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2 ‫ מתוך‬1 ‫עמוד‬
Multi-step synthesis: solid phase peptide synthesis (SPPS)
Bacteria use quorum-sensing molecules (QSMs) to communicate within as well as across
species. Bacillus subtilis, a Gram-positive bacterium, uses a pentapeptide to regulate competence
and sporulation. The structure of this pentapeptide is Glu-Arg-Gly-Met-Thr (ERGMT). In the
following experiment you will synthesize this peptide on solid phase (resin), cleave it from the
resin, and analyze it by HPLC and MS. For more information see the links below:
Please search the internet for the coupling and Fmoc/Boc deprotection mechanisms.
Learn about the SPPS in general and orthogonality of protecting groups.
And check out the slides summarizing the SPPS chemistry
Fmoc chemistry link:
http://web.whittier.edu/people/webpages/personalwebpages/hashemzadeh/solid%20phase%20pe
ptide%20synthesis.pdf
Boc chemistry link:
Presentation
:
http://www.google.co.il/url?sa=t&source=web&cd=12&ved=0CCIQFjABOAo&url=http://www
.chem.missouri.edu/chem2120/SPPS.ppt&rct=j&q=solid%20phase%20peptide%20synthesis%20
boc%20mechanism&ei=RFX2TL6_LcWbOsmFgJwI&usg=AFQjCNGi1P7HWevi7Hq_NETL39GE_x4IA&cad=rja
The following articles are for the general knowledge:
The role of the synthesized peptide in quorum-sensing:
http://www.bgu.ac.il/~meijler/literature/subtilis_QS.pdf
Bruce Merrifield paper 1963 in which he introduce the SPPS strategy:
http://pubs.acs.org/doi/pdf/10.1021/ja00897a025
Coupling reagents and mechanism:
http://www.bgu.ac.il/~meijler/literature/peptide_synth.pdf
Fmoc-SPPS
A) amino acids MW:
Amino acid
Fmoc-Thr(Trt)-OH
Fmoc-Arg(Pbf)-OH
Fmoc-Glu(tBu)-OH
Fmoc-Met-OH
Fmoc-Gly-OH
HBTU
MW(mg/mmol)
583.7
648.8
425.5
371.5
297.3
379.3
B) Calculate amino acids (AAs) that are needed for the synthesis based on resin loading (the
attached protocol is for 0.05 mmol synthesis) and Rink amide resin with loading of 0.6 mmol/gr.
For better understanding see the presentation. For attachment of the first amino acid to the resin,
see the following protocol:
I.
Follow the protocol below for further couplings. You will work in groups of two
students, five groups will synthesize ERGMT, while four groups will synthesize
7 ‫ניסוי‬
2 ‫ מתוך‬2 ‫עמוד‬
TMGRE. How will reversal of the sequence affect the properties and bioactivity of
the peptide?
Amino Acid: 4 eq---------- 0.2 mmol
(For each coupling we take 4 eq. of amino acid relatively to the scale which is the
same number as for NH2 groups on the resin)
HBTU 4 eq-- 0.2 mmol (0.5 mL solution of HBTU)
(For each coupling we take 4 eq. of HBTU in solution of DMF relatively to the scale
which is the same number as for NH2 groups on the resin.
In order to prepare this solution: Prepare 0.5 M solution of HBTU in 3.5 mL of DMF)
DIEA: 8 eq-------------- 70 microliter of pure DIEA
Prepare a 20% (v/v) of piperidine solution in DMF. (50 mL)
1. Swell the resin with DMF for 10 min (2 mL is enough DMF to cover
the resin)
The resin amines on the resin is protected also with Fmoc so you have resin-NHFmoc, that's why step 2 is needed.
2. To remove the Fmoc group: Treat the resin-Fmoc with 2 mL piperidine
solution, 3 times (3 minutes/ 7 minutes/ 3minutes each time fresh
portions is added).
3. Wash the resin with DMF, 3 mL x 4
4. Activate amino acid with HBTU solution (0.5 mL) and DIEA (70
microliter) for 4 min
4 minutes starts after the addition of DIEA.
5. After the 4 minutes are over, remove the DMF and add the activated
amino acid solution to the semi-dry resin and leave for 45 min (only for
first coupling).
Shake gently every 5 minutes
6.
After the coupling time has finished, wash the resin with DMF
3 mL x 5.
7. Remove the Fmoc protecting group with 2 mL piperidine, 3 mL x 3
(3/7/3 min each deprotection step) {like step 2}
8. Wash the resin with DMF 3 mL x 5
9. For coupling of the next amino acids, repeat steps (4-8), coupling time
30 min.
II.
Cleavage
1. Take the desired amount for cleavage.
2. remove the Fmoc from the N-terminal (step7).
3. Wash with DMF, MeOH, DCM, and dry.
4. Treat it with appropriate volume of cleavage mixture (2.5% TIS +2.5% H2O in
trifluoroacetic acid (TFA) for 1.5h. (prepared by instructor)
5. Collect the TFA mixture and wash the resin with another portion of TFA.
6. Remove the TFA by evaporating the TFA with air flush.
B) Analyze the products by HPLC and / or LC-MS
7 ‫ניסוי‬