Microwave Assisted Peptide Synthesis

Microwave Assisted Peptide SynthesisSanjukta GhoshGreen Chemistry – 671December 8, 2011

OverviewI.II.What are peptides and why are they importantConventional method of peptide synthesis : SPPS andchallengesIII. Microwave technology and its contributionIV. Conclusion1

OverviewI.II.What are peptides and why are they importantConventional method of peptide synthesis : SPPS andchallengesIII. Microwave technology and its contribution in SPPSIV. Conclusion2

Peptides Peptides are short amino acid sequences, linked together with a polaramide bond Every peptide has a N-terminus and C-terminus residue on the ends ofthe peptide except for cyclic peptideswater is eliminatedOtwo amino acidscondense to form.H2NCHOCOHH2NCHR1OHR2N or aminoterminusH2N.a dipeptide. Ifthere are more itbecomes a polypeptide.Short polypeptide chainsare usually called peptideswhile longer ones are calledproteins.COCHCR1ONHCHCC or carboxyterminusOH HOHR2peptide bond is formedresidue 1residue 23

Growing Importance of Peptides Biological building blocks of Proteins and Enzymes; Theyact as active regulators and information carriersPeptides have numerous therapeutic potential ashormones, enzymes, antibiotics, antitumor agents andneurotransmittersCompared with small molecule therapies, peptideshave higher specificity and lower toxicity, no accumulationin organs and no side effectsInsulin was the first peptide to be administeredtherapeutically40 – 50 commercially available chemically synthesizedpeptide based drugs, about 600 in clinical trial stagesWatching peptide drugs grow up. Chem. Eng. News 2005, 83,17Synthetic therapeutic peptides: science and market. Drug Discovery Today 2010, 15, 405

Peptide NCEs entering clinical study:Rapidly Increasing MarketCompany enFerringGeneric relixIndicationHeart failureOsteoporosisHIVProstate CancerSevere chronic painDiabetes Type 1 and 2Diabetes Type 2Neuroendocrine tumorsImmune 2005200720082008Annual global salesexceeding eleven billionUS dollars2010 Report Summary: Development trends for peptide therapeutics, Peptide Therapeutics Peptide-Chemistry6

OverviewI.II.What are peptides and why are they importantConventional method of peptide synthesis : SPPS andchallengesIII. Microwave technology and its contribution in SPPSIV. Conclusion7

Solid Phase Peptide SynthesisSolid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide",Journal of the American Chemical Society 85 (14): 2149 Discovered by Robert Bruce Merrifield in 1963 Fifth most cited article in journal’s history Won Nobel Prize in Chemistry in 1984July 15, 1921, TexasMay 14, 2006, N. jersey8

Deprotection Coupling CleavageElegant and EfficientChallenges : Green?9

Challenges in SPPSSteps of SPPSChemicals useda. Carbodiimides: dicyclohexylcarbodiimide(DCC) and diisopropylcarbodiimide (DIC),Phosphonium and Uronium saltsCouplingAromatic oximes: 1-hydroxybenzotriazole(HOBt), 1-hydroxy-7-aza-benzotriazole (HOAt)Challengesa. Skin irritations and respiratoryproblems.Toxic nitrogen containingphosphoric byproduct.b. Sensitive and potentialexplosivesProtection/Deprotectiont-Boc: (tert Butyloxy carbonyl) ; HF forremoval Fmoc: (Fluorenyl-methoxy-carbonyl);for removal : TFA ; 20% piperidine in DMFHarsh Reaction conditionsCleavageTrifluoroacetic acid (TFA)Harsh Reaction conditionsSolventsDimethylformamide H-C( O)N(CH3)2DichloromethaneCH2Cl2Excess of waterUndesirable solvents ; Byproductsin the form of ureas,phosphonium salts, scavengers.o Use of hazardous chemicals and solvents in large amountso Formation of toxic byroducts and wasteo Low atom efficiency and high E factor10

Challenges in SPPSo Yield : If each amino acid addition has a 90% yield then the overallyield of a 50 amino acid peptide is only 0.5%.o Purity : purity of the product formed is affected by deleterioussequences and racemizationo Not suitable for difficult peptides. Difficulties are mainly related to: Intra- and/or intermolecular aggregation Secondary structure formation Steric hindrance of protecting groups which cangenerate premature termination of the sequenceNeed and scope for Improvement11

OverviewI.II.What are peptides and why are they importantConventional method of peptide synthesis : SPPS andchallengesIII. Microwave technology and its contribution in SPPSIV. Conclusion12

History of Microwave Technology 1946 : Microwave radiation was discovered as a method ofheating by Dr. Percy Spencer1947 : Housed in refrigerator-sized cabinets, First commercialdomestic microwave oven was introduced1990s : Microwave chemistry emerged and developed as afield of study for its applications in chemical reactions1992 : First attempt to synthesize peptide in householdmicrowave oven 12000 : First commercial microwave synthesizer was introducedto conduct chemical synthesisRadarange: 6 Feet Tall,750 Pounds, Cost US 50001 J.Org.Chem. 1992, 57, 478114

Microwave Technology /Chemistry The fundamental mechanism of microwave heating involvesagitation of polar molecules or ions that oscillate under theeffect of an oscillating electric or magnetic field. Only materials that absorb microwave radiation are relevantto microwave chemistry Operation range : 1GHz - 100 GHz . Chosen based onregulatory and cost constraintsArrhenius Equation: k A e –Ea/RT13

Thermal effect : Reduction in reaction TimeFmoc/tBu RT and MW-assisted SPPS deprotection and coupling protocols usedfor the synthesis of PTHrP(1–34)NH2First deprotectionTime(min)ProtocolSecond deprotectionPower(W)Temperature( C)Time(min)Power(W)Temperature ( C)CouplingTime(min)Power(W)Temperature ( C)RTSPPS5—2010—2020—20MWassisted SPPS0.535753607553075Faster Reactions : Time decreased from 35 mins to 8.5 minsJournal of Peptide ScienceVolume 17, Issue 10, pages 708–714, October 201115

Electromagnetic effect : Synthesis of difficult peptidesLess reagents requiredSource : Journal of Peptide Science 2007; 13: 143–14816

Direct Solid-Phase Synthesis of the β-Amyloid(1-42) Peptide Using Controlled Microwave HeatingBernadett Bacsa, Szilvia Bosze and C. Oliver KappeJ. Org. Chem. Vol. 75, No. 6, 2010β-amyloid peptide : H-DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA300 W single-mode manual microwave peptide synthesizerReactionTemperatureRT (SPPS)860C (MW)Coupling (DIC, HOBt,NMP, Fmoc-AA)45 minsOver AllSynthesis Time54 hHPLCPurity %54IC5010 mins26 h785.125.48β-amyloid (1-42) peptide is the major component of the neurological plaquesin Alzheimer’s patientsRT Synthesis is difficult due to reported on-resin aggregation and folding dueto hydrophobic C-terminus(IC50 : Half maximal inhibitory concentration) : How effective a drug is ?17

Solid-Phase Peptide Synthesis in Water Using Microwave-Assisted HeatingOrganic Letters, 2009, 11 (20), pp 4488–4491Leu-enkephalin; neurotransmitter :Tyr-Gly-Gly-Phe-Leu (H-YGGFL-OH)Complete synthesis in water with water soluble reagents18

Results and DiscussionHPLC chromatograms of crude Leu-Enk synthesized by SPPS in water .Faster reaction with high yield and purity (81- 90%)Small amount of impurities were observed as a result of byproduct and deletionreactions which was minimized using Triton detergentChallenge : Compatibility of the peptide and reagents in waterPurity of the peptide obtained by microwave heatingand conventional heating on a hot plate was similar19

Solid-Phase Synthesis of Difficult Peptide Sequences at ElevatedTemperatures: A Critical Comparison of Microwave and ConventionalHeating TechnologiesJ. Org. Chem. 2008, 73, 7532–7542Bernadett Bacsa,† Kata Horva ti,‡ Szilvia Bo sze,‡ Fritz Andreae,§ and C. Oliver tBu)-Ile-Met-Asn(Trt)-Ser(tBu)-CONH2 Conventional heating was done using heating block (PLS 4 6 organic synthesizer fromAdvanced ChemTech.300 W single-mode manual microwave peptide synthesizer (Discover SPS)Fast responding fiberoptic probe system as accurate temperature measurement device20

Result : Synthesis of GILTVSVAV Using Microwave andConventional Heating at the Same Temperature21

ConclusionPeptide 1peptide 2Peptide 3 Number ofResidues91523RT SPPSConventionalHeating32- 37%91%87%48%MicrowaveIrradiation92-95%91%54%The observed enhancement effects in the microwave-assisted SPPS areof purely thermal nature and not related to the microwave fieldNo evidence for a proposed disaggregation of the peptide backbone viadirect interaction of the peptide chain with the microwave field wasobtained22

Advantages over SPPS Faster reactionsBetter yield and Purity of peptideBetter technology for difficultsequencesEfficient source of heatingUniform Heating of the sampleGreener solventsSolvent less synthesis is possibleLess solvent and Reagents UseGreater ReproducibilityDisadvantages Heating under elevatedtemperatures.Racemization of the Histidine andcysteineHeating EfficiencyLack of scalabilitySafety hazardsHealth HazardsMW have improved the peptide synthesis in pharmaceutical industry byreducing the significant amount of time, energy and waste generation23

Path Forward :Attempts to make SPPS Green Use of more environmentally safe solvents and reagents Use of alternative reaction media : Ionic liquids, scCO2 Biocatalysis : Enzyme catalyzed peptide synthesis24

Milestones : FlowsynthAccelbeamPeptide Scientific Inc. PSI 200- 600Saves 40% of solvents by recyclingFully and semi automated, scales upto 10kgAAPPTechBiotage : Initiator Peptide WorkstationFully automated ;Difficult sequences, :Single reactor vessel, 25 ml to 2 LCEM: Liberty 1Small scale, cost-effective,ideal for universities.25

ReferencesConventional and microwave-assisted SPPS approach: a comparative synthesis of PTHrP(1–34)NH2, October 2011 Journal of Peptide Science, Volume 17, Issue 10, pages 708–714,Direct Solid-Phase Synthesis of the β-Amyloid (1-42) Peptide Using Controlled Microwave HeatingJ. Org. Chem. Vol. 75, No. 6, 2010Solid-Phase Peptide Synthesis in Water Using Microwave-Assisted HeatingOrganic Letters, 2009, 11 (20), pages 4488–4491Solid-Phase Synthesis of Difficult Peptide Sequences at Elevated Temperatures: A CriticalComparison of Microwave and Conventional Heating TechnologiesJ. Org. Chem. 2008, 73, 7532–7542Microwave Assisted Peptide Synthesis – A Tool to Replace Classical SPPS ?Peptides (2005) Volume: 43, Issue: 2, Pages: 148-149Microwaves in organic and medicinal chemistry By C. Oliver Kappe, Alexander Stadler.Microwave Heating as a Tool for Sustainable Chemistry By Nicholas E. LeadbeaterNature Reviews Drug Discovery January 2006, 5, 51-63Watching peptide drugs grow up Chem. Eng. News 2005, 83, 17–2126

The protein alphabet--the 20 amino acid R H32 H3CCH2CH2CH2HCH2CHEFGHI1 1 H2CH2QRS21211CH21 H3C2CH3CH2CHNTVWY4

A microwave oven converts only part of its electrical input into microwaveenergy. A typical consumer microwave oven consumes 1100 W of electricityin producing 700 W of microwave power, an efficiency of 64%. The other 400W are dissipated as heat, mostly in the magnetron tube. Additional power isused to operate the lamps, AC power transformer, magnetron cooling fan,food turntable motor and the control circuits. Such wasted heat, along withheat from the product being microwaved, is exhausted as warm air throughcooling vents

Conventional and microwave-assisted SPPS approach: a comparative synthesis of PTHrP(1– 34)NH 2, October 2011 Journal of Peptide Science, Volume 17, Issue 10, pages 708–714, Direct Solid-Phase Synthesis of the β-Amyloid (1-42) Peptide Using Controlled Microwave Heating J. Org. Chem. Vol. 75, No. 6, 2010 Solid-Phase Peptide Synthesis in .