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Organic-chemical practical course Summer semester 2007






04. 04. 2007 - 13. 04. 2007

Stephan Steinmann 1

1. Formula Scheme[1]

The TFA salt of the tripeptide H-Pro-Pro-Asp-NH2 is synthesised by solid phase synthesis on Rink amide resin following the Fmoc Protocol.

O HO FmocNH O 1 Fmoc O N OH Boc N O OH 3 2 CF3COO 4 NH2 Solid phase synthesis O N O HN O O O OH


2. Mechanism

O H O HN O O - C5H12N+ 7 O 5 N Linker NH2 11 10 + C5H12N+ - CO2 9 Linker N H O O 8 O HN 6 Linker N H O

Scheme 1. Mechanism of Fmoc deprotection with piperidine.

The mechanism for a Fmoc deprotection is shown exemplary in Scheme 1. Mechanism of Fmoc deprotection with piperidine. Rink amide resin is functionalised and protected as a carbamate 5. Piperidine (6), a nucleophilic base, deprotonates 5, which then decomposes to 8 and 9. Carbamide acid 9 looses carbon dioxide, yielding in the free amine 11. Piperidine (6) as scavenger adds nucleophilic to the produced methylene fluorene (8), yielding 10.


A coupling with PyBOP and HOBt is shown in Scheme 2: Nucleophilic substitution at PyBOP (12) with the carboxylate 1 yields in 13. In a fast reaction, deprotonated HOBt (14) forms the active ester 16, which reacts slowly with the free amine 11, forming the amide 17.



O O O O 1 13 N N P N O O O NHFmoc N

N N O 14


FmocNH 12

N NP O N 15

O O H Linker N O 17 16

Scheme 2. Coupling with PyBOP and HOBt.


Linker NH2 11 - -OBt

FmocNH O O


The mechanism of coupling amino acids with HCTU is shown in Scheme 3: HCTU (18) is attacked by the carboxylate anion 2, building the active ester 19, which in turn is attacked by the amine 20 and decays into the urea derivative 22 and the triazole 23.

FmocN N N N Cl 18 O FmocN N Cl N O N N O O N H2N O O 20 N H Linker NFmoc O O HN Linker N H O O Cl N O 21 23 NH N N 22 O N N O N O O 2 N Fmoc Cl N O N N N 19 O O N


Scheme 3. Coupling with HCTU.


H N R O O O + CF3COOH - CF3COOR H O O 25 R 26 NH OH O 27 O 28 - CO2 - H+ R NH2 O 29



Scheme 4. Boc deprotection with trifluoro acetic acid.

Boc deprotection is shown in Scheme 4: The Boc protected carbamate 24 is protonated in trifluoro acetic acid. The protonated species 25 eliminates the tert.-carbocation 26, which undergoes elimination to 28. The carbamide acid 27 looses carbon dioxide, setting free the amine 29.



O OH OH HN 32 O O 33 O H 2N 34 R R

O N H 30



O N H 31



Scheme 5. Cleavage from the Rink Amide resin with trifluoro acetic acid.

A possible mechanism for the cleavage of a peptide from a Rink Amide resin is shown in Scheme 5: In trifluoro acetic acid 30 can be protonated to 31, which decays easily into the carbocation 33, which is very good stabilised and 32, which tautomerises into the amide 34.

3. Experimental

Rink amide resin (500 mg, 0.34 mmol, 1.0 eq) was shaken with 10 ml dichloromethane (DCM) for 5 min in a Merrifield flask. After removal of the DCM a mixture of DMF/Piperidine (4:1, 10 ml) was added and the suspension agitated for 3 min. The same was done for another 10 min. Then the solid was washed five times with DMF and three times with DCM. 4

The performed Kaiser test turned blue, indicating a successful removal of Fmoc protecting groups. A solution of absolute DMF (1 ml), absolute DCM (5 ml), PyBOP (535 mg, 1.03 mmol, 3.0 eq), HOBt (156 mg, 1.02 mmol, 3.0 eq), Fmoc-Asp(OtBu)-OH (422 mg, 1.03 mmol, 3 eq) and


Pr2NEt (0.35 ml, 20 mmol, 6.0 eq) was added to the polymer and the mixture agitated for 1.5

h. Then the solid was washed five times with DMF and six times with DCM. The performed Kaiser test turned again blue, indicating that the coupling had not worked. The solid was suspended in DCM and stored for some days. A solution of HCTU (423 mg, 1.02 mmol, 3.0 eq) and Fmoc-Asp(OtBu)-OH (421 mg, 1.02 mmol, 3.0 eq) in absolute DMF (2.5 ml) and iPr2NEt (0.55 ml, 3.21 mmol, 9.4 eq) was given to the dry solid and agitated for 1.5 h. The mixture turned from yellow to orange-brown. The solution was removed and the residue washed five times with DMF and three times with DCM. The Kaiser test didn't turn blue, indicating a successful coupling. Fmoc deprotection was performed as described above, after which the Kaiser test turned blue. The second coupling was done with a solution of Fmoc-Pro-OH (347 mg, 1.03 mmol, 3.0 eq) and HCTU (423 mg, 1.02 mmol, 3.0 eq) in absolute DMF (2.5 ml) and iPr2Net (0.55 ml, 3.21 mmol, 9.4 eq) within 75 min. After washing (five times with DMF, six times with DCM) the Kaiser test didn't turn blue anymore. Fmoc deprotection was carried out as usual and after washing (fife times with DMF, three times with DCM) the beads turned blue in a solution of chloranil in toluene (0.05 ml) and acetone (0.2 ml). The last coupling was done with a solution of Boc-Pro-OH (224 mg, 1.04 mmol, 3.1 eq) and HCTU (426 mg, 1.03 mmol, 3.0 eq) in absolute DMF (2.5 ml) and iPr2Net (0.55 ml, 3.21 mmol, 9.4 eq) within about 1.5 h. After washing (five times DMF, three times DCM) the beads remained colourless in a saturated solution of chloranil in toluene (0.05 ml) and acetone (0.2 ml), thus the coupling was successful. The peptide was cleaved from the polymer by addition of a solution of trifluoro acetic acid in DCM (2:1, 4 ml), which turned the solid and the solution deep red. After agitating for 50 min, the solution was pressed out of the Merrifield flask into a 50 ml flask and some DCM was rinsed over the solid. Trifluoro acetic acid in DCM (2:1, 4ml) was added again and the mixture agitated for another 20 min. The solution was pressed in the same flask and the resin rinsed with DCM. The resin was purple. 5

The red solution was partially evaporated at the rotary evaporator to about 1-2 ml. Addition of ether (25 ml) led to a complete loss of the red colour and a nearly white precipitate, which was put into the fridge. The main part of the clear solution from above the precipitate was removed with a pipette and the rest of the suspension put on the rotary evaporator. The resulting highly viscous liquid/oil was tried to dissolve in DCM and some methanol (20 drops), but a brown oozy residue remained. Therefore the solvents were removed at the rotary evaporator as completely as possible. The now even more viscous oil could be dissolved in a small amount of pure methanol. Addition of ether to the solution produced a precipitate, which was partly finely suspended in the solution, partly stuck to the flask. The suspension was transferred to tubes and put into a centrifuge. Only a tiny amount of solid was obtained like this. The residue in the flask was dried at the rotary evaporator, resulting in a white crystalline solid. (80 mg, 50 %)

4. Analytics[2]

MS (ESI): m/z: 327.1 [M+H]+ (100%) and 652.8 [2M+H]+ (15%).


H-NMR (250 MHz, D2O): /ppm = 4.55 (mc, 2H, -Pro); 4.39 (dd, 3JHH = 8.4 Hz, 3JHH = 7.8

Hz, 1H, -Asp); 3.40 (mc, 4H, -Pro); 2.78 (mc, 2H, -Asp); 2.48 (mc, 1H, -Pro); 2.25 (mc, 1H, -Pro); 1.99 (m, 6H, -Pro and -Pro).

5. References

[1] [2] Bodansky, Jakubke, Novabiochem Catalogue, 2003. P. Krattiger, R. Kovasy, J. D. Revell, S. Ivan and H. Wennemers, Organic Letters 2005, 7, 1101.




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