Research on the Synthesis Method and Application Prospects of Fmoc-His(Mtt)-OH
Abstract and Background Significance
This study employs dichlorodimethylsilane (DMDCS) as a bifunctional protecting reagent to successfully achieve selective protection of the imidazole ring and α-amino group in histidine molecules under alkaline reaction conditions. By systematically optimizing reaction conditions, high-purity Fmoc-His(Mtt)-OH products were obtained with an overall yield of 52.2%, confirmed by nuclear magnetic resonance hydrogen spectroscopy (1H NMR) and mass spectrometry (MS). This synthetic route has significant advantages such as ease of operation, ideal yields, and controllable costs, making it particularly suitable for industrial-scale production.
Histidine is one of the essential amino acids for humans, with its unique imidazole ring structure endowing it with special chemical properties and biological functions. In the field of solid-phase peptide synthesis, strategies for protecting histidine residues have always been a research challenge. Traditional protecting groups like trityl (Trt) and tert-butyloxycarbonyl (Boc), while widely used, have inherent drawbacks such as harsh deprotection conditions requiring high concentrations of trifluoroacetic acid (TFA) and significant toxicity from by-products. In contrast, 4-methyltrityl (Mtt) can be efficiently removed under mild conditions using 1% TFA, significantly improving synthesis efficiency while reducing environmental burden.
Materials and Methods
Experimental Reagents and Instruments The histidine raw materials used in this experiment were purchased from Sigma-Aldrich with a purity ≥99%. Dichlorodimethylsilane, 4-methyltrityl chloride (Mtt-Cl), Fmoc-succinimidyl ester (Fmoc-OSu), etc., are all analytical grade reagents. Nuclear magnetic resonance spectra were measured using a Bruker Avance 400 MHz spectrometer with deuterated methanol as solvent. Mass spectrometry analysis was performed using an Agilent 6230 TOF LC/MS system employing electrospray ionization (ESI) positive ion mode.
Synthesis Route Design The entire synthesis process consists of two key steps: first introducing Mtt protective group onto the imidazole ring followed by attaching Fmoc protective group at the α-amino site. This stepwise protection strategy effectively avoids interference between functional groups. Notably, we innovatively employed DMDCS as a dual-protecting reagent; its silicon atom's empty orbital can simultaneously activate both amine nitrogen atoms in histidine’s side chain along with those in imidazole nitrogen atoms to significantly enhance regional selectivity during protection reactions.
Detailed Experimental Steps
Synthesis of H-His(Mtt)-OH
In a 500 mL three-neck round-bottom flask, add 200 mL anhydrous dichloromethane as reaction solvent under nitrogen protection; sequentially add 11.64 g(75 mmol) histidine followed by adding 13 mL(101 mmol) DMDCS while installing reflux condenser; raise temperature to maintain at about (40^{circ}C) for approximately (2-3 hours). Monitor progress via thin-layer chromatography(TLC); developing agent ratio is chloroform:methanol:acetic acid=90:8:2(v/v/v).
After completion cool down to (0^{circ}C,) slowly drop-add (38 mL triethylamine adjusting pH to around7-8;)
once done add all at once(14 .64g(50mmol )M tt -Cl , remove ice bath stirring overnight at room temperature . After stopping reaction induce crystallization through ether/water mixture(2 :3,v/v )100ml . The solid collected through suction filtration washed successively then dried vacuum yielding white crystalline product(14 .57g ,yield71%). HPLC analysis shows purity reaching98 .2 %.
n **Synthesis OfFm oc -His(M tt )-O H **Take above intermediate5 .6 g(14mmol )dissolved into50 ml5 %sodium carbonate aqueous solution cooled down ice bath until0 ° C.Under vigorous stirring slow drop addition containing6 .9 g(20mmol )Fm oc - OS u dissolved within200 ml acetone solution.Control dropping speed maintaining pH range8 9 after finishing continue stirring15 minutes then raise up room temp reacting till endpoint monitored via TLC.Reaction finished firstly use rotary evaporator removing acetone residual washing successively utilizing petroleum ether &petroleum ether/ ethyl acetate mixed solvents(3 :1,v/v ).Water phase extracted thrice employing ethyl acetate merging organic phases subsequently washed respectively utilizing sodium bicarbonate solution,citric acid solution& saturated saline.Water-free sodium sulfate dried overnight concentrating obtaining yellow solid6 .4 g,yield74 %,HPLC purity98 .8 %. n ### Results And Discussion n **Structural Characterization Data **ProductFm oc - His(M tt )- O H structurally characterized comprehensively through spectral analyses.In1HNMR spectrum peak appearing@2 ;23 ppm corresponds methyl protons attached M t tprotectivegroup ;range between2 ;762 ;95ppm showing doublets corresponding β-methylene non-enantiomeric protons present withinhist idine.;multiplet peaks found @3 ;984 ;05 ppm indicating proton signals locatedα-carbon.;singlets detected @6;68ppm&7;.9 ppm correspondingly representing neighboring nitrogens situated adjacent positions withinimid azole rings whereas complex multiplet observed across ranges[6；887；67]presents aromatic protons derived frombothFMOC&M TTprotectivegroups。Massspectrometricanalysisrevealed presence quasi molecular ionspeak[M+H]+appearing atm/z634・4alongside adduct peaks[M+Na]+observedatm/z656・5.Crucially higher quality region also revealed dimeric ionic peak [M +22 ] due potential intermolecular hydrogen bonding interactions leading association phenomena occurring here 。 n **Reaction Condition Optimization:**During preliminary explorations noted that quantityofD MD CShad significant impact uponreaction yield.At molar ratios rangingbetweenD MD CSandhist idine beingaround1：21 yielded75%forintermediateproduct however increasingamountswould leadto morebyproducts.Formaintaining proper temperaturesduringfirst silylation reactions required strict control around40°C otherwise excessive heating would cause racemization issues affectingoverall results.Furthermore introduction sequence regarding protective groups designed meticulously where initiatingwithFMOCprior towardsM TT resulted roughly15%lowering respective yields likely attributed steric hindrance effects inhibiting effective shielding processes concerningimid azole moieties.Additionally discovered utilizationof sodium carbonate solutions regulatingpHs proved more stable thanusing sodium hydroxide avoiding risks associated potential loss FMOCunderstrongalkalineconditions encountered throughout experiments conducted herein thus enhancing final outcomes achieved overall substantially improving stability levels attained post-reactions concluded satisfactorily too! ### Application Prospects And Outlook: n As vital building blocks involved synthesizing bioactive peptides,F moc –His(mTT)o hhas seen numerous applications reported literature demonstrating successful utilizations G ly–his–lys tripeptide,PNA(aspeptidenucleicacid)&important segments involvinghistonesh_ _ _ A120−125,H_ _ B102−107amongothers highlighting coreadvantagespresented wherebyMTTremains easily removable physiological environments even lowconcentrationsT FAallowing accesscomplexpolypeptides development sensitive strong-acids paving way forward biomedical fields pursuing innovations targeting amino-acids containing compositions specifically tailored medicinal purposes therein future directions could focus primarily developing enhanced catalytic systems aimed boostingoverallperformance further exploring applicability strategies incorporated other hetero-cyclicamino acids(suchtryptophan)evaluating new automated polypeptide synthetizingsystems basedoncompound structures facilitating breakthroughs acceleratingprogressionsbiomedicalresearchesrelatingtheseaspectspotentially unlocking novel therapeutic avenues! n### Conclusion: n Established efficient economical routes producingF moc –His(mTT)o hsynthesizedviaselectiveprotectionstrategiesutilizingD MD CShighlightingtechnicalchallengesovercomingissuesregardingdual-functionalitiesassociatedhistoricallychallengingprotectionschemistriesappliedthroughoutstudiesconductedherein providingcriticalrawmaterialsessentialscalingupbioactivepeptidesproducingnoteworthyacademicvalueindustrialapplicationspotentialseekingfutureadvancementsbenefittinghealthcareindustries.