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1 SUPPRTIG IFRMATI Salicylaldehyde Ester-Induced Chemoselective Peptide Ligations Enabling Generation of atural Peptidic Linkages at the Serine/Threonine Sites Xuechen Li,* iu Yung Lam, Yinfeng Zhang, Chun Kei Chan Department of Chemistry, The University of ong Kong, Pokfulam Road ong Kong, P.R.China General Methods. All commercial materials were used as supplied unless otherwise noted. Amino acids and resins were purchased from GL Biochem-Shanghai and ovabiochem. All separations using PLC involved a mobile phase of 0.05% TFA (v/v) in water (solvent A)/0.04% TFA in acetonitrile (solvent B). LC-MS chromatographic separations were performed using a Waters 1525 Separations Module and a Waters 2998 otodiode Array Detector equipped with Varian Microsorb C18 column (2X 150 mm) at a flow rate of 0.2 ml/min or SunFire C18 column (4.6X 150 mm) at a flow rate of 0.6 ml/min. PLC separations were performed using Microsorb C18 column at a flow rate of 16.0 ml/min. UPLC chromatographic separations were performed using an ACQUITY Ultra Performance LC and SQ Detector equipped with either ACQUITY UPLC BE C8 1.7 µm 2.1X100 mm Column or ACQUITY UPLC BE C µm 2.1X100 mm Column at a flow rate of 0.3 ml/min. MR 1 and 13 C spectra were recorded on Bruker instruments in CDCl 3 at 500 or 600 M z for 1 and 125 or 150 Mz for 13 C. General procedure for preparation of -salicylaldehyde ester: The -salicylaldehyde esters of Ala, Val Ile and Pro were prepared by coupling Fmoc-Ala-, Fmoc-Val-, Fmoc-Ile-, Fmoc-Pro- and Fmoc-Lys(Boc)- respectively, with salicylaldehyde and EDCI in C 2 Cl 2. Fmoc-Val-Thr-salicylaldehyde ester 5 was prepared by coupling Fmoc-Val-Thr (ψ Me,Me pro)- with salicylaldehyde and EDCI in C 2 Cl 2, followed by treatment with 95% TFA in 2. It was also synthesized by using Fmoc SPPS chemistry where the Fmoc-Val-Thr-bz was prepared according to Dawson s protocol 1, followed by phenolysis of the salicylaldehyde. General procedure for ligation: The serine or threonine segment (1.1 equiv.) and the -salicylaldehyde ester segment (1.0 equiv.) were dissolved in pyridine/acetic acid (1:1 mole/mole) at a concentration of 0.05 M. The reaction was stirred at room temperature. The reaction was monitored using LCMS. After the completion of the reaction (within 5 hours), the solvent was removed by lyophilization. Then, the residue was treated with TFA/ 2 /i-pr 3 Si (94/5/1, v/v/v) for 5 min to give the coupled product with a natural peptide bond at the ligation site. The solvent could be blown off by a stream of air for purification. The products were purified by PLC (C18, 45% 85% MeC/ 2, 0 30 min, λ = 263 nm). The followings present LCMS profiles monitoring the process of the serine and threonine based ligation. For step 1, 5 µl of the reaction mixture was taken, diluted with acetonitrile/water and directly injected into LC-MS system at 30 min and 5 h, respectively. For step 2 (the deprotection), 5 µl of the reaction mixture was taken at 5 min, diluted with acetonitrile/water and directly injected into LC-MS. The Ac/pyridine peak was in the solvent front and cut off in the spectrum. The UV trace was derived from a otodiode Array. 1 Blanco-Canosa, J. B.; Dawson, P. E. Angew. Chem. Int. Ed. 2008, 47,

2 1) Ligation between Fmoc-Ala-salicylaldehyde ester and -Ser-Bn A C Chemical Formula: C Exact Mass: C 2 Bn C 2 Bn 1 2 Chemical Formula: C Exact Mass: B C C 2 Bn Chemical Formula: C Exact Mass: MR: 1 MR (CDCl 3, 600 Mz) δ 7.68 (2, d, J = 7.5 z), 7.49 (2, d, J = 4.9 z), (9, m), 6.81 (1, d, J = 5.3 z, -), 5.31 (1, d, J = 5.2 z, -), 5.13 (2, s, C 2 ), 4.60 (1, t, J = 3.8 z, Ser-α ), 4.30 (1, d, J = 6.8 ), 4.16 (1, m, Ala-β ), 4.12 (1, t, J = 6.8 z), (2, m, Ser-β ), 1.33 (3, d, J = 6.3 z); 13 C MR (CDCl 3, 150 Mz) δ 172.5, 170.0, 156.1, 143.7, 143.6, 141.3, 135.0, 128.7, 128.6, 128.2, 127.7, 127.0, 125.0, 120.0, 67.6, 67.2, 62.8, 54.9, 50.8, 47.1,

3 3

4 1 C C 2 Bn 1.3 equiv C 2 Me 1.3 equiv M 5 h Bn 2 C only equiv C 1.2 equiv S C 2 Bn 0.05 M 5 h Bn 2 C only Chemoselectivity studies on Ser-CL; PLC profiles of the competitive reaction: A, crude reaction mixture of reaction 1; B, crude reaction mixture of reaction 2; C, standard Fmoc-Ala-Ser-Bn as a reference. 4

5 2) Ligation between Fmoc-Val-salicylaldehyde ester and -Ser-Bn C Chemical Formula: C Exact Mass: A C 2 Bn C 2 Bn 1 2 Chemical Formula: C Exact Mass: B C C 2 Bn Chemical Formula: C Exact Mass:

6 3) Ligation between Fmoc-Pro-salicylaldehyde ester and -Ser-Bn C Fmoc Chemical Formula: C Exact Mass: C 2 Bn 1 Fmoc C 2 Bn Fmoc Chemical Formula: C A Exact Mass: B C 2 C 2 Bn Chemical Formula: C Exact Mass:

7 4) Ligation between Fmoc-Ile-salicylaldehyde ester and -Ser-Bn A C Chemical Formula: C Exact Mass: C 2 Bn 1 C 2 Bn B Chemical Formula: C Exact Mass: C 2 Bn Chemical Formula: C Exact Mass: C 7

8 5) Ligation between Fmoc-Val-Thr-salicylaldehyde ester and -Ser-Bn C Chemical Formula: C Exact Mass: A C 2 Bn 1 2 C 2 Bn Chemical Formula: C Exact Mass: B C 2 Bn Chemical Formula: C Exact Mass: C 8

9 Screening various LCMS conditions only show one peak on LCMS spectrum. Then, the isolation of this product with PLC was followed by MR studies. Its MR spectrum shows only one product, without any detectable epimerized diastereomer. 1 MR (600 Mz, CDCl 3 ) was taken at 50 o C (13, m), 6.77 (1, br s, ), 5.21 (1, d, J = 7.2 z, ), 5.15 and 5.12 (2, ABd, J = 12.3 z, C 2 ), 4.57 (1, m, ), (4, m), 4.12 (1, t, J = 6.7 z), 3.93 (1, m), (1, m), 2.07 (1, m), 1.06 (3, d, J = 6.4 z), 0.84 (6, m). 9

10 6) Ligation between Fmoc-Ala-salicylaldehyde ester and -Thr-e-Et C Chemical Formula: C Exact Mass: A 1 2 Chemical Formula: C Exact Mass: B Chemical Formula: C Exact Mass: C 10

11 7) Ligation between Fmoc-Val-salicylaldehyde ester and -Thr-e-Et A C Chemical Formula: C Exact Mass: Chemical Formula: C Exact Mass: B 2 C Chemical Formula: C Exact Mass:

12 8) Ligation between Fmoc-Pro-salicylaldehyde ester and -Thr-e-Et Fmoc C Chemical Formula: C Exact Mass: A 1 2 Fmoc Chemical Formula: C Exact Mass: B Fmoc C Chemical Formula: C Exact Mass:

13 9) Ligation between Fmoc-Ile-salicylaldehyde ester and -Thr-e-Et C Chemical Formula: C Exact Mass: Chemical Formula: C A Exact Mass: B C Chemical Formula: C Exact Mass:

14 10) Ligation between Fmoc-Val-Thr-salicylaldehyde ester and -Thr-e-Et C Chemical Formula: C Exact Mass: A 1 2 Chemical Formula: C Exact Mass: Fmoc B C Chemical Formula: C Exact Mass:

15 11) Ligation between Fmoc-Lys-salicylaldehyde ester and -Ser-Me C 2 Me TFA C Chemical Formula: C Exact Mass: A C 2 Me 1 B Chemical Formula: C Exact Mass: C 2 Me Chemical Formula: C Exact Mass: C 15

16 In order to identify the product of the salicylaldehyde ester reacting with the serine derivative under the reported condition, the study was performed on the reaction between Boc-Ala-salicylaldehyde ester and Ser-Me due to the simplicity of the MR spectrum. Me Me 2 C 2 C Me 2 C Boc C 2 C 2 Me Boc imine Boc * Boc ester amide 8 9 BocAla-sialyclaldehde ester was reacted with 2 -Ser-Me as described above. After 3 hours, the reaction mixture was diluted with C 2 Cl 2, and washed with 2 and brined, and dried over a 2 S 4. After removal of organic solvents, the MR spectrum of the residue in crude form was taken in CDCl 3. The 1 MR spectrum indicated a rather clean product without detectable starting materials and decomposed byproduct. Therefore, 13 C MR, 2D-CSY, 2D-MQC, and E spectrum were also collected. ne putative product of the reaction is the ester 8. If this is the case, the oxazolidine exists in equilibrium with hydroxyl Schiff base open chain forms (imine), which shows a peak at 8.3 ppm in CDCl 3 2. owever, no peak was observed above 7.5 ppm in the 1D 1 MR spectrum. Therefore, the coupled product is more likely the amide 9. Two sets of independent MR data were obtained based on the assignment of the 2D CSY. Corresponding peaks of these two sets of data were seen crossed with each other on the ne spectrum, which indicates that these two sets of MR data were from a rotamer mixture. ne rotamer minor (33%) 1 MR δ 7.10 (1, dd, J = 7.4, 8.0 z), 7.00 (1, t, J = 7.4 z), 6.83 (1, t, J = 7.4 z), 6.80 (1, s, C of oxazolidine), 6.68 (1, d, J = 8.0 z), 5.40 (1, d, J = 8.1 z, -), 4.74 (1, m, α-ser), 4.40 (1, m, α-ala), 4.39 (2, m, β-ser), 3.87 (3, s, C 3 ), 1.40 (9, s), 1.20 (3, d, J = 7.7 z); 13 C MR (CDCl 3 ) δ (C=), (C=), (- C(=)-), (aromatic-c-), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), 86.8 (-C-), 79.9(-C-(C 3 ) 3 ), 69.7(βC-Ser), 59.1 (αc-ser), 53.6 (C 3 ), 48.8 (αc-ala), 28.3 (C 3 ), 19.9 (βc-ala). The other rotamer major (66%) 7.07 (1, dd, J = 7.4, 8.0 z), 7.00 (1, t, J = 7.4 z), 6.78 (1, t, J = 7.4 z), 6.76 (1, s, C of oxazolidine), 6.51(1, d, J = 8.0 z), 5.47 (1, d, J = 8.1 z, -), 4.86 (1, dd, J = 2.0, 6.4 z, α-ser), 4.20 (1, dd, J = 6.4, 9.3 z, β-ser), 4.14 (1, dd, J = 2.0, 9.3 z, β-ser), 4.07 (1, m, α-ala), (3, s, C 3 ), 1.40 (9, s), 1.20 (3, d, J = 7.7 z); 13 C MR (CDCl 3 ) δ 170.7(C=), (C=), (-C(=)-), (aromatic-c-), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), 85.6 (-C-), 80.3(-C-(C 3 ) 3 ), 67.0 (βc-ser), 58.3 (αc-ser), 52.8 (C 3 ), 48.4 (αc-ala), 28.3 (C 3 ), 19.0 (βc-ala). The assignments were based on -Cosy, C-Cosy. * ne 3 C Boc Me ne Boc trans cis Based on ne study, we assigned the major rotamer as trans and the minor one as cis. The C stereochemistry of oxazolidine was determined by the ne spectrum. A comparatively strong ne cross-peak between an aromatic- and α-ser in both cis and trans rotamers, while no such a cross-peak between C of oxazolidine and α-ser was found. These results indicate the C stereocenter is an S rather than an R epimer. 1 MR (500 Mz, CDCl 3 ) Me C 3 ne 2 F. Fulop, K. Pihlaja, J. Mattinen, G. Bernath, Tetrahedron Lett. 1987, 43, ; M. E. Alva Astudillo,. C. J. Chokotho, T. C. Jarvis, C. D. Johnson, C. C. Lewis, P. D. McDonnell, Tetrahedron 1985, 41,

17 13 C MR (125 Mz, CDCl 3 ) 17

18 C-CSY -CSY 18

19 2D-E 19

20 Epimerization studies Another epimerization studies were conducted using Fmoc-Ala-e(L)-salicylaldehyde ester and Fmoc-Ala-e(D)- salicylaldehyde ester reacting serine-me respectively. Fmoc-Ala-e(L)-salicylaldehyde ester and Fmoc-Ala-e(D)-salicylaldehyde ester were prepared as below, using standard peptide coupling conditions. Boc L C DCC Boc C 2 Cl 2 C Cl Et 2 C DCC ac 3 C 2 Cl 2 10 C Boc D C DCC Boc C 2 Cl 2 C Cl Et 2 C DCC ac 3 C 2 Cl 2 11 C Both Fmoc-Ala-e(L)-salicylaldehyde ester (10) and Fmoc-Ala-e(D)-salicylaldehyde ester (11) reacted with serine- Me (1.5 equiv) in Ac/pyridine for 5 hours at room temperature. After the solvent was removed by a stream of air, the residue was treated with 95%TFA in 2 for 5 minutes. Then the mixture was diluted with C 2 Cl 2, and washed with 2, 1, sat. ac 3 and brine, dried over as 4. After removal of the solvent, the residue in crude form was directly subjected to MR. The spectra of Fmoc-Ala-e(L)-Ser-Me (12) and Fmoc-Ala-e(D)-Ser-Me (13) were shown and compared below. o epimerization was observed during the two-step serine coupling reactions. 20

21 Diastereomers 12 and 13 (from unpurified ligations) were also compared by using PLC Ligation between 5 and 6 S 2 Preparation of peptide 6. Boc-Ser-Ala-Gln(Trt)-Lys(Boc)-Arg(Pbf)-is(Trt)-e-Gly-C is prepared using an automatic solid phase peptide synthesizer employing Fmoc-Gly-ovaSyn TGT resin and standard Fmoc amino acids. After completion of the synthesis, the resin is treated with a mixture of TFE/C 2 Cl 2 /Ac (1/8/1, v/v/v) to give the crude protected peptide. The crude protected peptide (23 mg, 12 µmol), an alanine thiopheol ester (3.9 mg, 18 µmol), EDC (3.4 µl, 19 µmol) and Bt (1.0 mg, 6.1 µmol) are mixed in anhydrous TFE/chloroform (0.3 ml/0.6 ml). The reaction mixture is stirred at room temperature for 2 h and the solvent is blown off by a stream of air. Then the residue was treated with a deprotection mixture (TFA/ 2 //ipr 3 Si, 3.3 ml/136 µl/160 mg/ 60 µl). After stirring at room temperature for 2 h, the solvent is blown off by a stream of air, and then the peptide is precipitated out by ether, followed by RP-PLC (20-50%, MeC/ 2 over 30 min, Microsorb C18 column, 16 ml/min, 226 nm) purification to give the product -Ser-Ala-Gln-Lys-Arg-is-e-Gly-Ala-S 6 (7.0 mg, 53%). The chemical ligation between -salicylaldehyde ester 5 and peptide 6 Peptide 6 (0.5 mg, 0.46 µm) and -salicylaldehyde 5 (0.3 mg, 0.55 µm) were dissolved in a mixture of pyridine/acetic acid (1:1, mol/mol, 50 µl) to give a concentration of 9.0 mm. The reaction was stirred at room temperature and monitored by LCMS. The reaction was completed in 6 hours. Then, the solvent was removed by lyophilization. The residue was then subjected to a mixture of TFA/ 2 /i-pr 3 Si (94/5/1, v/v/v, 0.5 ml) for 5 min. The solvent was then blown off by nitrogen and purified by RP-PLC (45-21

22 85%, MeC/ 2 over 30 min, Microsorb C18 column, 16 ml/min, 264 nm). The fractions were collected, and lyophilized to provide peptide 7 as a white solid (0.5 mg, 72%). S 2 PLC analysis of the crude reaction of 5 and 6 to form 7 (after ligation and deprotection): gradient: 40-60% MeC/ 2 over 20 min at a flow rate of 0.6 ml/min, SunFire C18 column (4.6 X 150 mm). UV and MS traces from UPLC analysis of compound 7 after purification. Gradient 20-55% MeC/ 2 over 8 min at a flow rate of 0.3 ml/min, C8 column. ESI-MS calcd for C S [M2] , found :