Synthetic studies on kinase inihbitors and cyclic peptides : strategies towards new antibiotics

towards new antibiotics

Tuin, A.W.

Citation

Tuin, A. W. (2008, December 16). Synthetic studies on kinase inihbitors and cyclic peptides : strategies towards new antibiotics. Retrieved from https://hdl.handle.net/1887/13365

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Chapter6| Structural and Biological Evaluation of

NovelLoloatinCAnalogues



















Introduction

TheLoloatinsarecationicantimicrobialpeptides(CAPs)isolatedfromlaboratorycultures

of bacteria collected from the reefs of Loloata Island, Papua New Guinea.

 Four different

loloatinswerediscovered.ThesefourcyclicpeptidesarenamedLoloatinA,B,CandDand

their structures are depicted in Figure 1. Loloatins AD are active against Gram positive

bacteria with MIC values between 1 and 8g/mL against a panel of 8 organisms tested.

 Besidesbeingthemostpotentofthefourfamilymembers,LoloatinCwasalsotheonlyone

active against the Gram negative bacteria strain Escherichia Coli, making it an interesting

lead compound for further development towards a broad spectrum antibiotic. There is no

literature precedence concerning the hemolytical activity of these CAPs.



LoloatinCshowsaremarkableresemblancetoGramicidinS.Botharecyclicdecapeptides,

andtheyshare4outofthe10aminoacidsintheirprimarysequence.GSiscomposedoftwo

identicalpentapeptides,eachconsistingofastrand(ValOrnLeu)connectedtoatype II’

turn motif (

PhePro). Loloatin C has one closely related pentapeptide motif (with a

Tyr

insteadofa

Phe)supplementedwithaTrp

PheAsnmoietyandatype Iturnformedby

AspTrp. Whereas GS is likely to carry two positive charges in biological surroundings,

Loloatin C has a zwitterionic character. The solution structure of GS has been extensively

studied in a variety of solvents.

 In each of these solvents GS adopts a rigid sheet

conformation that is stabilized by four interstrand hydrogen bonds. This sheet

conformation is characterized by an amphiphilic orientation of the individual side chains,

withthehydrophobicValandLeuresiduesononesideofthemoleculeandthehydrophilic

Ornsidechainsontheother.ThesolutionstructureofLoloatinCismuchlessdefined.

The

secondarystructureisfoundtobesolventdependent.InDMSO,ahydrogenbondaccepting

solvent, the secondary structure of Loloatin C was analyzed by means of chemical shift

perturbation and NOESY analysis. A strand composed of LeuOrnVal and an opposing 

helical

 Trp

PheAsn strand are interconnected via two turn motifs composed of Pro

Tyr

and AspTrp. The lack of chemical shift reference data in water / trifluoroethanol (TFE, a

hydrogen bond donating solvent known to stabilize secondary structural elements)

 precluded solid conclusions about the conformation of Loloatin C. In a 30% TFE / H

O

mixture,NOESYanalysisrevealedalessdefinedstructurewithonlyonesecondarystructural

Figure1;ChemicalstructuresofLoloatinAD,GSandSAA6

element, namely, an inverse turn around the

TyrProTrp sequence. Increasing the TFE

contentinthesolventmixtureto70%inducedaremarkablestructuralchange.Adumbbell

likeconformationwasidentifiedcenteredaroundtheOrnand

Pheresidues.Interestingly,

thisconformationorientatesthehydrophilicsidechains(Orn,AsnandAsp)toonesideofthe

moleculeandtheremaininghydrophobicsidechainstotheother,resultinginanamphiphilic

overallstructure.

The flexibility of Loloatin C and the zwitterionic character present attractive features

amenable for optimization towards Loloatin C analogues with improved antibacterial

propertiesandreducedhemolyticactivity.

In the previous chapter, a study is described in which GS was modified by the

incorporation of a series of SAAs replacing the turn motif consisting of a Pro and a

Phe

residue.ItwasshownthatSAA6couldbeusedasaturninducingdipeptideisosterwitha

slightly distinct architecture compared to the natural turn. In the study described in this

chapter,theprimarystructureofLoloatinCismodifiedbyreplacingsingleaminoacidsand/

or incorporating SAA 6. The newly synthesized analogues were evaluated for their

antibacterial and hemolytical activities and their secondary structure were assessed by

meansofNMRanalysis.



ThetargetmoleculesaredepictedinFigure2.Ascanbeseen,SAA6replaceseachofthe

threeputativeturnmotifsinLoloatinC.Firstly,thetypeII’turncomprisedofthePro

Tyr

motif,similartotheturnsfoundinGS,isreplaced(7,Figure2).Secondly,theAsn

Phepart

ofthehelicalturnisreplaced(8,Figure2)andreplacingthetypeIturninducingAspTrp

motifwithSAA6(9,Figure2)completestheseries.Inanalogue10(Figure2)SAA6replaces

theAspTrpmotifandthe

Phewithinthehelicalturnisreplacedby

Phe.Theinfluenceof

using

Phe instead of

Phe is further studied in 11 and in 12 which has an additional

replacementofthe

Aspwitha

Aspintendedasmodifiedturninducingmotif(Figure2).











Figure2;LoloatinCandnewlydesignedderivatives 7  12

Resultsanddiscussion

ThesynthesisoftheLoloatinCanaloguesisexemplifiedinScheme1fortheconstruction

of compound 7. Standard Fmoc based solid phase peptide synthesis (SPPS) starting from

commercially available HMPBBHA resin preloaded with FmocLeucine is subjected to 7

coupling cycles under the agency of HCTU en DiPEA affording linear octapeptide 13.

Staudinger reduction of the terminal azide functionality in 14 using PMe

 in wet THF

followed by acidic cleavage from the resin afforded 15 which was subsequently cyclized

uponPyBOP/HOBt/DiPEAtreatmentinDMFunderdiluteconditionsanddeprotectedusing

50%TFA/DCM.HPLCpurificationyieldedthetargetcompoundin7.4%yield.Analogues8to

12 were prepared following the same strategy, and although the final yields varied

considerably, suitable quantities of each compound for the ensuing biological and

hemolyticalstudieswereobtained.



Scheme1;SynthesisofLolatinCderivativesusingSPPS.Reagentsandconditions:8cyclesofa:20%

pip/NMP,3x5min.;b:FmocAAOH(5eq.),HCTU(5eq.),DiPEA(10eq.)90min.(ii)6(1.5eq.),HCTU

(1.5eq.),DiPEA(3eq.),16h.(iii)a:PMe3(1Min9:1THF/H2O,25eq.),16h.b:1%TFA/DCM.(iv)a:

PyBOP(5eq.),HOBt(5eq.),DiPEA(10eq.),16h.b:50%TFA/DCM,1h.

NMRstudiesofLoloatinderivatives 712

Next, attention was focused on the structural analysis of the newly designed Loloatin C

analogues in solution by means of NMR spectroscopy. The structure of each of the

analogueswasassessedbytheanalysisofthevicinal

J

couplingconstant

,thechemical

shiftperturbation

andtheNOESYspectrum.Thevicinal

J

couplingconstantcorrelates

withthedihedralanglewithinanaminoacidresidue.Itcanbeusedinaqualitativemanner

to identify secondary structural elements within a peptide. Consecutive large values (8.5 –

9.5 Hz) correspond to dihedral angles commonly found in sheets, small values for

J

 (3.5–4.5Hz)correspondtothosefoundinanhelix.Isolatedoccurrencesofsmallvalues

for

J

 (< 4 Hz) indicate the presences of a turn within the peptide chain. Another

qualitative method to distinguish between secondary structural elements is the chemical

shiftperturbation.Thismethodreliesonthecomparisonofthemeasuredchemicalshiftof

theprotonofacertainresidueandthestandardvalueofthatsameresidueinarandom

coilconfiguration( H

=observed H

–randomcoil H

).Thisisbasedontheobservation

thatprotonsofresidueswithinanhelixexperienceanupfieldshiftandresidueswithina

sheet experience a downfield shift compared to the chemical shift of that residue in a

randomcoilconfiguration.

Theobservedvaluesforthevicinalcouplingconstants

J

fortheLoloatinCanalogues

are depicted in Figure 3. The chemical shift perturbations are depicted in Figure 4. The

secondary structure of all derivatives contain a turn motif around

Tyr (

J

 < 4 Hz),

flankedbytwostrandregions(8.5<

J

<9.5Hz).TheLeuOrnValsequenceformsa

strand in all analogues. However, the relatively low value for

J

 of Val in 7 indicates

distortionoftheresidue.Interpretationofthechemicalshiftperturbationunderlinesthese

findings.TheTrp



PheAsnmotifismorevariableamongthedifferentcompoundsand

no general conclusions can be drawn. In compound 9, the

Tyr, Trp

 and

Phe residues

appear to form a helical region, as evidencedby their negative chemical shift perturbation

values.Thepositionofresidues9and10inanaloguesareconnectedeitherbySAA6(in10)

or by

AspTrp motif (in 11 an 12 resp.). The structure of compounds 10 and 11 are

remarkablysimilarexceptfortheregionsroundthe

Pheresidues.The

HNMRspectraof8

and12showseverepeakbroadening,andthelimitednumberofsequentialNOEsignalsin

the2DNMRpreventedcompleteassignmentofthespectra.











Figure3;Valuesofthevicinal³JNHH(Hz)forLoloatinCanditsderivatives.

Figure4;ChemicalshiftperturbationforLoloatinCanditsderivatives.

Biologicalevaluation

Finally, the antimicrobial and hemolytical potencies of the novel Loloatin C derivatives

were determined (Table 1 and Figure 5). Whereas the native Loloatin C (3) shows some

antimicrobial potency against gram positive strains, it is inactive against the gram negative

strains used in this assay. Unfortunately, Loloatin C is also hemolytically active. As for the

analogues7,8,9and12boththeantimicrobialandhemolytical

activitiesarehighlyreduced

compared to Loloatin C. Only analogues 10 and 11 are marginally active in both assays.





 Grampos. Grampos. Grampos. Gramneg. Gramneg. Grampos.

 Staph.aureus Staph.epidermis Entrerococ.faecalis E.Coli P.auruginosa Bacillusereus

 ATCC29213 ATCC12228 ATCC29212 ATCC25922 ATCC27853 ATCC11778

3 8 8 8 >64 >64 8

7 >64 >64 >64 >64 >64 >64

8 >64 >64 >64 >64 >64 >64

9 >64 >64 >64 >64 >64 >64

10 >64 32 >64 >64 >64 64

11 64 32 >64 >64 >64 32

12 >64 >64 >64 >64 >64 >64

Table1;MICvaluesforcompounds3and7 13

Figure5;HemolyticalactivityoftheLoloatinCderivatives

Conclusion

Six novel analogues of Loloatin C were synthesized and analyzed by means of NMR

spectroscopy.Fouroftheseanaloguescontainsugaraminoacid6,replacing2naturalamino

acids. Replacing either the Pro – DTyr or the AspTrp motif resulted in analogues (7 – 10)

with defined secondary structures that could be analyzed based on 1D and 2DNMR

spectra.LoloatinCandanalogues11and12provedtobemuchmoreflexibleinCD

OH.The

biological activities of all analogues were rather low with only marginal activities of

analogues10and11inboththeantimicrobialandhemolyticalassay.Theactivityofnative

Loloatin C has been explained by the dumbbelllike conformation it adopts. This

conformation induces an amphiphilic character of the peptide.

 The reduced activity of

analogues 7 – 12 might be explained by inability of these peptides to adopt a similar

conformation.



Experimentalsection

Generalprocedureforpeptidesynthesis:

a)stepwiseelongation:FmocLeuHMPBBHAresinResin(196mg,0.51mmol/g,0.1mmol)wassubmitted

tosevencyclesofFmocsolidphasesynthesiswiththeappropriatecommerciallyavailableaminoacidbuilding

blocks FmocOrn(Boc)OH, FmocValOH, FmocTyr(tBu)OH, FmocProOH, Fmoc^DPheOH, FmocPheOH,

FmocAsp(tBu)OH,FmocAsn(Trt)OHandFmocTrp(Boc)OHasfollows:a)deprotectionwithpiperidine/NMP

(1/4,v/v,5mL,15min);b)washwithNMP(5mL,3x,3min);c)couplingoftheappropriateFmocaminoacid(5

eq.,0.5mmol)inthepresenceofHCTU(5eq.,0.5mmol,206mg)andDiPEA(10equiv.,1mmol,162L)which

was preactivated for 2 min in NMP (5 mL) and shaken for 90 min; d) wash with NMP (5 mL, 3x, 3 min).

Couplingsweremonitored for completionbytheKaiser test.^10Finally,theNterminal aminewas liberatedby

Fmocdeprotectionwithpiperidine/NMP(1/4,v/v,5mL,15min)followedbywashingwithNMP(5mL,3x,3

min).CouplingofSAA6wasperformedasfollows:Totheresinboundpeptide,apreactivatedsolutionofSAA6

(1.5eq.44mg,0.150mmol),HCTU(1.5eq.,62mg,0.150mmol)andDiPEA(3.0eq.,74PL,0.45mmol)inNMP

(3mL)wasaddedandtheresultingsuspensionwasshakenfor16h.TheresinwasfinallywashedwithNMP(5

mL,3x,3min)togivethetitlecompound.

b)onresinStaudingerreduction.Theappropriateresinboundazidewastreatedwithapremixedcocktail

ofH2O(0.5mL)andPMe3(3.5mL,1MinTHF)andshakenfor16h.Theresinwaswashedwithmethanol(4mL,

3x,3min)andDMF(4mL,3x,3min.)

d)cyclization:ThelinearnonapeptidewastakenupinDMF(5mL)andaddeddropwiseoverthecourseof

anhourtoasolutionofbenzotriazole1yloxytrispyrrolidinophosphoniumhexafluorophosphate(PyBOP)(5

equiv.,270mg,0.5mmol),HOBt(5equiv.,67mg,0.5mmol)andDiPEA(15equiv.,254PL,1.5mmol)inDMF

(70 mL) and allowed to stir for 16h. The solvent was removed in vacuo and the resulting mixture was used

withoutfurtherpurificationinthedeprotectionstep.

e)deprotection:Thecrudecyclisedpeptidewastreatedwith50%TFA/DCM(10mL)for1h.,beforeitwas

concentratedandpurifiedbyHPLCpurification.



cyclo[ValOrnLeu^DTyrProTrp^DPheAsnAspTrp] 3: Prepared according

tothegeneralprocedure.Yield:19.7mg,13.5mol,13.5%.¹HNMR(500

MHz, CD3OH)  10.25 (s, 1H), 10.22 (s, 1H), 9.43 (bs, 1H), 9.34 (bs, 1H),

9.23(bs,1H),8.93(bs,1H),8.80(d,J=9.3,1H),8.68(bs,1H),8.28(d,J=

2.6,1H),8.14(s,1H),8.00(d,J=8.6,1H),7.65(d,J=7.7,1H),7.55(dd,J=

13.3,29.2,4H),7.43–7.32(m,3H),7.28–7.13(m,5H),7.09–6.89(m,6H),6.84(s,1H),6.72(bs,1H),6.63(d,J

=8.1,2H),5.94–5.84(m,1H),5.52(d,J=5.9,1H),4.78–4.68(m,1H),4.68–4.60(m,2H),4.32(bs,2H),4.06

(d,J=7.1,1H),3.65–3.63(m,1H),3.45–3.35(m,2H),3.22–3.00(m,4H),3.00–2.79(m,4H),2.64(t,J=

13.5,1H),2.40–2.14(m,5H),2.14–2.05(m,1H),2.03–1.93(m,2H),1.93–1.84(m,2H),1.83–1.75(m,4H),

1.74–1.65(m,2H),1.61–1.48(m,2H),1.39–1.23(m,3H),1.20(s,3H),1.16(m,6H),1.12–1.04(m,6H),0.94

–0.82(m,1H),0.19(s,1H).



cyclo[ValOrnLeuSAATrp^DPheAsnAspTrp] 7: prepared according to the

general procedure. Yield: 9.73 mg, 6.76 mol, 6.8%.¹H NMR (500 MHz,

CD3OH) 10.39(s,1H),10.33(s,1H),8.88(d,J=7.1,1H),8.53(d,J=8.3,1H),

8.39(d,J=8.9,1H),8.34(d,J=3.4,1H),8.25(d,J=8.2,1H),8.19(d,J=9.3,

1H),8.08–8.03(m,1H),7.98–7.93(m,1H),7.75(d,J=8.9,1H),7.73(s,1H),

7.66(d,J=7.8,1H),7.52(d,J=7.9,1H),7.38(bs,1H),7.34–6.94(m,20H),5.48(d,J=7.7,1H),4.82(dd,J=

8.1,13.8,1H),4.71(dt,J=4.8,10.2,1H),4.67–4.62(m,1H),4.56(d,J=11.7,1H),4.52(d,J=11.6,1H),4.46–

4.39(m,1H),4.38–4.30(m,3H),4.21(d,J=10.0,2H),3.84(s,1H),3.76–3.68(m,1H),3.34(s,1H),3.24–3.15

(m,3H),3.11–3.04(m,3H),2.99–2.92(m,1H),2.91–2.79(m,3H),2.73(bs,1H),2.69(dd,J=3.9,17.4,2H),

2.30–2.22(m,1H),2.06–1.94(m,2H),1.85–1.76(m,1H),1.72–1.57(m,4H),1.57–1.46(m,2H),1.36–

1.34(m,1H),1.32–1.25(m,2H),1.25–1.16(m,2H),1.01(d,J=6.7,3H),0.97(d,J=6.6,3H),0.85(d,J=5.9,

3H),0.79(d,J=5.9,3H).



cyclo[ValOrnLeu^DTyrProTrpSAAAspTrp] 8. Prepared according to

thegeneralprocedure.Yield:29.8mg,22.5mol,22.5%.1HNMR(600

MHz, MeOD)  10.67 (s, 1H), 10.65 (s, 1H), 10.32 (s, 1H), 8.99 (s, 1H),

8.38(d,J=9.5,1H),7.85(bs,1H),7.82(bs,1H),7.74(d,J=7.7,2H),7.57

(d,J=8.1,1H),7.55(bs,1H),7.49(d,J=7.8,1H),7.45(s,1H),7.38(d,J=

7.6,3H),7.32(t,J=7.5,2H),7.27(t,J=7.9,2H),7.22–7.14(m,3H),7.08(s,1H),7.03(dd,J=7.2,12.4,2H),

6.96(t,J=7.4,1H),6.92(d,J=8.3,2H),6.83–6.73(m,1H),6.65(d,J=8.4,2H),4.65–4.59(m,2H),4.59–

4.50(m,3H),4.33(d,J=14.4,1H),4.29(d,J=13.2,2H),4.19(t,J=5.0,1H),4.15(bs,1H),4.11–4.06(m,2H),

4.06–4.00(m,1H),3.90(d,J=8.1,1H),3.88(bs,1H),3.72(s,1H),3.52–3.45(m,2H),3.45–3.35(m,1H),3.34

(s,1H),3.28–3.16(m,4H),2.90(t,J=12.6,1H),2.77(dt,J=5.4,17.6,1H),2.77(bs,1H),2.61(bs,1H),2.19–

2.06(m,2H),2.01–1.87(m,2H),1.78–1.69(m,1H),1.63(dd,J=11.0,22.2,2H),1.60–1.53(m,2H),1.47–

1.38(m,2H),1.38–1.25(m,4H),1.25–1.17(m,7H),1.15–1.06(m,2H),1.06–0.98(m,5H),0.98–0.81(m,

6H),0.74–0.62(m,1H),0.61–0.47(m,1H),0.44–0.29(m,1H).HRMScaldfor[C69H86N12O15+H]⁺1323.64084,

found1323.64236.



cyclo[ValOrnLeu^DTyrProTrp^DPheAsnSAA] 9. Prepared according to

thegeneralprocedure.Yield:4.0mg,3.0mol,3.0%.¹HNMR(600MHz,

CD3OH) 10.30(s,2H),8.95(s,1H),8.79(d,J=8.4,1H),8.59(d,J=8.1,

1H),8.55(d,J=7.7,1H),8.49(d,J=8.7,1H),8.18(bs,1H),8.14(d,J=7.7,

1H),8.02(d,J=9.0,1H),7.78(d,J=9.2,2H),7.73(d,J=7.9,1H),7.55(d,

J=7.9,1H),7.32(d,J=8.1,1H),7.27(d,J=8.2,1H),7.21–7.14(m,4H),

7.14–7.09(m,4H),7.09–6.96(m,10H),6.94(t,J=7.4,2H),6.66(d,J=8.4,2H),5.41–5.31(m,1H),4.82–

4.75(m,1H),4.69(dd,J=7.9,15.5,1H),4.60–4.54(m,1H),4.46(t,J=8.0,1H),4.39–4.33(m,1H),4.16(dd,J

=2.4,7.5,1H),4.12–4.07(m,1H),3.37(d,J=7.6,3H),3.26(d,J=9.3,1H),3.18(dd,J=4.4,14.4,1H),3.07

(dd,J=6.8,13.8,1H),3.05–2.98(m,2H),2.96(dd,J=4.7,12.7,2H),2.91–2.74(m,5H),2.72–2.55(m,3H),

2.27(dd,J=9.5,17.2,1H),2.20(dd,J=9.3,17.1,1H),2.13(dd,J=6.8,13.7,1H),1.96–1.88(m,1H),1.79–

1.72(m,1H),1.72–1.61(m,6H),1.61–1.54(m,2H),1.44–1.33(m,3H),1.31–1.24(m,1H),1.02–0.91(m,

18H),0.43–0.32(m,1H).HRMScaldfor[C69H86N14O14+H]⁺1335.65207,found1335.65402.



cyclo[ValOrnLeu^dTyrProTrpPheAsnSAA] 10. Prepared according to

the general procedure. Yield: 46.6 mg, 36.6 mol, 36.6%. 1H NMR (600

MHz, MeOD)  10.41 (bs, 1H), 8.99 (d, J = 3.1, 1H), 8.82 (d, J = 8.4, 1H),

8.78(d,J=9.1,2H),8.72(d,J=8.4,2H),8.64(d,J=7.6,2H),7.94(d,J=

8.4,1H),7.85–7.79(m,5H),7.70(d,J=8.6,2H),7.69–7.65(m,2H),7.63

–7.60(m,1H),7.57–7.53(m,1H),7.51–7.48(m,J=12.5,1H),7.40–7.37(m,5H),7.37–7.30(m,7H),7.29–

7.22(m,7H),7.18–7.14(m,1H),7.12(t,J=7.3,2H),7.07–6.99(m,5H),5.18(q,J=7.6,1H),5.02(s,1H),4.94

–4.85(m,1H),4.82–4.76(m,1H),4.76–4.71(m,1H),4.71–4.67(m,2H),4.62(d,J=11.7,1H),4.57–4.47

(m,5H),4.43–4.36(m,2H),4.28–4.25(m,1H),4.24(d,J=3.0,1H),4.17(dd,J=1.7,8.2,1H),4.12(t,J=10.6,

1H),3.92(d,J=2.8,2H),3.81(d,J=16.7,1H),3.76–3.70(m,1H),3.65(s,1H),3.31(dd,J=9.1,14.7,18H),

3.23–3.18(m,25H),3.03–2.96(m,3H),2.89–2.81(m,3H),2.80–2.73(m,1H),2.70(dd,J=5.7,15.5,1H),

2.55–2.47(m,1H),2.31–2.20(m,3H),2.09–2.02(m,11H),2.01–1.93(m,2H),1.93–1.87(m,21H),1.84–

–1.03(m,20H),1.03–0.93(m,5H),0.42–0.30(m,1H).HRMScaldfor[C67H86N12O14+H]⁺1283.64592,found

1283.64735.



cyclo[ValOrnLeu^DTyrProTrpPheAsnAspTrp] 11. Prepared

according to the general procedure. Yield: 39 mg, 30 mol, 30%. 1H

NMR(600MHz,MeOD) 10.32(s,1H),8.91(d,J=2.9,1H),8.75(d,J=

7.9,1H),8.71(d,J=9.0,1H),8.64(d,J=8.4,1H),8.57(d,J=7.5,1H),

7.86 (d,J = 8.4,1H),7.78 –7.71(m,3H),7.62 (d,J =8.6,1H),7.60(bs,

1H),7.55–7.50(m,1H),7.47(t,J=7.6,1H),7.42(s,1H),7.33–7.22(m,9H),7.21–7.14(m,5H),7.04(t,J=

7.5,1H),6.98–6.93(m,3H),6.67(s,1H),6.64(d,J=8.4,2H),5.10(d,J=5.7,1H),4.74–4.69(m,1H),4.66(dd,

J=7.8,16.2,1H),4.61(d,J=11.7,1H),4.54(d,J=11.7,1H),4.47(d,J=3.9,1H),4.45–4.40(m,2H),4.34–

4.29(m,1H),4.16(d,J=2.9,1H),4.09(d,J=8.3,1H),3.85(d,J=2.6,1H),3.73(d,J=14.3,1H),3.62(d,J=

11.1,1H),3.58–3.54(m,1H),3.29–3.17(m,8H),3.17–3.11(m,2H),2.95–2.88(m,2H),2.81–2.73(m,2H),

2.73–2.66(m,1H),2.62(dd,J=5.6,15.4,1H),2.47–2.38(m,1H),2.23–2.13(m,2H),2.01–1.93(m,4H),

1.92–1.80(m,1H),1.77–1.68(m,1H),1.68–1.59(m,1H),1.59–1.45(m,4H),1.45–1.31(m,2H),1.30–

1.24(m,1H),1.02–0.95(m,14H),0.94–0.85(m,2H),0.33–0.24(m,1H).





cycloValOrnLeu^DTyrProTrpPheAsn^DAspTrp] 12. Prepared

according to the general procedure. Yield: 7.2 mg 5.4mol, 5.4 %.¹H

NMR(600MHz,CD3OH) 10.32(s,2H),10.30(s,2H),9.05(bs,2H),8.72

(bs,2H),8.61(d,J=8.6,2H),8.53(d,J=6.6,2H),8.45(bs,3H),7.95(d,J

=10.2,2H),7.78(bs,2H),7.71(d,J=7.9,3H),7.66(d,J=8.8,2H),7.47

(bs,2H),7.28(d,J=8.0,7H),7.25–7.11(m,18H),7.10–6.96(m,20H),6.93(t,J=7.5,3H),6.69(d,J=8.2,5H),

5.45(s,1H),4.84–4.57(m,2H),4.49–4.24(m,3H),4.19(s,2H),3.45–3.32(m,6H),3.27–3.22(m,4H),3.10

(s,9H),3.03–2.83(m,6H),2.74(dd,J=4.2,17.3,4H),2.67(dd,J=5.2,16.7,5H),2.30–2.04(m,6H),1.88–

1.52 (m, 21H), 1.42 (s, 7H), 1.35 – 1.21 (m, 4H), 1.13 – 0.74 (m, 43H), 0.50 (s, 2H). HRMS cald for

[C69H86N14O14+H]⁺1335.65207,found1335.65430.

References



1^ J.M.Gerard,P.Haden,M.T.Kelly,R.J.Anderson,J.Nat.Prod.,1999,62,80–85.^ 2 J.Scherkenbeck,H.Chen,R.K.Haynes,Eur.J.Org.Chem.,2002,23502355

3 Selectedexamplesinclude:a)A.Stern,W.A.Gibbons,L.C.Graig,Proc.Nat.Acad.Sci,1968,61,734–741.b)

W.A.Gibbons,G.Némethy,A.Stern,L.C.Graig,Proc.Nat.Acad.Sci,1970,67,239–246.c)C.R.Jones,M.

Kuo,W.A.Gibbons,J.Biol.Chem.,1979,254,10307–10312.d)E.M.Krauss,S.I.Chan,J.Am.Chem.Soc.,

1982,104,6953–6961.e)D.Mihailescu,J.C.Smith,J.Phys.Chem.B,1999,103,1586–1594.



4 a)H.Chen,R.K.Haynes,J.Scherkenbeck,K.H.Sze,G.Zhu,Eur.J.Org.Chem.2004,31–37.b)H.Chen,XK.

Guo,Chin.J.Struct.Chem.,2005,24,273–278.

5 Although the values of the chemical shift perturbation indicates an helical conformation, geometric

constraintsinthissmalldecapeptidewilmostlikelyenforcetheTrp,DPhe,Asnsequenceinahelicalturn.

SeeG.D.Rose,L.M.Gierasch,J.A.Smith,Adv.Prot.Chem.1985,37,1–109.

6 Reviewedin:J.F.Povey,C.MarkSmales,S.J.Hassard,M.J.Howard,J.Struct.Biol.,2007,157,329–338.



7





8 D.S.Wishart,B.D.Sykes,F.M.Richards,Biochemistry,1992,31,1647–1651.

9 Compounds8,10,11and12didnotfullydissolveforthehemolyticalassayinupto30%DMSO.Sincetheir

antimicrobialactivitieswerealsolow,thiswasnotfurtherpursued.

10