(2S)-6-amino-2-[(2-methylpropan-2-yl)oxycarbonylamino]hexanoic acid

Conditions: temp:55.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Attachment of N--Boc--Lys(Cl--z) to chloromethyl resin was performed by the cesium salt method. A sample of chloromethyl resin (200 g.) containing 0.74 mmol chloride per gram is treated with the cesium salt of Boc--Lys--(Cl--z) resulting from the neutralization of Boc--Lysine with cesium carbonate. About 73.8 grams of Boc--Lysine is dissolved in 80% methanol and 20% water and adjusted to pH 7.0 with about 29 grams of cesium carbonate. The resulting solution is dried on a rotary evaporator, then dried three more times after three additions of 100 milliliters of xylene. To the cesium salt of Boc--Lys(Cl--z) was added 200 grams of chloromethyl resin as above and sufficient 1-Methyl-2-Pyrrolidinone to make about 1.90 liters total volume. The resulting mixture is stirred at 55° C. for 48 hours. The resin was then washed extensively with methanol, then water, then again with methanol. The resin was air dried, then dried under vacuum. After cleavage of lysine from the resin with HF, amino acid analysis gave one peak corresponding to 0.36 mmol/gm. The resulting solution is dried on a rotary evaporator dried three more times after three additions of 100 milliliters of xylene To the cesium salt of Boc--Lys(Cl--z) was added 200 grams of chloromethyl resin as above and sufficient 1-Methyl-2-Pyrrolidinone The resin was then washed extensively with methanol dried dried under vacuum

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: To a suspension of 321 (0.26 g, 0.58 mmol) and N-Boc-lysine (1.1 g, 4.6 mmol) in dichloromethane (75 mL) and was added acetic acid (0.066 mL, 1.2 mmol). The reaction mixture was stirred for 1 h, then sodium triacetoxyborohydride (0.37 g, 1.7 mmol) was added and the resulting mixture stirred for 18 h. The mixture was then partitioned between water and dichloromethane, and the solid precipitate removed by suction filtration through celite. The product was mostly in the solid filter cake, so this was solubilized by washing with 1:1 dichloromethane/methanol. This solution was concentrated and the residue was purified by Gilson reverse phase HPLC (35-75% MeOH/H2O, Aquasil C18, 30 min) and lyophilized to yield BOC-protected product. This was dissolved in dichloromethane (75 mL) and trifluoroacetic acid (3 mL), and stirred at r.t. for 3 h. The mixture was concentrated and the residue was purified by Gilson reverse phase HPLC (35-75% MeOH/H2O, Aquasil C18, 30 min) and lyophilized to yield title compound 324 (44 mg, 69% yield). 1H NMR (DMSO-d6) δ (ppm) 1H: 9.02 (s, 1H); 8.66 (s, 1H); 8.53 (d, J=5.3, 1H); 8.35 (s, 1H); 8.28 (d, j=8.4, 1H); 7.98 (d, J=6.3, 1H); 7.72 (dd, J=13.5, 2.3, 1H); 7.37 (t, J=9.0, 1H); 7.21 (d, J=10.0, 1H); 6.89 (s, 1H); 6.68 (d, J=5.3, 1H); 4.00 (s, 2H); 2.75-2.70 (m, 2H); 2.55-2.52 (m, 1H); 2.45 (m, 1H); 1.70-1.30 (m, 6H); 0.67-0.62 (m, 2H); 0.44-0.40 (m, 2H). LRMS: 579.5 (M+H). the resulting mixture stirred for 18 h The mixture was then partitioned between water and dichloromethane the solid precipitate removed by suction filtration through celite filter cake by washing with 1:1 dichloromethane/methanol This solution was concentrated the residue was purified by Gilson reverse phase HPLC (35-75% MeOH/H2O, Aquasil C18, 30 min) to yield BOC-protected product The mixture was concentrated the residue was purified by Gilson reverse phase HPLC (35-75% MeOH/H2O, Aquasil C18, 30 min)

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: N-α-CBZ, N- -BOC-L-lysine and ethyl-2-pyrrolidineacetate are condensed by the procedure of Example 1, Step C to give N-α-CBZ, N- -BOC-L-lysyl-(S)-2-pyrrolidine acetic acid ethyl ester. The N-α-CBZ group is removed by hydrogenolysis in ethanol containing one equivalent of acetic acid over 10% palladium on charcoal utilizing the procedure of P. G. Katsoyannis and G. P. Schwartz, Methods in Enzymology, 47, 546 (1974), to give N- -BOC-L-lysyl-(S)-2-pyrrolidineacetic acid ethyl ester. condensed by the procedure of Example 1, Step C

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: N-α-CBZ, N- -BOC-L-lysine and ethyl-2-pyrrolidineacetate are condensed by the procedure of Example 1, Step C to give N-α-CBZ, N- -BOC-L-lysyl-(S)-2-pyrrolidine acetic acid ethyl ester. The N-α-CBZ group is removed by hydrogenolysis in ethanol containing one equivalent of acetic acid over 10% palladium on charcoal utilizing the procedure of P. G. Katsoyannis and G. P. Schwartz, Methods in Enzymology, 47, 546 (1974), to give N- -BOC-L-lysyl-(S)-2-pyrrolidineacetic acid ethyl ester. condensed by the procedure of Example 1, Step C

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: To the product of Step (B) above, the P-nitrophenyl ester of Boc-L-Glutamine [Gln (ONp), Peninsula, supra.] is coupled following the procedure of Step (E), Example 1, supra.

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: To the product of Step (B) above, the P-nitrophenyl ester of Boc-L-Glutamine [Gln (ONp), Peninsula, supra.] is coupled following the procedure of Step (E), Example 1, supra.

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Boc-Lys(N3)OH was synthesized from Boc-LysOH by using procedures for Cu(II)-catalyzed diazo-transfer to amines. (Alper, P. B.; Hung, S.-C.; Wong, C.-H. Tetrahedron Lett. 1996, 37, 6029-6032.) Boc-LysOH (4.38 g, 17.8 mmol), CuSO4.5H2O (44.7 mg, 178.2 □mol) and K2CO3 (3.69 g, 26.8 mmol) were dissolved in H2O (57 mL) and MeOH (114 mL). N3Tf (triflyl azide, 17.8 mmol in 150 mL CH2Cl2) was added, and the reaction mixture was stirred overnight. MeOH was removed under reduced pressure. The resulting aqueous solution (50 mL) was diluted with H2O (0.10 L), and the pH was adjusted to 6.0 with HCl. Sodium phosphate buffer (150 mL, 0.25 M, pH 6.2) was added, and the solution was washed with EtOAc (3×) to remove the triflic amine byproduct. The pH of the washed aqueous layer was adjusted to 2.0 with HCl, and the resulting solution was washed with EtOAc (4×) to extract the desired product. The organic layer was then dried over MgSO4(s) and filtered, and the solvent was removed under reduced pressure to yield Boc-Lys(N3)OH as a clear oil (50%). Spectral data. 1H NMR (500 MHz, CDCl3) δ:10.93 (broad s, 1H), 5.11 (d, J=8.3 Hz, 1H), 4.34 (m, 1H), 3.30 (d, J=7.0 Hz, 2H), 1.80 (m, 2H), 1.65 (m, 2H), 1.49 (m, 2H), 1.46 (s, 9H) ppm; 13C NMR (125 MHz, CDCl3) δ:179.96, 156.30, 97.44, 55.79, 53.79, 34.66, 31.07, 30.96, 25.20 ppm; MS (ESI) m/z 295.1383 (MNa+[C11H20N4O4Na]=295.1382). MeOH was removed under reduced pressure The resulting aqueous solution (50 mL) was diluted with H2O (0.10 L) Sodium phosphate buffer (150 mL, 0.25 M, pH 6.2) was added the solution was washed with EtOAc (3×) to remove the triflic amine byproduct the resulting solution was washed with EtOAc (4×) to extract the desired product The organic layer was then dried over MgSO4(s) filtered the solvent was removed under reduced pressure

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Boc-Lys(N3)OH was synthesized from Boc-LysOH by using procedures for Cu(II)-catalyzed diazo-transfer to amines. (Alper, P. B.; Hung, S.-C.; Wong, C.-H. Tetrahedron Lett. 1996, 37, 6029-6032.) Boc-LysOH (4.38 g, 17.8 mmol), CuSO4.5H2O (44.7 mg, 178.2 □mol) and K2CO3 (3.69 g, 26.8 mmol) were dissolved in H2O (57 mL) and MeOH (114 mL). N3Tf (triflyl azide, 17.8 mmol in 150 mL CH2Cl2) was added, and the reaction mixture was stirred overnight. MeOH was removed under reduced pressure. The resulting aqueous solution (50 mL) was diluted with H2O (0.10 L), and the pH was adjusted to 6.0 with HCl. Sodium phosphate buffer (150 mL, 0.25 M, pH 6.2) was added, and the solution was washed with EtOAc (3×) to remove the triflic amine byproduct. The pH of the washed aqueous layer was adjusted to 2.0 with HCl, and the resulting solution was washed with EtOAc (4×) to extract the desired product. The organic layer was then dried over MgSO4(s) and filtered, and the solvent was removed under reduced pressure to yield Boc-Lys(N3)OH as a clear oil (50%). Spectral data. 1H NMR (500 MHz, CDCl3) δ:10.93 (broad s, 1H), 5.11 (d, J=8.3 Hz, 1H), 4.34 (m, 1H), 3.30 (d, J=7.0 Hz, 2H), 1.80 (m, 2H), 1.65 (m, 2H), 1.49 (m, 2H), 1.46 (s, 9H) ppm; 13C NMR (125 MHz, CDCl3) δ:179.96, 156.30, 97.44, 55.79, 53.79, 34.66, 31.07, 30.96, 25.20 ppm; MS (ESI) m/z 295.1383 (MNa+[C11H20N4O4Na]=295.1382). MeOH was removed under reduced pressure The resulting aqueous solution (50 mL) was diluted with H2O (0.10 L) Sodium phosphate buffer (150 mL, 0.25 M, pH 6.2) was added the solution was washed with EtOAc (3×) to remove the triflic amine byproduct the resulting solution was washed with EtOAc (4×) to extract the desired product The organic layer was then dried over MgSO4(s) filtered the solvent was removed under reduced pressure

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A mixture of 2.37 g (10 mmol) of Nα -BOC-L-lysine and 1.65 g (11.0 mmol) of 3-fluoro-4-nitrotoluene in 10 mL of 1N NaOH and 10 mL of ethanol were heated at reflux for 48 hours, then cooled to room temperature. A precipitate was filtered off and discarded. Removal of the solvent/n vacuo gave an oil which was dissolved in 50 % ethanol/3M NH3 and taken to dryness. The residue was dissolved in 12 mL of 4N HCl in 33% acetic acid and stirred for four hours, then concentrated to dryness and purified by flash chromatography (eluting with 12:1:1 acetonitrile/water/HOAc). The resulting oil was recrystallized from water/methanol to afford 470 mg (42% yield) of the title product as bright orange prisms.: mp 234°-235° (dec.); MS M/Z (DCl/NH3): 282 [M+H]+ ; 1H NMR (300 MHz, D2O) δ: 1.1-1.45 (m, 6H), 2.78 (t, J=6.6 Hz, 2H), 2.99 (t, J=6.0 Hz, 1H), 5.88 (d, J=7.8 Hz, 1H), 6.07 (s, 1H), 7.32 (d, J=7.8 Hz, 1H). 13C NMR (75.5 MHz, D2O) δ: 185.73, 151.31,148.37, 131.02, 119.60, 116.09, 58.83, 45.20, 37.87, 31.19, 25.86; Analysis calc'd for C13H19N3O4 : C, 55.51;H, 6.81;N, 14.94; found: C, 55.25;H, 6.94; N, 14.75. were heated at reflux for 48 hours A precipitate was filtered off Removal of the solvent/n vacuo gave an oil which concentrated to dryness purified by flash chromatography (eluting with 12:1:1 acetonitrile/water/HOAc) The resulting oil was recrystallized from water/methanol

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Hexadecanedioic acid mono-tert-butyl ester can be activated with TSTU in similar fashion to the method described in Example 1, Step 6. The product can be reacted with Boc-Lys-OH in DMF at RT for 16 h. After concentrating under vacuum, AcOEt can be added the residue and the mixture can be washed with 0.2 N HCl. The organic phase is then dried over MgSO4 and concentrated under vacuum to yield the crude product, 15-((S)-5-tert-Butoxycarbonylamino-5-carboxypentylcarbamoyl)pentadecanoic acid tert-butyl ester. This can either be used without further purification or purified by flash chromatography. This compound can be activated with TSTU in similar fashion to the method described in Example 1, Step 6 to yield 15-[(S)-5-tert-butoxycarbonylamino-5-(2,5-dioxopyrrolidin-1-yloxycarbonyl) pentylcarbamoyl]pentadecanoic acid tert-butyl ester, and this compound can be coupled to insulin and deprotected to yield the title compound as described in Example 1, Step 7 using prepatative HPLC to isolate the product. After concentrating under vacuum, AcOEt can be added the residue the mixture can be washed with 0.2 N HCl The organic phase is then dried over MgSO4 concentrated under vacuum

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Hexadecanedioic acid mono-tert-butyl ester can be activated with TSTU in similar fashion to the method described in Example 1, Step 6. The product can be reacted with Boc-Lys-OH in DMF at RT for 16 h. After concentrating under vacuum, AcOEt can be added the residue and the mixture can be washed with 0.2 N HCl. The organic phase is then dried over MgSO4 and concentrated under vacuum to yield the crude product, 15-((S)-5-tert-Butoxycarbonylamino-5-carboxypentylcarbamoyl)pentadecanoic acid tert-butyl ester. This can either be used without further purification or purified by flash chromatography. This compound can be activated with TSTU in similar fashion to the method described in Example 1, Step 6 to yield 15-[(S)-5-tert-butoxycarbonylamino-5-(2,5-dioxopyrrolidin-1-yloxycarbonyl) pentylcarbamoyl]pentadecanoic acid tert-butyl ester, and this compound can be coupled to insulin and deprotected to yield the title compound as described in Example 1, Step 7 using prepatative HPLC to isolate the product. After concentrating under vacuum, AcOEt can be added the residue the mixture can be washed with 0.2 N HCl The organic phase is then dried over MgSO4 concentrated under vacuum

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Hexadecanedioic acid mono-tert-butyl ester can be activated with TSTU in similar fashion to the method described in Example 1, Step 6. The product can be reacted with Boc-Lys-OH in DMF at RT for 16 h. After concentrating under vacuum, AcOEt can be added the residue and the mixture can be washed with 0.2 N HCl. The organic phase is then dried over MgSO4 and concentrated under vacuum to yield the crude product, 15-((S)-5-tert-Butoxycarbonylamino-5-carboxypentylcarbamoyl)pentadecanoic acid tert-butyl ester. This can either be used without further purification or purified by flash chromatography. This compound can be activated with TSTU in similar fashion to the method described in Example 1, Step 6 to yield 15-[(S)-5-tert-butoxycarbonylamino-5-(2,5-dioxopyrrolidin-1-yloxycarbonyl) pentylcarbamoyl]pentadecanoic acid tert-butyl ester, and this compound can be coupled to insulin and deprotected to yield the title compound as described in Example 1, Step 7 using prepatative HPLC to isolate the product. After concentrating under vacuum, AcOEt can be added the residue the mixture can be washed with 0.2 N HCl The organic phase is then dried over MgSO4 concentrated under vacuum