Heinis et al 2009. In brief, soluble recombinant human factor XIIA (Baeriswyl et al. 2015) was incubated with several dilutions of tetracyclic or bicyclic peptide (7 dilutions from 50 μM to 12 nM). After 15 min, a tripeptide with a cleavable fluorescent AMC group on the C-terminus (hence called as “substrate”) was added to the mixture. The enzyme factor XIIA is known to cleave the AMC group when no enzyme inhibitors are present leading to increased fluorescent signal. However, binding between the tetracyclic or bicyclic peptide will hinder the cleavage of the fluorescent label of the substrate by the enzyme, therefore leading to less fluorescent signal. Via non-linear regression an IC.sub.50 can be determined (which was carried out by the software GraphPath). A variant of a known bicyclic peptide inhibitor of coagulation factor XIIA (FXIIA618, Baeriswyl et al. 2015) having the same peptide sequence but attached to scaffold 1,3,5-tris-(bromomethyl)benzene (TBMB) instead of TATA was used for comparison of biological activity.
(238) As shown in
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