Wash?0.1?mmol?resin?with?DMF.
Dissolve?0.244?g?(+)-biotin?(1?mmol,?MW?244.3)?in?5?mL?DMF-DMSO?(1:1)?solution.?A?little?warming?is?necessary.
Add?2.1?mL?0.45?M?HBTU/HOBt?solution?and?0.3?mL?DIEA?to?the?solution?prepared?in?step?2.
Add?the?activated?biotin?solution?to?the?resin?and?let?stir?overnight.
Check?resin?to?make?sure?coupling?is?complete?as?evidenced?by?negative?ninhydrin?test?(colorless).
Wash?resin?with?DMF-DMSO?(1:1)?(2x)?to?remove?excess?(+)-biotin.
Wash?resin?with?DMF?(2x)?and?DCM?(2x).
Let?the?resin?dry?before?proceeding?to?cleavage.
在2-氯TRT樹脂上掛第一個FMOC氨基酸的方法

Weigh?10?g?2-chlorotrityl?chloride?resin?(15?mmol)?in?a?reaction?vessel,?wash?with?DMF?(2x),?swell?the?resin?in?50?mL?DMF?for?10?min,?drain?vessel.
Weigh?10?mmol?Fmoc-amino?acid?in?a?test?tube,?dissolve?Fmoc-amino?acid?in?40?mL?DMF,?transfer?the?solution?into?the?reaction?vessel?above,?add?8.7?mL?DIEA?(50?mmol),?swirl?mixture?for?30?min?at?room?temperature.
Add?5?mL?methanol?into?the?reaction?vessel?and?swirl?for?5?min.
Drain?and?wash?with?DMF?(5x).
Check?substitution.
Add?50?mL?20%?piperidine?to?remove?the?Fmoc?group.?Swirl?mixture?for?30?min.
Wash?with?DMF?(5x),?DCM?(2x),?put?resin?on?tissue?paper?over?a?foam?pad?and?let?dry?at?room?temperature?overnight?under?the?hood.?Cover?the?resin?with?another?piece?of?tissue?paper,?press?lightly?to?break?aggregates.
Weigh?loaded?resin.
Pack?in?appropriate?container.
檢測樹脂上面是否掛上第一個FMOC氨基酸的方法

Weigh?duplicate?samples?of?5?to?10?mg?loaded?resin?in?an?eppendorf?tube,?add?1.00?mL?20%?piperidine/DMF,?shake?for?20?min,?centrifuge?down?the?resin.
Transfer?100?μL?of?the?above?solution?into?a?tube?containing?10?mL?DMF,?mix?well.
Pipette?2?mL?DMF?into?each?of?the?two?cells?(reference?cell?and?sample?cell),?set?spectrophotometer?to?zero.?Empty?the?sample?cell,?transfer?2?mL?of?the?solution?from?step?2?into?the?sample?cell,?check?absorbance.
Subs?=?101(A)/7.8(w)
A?=?absorbance
w?=?mg?of?resin
Check?absorbance?three?times?at?301?nm,?calculate?average?substitution.
FMOC方法合成多肽操作步驟?(0.25?mmol)

Wash?resin?with?DMF?(4x)?and?then?drain?completely.
Add?approximately?10?mL?20%?piperidine/DMF?to?resin.?Shake?for?one?min?and?drain.
Add?another?10?mL?20%?piperidine/DMF.?Shake?for?30?min.
Drain?reaction?vessel?and?wash?resin?with?DMF?(4x).?Make?sure?there?is?no?piperidine?remaining.?Check?beads?using?ninhydrin?test,?beads?should?be?blue.
Coupling?Step?-?Prepare?the?following?solution:
1?mmol?Fmoc-amino?acid
2.1?mL?0.45?M?HBTU/HOBT?(1mmol)
348?μL?DIEA?(2?mmol)
Add?above?solution?to?the?resin?and?shake?for?a?minimum?of?30?min.?This?coupling?step?can?be?longer?if?desired.
Drain?reaction?vessel?and?wash?resin?with?DMF?(4x).
Perform?Ninhydrin?test:
If?negative?(colorless),?proceed?to?step?2?and?continue?synthesis.
If?positive?(blue),?return?to?step?5?and?re-couple?the?same?Fmoc-amino?acid.?Increase?the?coupling?time?if?necessary.
合成含有磷酸化酪氨酸多肽的方法
Reagent:?N-a-Fmoc-O-phosphotyrosine

For?0.1?mmol?or?0.25?mmol?synthesis,?use?0.483?g?Fmoc-Tyr(PO3H2)-OH?(1?mmol,?MW?483.4)?.?For?ABI?synthesizers,?pack?Fmoc-Tyr(PO3H2)-OH?in?a?cartridge.
The?cycle?program?for?coupling?Fmoc-Tyr?(PO3H2)-OH?is?the?same?as?for?other?Fmoc-amino?acids?except?for?the?coupling?time?(see?step?3).?(Note:?ABI?synthesizers?use?HBTU/HOBT?as?the?activating?reagent.)
The?coupling?time?for?Fmoc-Tyr(PO3H2)-OH?needs?to?be?increased.?For?ABI?model?430A?peptide?synthesizer,?insert?several?steps?(i.e.,?vortex?on,?wait?990?sec,?vortex?off,?to?increase?the?coupling?time).?For?ABI?model?431A?peptide?synthesizer,?add?additional?"I"s.?Overnight?coupling?may?be?necessary?for?some?sequences.
After?the?coupling?step?for?Fmoc-Tyr(PO3H2)-OH,?perform?ninhydrin?test?to?ensure?complete?coupling.?Negative?(colorless)?ninhydrin?test?indicates?complete?coupling,?while?a?positive?(blue)?ninhydrin?test?indicates?incomplete?coupling.
Increase?the?coupling?time?of?the?amino?acid?residues?after?the?phosphotyrosine?or?perform?double?coupling.?(Note:?The?coupling?of?amino?acids?after?the?phosphotyrosine?can?be?difficult.)
There?is?a?limit?on?the?number?of?amino?acid?residues?that?can?be?coupled?after?the?hosphotyrosine.?Since?the?phospho?group?is?unprotected,?side?reactions?are?likely?to?ccur.?(Note:?Peptides?have?been?successfully?coupled?with?sequences?containing?up?o?ten?additional?amino?acids?following?the?phosphotyrosine?residue.)
用Wang樹脂和2-氯TRT樹脂同時合成多肽
Peptides?which?differ?in?the?C-termini?can?be?simultaneously?synthesized?in?one?reaction?vessel?by?employing?resins?that?possess?different?cleavage?properties.?The?resins?used?were?the?weak?acid?labile?2-chlorotrityl?resins?and?the?TFA?labile?Wang?resins.?The?success?of?this?approach?was?shown?by?the?co-synthesis?of?ACTH?(4-10)?with?ACTH?(4-11)?and?Neuropeptide?Y,?a?C-terminal?amide?peptide?with?its?corresponding?C-terminal?free?acid?analog.
*Hong?A.,?Le?T.,?and?Phan?T.?Techniques?in?Protein?Chemistry?VI,?531-562?(1995).
切割程序
Starting?Resin:?Chlorotrityl?resins
Reagents?for?1?g?Peptide-Resin:
1?mL?acetic?acid?(AcOH)
2?mL?trifluoroethanol?(TFE)
7?mL?dichloromethane?(DCM)

Prepare?above?mixture.
Add?peptide-resin?to?the?mixture?and?let?it?stir?at?room?temperature?for?1?h.
Filter?and?wash?resin?with?10?mL?TFE:DCM?(2:8)?(2x)?to?ensure?that?all?of?the?product?is?recovered.
Evaporate?the?solvent?until?there?is?less?than?5?mL?of?liquid.
Add?ether?to?a?test?tube?containing?about?100?mL?of?the?above?solution.?Check?solubility?of?the?fully?protected?peptide?in?ether.?If?the?product?precipitates,?proceed?to?step?6.?If?no?precipitate?is?observed,?proceed?to?step?7.
Add?cold?ether?to?the?residual?liquid?in?step?4?to?precipitate?the?fully?protected?peptide.?Filter?through?a?fine?sintered?funnel?to?obtain?the?product.
Some?fully?protected?peptides?are?soluble?in?ether.?In?this?case,?add?water?to?precipitate?them?out.?Filter?through?a?fine?sintered?funnel?to?obtain?the?product.
從Fmoc-Lys(Mtt)氨基酸上脫去Mtt的方法
Reagent:
Fmoc-Lys(Mtt)-OH

Swell?resin?in?DCM.
Wash?resin?with?3%?TFA/DCM?(2x)?(since?the?resin?is?swollen?in?DCM,?this?step?of?washing?the?resin?quickly?with?3%?TFA/DCM?ensures?that?the?actual?concentration?of?TFA?is?3%).
Shake?the?resin?in?3%?TFA?for?10?min.
Repeat?step?3.
Wash?resin?with?DCM?(3x),?DMF?(3x),?isopropanol?(3x),?and?DCM?(3x).
Let?the?resin?dry?in?air.
多肽標記FITC方法
Reagents:
FITC
Fmoc-e-Ahx-OH

Couple?Fmoc-e-Ahx-OH?to?the?amino?terminal?of?the?peptide-resin?using?standard?coupling?conditions.
"De-Fmoc"?with?piperidine?using?the?standard?20%?piperidine?procedure.
Wash?resin?with?DMF?(3-4x).
Swell?resin?with?DCM?and?drain.
Prepare?solution?of?1.1?equivalent?of?FITC?in?pyridine/DMF/DCM?(12:7:5).?Use?just?enough?solution?to?form?a?slurry?with?the?resin.?Do?not?use?too?much?solution?since?the?rate?of?the?reaction?is?proportionate?to?the?concentration?of?the?solution.
Add?the?solution?prepared?in?step?2?to?the?resin.
Let?mix?overnight.
Check?the?completion?of?the?reaction?using?ninhydrin?test.
If?the?coupling?of?FITC?to?the?amino?group?is?not?complete,?ninhydrin?test?will?give?a?blue?color.?Repeat?the?coupling?with?FITC?(steps?5-7)?if?necessary.
Wash?resin?with?DMF?(2x),?isopropanol?(2x),?and?DCM?(2x).
多肽標記FAM方法
Reagent:
5-carboxyfluorescein?(5-FAM)

Use?standard?coupling?method?to?couple?5-carboxyfluorescein?to?the?amino?group?of?the?peptide.?For?cost?saving?purposes,?use?2x?excess?compared?to?the?mmol?of?resin,?instead?of?the?standard?4x?excess?used?for?Fmoc-amino?acids.?For?0.1?mmol?synthesis,?use?75?mg?5-carboxyfluorescein,?76?mg?HBTU,?and?70?mL?DIEA

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