Inhibition of glyoxalase I: the first low-nanomolar tight-binding inhibitors. Swati S. More ξ and Robert Vince*

S1 Inhibition of glyoxalase I: the first low-nanomolar tight-binding inhibitors Swati S. More ξ and Robert Vince* Center for Drug Design, Academic ealth Center, and Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota, 8-123A WD, 308 arvard St SE, Minneapolis, M 55455, USA Table of Contents General Experimental Procedures Boc-Glu(Bn)-Gly-tBu (7) Cl. 2 -Glu(Bn)-Gly-tBu (8) Boc-γ-Glu[-Glu(Bn)-Gly-tBu]-tBu (9) Boc-γ-Glu[-Glu()-Gly-tBu]-tBu (10) Boc-γ-Glu[-Glu(C()-p-bromophenyl)-Gly-tBu]-tBu (11) Cl. 2 -Glu(C 3 )- (13) Boc-Glu(C 3 )- (14) Boc-Glu(C 3 )-tbu (15) Boc-Glu()-tBu (16) p-br--phenylhydroxylamine (18) Boc-D-Glu(C 3 )- (20) Boc-D-Glu(C 3 )-Gly-tBu (21) Cl. 2 -D-Glu(C 3 )-Gly-tBu (22) Boc-γ-Glu[-D-Glu(C 3 )-Gly-tBu]-tBu (23) Boc-γ-Glu[-D-Glu(C()-p-bromophenyl))-Gly-tBu]-tBu (25) Boc-Glu(Bn)-All (27) Cl. 2 -Glu(Bn)-All (28) Boc-γ-Gla[-Glu(Bn)-All]-tBu (29) Boc-γ-Gla[-Glu(Bn)-Gly-tBu]-tBu (31) Boc-γ-Gla[-Glu()-Gly-tBu]-tBu (32) Boc-γ-Gla[-Glu(C()-p-bromophenyl)Gly-tBu]-tBu (33) S3 S3 S4 S4 S5 S5 S6 S6 S7 S7 S8 S8 S9 S10 S10 S11 S12 S12 S13 S14 S14 S15

S2 -(p-bromobenzoyl)--benzylhydroxylamine (41) Boc-se-All (42) Boc-al(γ-bromo)-All (43) Boc-al(γ--benzyloxy-4-bromobenzimidyl)-All (45) (S)-allyl 2-(tert-butoxycarbonylamino)-4-oxobutanoate (46) -Cbz--benzylhydroxylamine (49) Boc-Dab(-Cbz- -benzyloxy)-all (50) Cl. 2 -Dab(-Cbz- -benzyloxy)-all (51) Boc-γ-Glu[-al(γ-bromo)-All]-tBu (54) Boc-γ-Glu[-Dab(-Cbz- -benzyloxy)-all]-tbu (55) Boc-γ-Glu[-Dab(-Cbz- -benzyloxy)-gly-tbu]-tbu (57) Boc-γ-Glu[-Dab( 2 )-Gly-tBu]-tBu (58) Boc-Dab-C (60) Boc-Dab(Cbz)-C (61) Boc-Dab(Cbz)-Gly-tBu (62) Boc-γ-Glu[-Dab(Cbz)-Gly-tBu]-tBu (64) Boc-γ-Glu[-Dab( 2 )-Gly-tBu]-tBu (58) Boc-γ-Glu[-Dab(-(p-bromobenzoyl)- -benzoyloxy)-gly-tbu]-tbu (66) Boc-γ-Glu[-Dab(-(p-bromobenzoyl)- -hydroxyl)-gly-tbu]-tbu (67) Figure S1 Thin layer chromatograms of incubation aliquots of compounds 5 8 with equine kidney γ-glutamyltranspeptidase S16 S16 S17 S17 S18 S19 S19 S20 S20 S21 S22 S23 S23 S23 S24 S25 S25 S26 S27 S28

S3 General Experimental Procedures: All commercial chemicals were used as supplied unless otherwise indicated. Dry solvents (TF, Et 2, C 2 Cl 2 and DMF) were dispensed under argon from an anhydrous solvent system with two packed columns of neutral alumina or molecular sieves. Flash chromatography was performed with Silia-P flash silica gel (silicycle, 230-400 mesh) with indicated mobile phase. All reactions were performed under inert atmosphere of ultra-pure argon with oven-dried glassware. 1 and 13 C MR spectra were recorded on a Varian 300 Mz spectrometer. igh resolution mass data were acquired on Bruker Biotof II ESI-MS spectrometer capable of positive and negative ion sources, using PPG or PEG as internal standards. Boc-Glu(Bn)-Gly-tBu (7). To an ice cold C 2 Cl 2 (25 ml) solution of Boc-Glutamic acid 5-benzyl ester (3.00 g, 8.90 mmol), Bt ( 1.44 g, 10.68 mmol), EDC (2.05 g, Boc 7 CBn CtBu 10.68 mmol), Cl.Gly-tBu (2.23 g, 13.35 mmol) and MM (2.93 ml, 26.70 mmol) were added and the reaction mixture was allowed to warm to room temperature overnight. The reaction mixture was then diluted with dichloromethane (20 ml) and washed consecutively with 10% citric acid solution (30 ml), sat. ac 3 solution (2 20 ml) and brine; dried over a 2 S 4 and the residue obtained after evaporation of C 2 Cl 2 was purified by column chromatography with 20% EtAc/hexanes to isolate amide 7 as a colorless oil (3.64 g, 91% yield). R f 0.45 (EtAc/hexanes, 1:2); [α] D 5.6 (c 0.5, CCl 3 ); 1 MR (300 Mz, CDCl 3 ) δ 7.37 7.28 (m, 5, Ar), 6.83 (s, 1, ), 5.41 (d, J = 6.9 z, 1, ), 5.10 (s, 2, C 2 Ph), 4.24 (q, J = 7.2, 4.5 z, 1, α-c:glu), 3.97 3.81 (m, 2, C 2 :Gly), 2.59 2.39 (m, 2, γ-c 2 :Glu), 2.23 2.09 (m, 1, β-c A B :Glu), 2.05 1.87 (m, 1, β-c A B :Glu), 1.43, 1.40 (2s, 18, C(C 3 ) 3 ); 13 C MR (75 Mz, CDCl 3 ) δ 173.2 (CBn), 171.9 (C(=)Gly), 168.7 (CtBu), 155.8 (C(=)), 135.9, 128.7, 128.44, 128.40 (C Ar ), 82.4, 80.3 (C(C 3 ) 3 ), 66.8 (C 2 Ph), 53.9 (α-c), 42.3 (C 2 :Gly),30.8 (γ-c), 28.7, 28.4 (C(C 3 ) 3 ), 28.3 (β-c); ESI-RMS m/z 473.2260 (M+a) +, C 23 34 2 7 + a + requires 473.2264.

S4 Cl. 2 -Glu(Bn)-Gly-tBu (8). A solution of Cl/dioxane (12 ml, 4M) was added in one portion to a 25 ml round bottom flask containing 7 (1.00 g, 2.22 mmol); cooled CBn 3 Cl CtBu 8 with an ice-water bath. The ice-bath was then removed and the mixture was stirred for 20 min after which TLC indicated completion of the reaction. The reaction mixture was concentrated under high vacuum at room temperature. The residue obtained was triturated with dry ethyl ether and dried to obtain a fluffy white solid. (0.85 g, 99% yield); 1 MR (300 Mz, CDCl 3 ) δ 8.45 (s, 1, ), 8.24 (s, 3, 3 ), 7.29 7.24 (m, 5, Ar), 5.07 (s, 2, C 2 Ph), 4.62 4.52 (m, 1, α-c:glu), 4.01 3.71 (m, 2, C 2 :Gly), 2.77 2.62 (m, 2, γ-c 2 :Glu), 2.46 2.23 (m, 2, β-c 2 :Glu), 1.38 (s, 9, C(C 3 ) 3 ); 13 C MR (75 Mz, CDCl 3 ) δ 173.5 (CBn), 169.1 (C(=)Gly), 168.7 (CtBu), 135.7, 128.7, 128.5, 128.4 (C Ar ), 82.5 (C(C 3 ) 3 ), 67.4 (C 2 Ph), 53.0 (α- C:Glu), 42.5 (C 2 :Gly), 30.1 (γ-c:glu), 28.4 (C(C 3 ) 3 ), 26.7 (β-c:glu). Boc-γ-Glu[-Glu(Bn)-Gly-tBu]-tBu (9). EDC (0.76 g, 3.96 mmol) and Bt (0.54 g, 3.96 mmol) and MM (1.1 ml, 9.90 mmol) were added to an ice-cold solution of 16 Boc CtBu 9 CBn CtBu (1.00 g, 3.30 mmol) and Cl.Glu(Bn)-Gly-tBu (8) (1.28 g, 3.30 mmol) in C 2 Cl 2 (30 ml). The solution was allowed to stir for 12 h and was then poured into water (20 ml). The dichloromethane layer was washed with 10% citric acid solution (25 ml), sat. ac 3 solution (2 20 ml) and brine; dried over a 2 S 4 and the residue obtained after evaporation of C 2 Cl 2 was purified by column chromatography with 50% EtAc/hexanes to isolate product as a colorless oil (1.76 g, 84% yield). R f 0.50 (EtAc/hexanes, 1:1); [α] D 8.9 (c 1.048, CCl 3 ); 1 MR (300 Mz, CDCl 3 ) δ 7.29 7.21 (m, 5, Ar), 7.49 (s, 1, ), 7.19 (d, J = 8.1 z, 1, ), 5.41 (d, J = 7.8 z, 1, ), 4.64 (q, J = 8.1, 5.4 z, 1, α- C:Glu), 4.14 4.07 (m, 1, α-c:glu), 3.97 3.79 (m, 2, C 2 :Gly), 2.47 2.41 (m, 2, γ-c 2 :Glu), 2.36 2.29 (m, 2, γ-c 2 :Glu), 2.16 2.10 (m, 2, β-c 2 :Glu), 2.02 1.82 (m, 2, β-c 2 :Glu), 1.42 1.40 (3s, 27, C(C 3 ) 3 ); 13 C MR (75 Mz, CDCl 3 ) δ 173.1, 172.5, 171.6, 171.5, 168.7 (C(=)), 155.8 (C(=)), 135.9, 128.6, 128.5, 128.3

S5 (C Ar ), 82.1, 79.8 (C(C 3 ) 3 ), 66.6 (C 2 Ph), 53.8, 52.4 (α-c:glu), 42.2 (C 2 :Gly), 32.5, 30.6 (γ-c:glu), 28.6, 28.3, 28.2 (C(C 3 ) 3 ), 27.8, 27.1 (β-c:glu); ESI-RMS m/z 658.3347 (M+a) +, C 32 49 3 10 + a + requires 658.3316. Boc-γ-Glu[-Glu()-Gly-tBu]-tBu (10). A hundred milligrams of 10% Pd C was added to the methanolic solution of 9 (1.00 g, 1.57 mmol) which was hydrogenated on a Boc CtBu 10 parr-hydrogenator at 40 psi for 90 min. The reaction mixture was then filtered over celite and evaporated to dryness to obtain the product as an oil (0.82 g, 86% yield). 1 MR (300 Mz, CDCl 3 ) δ 9.49 (bs, 1, C), 7.49 (s, 1, ), 7.19 (d, J = 8.1 z, 1, ), 5.41 (d, J= 7.8 z, 1, ), 4.64 (q, J = 8.1, 5.4 z, 1, α-c:glu), 4.14 4.07 (m, 1, α- C:Glu), 3.97 3.79 (m, 2, C 2 :Gly), 2.47 2.41 (m, 2, γ-c 2 :Glu), 2.36 2.29 (m, 2, γ-c 2 :Glu), 2.16 2.10 (m, 2, β-c 2 :Glu), 2.02 1.82 (m, 2, β-c 2 :Glu), 1.42 1.40 (3s, 27, C(C 3 ) 3 ); 13 C MR (75 Mz, CDCl 3 ) δ 176.1 (C), 173.0, 172.0, 171.7, 168.8 (C(=), 156.1 (C(=)), 82.4, 80.2 (C(C 3 ) 3 ), 53.8, 52.4 (α-c:glu), 42.4 (C 2 :Gly), 32.6, 30.4 (γ-c:glu), 28.9, 28.7, 28.4 (C(C 3 ) 3 ), 28.3, 27.6 (β-c:glu); ESI- LRMS m/z 546.2 (M+) +. C CtBu Boc-γ-Glu[-Glu(C()-p-bromophenyl)-Gly-tBu]-tBu (11). To an ice cold C 2 Cl 2 (10 ml) solution of 10 (0.20 g, 0.37 mmol), p-br-ar 18 (0.21 g, 1.10 Boc CtBu 11 CtBu Br mmol) and EDC.Cl (0.11 g, 0.55 mmol) were added in one portion. The reaction mixture was allowed to stir at 4 C for 5 h. The residue obtained after evaporation of C 2 Cl 2 was partitioned between EtAc and 1 Cl; the EtAc layer was washed with brine; dried over a 2 S 4 and the crude product obtained after evaporation of EtAc was purified by silica gel chromatography with 60% EtAc/hexanes to isolate product as a light yellow oil (0.14 g, 52% yield). R f 0.15 (Acetone/hexanes, 1:2); [α] D 9.7 (c 0.35, CCl 3 ); 1 MR (300 Mz, CDCl 3 ) δ 9.23

S6 (bs, 1, ), 7.53, 7.38 (2bd, 4, Ar), 7.19 6.80 (m, 2, ), 5.19 (bs, 1, ), 4.52 (m, 1, α-c:glu), 4.09 (m, 1, α-c:glu), 3.89 3.80 (q, J = 8.7 z, 19.5 z, 2, C 2 :Gly), 2.57 1.97 (m, 8, γ-c 2 :Glu, β-c 2 :Glu), 1.39, 1.38 (s, 27, C(C 3 ) 3 ); 13 C MR (75 Mz, CDCl 3 ) δ 173.2, 172.8, 171.7, 171.6, 169.0 (C(=), 156.1 (C(=)), 140.0, 131.6, 122.6, 118.7 (C Ar ), 82.6, 80.4 (C(C 3 ) 3 ), 53.5, 52.5 (α-c:glu), 42.2 (C 2 :Gly), 32.2, 30.6, 29.5, 28.7 (β-c:glu, γ-c:glu), 28.5, 28.2, 28.1 (C(C 3 ) 3 ); ESI- RMS m/z 715.2515 (M+) +, C 31 47 Br 4 10 + + requires 715.2554. Cl. 2 -Glu(C 3 )- (13). To a suspension of L-Glutamic acid (12) (7.36 g, 0.05 mol) in dry methanol (150 ml) under argon was added chlorotrimethylsilane dropwise CC 3 3 C Cl 13 (11.95 g, 0.11 mol) over 5 min. The clear solution thus obtained was stirred at room temperature for 10 min. The methanolic solution was then evaporated to dryness to obtain a white solid which was dried under vacuum. 1 MR of the crude product (9.72 g, 97% yield) showed monomethylation as the major product with a trace (3%) of the dimethylated product. The crude product was used in the next step without further purification. Analytical data was obtained on pure product crystallized from hot methanol. mp 181 C (dec.) (lit. 182 C (dec.)); [α] D +29.2 (c 2.0, 6 Cl); 1 MR (300 Mz, CD 3 D) δ 4.04 (t, J = 6.9 z, 1, α-c), 3.69 (s, 3, CC 3 ), 2.58 (dt, J = 7.2, 2.4 z, 2, γ-c 2 ), 2.29-2.10 (m, 2, β-c 2 ). Boc-Glu(C 3 )- (14). In a round bottom flask, 5.00 g (25.30 mmol) of - Glu(Me)- (13) was dissolved in water (25 ml) at 0 C. Subsequently, 50 ml of 1, CC 3 Boc C 14 4-dioxane was added to the solution and stirred for 5 min. At 0 C, Boc 2 (6.64 g, 30.36 mmol) and ac 3 (5.33 g, 63.25 mmol) were added to the solution and stirred at rt overnight. The mixture was dried under reduced pressure, and the residue was dissolved in 10% aqueous ac 3, and washed with diethyl ether. The aqueous layer was neutralized to p 4 with solid citric acid from which -terminal protected Glu(Me) (14) was extracted with EtAc (3 75 ml). Combined EtAc layers were washed with water and brine; dried over a 2 S 4 ; evaporated to dryness to obtain the acid 14 (6.03 g, 91% yield). Rotamers

S7 are seen in proton and carbon MR. R f 0.6 (methanol); 1 MR (300 Mz, CDCl 3 ) δ 10.56 (bs, 1, C), 6.74, 5.21 (2s, 1, ), 4.35, 4.25 (2q, J = 7.5, 5.4 z, α-c), 3.68 (s, 3, CC 3 ), 2.56 2.37 (m, 2, γ-c 2 ), 2.29 2.18 (m, 1, β-c A B ), 1.98-2.09 (m, 1, β-c A B ), 1.44 (s, 9, C(C 3 ) 3 ); 13 C MR (75 Mz, CDCl 3 ) δ 175.6 (C), 173.6 (CC 3 ), 155.8 (C(=)), 80.4 (C(C 3 ) 3 ), 52.9 (α-c), 52.1 (C 3 ), 30.4 (γ-c), 28.6 (C(C 3 ) 3 ), 27.9 (β-c). Boc-Glu(C 3 )-tbu (15). To an ice-cold C 2 Cl 2 (100 ml) solution of DCC (4.74 g, 22.99 mmol), DMAP (0.23 g, 10 mol%) and tert-butyl alcohol ( 14.17 g, 0.192 mol) were CC 3 Boc Ct Bu 15 added. A C 2 Cl 2 (10 ml) solution of 14 (5.00 g, 19.16 mmol) was then added to the above solution dropwise over 15 min. After stirring the reaction mixture for 1 hr at 0 C and overnight at rt, the reaction mixture was concentrated in vacuo, the residue was taken up in EtAc (100 ml) and the suspension was filtered through Celite to remove DCU. The EtAc layer was then extracted with 10% citric acid solution (50 ml), sat. ac 3 solution (2 50 ml) and brine; dried over a 2 S 4. The solution was concentrated in vacuo, and the residue obtained was purified by flash column chromatography with 20% EtAc/hexanes (5.71 g, 94% yield). R f 0.6 (EtAc/hexanes, 1:2); 1 MR (300 Mz, CDCl 3 ) δ 5.10 (d, J = 7.8 z, 1, ), 4.11 (q, J = 7.2, 13.2 z, 1, α-c), 3.60 (s, 3, C 3 ), 2.42 2.23 (m, 2, γ-c 2 ), 2.13 2.02 (m, 1, β-c A B ), 1.90 1.78 (m, 1, β-c A B ), 1.39, 1.36 (2s, 18, C(C 3 ) 3 ); 13 C MR (75 Mz, CDCl 3 ) δ 173.3, 171.3 (C(=)), 155.4 (C(=)), 82.3, 79.9 (C(C 3 ) 3 ), 53.6 (α-c), 51.9 (C 3 ), 30.4 (γ-c), 28.6, 28.3 (C(C 3 ) 3 ), 28.1 (β-c); ESI-LRMS m/z 340.1 (M+a) +. Boc-Glu()-tBu (16). To a solution of 15 (0.3g, 0.66 mmol) in TF (3ml) was added 1M aqueous lithium hydroxide (1.3 ml, 1.3 mmol). The resulting solution was stirred at C Boc CtBu 16 room temperature for 45 minutes, after which the TLC (30% EtAc/hexanes) showed the absence of compound 15. The reaction mixture was evaporated to remove TF and then partitioned between diethyl ether and water. The aqueous layer was then neutralized to p 4 by solid

S8 citric acid and the precipitated acid was taken up in EtAc (3 50 ml). Combined EtAc layers were washed with water, brine; dried over MgS 4, and evaporated in vacuo, to leave 16 as a colorless oil which was used without further purification. Rotamers were seen in proton and carbon MR. The yield was 0.25g (84%). 1 MR (300 Mz, CDCl 3 ) δ 11.07 (bs, 1, C), 6.16, 5.26 (2d, J = 7.8 z, 1, ), 4.17, 4.00 (2q, J = 7.5 z, 5.1 z, 1, α-c), 2.48 2.29 (m, 2, γ-c 2 ), 2.17 2.05 (m, 1, β- C A B ), 1.94 1.80 (m, 1, β-c A B ), 1.43, 1.39 (2s, 18, C(C 3 ) 3 ); 13 C MR (75 Mz, CDCl 3 ) δ 178.3 (C), 171.4 (CtBu), 155.7 (C(=)), 82.7, 80.3 (C(C 3 ) 3 ), 53.5 (α-c), 30.5 (γ-c), 28.6, 28.3 (C(C 3 ) 3 ), 28.1 (β-c). p-br--phenylhydroxylamine (18). Wet, 5% rhodium on carbon (0.125 mg), TF (50 ml), and 4-bromonitrobenzene (17) (5.00 g) were introduced into a 250-mL, threenecked, round-bottomed flask fitted with a thermometer, and a Br 18 condenser. The mixture was cooled to 15 C and hydrazine hydrate (1.26 ml) was introduced into the reaction mixture dropwise over 10 min. The temperature of the mixture was maintained at 25 30 C throughout the addition by means of an ice water bath. The reaction mixture was allowed to stir for an additional 2 h at 25 30 C after which TLC indicated the completion of the reaction. The mixture was then filtered over celite and the catalyst was washed with a little TF. An equal volume of C 2 Cl 2 was added to the TF solution which was then dried over MgS 4 and concentrated to low volume under reduced pressure. Addition of a little petroleum ether precipitated -phenylhydroxylamine, which was filtered and washed with petroleum ether to obtain product as a white shiny solid (3.30 g, 71% yield). mp. 90 92 ºC (lit. 91 92 ºC); R f 0.6 (EtAc/hexanes 1:2); 1 MR (300 Mz, CDCl 3 ) δ 7.38 (d, J = 8.4 z, 2, Ar), 6.88 (d, J= 8.4 z, 2, Ar), 6.75 (s, 1, ), 5.45 (bs, 1, - ); 13 C (75 Mz, CDCl 3 ) δ 148.9, 131.9, 116.2, 114.6 (C Ar ). Boc-D-Glu(C 3 )- (20). To a suspension of D-Glutamic acid (3.68 g, 0.03 mol) in dry methanol (75 ml) under argon was added chlorotrimethylsilane dropwise (5.97 g, 0.05 mol) over 5 min. The clear solution thus formed was stirred at room temperature for 10 min. The methanol solution was then evaporated to dryness to obtain a white solid

S9 (4.84 g, 98% yield) which was dried under vacuum. The crude product was used in the next step without further purification. In a round bottom flask, 4.84 g (24.49 mmol) of -Glu(Me)- was dissolved in 1,4- dioxane/water (2:1, 75 ml) at 0 C. Subsequently, Boc 2 (6.43 g, 29.48 mmol) and C Boc CC 3 20 ac 3 (4.11 g, 48.98 mmol) were added to the solution and stirred at rt overnight. The mixture was dried under reduced pressure, and the residue was dissolved in 10% aqueous ac 3, and washed with diethyl ether. The aqueous layer was neutralized to p 4 with solid citric acid from which -terminal protected Glu(Me) (20) was extracted with EtAc. Rotamers are seen in proton and carbon MR (5.50 g, 86% yield). R f 0.6 (methanol); 1 MR (300 Mz, CDCl 3 ) δ 10.58 (bs, 1, C), 6.72, 5.19 (2s, 1, ), 4.31, 4.23 (2q, J = 7.5, 5.4 z, α-c), 3.65 (s, 3, C 3 ), 2.53 2.35 (m, 2, γ-c 2 ), 2.26 2.17 (m, 1, β-c A B ), 2.09 1.98 (m, 1, β-c A B ), 1.41 (s, 9, C(C 3 ) 3 ); 13 C MR (75 Mz, CDCl 3 ) δ 175.0 (C), 172.6 (CC 3 ), 155.5 (C(=)), 80.1 (C(C 3 ) 3 ), 52.5 (α-c), 52.4 (C 3 ), 30.1 (γ-c), 28.9 (C(C 3 ) 3 ), 27.6 (β-c). Boc-D-Glu(C 3 )-Gly-tBu (21). To an ice-cold C 2 Cl 2 (25 ml) solution of Boc- Glutamic acid 5-methyl ester (20) (3.00 g, 11.48 mmol), Bt ( 1.86 g, 13.77 mmol), Boc 21 CC 3 Ct Bu EDC (2.64 g, 13.77 mmol), Cl.Gly-tBu ( 2.88 g, 17.22 mmol) and MM (3.78 ml, 34.44 mmol) were added and the reaction mixture was allowed to warm to rt overnight. Then reaction mixture was diluted with C 2 Cl 2 (20 ml) and washed consecutively with 10% citric acid solution (30 ml), sat. ac 3 solution (2 20 ml) and brine; dried over a 2 S 4 and the residue obtained after evaporation of C 2 Cl 2 was purified by column chromatography with 20% EtAc/hexanes to isolate product as a colorless oil. (3.78 g, 88% yield); R f 0.3 (EtAc/hexanes, 1:2); [α] D +4.5 (c 0.64, CCl 3 ); 1 MR (300 Mz, CDCl 3 ) δ 6.99 (s, 1, ), 5.51 (d, J = 6.9 z, 1, ), 4.24 4.16 (m, 1, α-c:glu), 3.92 3.75 (m, 2, C 2 :Gly), 3.59 (s, 3, C 3 ), 2.48 2.33 (m, 2, γ-c 2 :Glu), 2.17 2.03 (m, 1, β-c A B :Glu), 1.93 1.83 (m, 1, β- C A B :Glu), 1.37, 1.35 (2s, 18, C(C 3 ) 3 ); 13 C MR (75 Mz, CDCl 3 ) δ 173.8, 171.9,

S10 168.7 (C(=)), 155.8 (C(=)), 82.2, 80.1 (C(C 3 ) 3 ), 53.8 (α-c:glu), 51.9 (C 3 ), 42.2 (C 2 :Gly), 30.5 (γ-c:glu), 28.6, 28.4 (C(C 3 ) 3 ), 28.3 (β-c:glu); ESI-RMS m/z 375.2148 (M+) +, C 17 30 2 7 + + requires 375.2131. Cl. 2 -D-Glu(C 3 )-Gly-tBu (22). A solution of Cl/dioxane (12 ml, 4M) was added in one portion to a 25 ml round bottom flask containing 21 (2.00 g, 5.34 mmol); 3 Cl 22 CC 3 C tbu cooled with an ice-water bath. Then the ice-bath was removed and the mixture was stirred for 20 min after which the TLC indicated the completion of the reaction. The reaction mixture was concentrated under high vacuum at room temperature. The residue obtained was triturated with dry ethyl ether and dried to obtain a fluffy white solid which was used immediately in the next step. (1.57 g, 95% yield); 1 MR (300 Mz, CD 3 D) δ 8.45 (s, 1, ), 8.24 (s, 3, 3 ), 4.62 4.52 (m,1, α-c:glu), 4.01 3.71 (m, 2, C 2 :Gly), 3.72 (s, 3, C 3 ), 2.77 2.62 (m, 2, γ-c 2 :Glu), 2.46 2.23 (m, 2, β-c 2 :Glu), 1.41 (s, 9, C(C 3 ) 3 ); 13 C MR (75 Mz, CD 3 D) δ 174.0, 169.1, 168.7 (C(=)), 82.4 (C(C 3 ) 3 ), 53.0 (α-c:glu), 52.4 (C 3 ), 42.5 (C 2 :Gly), 29.9 (γ-c:glu), 28.4 (C(C 3 ) 3 ), 26.8 (β-c:glu); ESI-LRMS m/z 275.1 (M+) +. Boc-γ-Glu[-D-Glu(C 3 )-Gly-tBu]-tBu (23). EDC (0.76 g, 3.96 mmol) and Bt (0.54 g, 3.96 mmol) and MM (1.1 ml, 9.90 mmol) were added to an ice-cold solution Boc CtBu 23 CC 3 CtBu of 16 (1.00 g, 3.30 mmol) and Cl.Glu(Me)-Gly- tbu (22) (1.23 g, 3.96 mmol) in C 2 Cl 2 (30 ml). The solution was allowed to stir for 12 h before being poured into water (20 ml). The dichloromethane layer was washed with 10% citric acid solution (25 ml), sat. ac 3 solution (2 20 ml) and brine; dried over a 2 S 4 and the residue obtained after evaporation of C 2 Cl 2 was purified by column chromatography with 50% EtAc/hexanes to isolate product 23 as a colorless oil. (0.63 g, 34% yield); R f 0.35 (EtAc/hexanes, 1:1); [α] D +14.7 (c 1.05, CCl 3 ); 1 MR (300 Mz, CDCl 3 ) δ 7.29 7.21 (m, 5, Ar), 7.49 (s, 1, ), 7.19 (d, J = 8.1 z, 1, ), 5.41 (d, J = 7.8 z, 1, ), 4.64 (q, J = 8.1 z, 5.4 z, 1, α-c:glu), 4.14 4.07 (m, 1, α-c:glu), 3.97

S11 3.79 (m, 2, C 2 :Gly), 2.47 2.41 (m, 2, γ-c 2 :Glu), 2.36 2.29 (m, 2, γ-c 2 :Glu), 2.16 2.10 (m, 2, β-c 2 :Glu), 2.02 1.82 (m, 2, β-c 2 :Glu), 1.42 1.40 (3s, 27, C(C 3 ) 3 ); 13 C MR (75 Mz, CDCl 3 ) δ 174.6, 172.2, 171.6, 171.4, 168.7 (C(=)), 156.0 (C(=)), 82.4, 82.0, 80.1 (C(C 3 ) 3 ), 53.3, 53.1 (α-c:glu), 52.1 (C 3 ), 42.2 (C 2 :Gly), 32.4, 30.6 (γ-c:glu), 29.3, 28.6, 28.4 (C(C 3 ) 3 ), 28.3, 27.5 (β-c:glu); ESI- RMS m/z 560.3210 (M+) +, C 26 46 3 10 + + 560.3183. Boc-γ-Glu[-D-Glu(C()-p-bromophenyl))-Gly-tBu]-tBu (25). 2 Li (0.9 ml, 1.78 mmol) in 2 was added to the clear solution of tripeptide 23 (0.50 g, 0.89 mmol) in 1:1 TF/ 2 (10 ml). The resulting twophase mixture was vigorously stirred for 2 h after Boc CtBu 25 CtBu Br which TF was evaporated in vacuo, and the resulting aqueous layer was poured into 30 ml of diethyl ether and 30 ml of 10% a 2 C 3 solution. The aqueous layer was separated and then neutralized with solid citric acid. The precipitated acid was extracted with EtAc (3 40 ml). Combined EtAc layers were washed with brine; dried over a 2 S 4 and evaporated to give the product acid 24 as a colorless oil (0.44 g, 91% yield). The product was used immediately in the next coupling step. To a solution of carboxylic acid 24 (400 mg, 0.73 mmol) and p-bromophenyl hydroxylamine 18 (0.68 g, 3.66 mmol) in C 2 Cl 2 (10 ml) cooled to 0 ºC was added EDC (280 mg, 1.46 mmol) in one portion and the reaction was stirred in ice-water bath for 6 h. The reaction mixture was then diluted with C 2 Cl 2 and washed with 1 Cl and brine. The organic layer was separated; dried over a 2 S 4 and the crude product obtained after evaporation of C 2 Cl 2 was purified by silica gel chromatography with 60% EtAc/hexanes to isolate product as a light yellow oil (0.32 g, 62% yield). R f 0.35 (EtAc/hexanes, 7:3); [α] D +12.0 (c 0.25, CCl 3 ); 1 MR (300 Mz, CDCl 3 ) δ 9.23 (bs, 1, ), 7.58 (d, J = 3.9 z, 2, Ar), 7.42 (d, J = 4.2 z, 2, Ar), 7.01 (m, 2, ), 5.35 5.27 (bs, 1, ), 4.60 (q, J = 4.2 z, 6.6 z, 1, α-c:glu), 4.14 (m, 1, α-c:glu), 3.98 3.75 (m, 2, C 2 :Gly), 2.49 1.68 (m, 8, γ-c 2 :Glu, β-c 2 :Glu),

S12 1.44, 1.43, 1.40 (3s, 27, C(C 3 ) 3 ); 13 C MR (75 Mz, CDCl 3 ) δ 173.2, 172.8, 171.7, 171.6, 169.0 (C(=)), 156.1 (C(=)), 140.0, 131.6, 122.6, 118.7 (C Ar ), 82.6, 80.4 (C(C 3 ) 3 ), 53.5, 52.5 (α-c:glu), 42.2 (C 2 :Gly), 32.2, 30.6, 29.5, 28.7 (β-c:glu, γ- C:Glu), 28.5, 28.2, 28.1 (C(C 3 ) 3 ); ESI-RMS m/z 715.2518 (M+) +, C 31 47 Br 4 10 + + requires 715.2554. Boc-Glu(Bn)-All (27). ünig s base (3.1 ml, 17.80 mmol) and allyl bromide (3.87 ml, 44.51 mmol) were added to a solution of Boc-Glu(Bn)- (6) (3.00 g, 8.90 mmol) BnC AllC 27 Boc in dry benzene (50 ml), and the mixture was heated at reflux for 4 h. After being cooled to rt, the reaction mixture was evaporated to dryness. The residue obtained after evaporation of benzene was dissolved in EtAc (200 ml); washed with 10% aqueous citric acid (3 50 ml), 5% aqueous ac 3 (3 50 ml), brine (3 50 ml), dried (a 2 S 4 ) and concentrated in vacuo. The residue was dissolved in hexanes/etac (2:1, 50 ml) and passed through a silica gel plug (10 g). The plug was eluted further with a 100 ml of the same solvent mixture. Combined eluents were evaporated to give 27 as a viscous oil (5.2 g, 92% yield). R f 0.5 (EtAc/hexanes, 1:4); 1 MR (300 Mz, CDCl 3 ) δ 7.35 (s, 5, Ar), 5.91 5.83 (m, 1, C=C 2 ), 5.31 (dd, J= 5.6 z, 2, C=C 2 ), 5.12 (s, 2, C 2 Ph), 4.62 (d, J = 5.8 z, 2, C 2 C), 4.38 (m, 1, α-c), 2.50 2.46 (m, 2, γ- C 2 ), 2.24 (m, 1, β-c A B ), 1.96 (m, 1, β-c A B ), 1.43 (s, 9, C(C 3 ) 3 ); 13 C MR (75 Mz, CDCl 3 ) δ 172.6, 171.9 (C(=)), 155.5 (C(=)), 135.9, 131.7, 128.7, 128.4, 128.4 (C Ar, C=C 2 ), 119.1 (C=C 2 ), 80.2 (C(C 3 ) 3 ), 66.7 (C 2 Ph), 66.3 (C 2 C), 53.2 (α-c), 30.6 (γ-c), 28.6 (C(C 3 ) 3 ), 28.0 (β-c); ESI-LRMS m/z 378.1 (M+) +. Cl. 2 -Glu(Bn)-All (28). The Boc-protected amine 27 (2.0 g, 5.30 mmol) was dissolved in 4 Cl in dioxane (10 ml) and stirred at room temperature for 1 h. After BnC removal of all volatiles at reduced pressure, the residue was triturated AllC 28 3 Cl with Et 2 (3 10 ml) providing the hydrochloride salt 28 (1.59 g, 96% yield). 1 MR (300 Mz, CD 3 D) δ 7.47 7.35 (s, 5, Ar), 6.03 5.93 (m, 1, C=C 2 ), 5.42 (m, 2, C=C 2 ), 5.21 (s, 2, C 2 Ph), 4.75 (d, J = 6.0 z, 2, C 2 C), 3.45 (m, 1, α-c), 2.45 2.35 (m, 2, γ-c 2 ), 2.29 (m, 1, β-

S13 C A B ), 2.01 (m, 1, β-c A B ), 1.43 (s, 9, C(C 3 ) 3 ); 13 C MR (75 Mz, CD 3 D) δ 173.1, 172.3 (C(=)), 135.9, 131.7, 128.7, 128.4, 128.4 (C Ar, C=C 2 ), 118.9 (C=C 2 ), 80.1 (C(C 3 ) 3 ), 66.4 (C 2 Ph), 65.7 (C 2 C), 53.1 (α-c), 30.6 (γ-c), 29.1 (β-c); ESI-LRMS m/z 278.1 (M+) +. Boc-γ-Gla[-Glu(Bn)-All]-tBu (29). An ice-cold dichloromethane solution of CDI (0.57 g, 3.50 mmol) was stirred under argon for 5 min before adding via cannula a ButC Boc 29 CBn dichloromethane solution of 28 (1.00 g, 3.18 mmol) and 0.87 ml of MM (7.96 mmol) over 20 min. The reaction mixture was stirred further for 15 min. A C 2 Cl 2 solution of the second amine 38 (1.24 g, 4.78 mmol) and 0.87 ml of MM (7.96 mmol) was then added to the reaction mixture in one portion and the resulting solution was then allowed to stir at rt overnight. The reaction mixture was then poured in separating funnel and washed with 10% citric acid solution (2 20 ml) and brine. The organic layer was then dried over a 2 S 4 and the residue obtained was purified by silica gel chromatography with 60% EtAc/hexanes, to obtain product as a colorless oil (1.47 g, 82% yield). R f 0.3 (EtAc/hexanes, 2:3); [α] D 9.3 (c 1.0, CCl 3 ); 1 MR (300 Mz, CDCl 3 ) δ 7.33 (s, 5, Ar), 5.93 5.79 (m, 1, C=C 2 ), 5.61 (d, J = 7.2 z, 1, ), 5.47 (d, J = 7.8 z, 1, ), 5.32 5.20 (m, 2, C=C 2 ), 5.09 (s, 2, C 2 Ph), 4.58 (d, J = 5.7 z, 2, C 2 C), 4.52 4.45 (m, 1, α-c:gla), 4.21 4.12 (m, 1, α-c:glu), 3.51 (m, 2, β-c 2 :Gla), 2.50 2.45 (m, 2, γ-c 2 :Glu), 2.25 2.13 (m, 1, β-c A B :Glu), 2.03 1.94 (m, 1, β-c A B :Glu), 1.43, 1.41 (2s, 18, C(C 3 ) 3 ); 13 C MR (75 Mz, CDCl 3 ) δ 172.9, 172.7, 170.1 (C(=)), 157.7, 155.9 (C(=)), 135.9, 131.7, 128.7, 128.4, 124.5 (C Ar, C=C 2 ), 119.0 (C=C 2 ), 82.7, 80.2 (C(C 3 ) 3 ), 66.7 (C 2 Ph), 66.3 (C 2 ), 55.4 (α-c:gla), 52.9 (α- C:Glu), 42.9 (β-c:gla), 30.7 (β-c:glu), 28.6, 28.3 (C(C 3 ) 3 ), 28.2 (β-c:glu); ESI- RMS m/z 564. 2940 (M+) +, C 28 41 3 9 + + requires 564.2920. Boc-γ-Gla[-Glu(Bn)-Gly-tBu]-tBu (31). (Ph 3 P) 4 Pd (0.20 g, 10 mol%) was added under nitrogen to a solution of allyl ester 29 (1.00 g, 1.77 mmol) in C 2 Cl 2 (20 ml) at

S14 room temperature and the resulting mixture was treated dropwise with (1.58 ml, 17.76 mmol) morpholine. After completion of the reaction (1 h) as judged by TLC, solvent was removed in vacuo. The residue was redissolved in EtAc (20 ml) and the resulting solution was washed with 1 Cl (15 ml) and water (15 ml). The organic layer was dried over a 2 S 4 and evaporated in vacuo; purified by flash chromatography with C 2 Cl 2 /C 3 (4:1) to obtain the acid 30 which was used in the next coupling step. EDC (0.41 g, 2.13 mmol), Bt (0.29 g, 2.13 mmol) and MM (0.58 ml, 5.33 mmol) were added to an ice-cold solution of 30 (0.50 g, 1.01 mmol) and Cl.Gly-tBu (0.59 g, But C Boc 31 CBn Ct Bu 3.55 mmol) in C 2 Cl 2 (20 ml). The solution was allowed to stir for 12 h before being poured into water (20 ml). The dichloromethane layer was washed with 10% citric acid solution (25 ml), sat. ac 3 solution (2 20 ml) and brine; dried over a 2 S 4 and the residue obtained after evaporation of C 2 Cl 2 was purified by column chromatography with 50% EtAc/hexanes to isolate product as a colorless oil (0.85 g, 76% yield). R f 0.50 (EtAc/hexanes, 3:2); [α] D 5.1 (c 1.0, CCl 3 ); 1 MR (300 Mz, CDCl 3 ) δ 7.32 (s, 5, Ar), 7.07 (s, 1, ), 6.09 (s, 1, ), 5.89 (s, 1, ), 5.74 (d, J = 7.2 z, 1, ), 5.09 (s, 2, C 2 Ph), 4.38 (q, J = 7.8, 13.2 z, 1, α-c:gla), 4.18 4.12 (m, 1, α-c:glu), 3.87 (t, J = 5.7 z, 2, C 2 :Gly), 3.54 3.44 (m, 2, β-c 2 :Gla), 2.58 2.44 (m, 2, γ-c 2 :Glu), 2.20 2.09 (m, 1, β- C A B :Glu), 1.98 1.91 (m, 1, β-c A B :Glu), 1.46, 1.43, 1.40 (3s, 27, C(C 3 ) 3 ); 13 C MR (75 Mz, CDCl 3 ) δ 173.4, 172.8, 170.3, 168.8 (C(=)), 158.3, 155.9 (C(=)), 135.9, 128.7, 128.4 (C Ar ), 82.5, 82.3, 80.0 (C(C 3 ) 3 ), 66.3 (C 2 Ph), 55.4 (α-c:gla), 53.5 (α-c:glu), 42.7 (β-c:gla), 42.4 (C 2 :Gly), 30.8 (γ-c:glu), 28.7, 28.4 (C(C 3 ) 3 ), 28.3 (β-c:glu); ESI-RMS m/z 637.3440 (M+) +, C 31 48 4 10 + + requires 637.3449. Boc-γ-Gla[-Glu()-Gly-tBu]-tBu (32). A solution of benzyl ester tripeptide 31 (0.60 g, 0.94 mmol) in anhydrous Me (25 ml) was treated with Pd C (100 mg) and stirred under hydrogen pressure 55 psi. at rt for 2 h. The suspension was filtered through a fine-porosity filter paper; then through a 45 µm PFTE filter and washed twice with

S15 methanol. Combined organic layers were concentrated under reduced pressure to obtain the tripeptide acid 32 as a white foam (0.46 g, 89% yield). 1 MR (300 Mz, CDCl 3 ) δ ButC Boc 32 C 10.72 (s, 1, C), 7.56 (s, 1, ), 6.40, 6.27 (2s, 1, ), 5.91 5.80 (m, 2, ), 4.48 (m, 1, α-c:gla), 4.18, 4.03 (2m, 1, α-c:glu), 3.93 3.82 (m, 2, C 2 :Gly), 3.58 3.46 (m, 2, β- C 2 :Gla), 2.52 2.42 (m, 2, γ-c 2 :Glu), 2.16 2.12 (m, 1, β-c A B :Glu), 1.98 1.91 (m, 1, β-c A B :Glu), 1.46 1.39 (3s, 27, C(C 3 ) 3 ); 13 C MR (75 Mz, CDCl 3 ) δ 176.4 (C), 173.5, 170.5, 168.9 (C(=)), 158.7, 156.2 (C(=)), 82.6, 82.4, 80.1 (C(C 3 ) 3 ), 55.4 (α-c:gla), 53.4 (α-c:glu), 42.5 (β-c:gla), 42.3 (C 2 :Gly), 30.4 (γ- C:Glu), 28.5 (β-c:glu), 28.2, 28.1 (C(C 3 ) 3 ). CtBu Boc-γ-Gla[-Glu(C()-p-bromophenyl)Gly-tBu]-tBu (33). To a solution of carboxylic acid 32 (0.40 g, 0.73 mmol) and p-bromophenyl hydroxylamine 18 (0.68 g, ButC Boc 33 CtBu Br 3.66 mmol) in dichloromethane (10 ml) cooled to 0 ºC was added EDC (0.28 g, 1.46 mmol) in one portion and the reaction was stirred in icewater bath for 6 h. The reaction mixture was then diluted with C 2 Cl 2 and washed with 1 Cl and brine; the organic layer was separated; dried over a 2 S 4 and the crude product obtained after evaporation of C 2 Cl 2 was purified by silica gel chromatography with 60% EtAc/hexanes to isolate product as a light yellow oil (0.33 g, 64% yield). R f 0.25 (EtAc/hexanes, 7:3); [α] D 15.0 (c 0.5, CCl 3 ); 1 MR (300 Mz, CDCl 3 ) δ 9.77, 9.58 (bs, 1, ), 7.51, 7,37 (m, 4, Ar), 6.40, 6.03, 5.82 (m, 3, ), 4.61 (m, 1, α-c:gla), 4.22 (m, 1, α-c:glu), 3.98 3.76 (m, 2, C 2 :Gly), 3.60 3.42 (m, 2, β- C 2 :Gla), 2.42 1.94 (m, 4, γ-c 2 :Glu, β-c 2 :Glu), 1.41, 1.40, 1.37 (3s, 27, C(C 3 ) 3 ); 13 C MR (75 Mz, CDCl 3 ) δ 173.2, 172.8, 171.7, 171.6, 169.0 (C(=), 156.1 (C(=)), 140.0, 131.6, 122.6, 118.7 (C Ar ), 82.6, 80.4 (C(C 3 ) 3 ), 53.5 (α-c:gla), 52.5 (α-c:glu), 43.1 (β-c:gla), 42.2 (C 2 :Gly), 32.2, 30.6, 29.5, 28.7 (β-c:glu, γ-

S16 C:Glu), 28.5, 28.2, 28.1 (C(C 3 ) 3 ); ESI-RMS m/z 716.2480 (M+) +, C 30 46 Br 5 10 + + requires 716.2506. -(p-bromobenzoyl)--benzylhydroxylamine (41). To a stirred solution of p- bromobenzoyl chloride (2.50 g, 11.39 mmol) in C 2 Cl 2 (360 ml) was added - Br Bn 41 benzylhydroxylamine hydrochloride (2.00 g, 12.53 mmol) under a nitrogen atmosphere at room temperature. After the solution was stirred at the same temperature for 15 min, pyridine (2.02 ml, 25.06 mmol) was added dropwise to the reaction mixture at 0 ºC. After 2 h at room temperature, the reaction mixture was diluted with C 2 Cl 2 and washed with 1 Cl and brine. The organic phase was dried over a 2 S 4 and concentrated under reduced pressure. Purification of the residue by flash chromatography (hexane/acet 2:1) afforded the hydroxamate 41. (2.54 g, 73% yield); R f 0.6 (EtAc/hexanes, 1:4); 1 MR (300 Mz, CDCl 3 ) δ 7.48 7.24 (m, 9, Ar), 4.86 (s, 2, C 2 Ph), 3.83 (s, 1, ); 13 C MR (75 Mz, CDCl 3 ) δ 165.7 (C(=)), 135.3 126.6 (C Ar ), 78.2 (C 2 Ph); ESI- LRMS m/z 328.0 (M+a) +. Boc-se-All (42). To a solution of L-omoserine 40 (1.00 g, 8.40 mmol) in 2 (10 ml) and acetonitrile (10 ml), a (336 mg, 8.40 mmol) and tert-butyl dicarbonate Boc CAll 42 (2.02 g, 9.24 mmol) were added at room temperature. The resulting solution was allowed to stir for 14 h. Then reaction mixture was evaporated to dryness to remove all the solvents. The resulting white solid was triturated with diethyl ether twice and dried under vacuum for 1 h. The solid thus obtained was dissolved in DMF (25 ml) to which allyl bromide (798 µl, 9.24 mmol) was added and the reaction mixture was stirred for 24 h. After reducing the solvent volume, the residue was dissolved in EtAc and washed with sat. ac 3 (2 30 ml), 0.1 M KS 4 solution (2 30 ml) and brine. The EtAc layer was dried over a 2 S 4, filtered and evaporated to obtain product 42 as a viscous oil (1.3 g, 60 % yield) which was used in the next step without further purification. R f 0.4 (EtAc/hexanes, 3:7); [α] D 31.1 (c 1.0, C 3 ); 1 MR (300 Mz, CDCl 3 ) δ 5.79 (m, 1, C=C 2 ), 5.40 (d, J = 3.6 z, 1, ), 5.23 (d, J = 9.3 z, 1, C=C 2 ), 5.14 (d, J = 5.4 z, 1,

S17 C=C 2 ), 4.53 (m, 2, C 2 C), 4.36 (m, 1, α-c), 3.60 (m, 2, γ-c 2 ), 3.42 (bs, 1, ), 2.02 (m, 1, β-c A B ), 1.63 (m 1, β-c A B ), 1.33 (s, 9, C(C 3 ) 3 ); 13 C MR (75 Mz, CDCl 3 ) δ 172.7, 156.4 (C(=)), 131.7 (C=C 2 ), 118.8 (C=C 2 ), 80.3 (C(C 3 ) 3 ), 66.1 (C 2 C), 58.5 (C 2 ), 51.1 (α-c), 35.6 (β-c), 28.4 (C(C 3 ) 3 ). Boc-al(γ-bromo)-All (43). To a C 2 Cl 2 (25 ml) solution of homoserine alcohol 42 (2.00 g, 7.72 mmol), methanesulfonyl chloride (717 µl, 9.26 mmol) and triethylamine Br Boc CAll 43 (1.39 ml, 10.0 mmol) were added. The resulting solution was stirred at room temperature for 95 min. Lithium bromide (6.71 g, 77.2 mmol) and acetone (25 ml) were then added, and the mixture was stirred for an additional 14 h. The solvents were removed; the residue was dissolved in EtAc (200 ml) and poured into a separatory funnel. The organic layer was washed with 2 (3 80 ml), sat. ac 3 solution (80 ml), and brine (80 ml) and then dried. The solvent was removed, and column chromatography of the residue (EtAc/hexanes, 20:80 to 30:70) afforded 2.34 g (7.25 mmol, 94% yield) of 43 as a white solid. mp 50 51 ºC; [α] D 28.6 (c 1.0, CCl 3 ); R f 0.6 (EtAc/hexanes, 3:7); 1 MR (300 Mz, CDCl 3 ) δ 5.87 (m, 1, C=C 2 ), 5.27 (dd, J = 1.2 z, 16.8 z, 2, C=C 2 ), 5.23 (bd, J = 6.3 z, 1, ), 4.62 (d, J = 6 z, 2, C 2 C), 4.41 (m, 1, α-c), 3.42 (t, J = 7.2 z, 2, γ-c 2 ), 2.39 (m, 1, β-c A B ), 2.20 (m, 1, β-c A B ), 1.42 (s, 9, C(C 3 ) 3 ); 13 C MR (75 Mz, CDCl 3 ) δ 171.6 (CAll), 155.4 (C(=)), 131.5 (C=C 2 ), 119.3 (C=C 2 ), 80.5 (C(C 3 ) 3 ), 66.5 (C 2 C), 52.8 (α-c), 36.1 (β-c), 28.6 (C(C 3 ) 3 ); ESI-LRMS m/z 344.1 (M+a) +. Boc-al(γ--benzyloxy-4-bromobenzimidyl)-All (45). A flask was charged with 41 (500 mg, 1.63 mmol) and anhydrous DMF (5 ml) to which sodium hydride (60% dispersion in mineral oil, 63 mg, 1.57 mmol) was added, and the mixture was stirred for 30 min at room temperature. The clear solution thus obtained was cooled to 0 ºC followed by the addition of compound 43 (438 mg, 1.36 mmol). The sides of the flask were rinsed with an additional DMF (5 ml), and the reaction mixture was stirred for 30 min at 0 ºC. The ice bath was removed, and the reaction was allowed to warm to rt;

S18 stirred for an additional 3.5 h. The reaction mixture was poured into a separatory funnel and diluted with EtAc (125 ml). The organic layer was washed with 10% citric acid (3 Bn Boc CAll Br 45 50 ml) and brine (50 ml) and then dried. The solvent was removed, and column chromatography of the residue (EtAc/hexanes, 20:80 to 30:70) afforded 0.48 g (yield 64%) of the -alkylated product 45. R f 0.55 (EtAc/hexanes, 3:7); [α] D 21.4 (c 1.0, CCl 3 ); 1 MR (300 Mz, CDCl 3 ) δ 7.56 7.26 (Ar), 5.91 5.78 (m, 1,C=C 2 ), 5.51 (bd, J = 8.4 z, 1, ), 5.26 (dd, J = 1.5 z, 17.4 z, 2, C=C 2 ), 5.12 (s, 2, C 2 Ph), 4.67 4.54 (m, 2, C 2 C), 4.50 (m, 1, γ-c A B ), 4.41 4.34 (m, 1, α-c), 4.28 (m,1, γ-c A B ), 2.25 (m, 1, β- C A B ), 2.16 (m, 1, β-c A B ), 1.42 (s, 9, C(C 3 ) 3 ); 13 C MR (75 Mz, CDCl 3 ) δ 171.9 (CAll), 155.6 (C), 153.23 (C=), 137.6 (C=C 2 ), 131.7 124.6 (C Ar ), 118.9 (C=C 2 ), 80.2 (C(C 3 ) 3 ), 76.9 (C 2 Ph), 68.7 (γ-c), 66.3 (C 2 C), 51.6 (α- C), 32.8 (β-c), 28.7 (C(C 3 ) 3 ); ESI-LRMS m/z 569.2 (M+a) +. (S)-allyl 2-(tert-butoxycarbonylamino)-4-oxobutanoate (46). Dess-Martin periodinane (450 mg, 1.06 mmol) was added to a solution of alcohol 42 (250 mg, 0.96 mmol) in C Boc CAll 46 C 2 Cl 2. The reaction mixture was stirred for 2 h at room temperature and evaporated to dryness. The residue was suspended in diethyl ether (20 ml), extracted with aqueous ac 3 /a 2 S 2 3 solution (100 ml of saturated ac 3 containing 25 g of a 2 S 2 3 ) and brine, and dried over a 2 S 4. The organic layer was passed through a short silica gel plug to obtain 46 (0.20 g) with 82% yield. R f 0.60 (EtAc/hexanes, 2:3); 1 MR (300 Mz, CDCl 3 ) δ 9.71 (s, 1, C), 5.82 (m, 1, C=C 2 ), 5.45 (bs, 1, ), 5.24 (m, 2, C=C 2 ), 4.61 (d, J = 11.4 z, 2, C 2 C), 4.54 (m, 1, α-c), 3.18 2.90 (m, 2, β-c 2 ), 1.43 (s, 9, C(C 3 ) 3 ); 13 C MR (75 Mz, CDCl 3 ) δ 199.5 (C), 170.8 (CAll), 155.5 (C(=)), 131.5 (C=C 2 ), 119.1 (C=C 2 ), 80.5 (C(C 3 ) 3 ), 66.6 (C 2 C), 48.9 (α-c), 46.3 (β-c), 28.6 (C(C 3 ) 3 ); ESI-LRMS m/z 258.1 (M+) +. -Cbz--benzylhydroxylamine (49). To a stirred suspension of - benzylhydroxylamine (4.00 g, 25 mmol) in C 2 Cl 2 (25 ml) were added,-

S19 (diisopropyl)ethylamine (8.7 ml, 50 mmol) and benzyl chloroformate (3.6 ml, 25 mmol) at 0 ºC. The reaction mixture was stirred at 0 ºC for 2 h before being concentrated under Bn 49 reduced pressure. The residue obtained after evaporation was taken up in EtAc (100 ml) and was washed with 2 Cl (2 50 ml), brine and dried over a 2 S 4. Removal of the volatiles in vacuo provided an oil, which was triturated with hexanes to give 49 (5.45 g, 85% yield) as a white powder. mp 64 65 ºC (lit. 64 66 ºC); R f 0.25 (EtAc/hexanes, 1:4); 1 MR (300 Mz, CDCl 3 ) δ 7.42 7.24 (m, 10, Ar), 5.21 (s, 2, C 2 Ph), 4.89 (s, 2, C 2 Ph), 2.03 (s, 1, ); 13 C MR (75 Mz, CDCl 3 ) δ 156.1 (C(=)), 129.4 128.5 (C Ar ), 78.9 (C 2 Ph), 67.8 (C 2 Ph); ESI-LRMS m/z 280.1 (M+a) +. Boc-Dab(-Cbz- -benzyloxy)-all (50). To a solution of carbamate 49 (0.500 g, 1.94 mmol) in anhydrous DMF (5 ml), sodium hydride (60% dispersion in mineral oil, 74 Bn Cbz Boc CAll 50 mg, 1.85 mmol) was added at rt, and the mixture was stirred for 30 min and then cooled to 0 ºC. To this solution bromo-derivative of homoserine 43 (0.42 g, 1.30 mmol) dissolved in dry DMF (5 ml) was added at 0 ºC. The reaction mixture was allowed to warm to rt overnight before being poured into a separatory funnel and diluted with EtAc (125 ml). The organic layer was washed with 10% citric acid (3 50 ml) and brine (50 ml) and then dried over a 2 S 4. The solvent was removed, and column chromatography of the residue (EtAc/hexanes, 3:7 to 1:1) afforded 0.57 g (yield 89%) of the -alkylated product 50. R f 0.45 (EtAc/hexanes, 2:3); [α] D 19.1 (c 1.024, CCl 3 ); 1 MR (300 Mz, CDCl 3 ) δ 7.41 7.14 (m, 10, Ar), 5.74 (m, 1, C=C 2 ), 5.21 4.96 (m, 5,, C=C 2, C 2 Ph), 4.61 (s, 2, C 2 C), 4.45 (m, 2, C 2 C), 4.22 (m, 1, α-c), 3.42 (m, 2, γ-c 2 ), 2.14 1.78 (m, 2, β-c 2 ), 1.43 (s, 9, C(C 3 ) 3 ); 13 C MR (75 Mz, CDCl 3 ) δ 171.2 (CAll), 156.7, 154.8 (C(=)), 135.2 127.9 (C Ar, C=C 2 ), 118.3 (C=C 2 ), 79.4 (C(C 3 ) 3 ), 78.1 (C 2 C), 66.6 (C 2 Ph), 65.4 (C 2 C), 50.8 (α-c), 45.9 (γ-c), 29.1 (β-c), 27.7 (C(C 3 ) 3 ); ESI-LRMS m/z 499.1 (M+) +. Cl. 2 -Dab(-Cbz- -benzyloxy)-all (51). A solution of 50 (0.20 g, 0.40 mmol) in 4 Cl in 1,4-dioxane (20 ml) was stirred at room temperature for 1 h. The reaction

S20 mixture was evaporated in vacuo to give an oily residue, which was triturated with anhydrous Et 2 to give 51 (160 mg, 95% yield) as a white powder; which was used in the next step without further purification. 1 MR (300 Mz, CDCl 3 ) δ 8.81 (bs, 3, 3 ), 7.34 7.25 (m, 10, Ar), 5.76 (m, 1, C=C 2 ), 5.25 5.12 (m, 4, C=C 2, C 2 Ph), 4.83 (s, 2, C 2 C), 4.55 (m, 2, C 2 C), 4.26 (m, 1, α-c), 3.88 3.73 (m, 2, γ-c 2 ), 2.54 2.32 (m, 2, β-c 2 ); 13 C MR (75 Mz, CDCl 3 ) δ 168.7 (CAll), 157.3 (C(=)), 135.8 128.4 (C Ar, C=C 2 ), 119.8 (C=C 2 ), 77.3 (C 2 C), 68.6 (C 2 Ph), 67.5 (C 2 C), 51.4 (α-c), 45.7 (γ-c), 28.2 (β-c); ESI-LRMS m/z 399.1 (M+) +. Bn Cbz 2 CAll Cl 51 Boc-γ-Glu[-al(γ-bromo)-All]-tBu (54). A solution of Boc-protected amine 43 (0.50 g, 1.55 mmol) in 4 Cl in 1,4-dioxane (15 ml) was stirred at room temperature for 1 h. The reaction mixture was evaporated to give an oily residue, which was triturated with anhydrous Et 2 to give the crude amine (0.39 g, 97% yield) as a white foam; which was used in the next step immediately without further purification. To a stirred solution of carboxylic acid 16 (0.54 g, 1.81 mmol) in C 2 Cl 2 (15 ml) at ambient temperature was added a solution of the crude amine obtained before (0.39 g, Boc CtBu 54 Br CAll 1.50 mmol) in C 2 Cl 2 (5 ml), followed by addition of Et 3 (0.63 ml, 4.52 mmol) and PyAP (0.94 g, 1.81 mmol). After stirring the reaction mixture for 18 h at ambient temperature, the reaction mixture was quenched with sat. 4 Cl solution (25 ml) and extracted with EtAc (3 50 ml). The combined organic layers were washed with brine (50 ml), dried over MgS 4, concentrated and the residue obtained was purified by flash column chromatograph to afford bromodipeptide 54 (0.62 g, 82% yield) as a viscous oil. R f 0.40 (EtAc/hexanes, 2:3); [α] D 11.3 (c 0.86, CCl 3 ); 1 MR (300 Mz, CDCl 3 ) δ 6.91 (bd, J = 7.5 z, 1, ), 5.94 5.81 (m, 1, C=C 2 ), 5.94 5.81 (m, 3, C=C 2, ), 4.72 4.59 (m, 3, C 2 C, α-c:glu), 4.15 4.04 (m, 1, α-c:al), 3.41 (t, J = 6.9 z, 2, γ- C 2 :al), 2.48 2.10 (m, 5, β-c 2 :Glu, β-c 2 :al, γ-c A B :Glu), 1.86 1.78 (m, 1,

S21 γ-c A B :Glu); 13 C MR (75 Mz, CDCl 3 ) δ 172.3, 171.4, 171.2 (C(=)), 156.1 (C(=)), 131.5 (C=C 2 ), 119.2 (C=C 2 ), 82.6, 80.3 (C(C 3 ) 3 ), 66.5 (C 2 C), 53.6 (α-c:glu), 51.7 (α-c:al), 35.6 (β-c:al), 32.7 (γ-c:glu), 31.8 (γ- C:al), 29.8 (β-c:glu), 28.6, 28.3 (C(C 3 ) 3 ); ESI-LRMS m/z 507.2 (M+) +. Boc-γ-Glu[-Dab(-Cbz- -benzyloxy)-all]-tbu (55). a (22.5 mg, 0.56 mmol, Boc CtBu 55 50% in mineral oil) was added to a solution of -(benzyloxycarbonyl)-benzylhydroxylamine (49) (152 mg, 0.59 mmol) in anhydrous DMF (5 ml), and the mixture was stirred for 30 min at room temperature. A solution of 54 (200 mg, 0.39 mmol) in dry DMF (5 ml) was added dropwise to the solution containing the deprotonated carbamate cooled to 0 ºC, and the reaction mixture was stirred for 30 min at 0 ºC. After being stirred overnight at rt, the reaction was diluted with EtAc and the organic layer was washed with 10% citric acid (3 50 ml), brine (50 ml) and then dried over a 2 S 4. The solvent was removed, and column chromatography of the residue afforded 0.21 g (yield 76%) of the -alkylated dipeptide 55. R f 0.4 (EtAc/hexanes, 1:1); [α] D 7.3 (c 0.62, CCl 3 ); 1 MR (300 Mz, CDCl 3 ) δ 7.39 7.25 (m, 10, Ar), 6.56 (d, J = 7.2 z, 1, ), 5.88 5.75 (m, 1, C=C 2 ), 5.28 5.13 (m, 4, C=C 2, CC 2 Ph), 4.86 4.78 (dd, J = 9.9, 12.0 z, 2, C 2 Ph), 4.64 4.53 (m, 3, α-c:glu, C 2 C), 4.14 (m, 1, α-c:dab), 3.63 3.44 (m, 2, γ-c 2 :Dab), 2.25 1.81 (m, 6, β-c 2 :Glu, γ-c 2 :Glu, β-c 2 :Dab), 1.44 (s, 18, C(C 3 ) 3 ); 13 C MR (75 Mz, CDCl 3 ) δ 172.0, 171.5 (C(=)), 157.1, 155.8 (C(=)), 136.0 128.4 (C Ar, C=C 2 ), 119.1 (C=C 2 ), 82.4, 80.1 (C(C 3 ) 3 ), 77.5 (C 2 Ph), 68.2 (CC 2 Ph), 66.4 (C 2 C), 53.8 (α-c:glu), 50.4 (α-c:dab), 46.3 (γ-c:dab), 32.7 (γ-c:glu), 29.7 (β-c:glu), 29.2 (γ-c:dab), 28.7, 28.3 (C(C 3 ) 3 ); ESI- LRMS m/z 684.3 (M+) +. Bn Cbz CAll Boc-γ-Glu[-Dab(-Cbz- -benzyloxy)-gly-tbu]-tbu (57). (Ph 3 P) 4 Pd (17 mg, 10 mol%) was added under nitrogen to a solution of the allyl ester 55 (0.10 g, 0.15 mmol) in C 2 Cl 2 (20 ml) at room temperature and the resulting mixture was treated dropwise with

S22 morpholine (0.13 ml, 1.46 mmol). After completion of the reaction (1 h) as judged by TLC, solvent was removed in vacuo. The residue was redissolved in EtAc (20 ml) and the resulting solution washed with 1 Cl (15 ml) and water (15 ml). The organic layer was dried over a 2 S 4 and evaporated in vacuo. The crude acid 56 (88 mg, 94% yield) thus obtained was immediately purified by flash column chromatography and used in the next coupling step. EDC (40 mg, 0.21 mmol), Bt (28 mg, 0.21 mmol) and MM (46 µl, 0.41 mmol) were added to an ice-cold solution of acid 56 (88 mg, 0.14 mmol) and Cl.Gly-tBu Boc CtBu (0.25 g, 1.51 mmol) in C 2 Cl 2 (20 ml). The solution was allowed to stir for 12 h before being poured into water (20 ml). The dichloromethane layer was washed with 10% citric acid solution (25 ml), sat. ac 3 solution (2 20 ml) and brine; dried over a 2 S 4 and the residue obtained after evaporation of C 2 Cl 2 was purified by column chromatography with 50% EtAc/hexanes to isolate product 57 as a colorless oil (79 mg, 72% yield). R f 0.45 (EtAc/hexanes, 3:2); [α] D 13.1 (c 0.91, CCl 3 ); 1 MR (300 Mz, CDCl 3 ) δ 7.45 7.26 (m, 10, Ar), 7.16 (t, J = 5.4 z, 1, ), 6.68 (d, J = 7.5 z, 1, ), 5.26 (d, J = 8.1 z, ), 5.18 (s, 2, CC 2 Ph), 4.92 4.84 (m, 2, C 2 Ph), 4.52 4.43 (m, 1, α-c:glu), 4.13 (m, 1, α-c:dab), 3.94 3.72 (m, 3, C 2 :Gly, γ-c A B :Dab), 3.56 3.47 (m, 1, γ-c A B :Dab), 2.27 2.02 (m, 5, β- C 2 :Glu, γ-c 2 :Glu, β-c A B :Dab), 1.88 1.80 (m, 1, β-c A B :Dab), 1.42, 1.41, 1.39 (3s, 27, C(C 3 ) 3 ); 13 C MR (75 Mz, CDCl 3 ) δ 172.1, 171.5, 171.2, 168.7 (C(=)), 157.5, 155.8 (C(=)), 135.9 128.3 (C Ar ), 82.3, 80.1 (C(C 3 ) 3 ), 77.7 (C 2 Ph), 68.3 (CC 2 Ph), 53.7 (α-c:glu), 51.1 (α-c:dab), 47.0 (γ-c:dab), 42.3 (C 2 :Gly), 32.6 (γ- C:Glu), 30.5 (β-c:glu), 28.7, 28.4, 28.3 (C(C 3 ) 3 ), 23.0 (γ-c:dab); ESI-LRMS m/z 757.4 (M+) +. 57 Cbz Bn CtBu Boc-γ-Glu[-Dab( 2 )-Gly-tBu]-tBu (58). A solution of Cbz-protected tripeptide 57 (50 mg, 0.07 mmol) in anhydrous Me (10 ml) was treated with Pd C (10 mg) and stirred under a 2 balloon for 1 h at rt. The reaction mixture was filtered through Celite

S23 and was washed with methanol. The combined organic layers were concentrated under reduced pressure to obtain the tripeptide amine 58 as a colorless oil (30 mg, 90% yield). 2 Boc CtBu Ct Bu 58 1 MR (300 Mz, CDCl 3 ) δ 8.18 (bs, 1, ), 7.42 (bs, 1, ), 5.57 (d, J = 7.8 z, 1, ), 4.59 (m, 1, α-c:glu), 4.15 (m, 1, α-c:dab), 3.94 3.78 (m, 2, C 2 :Gly), 3.68 (bs, 2, 2 ), 2.95 2.83 (m, 2, γ-c 2 :Dab), 2.36 (m, 2, β-c 2 :Dab), 2.18 1.78 (m, 4, β-c 2 :Glu, γ-c 2 :Glu), 1.39, 1.38, 1.37 (3s, 27, C(C 3 ) 3 ); 13 C MR (75 Mz, CDCl 3 ) δ 172.4, 171.5, 171.4, 168.7 (C(=)), 157.3, 155.8 (C(=)), 136.6, 128.6, 128.2 (C Ar ), 82.4, 80.1 (C(C 3 ) 3 ), 67.0 (C 2 Ph), 53.7 (α-c:glu), 50.7 (α-c:dab), 42.3 (C 2 :Gly), 37.6 (γ- C:Dab), 34.0 (γ-c:glu), 32.6 (β-c:dab), 29.1 (β-c:glu), 28.6, 28.4, 28.3 (C(C 3 ) 3 ); ESI- LRMS m/z 517.4 (M+) +. Boc-Dab-C (60). To solution of -Boc-L-glutamine (59) (3.69 g, 15 mmol) in TF (36 ml) and water (9 ml), PIDA (5.79 g, 18 mmol) was added at 4 C. After being 2 Boc C 60 stirred for 6 h, the reaction mixture was evaporated to dryness; the resulting residue was dissolved in water (20 ml) and extracted with EtAc (3 20 ml). The organic layer was discarded and the aqueous layer was evaporated to dryness. The crude solid obtained was washed several times with cold chloroform and dried in vacuo to afford 60 (2.48 g, 76% yield). mp 207 C (lit. 208 209 C); [α] D 10.0 (c2, Ac) [lit. [α] D 8.0 (c2, Ac)]; 1 MR (300 Mz, DMS-d 6 and a drop of TFA) δ 3.51 3.47 (m, 1, α-c), 3.38 3.28 (bs, 1, ), 2.90 2.88 (m, 2, 2 ), 1.90 1.85 (m, 2, γ-c 2 ), 1.79 1.65 (m, 2, β-c 2 ), 1.37 (s, 9, C(C 3 ) 3 ). Boc-Dab(Cbz)-C (61). A stirring mixture of the -Boc-Dab- (60; 2.00 g, 9.17 mmol), TF (30 ml), water (10 ml), K 2 C 3 (2.53 g, 18.35 mmol) and K (0.51 g, 9.17 mmol) was cooled to 5 C. Cbz-Cl solution (50 wt% in toluene, 4.70 ml, 13.76 mmol) was then added dropwise over a period of 1 h. The mixture was allowed to stir for 8 h before being concentrated under reduced pressure to remove the TF. The aqueous

S24 residue was extracted with diethyl ether (50 ml), acidified to p 4 by solid citric acid and the resulting precipitate was extracted by C 2 Cl 2 (2 50 ml). The combined organic Cbz Boc C 61 layers were dried (a 2 S 4 ) and evaporated to furnish the free acid 61 (2.61 g, 81% yield) as a viscous oil which was used without further purification. 1 MR (300 Mz, CDCl 3 ) δ 11.05 (bs, 1, C), 7.26 7.20 (m, 5, Ar), 5.78 (m, 1, ), 5.49 (d, J = 8.1 z, 1, ), 5.16 4.96 (m, 2, C 2 Ph), 4.32 4.25 (m, 1, α-c), 3.42 3.02 (m, 2, γ-c 2 ), 2.06 1.65 (m, 2, β-c 2 ), 1.36 (s, 18, C(C 3 ) 3 ); 13 C MR (75 Mz, CDCl 3 ) δ 175.2 (C), 156.9, 156.1 (C(=)), 136.6, 128.6, 128.2 (C Ar ), 80.3 (C(C 3 ) 3 ), 66.9 (C 2 Ph), 51.2 (α-c), 37.5 (γ-c), 33.4 (β-c), 28.5 (C(C 3 ) 3 ). Boc-Dab(Cbz)-Gly-tBu (62). To a solution of the acid 61 (1.00 g, 2.84 mmol) in C 2 Cl 2 (20 ml) at 0 ºC was added glycine tert-butyl ester (0.71 g, 4.26 mmol), MM Boc 62 Cbz CtBu (0.94 ml, 8.52 mmol), Bt (0.46 g, 3.41 mmol), and EDC (0.65 g, 3.41 mmol). Reaction mixture was then slowly allowed to warm to rt. After stirring overnight at rt, the mixture was concentrated. The residue was taken up in EtAc and washed with 10% citric acid (2 30 ml), sat. ac 3 (2 30 ml) and brine. The EtAc layer was dried over a 2 S 4 and the crude product obtained after evaporation of EtAc was purified by silica gel column chromatography to afford the dipeptide 62 (1.16 g, 88% yield) as oil. R f 0.45 (EtAc/hexanes, 2:3); [α] D 5.4 (c 0.48, CCl 3 ); 1 MR (300 Mz, CDCl 3 ) δ 7.33 7.25 (m, 6, Ar, ), 5.69 (bs, 1, ), 5.54 (bs, 1, ), 5.12 5.00 (m, 2, C 2 Ph), 4.26 (m, 1, α-c), 3.99 3.76 (m, 2, C 2 :Gly), 3.48, 3.09 (2m, 2, γ-c 2 ), 1.97 1.75 (m, 2, β-c 2 ), 1.45, 1.39 (2s, 18, C(C 3 ) 3 ); 13 C MR (75 Mz, CDCl 3 ) δ 172.0, 168.8 (C(=)), 157.1, 155.9 (C(=)), 136.7, 128.6, 128.2 (C Ar ), 82.4, 80.3 (C(C 3 ) 3 ), 67.0 (C 2 Ph), 51.8 (α-c), 42.3 (C 2 ), 37.6 (γ-c), 34.3 (β-c), 28.6, 28.4 (C(C 3 ) 3 ); ESI-LRMS m/z 466.6 (M+) +. Boc-γ-Glu[-Dab(Cbz)-Gly-tBu]-tBu (64). A solution of Cl/dioxane (12 ml, 4M) was added in one portion to a 25 ml round bottom flask containing 62 (1.00 g, 2.15 mmol); cooled with an ice-water bath. The ice-bath was then removed and the mixture

S25 was kept stirred. After 20 min, TLC indicated the completion of the reaction. The reaction mixture was concentrated under high vacuum at room temperature. The residue obtained was triturated with dry ethyl ether and dried to obtain amine 63 as a sticky white solid which was employed directly for the coupling reaction. EDC (0.62 g, 3.22 mmol) and Bt (0.42 g, 3.22 mmol) and MM (0.71 ml, 6.45 mmol) were added to an ice-cold solution of 63 and Boc-Glu()-tBu (16) (0.72 g, Cbz CtBu Boc 64 CtBu 2.36 mmol) in C 2 Cl 2 (40 ml). The solution was allowed to stir for 12 h and was then poured into water (30 ml). The C 2 Cl 2 layer was washed with 10% citric acid solution (30 ml), sat. ac 3 solution (2 25 ml) and brine; dried with a 2 S 4 and the residue obtained after evaporation of C 2 Cl 2 was purified by column chromatography with 50% EtAc/hexanes to isolate tripeptide product 64 as a colorless oil (1.06 g, 76% yield). R f 0.50 (EtAc/hexanes, 3:2); [α] D 9.6 (c 0.84, CCl 3 ); 1 MR (300 Mz, CDCl 3 ) δ 7.33 7.25 (m, 6, Ar), 6.85 (bs, 1, ), 5.72 (bs, 1, ), 5.27 (m, 1, ), 5.12 5.00 (m, 2, C 2 Ph), 4.53 (m, 1, α-c:glu), 4.13 (m, 1, α-c:dab), 4.00 3.76 (m, 2, C 2 :Gly), 3.51, 3.09 (2m, 2, γ-c 2 :Dab), 2.28 (t, J = 7.5 z, 2, β-c 2 :Dab), 2.18 1.79 (m, 4, β-c 2 :Glu, γ-c 2 :Glu), 1.46, 1.43, 1.40 (3s, 27, C(C 3 ) 3 ); 13 C MR (75 Mz, CDCl 3 ) δ 172.4, 171.5, 171.4, 168.7 (C(=)), 157.3, 155.8 (C(=)), 136.6, 128.6, 128.2 (C Ar ), 82.4, 80.1 (C(C 3 ) 3 ), 67.0 (C 2 Ph), 53.7 (α-c:glu), 50.7 (α-c:dab), 42.3 (C 2 :Gly), 37.6 (γ-c:dab), 34.0 (γ-c:glu), 32.6 (β-c:dab), 29.1 (β-c:glu), 28.6, 28.4, 28.3 (C(C 3 ) 3 ); ESI-LRMS m/z 651.3 (M+) +, ESI-RMS m/z 651.3575 (M+) +, C 32 50 4 10 + + requires 651.3599. Boc-γ-Glu[-Dab( 2 )-Gly-tBu]-tBu (58). A solution of tripeptide 64 (0.70 g, 1.07 mmol) in methanol (25 ml) was placed in a 100 ml two-neck round bottom flask and Boc CtBu 58 2 CtBu purged with argon. Pd C (10% mol, 70 mg) was added under a stream of argon. The flask was evacuated under vacuum and hydrogenated under hydrogen balloon at about 1 psi of hydrogen shaken for 60 minutes before being diluted with 200 ml of C 2 Cl 2. The suspension was filtered through a short pad of celite and