Supporting Information
A Tetra-Orthogonal Strategy for the Efficient Synthesis of Scaffolds Based on Cyclic Peptides Nitin Jain and Simon H. Friedman* *e-mail:
[email protected] Contents Materials and Methods ..................................................................................................... 2 Materials ....................................................................................................................................... 2 Analytical Characterization .......................................................................................................... 6
Analytical results............................................................................................................... 7 HPLC chromatograms .................................................................................................................. 7 ESI-MS analysis ......................................................................................................................... 15
References ........................................................................................................................ 25
1
Materials and Methods Materials Fmoc-Asp(Opp)-OH and Fmoc-Dap(Dde)-OH were purchased from Chem-Impex International Inc. (Wood Dale, IL). Fmoc-Dap(Boc)-OH and NMP (N-methyl-2pyrrolidone) were obtained from Advanced ChemTech (Louisville, KY, USA). HATU (O- (7-azabenzotriazole-1-yl)-N, N, N’ N’-tetramethyluronium hexafluorophosphate) was purchased from Novabiochem. DIEA (N, N-diisopropylethylamine) and piperidine (99%) were obtained from Sigma Aldrich (St. Louis, MO, USA). Rink amide MBHA resin was obtained from EMD Biosciences (San Diego, CA, USA). TFA (99%) was purchased from Acros Organics (New Jersey, USA).
Method Overview of approach: The synthesis uses four degrees of orthogonality using the protecting groups Opp, Dde, Fmoc and Boc. The Opp group was deprotected using 1% TFA in DCM. The Dde group was selectively deprotected in the presence of Fmoc using NH2OH-Imidazole solution. The Fmoc group was selectively deprotected using 20% piperidine solution and finally the Boc group was deprotected using 95% TFA during the final cleavage. The final molecules had a net +1 charge. Synthesis was performed on 15 mg of rink amide MBHA (0.7 mmole/gm.) resin in a 1.5 mL Eppendorf® microcentrifuge tube. Resin was first washed with NMP (300µL x 5 x 5 min). Fmoc deprotection was then performed using 300µL of 20% piperidine in two rounds of 7 min and 10 min respectively. Post deprotection, thorough washings (300µL NMP x 5 x 5 min) were done to remove excess piperidine. The free amine group 2
on the resin was then coupled with Fmoc-Asp(Opp)-OH. A concentration of 300 mM and a volume of 300 µL were used for the coupling step. HATU (300 mM) and DIEA (600 mM) were used as coupling reagents. The carboxylic acid of Fmoc-Asp(Opp)-OH was preactivated for 10 min following which acylation was done for 3 hours. After 3 hours, the reaction mixture was aspirated and the resin was washed with NMP (300µL x 5 x 5 min). Any unreacted amine on the resin was capped using freshly prepared capping solution containing 10% acetic anhydride and 5% DIEA in NMP for 15 min. NMP washes (300µL x 5 x 5 min) were done to remove any excess capping solution. The Nα Fmoc group was then deprotected using 20% piperidine followed by NMP washes as mentioned above. At this stage the side chain carboxylic acid group was still protected with the phenylisopropyl ester group. Depending on the desired sequence, either FmocGly-OH or Fmoc-Dap(Dde)-OH was then coupled using the conditions described above for Fmoc-Asp(Opp)-OH. Following Dde couplings, the side chain protecting Dde group was selectively deprotected and acylated with quinoxaline-2-carboxylic. This was intentionally done to prevent the migration of the Dde group to α- position as has been reported in the literature. The Dde group was selectively deprotected using conditions that were developed by Bradley et al. for the selective deprotection of the Dde group in the presence of Fmoc group [1]. For this a stock solution of NH2OH (1250mg) and imidazole (918mg) was prepared in 5 mL of NMP. The solution was sonicated to help dissolve the chemicals in the highly viscous solution. Once a clear solution was obtained, 5 volumes of this stock solution was mixed with 1 volume of methylene chloride. For the deprotection step, 250 µL of NH2OH-Imidazole stock solution was mixed with 50 µL of methylene chloride and thoroughly mixed. The resulting 300 µL solution was then added
3
to the resin and the deprotection of Dde group was carried out for 3 hours. Post deprotection, NMP washes (300 µL x 10 x 5 min) were done to remove traces of NH2OH-Imidazole solution. A total of 10 washes were done to accomplish this. Once the side chain amine was deprotected, it was then acylated with quinoxaline-2-carboxylic acid. The coupling solution (300µl) comprised quinoxaline-2-carboxylic acid (300mM), HATU (300 mM) and DIEA (600 mM). Preactivation was performed for 10 minutes followed by overnight (~12-15 hours) acylation. Post coupling, standard NMP washes were performed followed by capping and NMP washes. The Fmoc group was then selectively deprotected using standard 20% piperidine conditions. Additional rounds as needed of Fmoc-Gly-OH or Fmoc-Dap(Dde)-OH were then repeated using conditions mentioned above. The final residue incorporated was Fmoc-Dap(Boc)-OH and was used to achieve side chain to backbone cyclization. The Nα amine group of this terminal Dap was used for the side chain- backbone cyclization while the side chain amine group was used to help provide a net +1 charge to the molecule. To achieve the on-resin cyclization, the Fmoc-Dap(Boc)-OH terminal α-NH2 was first selectively deprotected using 300 µL of 20% piperidine in two rounds of 7 min and 10 min respectively. NMP (300 µL x 5 x 5 min) washes were done to remove any piperidine residue, followed by 3 DCM washes. The DCM washes were done to remove NMP and prepare the resin for the aspartic acid side chain deprotection. Finally, the phenylisopropyl ester group on the aspartic acid side chain carboxylic acid group was selectively deprotected using 1% TFA in DCM (300 µL x 13 x 2 min). Multiple DCM washes were done to remove traces of TFA followed by NMP washes (300µL x 5 x 5 min).
4
Once the aspartic acid side chain carboxylic acid and terminal Dap α-amine were deprotected, on-resin side chain to backbone cyclization was achieved using HATU and DIEA as coupling agents. 57mM peptide, 59.5mM HATU, 119mM DIEA. Unlike, normal couplings where the ratio of amino acid: HATU: DIEA were 1:1:2, a ratio of 1:1.05:2.1 was used for the cyclization step. Required amounts of HATU and DIEA were taken in 200 µL NMP. The carboxylic acid was activated in situ and cyclization reaction was carried out overnight. After overnight cyclization, resin was thoroughly washed with NMP and then with DCM. After DCM washes, the resin was air dried and the test molecule was finally cleaved from the resin using 300 µL of cleavage cocktail that consisted of TFA: H2O: TIS (95: 2.5: 2.5 volume ratio) for 3 hours. Post cleavage, the TFA solution was transferred to a microcentrifuge. TFA was removed using a stream of nitrogen followed by three rounds of ether trituration using ice cold ether to remove any traces of TFA. The resulting residue was dissolved in 50 µL DMSO for analytical characterization.
5
Analytical Characterization Spectroscopy UV-Vis analysis of the molecules was performed using a USB-2000 fiber optic spectrometer (Ocean Optics, Inc.) with a DT-Mini-B lamp source. The final yield of the molecules was calculated using a molar extinction coefficients of 7722 M-1 cm-1; 15,444 M-1 cm-1 and 23,166 M-1 cm-1 for the mono, bis and tris molecules respectively. HPLC analysis HPLC analysis was performed on crude reaction products using a Hewlett Packard 1090 instrument with diode array detection. A Microsorb C8 (5 µm, 150 × 2.0 mm, Varian) column was used. A gradient from 0-100% solvent B (solvent A: 0.1% TFA/water, solvent B: acetonitrile) with a flow rate of 0.3ml/min over 30 min was used. Electrospray MS of the collective crude was performed in the positive ion mode using an ABI Q-Trap 2000 mass spectrometer. Reaction yields were assessed by determining the moles of quinoxaline present in the product and comparing these with the expected moles based on resin derivatization. Purity was estimated by comparing the HPLC integration of the tallest peak or peak cluster with the integration for the total chromatogram.
6
Analytical results HPLC Chromatograms 210nm Chromatogram of Molecule 10
210nm chromatogram Molecule 10 (Asp-Dap-Gly-Dap-Gly-Gly-Dap)
2500
Absorbance(mAu)
2000
1500
1000
500
0
0
5
10
15
20
25
30
Time (min)
7
HPLC chromatograms: 21 crude products obtained at 320nm after TFA cleavage of the cyclic heptapeptide library.
Molecule 1: Asp-Gly-Gly-Dap-Gly-Gly-Dap DAD1 B, Sig=320,4 Ref=450,80 (NITIN\MR180003.D) 7.958
mAU
200
150
7.117
50
9.431
100
0 5
10
15
20
25
min
15
20
25
min
15
20
25
min
Molecule 2: Asp-Gly-Gly-Gly-Dap-Dap-Dap DAD1 B, Sig=320,4 Ref=450,80 (NITIN\MR180004.D) 10.062
mAU 350
300
250
100
10.860
9.006 9.266
150
50
11.863
200
0 5
10
Molecule 3: Asp-Gly-Gly-Dap-Gly-Dap-Dap DAD1 B, Sig=320,4 Ref=450,80 (NITIN\MR180005.D) 10.417 10.313
mAU 350
300
250
200
150
11.877
100
50
0 5
10
8
Molecule 4: Asp-Gly-Gly-Dap-Dap-Gly-Dap DAD1 B, Sig=320,4 Ref=450,80 (NITIN\MR180006.D) 9.887
mAU
400
300
9.015 9.138
100
11.877
200
0 5
10
15
20
25
min
15
20
25
min
15
20
25
min
Molecule 5: Asp-Gly-Dap-Gly-Gly-Dap-Dap DAD1 B, Sig=320,4 Ref=450,80 (NITIN\MARCH161.D) 10.297
mAU
400
300
200
9.095
100
0 5
10
Molecule 6: Asp-Gly-Dap-Gly-Dap-Gly-Dap DAD1 B, Sig=320,4 Ref=450,80 (NITIN\MR180007.D) 10.073
mAU
300
250
200
150
11.630
100
50
0 5
10
9
Molecule 7: Asp-Gly-Dap-Dap-Gly-Gly-Dap DAD1 B, Sig=320,4 Ref=450,80 (NITIN\MR180008.D) 9.845
mAU
400
300
200
9.053
100
0 5
10
15
20
25
min
15
20
25
min
15
20
25
min
Molecule 8: Asp-Dap-Gly-Gly-Gly-Dap-Dap DAD1 B, Sig=320,4 Ref=450,80 (NITIN\MR180009.D) 9.943
mAU 400 350 300 250 200 150
8.995
11.663 11.920
100 50 0 5
10
Molecule 9: Asp-Dap-Gly-Gly-Dap-Gly-Dap DAD1 B, Sig=320,4 Ref=450,80 (NITIN\MR180010.D) 10.150
mAU
350 300 250 200
9.745
100 50
11.572 11.855
150
0 5
10
10
Molecule 10: Included in main paper (Representative data)
Molecule 11: Asp-Dap-Dap-Gly-Gly-Gly-Dap DAD1 B, Sig=320,4 Ref=450,80 (NITIN\MR180012.D) 9.909
mAU
250
200
150
11.738
100
50
0 5
10
15
20
25
min
15
20
25
min
15
20
25
min
Molecule 12: Asp-Gly-Dap-Dap-Gly-Dap-Dap DAD1 B, Sig=320,4 Ref=450,80 (NITIN\MR180013.D) 11.535
mAU
350 300 250 200 150 100 50 0 5
10
Molecule 13: Asp-Gly-Dap-Dap-Dap-Gly-Dap DAD1 B, Sig=320,4 Ref=450,80 (NITIN\MR180014.D) 11.426
mAU
400
300
10.311 10.438
200
100
0 5
10
11
Molecule 14: Asp-Gly-Gly-Dap-Dap-Dap-Dap DAD1 B, Sig=320,4 Ref=450,80 (NITIN\MR180015.D) 11.495
mAU 300
250
200
10.387 10.569
150
100
50
0 5
10
15
20
25
min
15
20
25
min
15
20
25
min
Molecule 15: Asp-Gly-Dap-Gly-Dap-Dap-Dap DAD1 B, Sig=320,4 Ref=450,80 (NITIN\MR180016.D) 11.594
mAU
11.335
200 175 150 125 100 75 50 25 0 5
10
Molecule 16: Asp-Dap-Gly-Gly-Dap-Dap-Dap DAD1 B, Sig=320,4 Ref=450,80 (NITIN\MR180017.D) 11.870
mAU
11.634
250
200
150
13.805
11.432
10.235
50
10.786
100
0 5
10
12
Molecule 17: Asp-Dap-Dap-Gly-Gly-Dap-Dap
11.887
10.606
11.710
DAD1 B, Sig=320,4 Ref=450,80 (NITIN\MR180018.D) mAU 300
250
200
150
13.121
50
13.692
11.307
12.399
100
0 5
10
15
20
25
min
15
20
25
min
15
20
25
min
Molecule 18: Asp-Dap-Gly-Dap-Gly-Dap-Dap DAD1 B, Sig=320,4 Ref=450,80 (NITIN\MR180019.D)
11.651
12.203
mAU
175 150
11.794
125 100
13.026 13.335 13.540
75
10.639
50 25 0 5
10
Molecule 19: Asp-Dap-Gly-Dap-Dap-Gly-Dap DAD1 B, Sig=320,4 Ref=450,80 (NITIN\MR180020.D) 11.518
mAU 400 350 300
10.328 10.475 10.691 10.983
150 100 50
13.041
200
11.712
250
0 5
10
13
Molecule 20: Asp-Dap-Dap-Gly-Dap-Gly-Dap DAD1 B, Sig=320,4 Ref=450,80 (NITIN\MR180021.D) 11.387
mAU
500
400
10.245 10.412
300
100
10.862
10.073
200
0 5
10
15
20
25
min
15
20
25
min
Molecule 21: Asp-Dap-Dap-Dap-Gly-Gly-Dap DAD1 B, Sig=320,4 Ref=450,80 (NITIN\MR180022.D) 11.545
mAU 400
10.516
350 300 250 200 150
10.955
100 50 0 5
10
14
ESI-MS analysis 21 crude products obtained after TFA cleavage of the cyclic heptapeptide library. Molecule 1
Molecule 2
15
Molecule 3
Molecule 4
16
Molecule 5
Molecule 6
17
Molecule 7
Molecule 8
18
Molecule 9
Molecule 10
19
Molecule 11
Molecule 12
20
Molecule 13
Molecule 14
21
Molecule 15
Molecule 16
22
Molecule 17
Molecule 18
23
Molecule 19
Molecule 20
24
Molecule 21
References [1] J.J. Diaz-Mochon, L. Bialy, M. Bradley, Full orthogonality between Dde and Fmoc: The direct synthesis of PNA-peptide conjugates, Organic Letters 6(7) (2004) 1127-1129.
25
