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Title Low pH-triggering changes in peptide secondary structures

Author(s) Furukawa, Kaori; Oba, Makoto; Toyama, Kotomi; Opiyo, George Ouma;Demizu, Yosuke; Kurihara, Masaaki; Doi, Mitsunobu; Tanaka, Masakazu

Citation Organic and Biomolecular Chemistry, 15(30), pp.6302-6305; 2017

Issue Date 2017-08-14

URL http://hdl.handle.net/10069/37739

Right © 2017 Royal Society of Chemistry

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Received00thJanuary20xx,Accepted00thJanuary20xx

DOI:10.1039/x0xx00000x

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LowpH-triggeringChangesinPeptideSecondaryStructuresKaoriFurukawa,aMakotoOba,*aKotomiToyama,aGeorgeOumaOpiyo,aYosukeDemizu,bMasaakiKurihara,cMitsunobuDoi,dMasakazuTanaka*a

Wedevelopedanovelmethodologyusingcyclica,a-disubstituteda-aminoacids(dAAs)withanacetal-sidechaintocontrolpeptidesecondarystructures.TheintroductionofcyclicdAAsintopeptidescontributedtothestabilizationofpeptidesecondarystructuresasahelix,whileanacidictreatmentofpeptidesresultedinamarkedconformationalchange.

Foldamers, which were defined by S. H. Gellman as anypolymerswitha strong tendency toadopta specific compactconformation,1 have been the focus of studies for the pastdecadedue to theiruniqueproperties2–4andareexpected tobecomedrugcandidates.5Inorderforthefunctionsofsyntheticfoldamers to become similar to those of natural proteins,foldamerneedtomakedistinctchangesintheirconformationinresponsetoanexternalstimulusorenvironmentalchange.6Proteinsoftenstarttofunctionafteranappropriatestructuralchange in a suitableenvironment.a,a-Disubstituteda-aminoacids(dAAs)arepromisingtoolsforthedesignoffoldamers.7,8Peptides with cyclic dAAs are more likely to adopt a helicalstructure, even those with short sequences.9–13 These helicalpeptide foldamers have been reported to function asantimicrobial peptides,14,15 peptide catalysts,16,17 and drugdeliverypeptides.18–20Incontrast,peptidescomposedofacyclicdAAswithtwobulkysubstituentsequaltoorlargerthanethylgroups are more likely to form extended planar C5conformations.4,21–25 Therefore, the side chains of dAAs inpeptideschangefromacyclictoanacyclicstructurewithtwobulky substituents, as a consequence, peptide secondary

structures may change from a helical to a planar or randomstructure.

Fig. 1. Conformational changes in peptides containing cyclicdAAsbyanacidictreatment.Inthepresentstudy,wedesigneddAAswithacyclicacetal-sidechain.Acyclicacetalishydrolyzedunderacidicconditionsandgeneratesanacyclicdiol.Besidesstructuralchangesfromacyclictoanacyclicstructure,thehydrolysisofanacetaltoadiolincreases its hydrophilicity and produces two hydrogen bonddonors, which may break intra- and intermolecular peptideinteractions. We assumed that the synergistic effects ofstructuralchanges(fromcyclictoacyclic),hydrophilicchanges(fromanacetaltoadiol),andtheproductionofhydrogenbonddonorscontributetoconformationalchangesinpeptideswithdAAs(Fig.1).WepreparedtwotypesofdAAswithacyclicacetaland evaluated their lowpH-triggering structural changes.Wealso introduced dAAs into L-leucine (Leu) sequences andexaminedtheirpeptidesecondarystructuresbeforeandafteranacidictreatmentinordertoclarifyconformationalchangesinpeptides.

H2N CO2HH2N CO2H

OH OHOO

R R'Low pH

Introductioninto the peptide

Introductioninto the peptide

Acidic treatmentCyclic dAA Acyclic dAA

: L-Leu

· From cyclic to acyclic· Increase in hydophilicity· Hydrogen bond donors

Conformational change

CyclicdAA

AcyclicdAA

Cbz-Hms(Ipr)-OMe1a

Cbz-Hms-OMe2

H2O99%

TFA i) HMDS, TMSOTfO O

Cbz-HN CO2Me

OH OH

Cbz-HN CO2MeCbz-Hms(c-Hex)-OMe

1b

O O

Cbz-HN CO2Meii) Cyclohexanone TMSOTf

50% (2 steps)

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Scheme1.SynthesisofacyclicdAAwithanacetalsidechain.Hms:a-hydroxymethylserine; Hms(Ipr):O,O-isopropylidine-a-hydroxymethylserine; Hms(c-Hex): O,O-cyclohexylidine-a-hydroxymethylserine.TheacyclicdAAwithadiol-sidechain,Cbz-Hms-OMe2,wassynthesizedviaCbz-Hms(Ipr)-OMe1a(Scheme1).Acetalizationusing cyclohexanone was achieved according to previouslyreported methods,26 giving Cbz-Hms(c-Hex)-OMe 1b in amoderateyield.WethenpreparedoctapeptideswithHms(Ipr),Hms(c-Hex),orHmsintheL-Leusequences(Scheme2),inorderto evaluate the effects of side chain structures on peptidesecondary structures and low pH sensitivity. The synthesis ofpeptideswasachievedusing fragmentcondensationsolution-phase methods with HATU/HOAt or HBTU/HOBt as couplingreagents.Thecouplingyieldstoobtainoctapeptides7-10wererelativelylow,whichwasprobablyduetoasterichindranceofcyclic dAA at N-terminus of hexapeptides. The Ac-protectedHms(Ipr) octapeptide 8 was converted into the Hmsoctapeptide11notbyTFA/H2Obut2MHCl/MeOHinmoderateyield.

Scheme2.SynthesisofpeptideshavingHms(Ipr),Hms(c-Hex),orHmsinL-Leusequences.Low pH-triggering changes in the side chain structures ofcyclicdAAs1aand1bwereevaluatedby1HNMRmeasurements(Fig. S1). ThecyclicdAA1wasdissolved inpH2orpH3DCl-D2O/CD3OD (50/50). After an incubation at 25˚C for theindicatedtimes,1HNMRweremeasuredandtheconversion(%)

ofcyclicdAA1 intotheacyclicdAA2wascalculatedbasedonthepeakintensityofmethyleneprotonsinthesidechain.Fig.S1reveals thatsidechainstructureshaveasignificant impactonlowpH-triggeringchangesfromacyclicacetaltoanacyclicdiol. Fig. S2 showed the time-dependent MALDI-TOF-MSspectra of the Hms(Ipr) octapeptide 7 and Hms(c-Hex)octapeptide9inpH7andpH2TFE/H2OatroomtemperatureinordertodemonstratelowpH-triggeringsidechainstructuralchanges indAAs inpeptides.TheHms(Ipr)octapeptide7wascompletely transformed into theHmsoctapeptide11 atpH2after1day(Fig.S2b),butremainedintactatpH7(Fig.S2a).InthecaseoftheHms(c-Hex)octapeptide9atpH2(Fig.S2d),twoacetal moieties in the peptides were gradually changed intodiols and completely deprotected after 4 days. On the otherhand,nochangewasobserved inthespectrumatpH7,evenafter4days(Fig.S2c).Theseresultswereconsistentwiththoseshown in Fig. S1, in which the Hms(Ipr) dAA 1a was moresensitive to low pH than the Hms(c-Hex) dAA 1b. PeptideshavingdAAswithacyclicacetaldesignedhereinwerestableatneutralpH,butreactiveatacidicpH.

Fig.2. CD spectraof theHms(Ipr)octapeptide8,Hms(c-Hex)octapeptide 10, and Hms octapeptide 11. The peptideconcentrationwas0.1mMinTFE.We then investigated the secondary structures ofoctapeptides inTFE solutionusingCDspectralmeasurements(Figs.2andS3).Negativemaximaat205–209nm(p→p*)and222–225nm(n→p*)arediagnosticofright-handed(P)helicalstructures.27–29TheratioofR(qn→p*/qp→p*)hasbeenusedasaparametertodistinguisha-helicalfrom310-helicalstructures(R≈1:a-helix;R≤0.4:310-helix).

30,31Atypicalrandomstructureshows a negative maximum at 195–200 nm (p→p*) and apositivemaximum at 217 nm (n→p*). Cbz- andAc-protectedHms(Ipr)andHms(c-Hex)octapeptidesshowedsimilarresults,inwhichnegativemaximaatapproximately205nmand225nmwereobserved(Fig.S3).BasedontheRvalues(7:0.44;8:0.41;9: 0.23;10: 0.31), they adopteda right-handed (P) 310-helicalstructure. The FT-IR absorption spectra (the 3250–3500 cm–1regioninCDCl3)ofHms(Ipr)peptides5a/6a/7andHms(c-Hex)peptides 5b/6b/9 supported these results (Fig. 3). The low

Cbz-HN CO2H3a: Hms(Ipr)3b: Hms(c-Hex)

H-(L-Leu)2-OMeHATU, HOAt

DIPEA

Cbz-HNO

HN N

H

O

CO2Me

4a: Hms(Ipr) 86%4b: Hms(c-Hex) 94%

i) H2, 5% Pd/C or Pd(OH)2/C

ii) Cbz-(L-Leu)2-OH HBTU, HOBt or HATU, HOAt DIPEA

NH O

HN N

H

O

CO2Me

5a: Hms(Ipr) 71%5b: Hms(c-Hex) 53%

HN

OCbz-HN

i) H2, 10% Pd/C or Pd(OH)2/C

ii) 3a HBTU, HOBt, DIPEA or 3b HATU, HOAt, DIPEA

NH O

HN N

H

O

CO2Me

6a: Hms(Ipr) 83%6b: Hms(c-Hex) 40%

HN

ONH

OCbz-HN

i) H2, 10% Pd/C, or Pd(OH)2/C

ii) Pg-(L-Leu)2-OH HBTU, HOBt or HATU, HOAt DIPEA

NH O

HN N

H

O

CO2Me

7: Hms(Ipr) Pg: Cbz 25%8: Hms(Ipr) Pg: Ac 20%9: Hms(c-Hex) Pg: Cbz 12%10: Hms(c-Hex) Pg: Ac 5%

HN

ONH

OHNN

H

OPg-HN

O

O

O

O

Hms(Ipr): R, R' = MeHms(c-Hex): R, R' =

OOOO

OO

O O

OO

O O

OO

R R'R R'

R R'

R R'

R R'

R R'

R R'

NH O

HN N

H

O

CO2MeHN

ONH

OHNN

H

OAc-HN

O

O

OH OH

OHOH

11

2 M HCl/MeOH

39%

-7000

-5000

-3000

-1000

1000

3000

5000

7000

190 200 210 220 230 240Wavelength (nm)

[Θ] R

(deg

·cm

-2·d

mol

-1)

– Hms(Ipr) peptide 8– Hms(c-Hex) peptide 10– Hms peptide 11

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frequencybandat3350cm–1inpentapeptides5a/5bshiftedtoalowerwavenumberof3330cm–1inoctapeptides7/9,anditsintensity increased with the elongation of peptide lengths,suggestingthatthedominantsecondarystructurewasahelix.Hexapeptides 6a/6b showed bands assigned to peptide NHgroups intramolecularly hydrogen bonded to acetal oxygens,whichisconsistentwithapreviousstudy.26,32Ontheotherhand,theHmsoctapeptide1133showedthetypicalCDspectrumofarandom structure with a negative maximum at 195 nm andpositive maximum at 215 nm (Fig. 2). Furthermore, theconformationsofoctapeptides7and11wereanalyzedviaCDinMeOH/H2O(50/50)(Fig.S4).ThepreferredconformationofHms octapeptide 11 was a random structure, while that ofHms(Ipr) octapeptide7was ana-helical structure (R = 0.86).Changes in the side chain structures from cyclic acetals toacyclic diols led to marked changes in peptide secondarystructuresfromahelicaltoarandomstructure.Wepreviouslyreported that the preferred conformation of L-Leu-basedoctapeptides containing two cyclic dAAs with a seven-memberedringwassimilartothathavingtwodipropylglycinesasacyclicdAAs,aright-handed(P)310-helicalstructure.

34Thesefindings implied that a structural change in the side chain ofdAAs from cyclic to acyclic is not sufficient to induceconformational changes in dAA-containing L-Leu peptides.However, in the present study, a marked conformationalchangewasobservedinpeptideswithHms(Ipr/c-Hex)andHms,suggestingthatnochangeinthesidechainfromcyclictoacylic,but a change in the hydrophilicity and/or generation ofhydrogenbondacceptors is important for changes inpeptidesecondarystructures.

Fig.3.FTIRabsorptionspectraofa)Hms(Ipr)pentapeptide5a,hexapeptide 6a, and octapeptide 7, and b) Hms(c-Hex)pentapeptide 5b, hexapeptide 6b, and octapeptide 9. Thepeptideconcentrationwas5mMinCDCl3.TheHms(Ipr)octapeptide7formedgoodcrystalsforanX-raycrystallographicanalysisduetoslowevaporationofthesolvent(EtOH/H2O) at room temperature.35 Crystal and diffractionparametersaresummarizedinTableS1.Relevantbackboneandside-chain torsion angles aswell as intra- and intermolecularhydrogen bond parameters are listed in Tables S2 and S3,respectively.Themolecularstructuresof7areshowninFigs.4aand 4b. Two right-handed (P) a-helices were present in theasymmetricunitalongwith threeethanolmolecules (Fig.4a).Theconformationsof the twomolecules (moleculesAandB)weregenerallysimilar.Themeanϕandψtorsionanglesoftheaminoacidresidues(1–6)were–60.0˚and–43.4˚formolecule

Aand–58.1˚and–44.3˚formoleculeB,respectively,whichareclosetothoseforanidealright-handed(P)a-helix(–60˚and–45˚,respectively).Fiveintramolecularhydrogenbondsofthe i⟵i+4type,whichcorrespondedtothea-helicalconformation,are shown in molecules A and B (Table S3). Besides thehydrogenbondofthei⟵i+4type,onehydrogenbondofthei⟵ i+3 type (310-helical conformation) was observed inmoleculeB [N(7)···O(4)], indicating that this specifichydrogenbond was of the bifurcated type. In the packing mode, twomolecules, A and B, were alternatively connected byintermolecular hydrogen bonds along the backbone, forminghead-to-tail aligned chains; i.e., ···A···B···A···B···. These resultsslightlydifferedfromtheCDspectraldatainTFE(Fig.S3),butwere similar to that inMeOH/H2O (50/50) (Fig. S4). ThismayhavebeenbecausethesinglecrystalfortheX-rayanalysiswasobtainedbyrecrystallizationfromEtOH/H2OandtheCDspectraweremeasuredinTFEorMeOH/H2O,or,alternatively,aminor(P) a-helical structure existing in solution may have beenpreferentiallyinducedinthecrystalstateasaconsequenceofnucleation events. It is important to note that no hydrogenbondfromtheacetaloxygenswasobservedinthesidechainofHms(Ipr). Acetal oxygens did not suppress the formation ofhydrogenbondsinthepeptidebackbone.Unfortunately,goodcrystalsoftheHmsoctapeptide11havenotyetbeenobtainedforanX-rayanalysis.Conformationalcalculationsfora-helicaloctapeptides 8 and 10 were performed using the statisticalsoftwareMacroModel 10.0 (MCMMmethod; AMBER* (H2O);20,000 calculations) (Fig. S5). The global minimum energyconformation of the calculated a-helical structure of theHms(Ipr)octapeptide8closelymatchedtheX-rayconformationof 7 (Fig. 4c), although these peptides had a different N-terminal-protectinggroup(7:Ac;8:Cbz).

Fig.4.a)Crystal-statestructuresoftheHms(Ipr)octapeptide7.b) Side and top views ofmoleculeA. c) Superimposition of7

0

2

4

6

8

10

12

32003250330033503400345035000

2

4

6

8

10

12

14

16

3200325033003350340034503500Wavenumbers (cm–1) Wavenumbers (cm–1)

Abso

rptio

n

Abso

rptio

n

– pentapeptide 5a– hexapeptide 6a– octapeptide 7

– pentapeptide 5b– hexapeptide 6b– octapeptide 9

(a) Hms(Ipr) peptide (b) Hms(c-Hex) peptide

Molecule A Molecule B

EtOH

EtOH

EtOH

(a)

(b)

(c)

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(molecule A) assessed by an X-ray analysis (gray) and thecalculateda-helicalstructureof8(green).

ConclusionsInsummary,wehereindevelopedastrategyusingcyclicdAAsfor conformational changes in peptides from a helical to arandomstructurebyanacidictreatment.Thecyclicacetal-sidechainwasconvertedintotheacyclicdiol-sidechaininresponseto low pH. The acidic treatment of the cyclic dAA-containingpeptide afforded an acyclic dAA-containing peptide, and,consequently,peptidesecondarystructuresmarkedlychangedfrom a helical to a random structure. These results willcontributetothedesignoffunctionalfoldamersinresponsetoanexternalstimulusorenvironmentalchange.

AcknowledgementThis work was supported in part by JSPS KAKENHI GrantNumbers25560226and17H03998,theSumitomoFoundation,andtheTakedaScienceFoundation.

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33 Hmsoctapeptide11wasdissolvedinMeOHandTFE,butnotinCHCl3andCH3CN.Therefore,conformationalanalysisofitbyFTIRspectrawasimpossible.

34 M.Oba,H.Nonaka,M.Doi,M.Tanaka,Biopolymers(Pept.Sci.),2016,106,210.

35 CCDC-1543113containsthesupplementarycrystallographicdataforthispaper.ThesedatacanbeobtainedfreeofchargefromTheCambridgeCrystallographicDataCentrevia:Availableat:www.ccdc.cam.ac.uk/data_request/cif.