Lit. Sem. 2010/7/14
Shunsuke Sakaoka (M2)

Cross-Linking System to Stabilize Alpha Helics Conformation in Short Peptides

Contents

1 alpha helics conformation
2 some strategies to stabilize alpha helics conformation
3 cross-linking with lactam bridge
4 cross-linking with all hydrocarbon staple
5 cross-linking with acetylenic cross-linking agent

1 alpha helics conformation
Alapha helics, a common motif in the secondary structure of peptides, is right-handed coiled conformation.
• Every backbone N-H group donates a hydrogen bond to the backbone C=O group. (i-I+4 hydrogen bonding)
Oi Cα• Each amino acid residues corresponds to a 100º trun in the helics.
i+4100ºN
Ri+3 CαONi+1 CαO O• The pitch of the alpha-helics (the vertical distance between two100ºH
consecutive turns of the helics) is 5.4Å.
100ºNi+2 CαH• Residues in alpha helics typically adopt backbone (ϕ,ψ) dihedral
R NO
Hangles around (-60º, -40º).
Ramachandran diagram
O HNH
NCαi-1OCα5.4Å

iNNHCαi+1HO i-1Ri-40ºiR-60º• Alpha helics is composed of 4 ~ 40 amino acid residues. (average 10 residues, 3 turns)• Many of alpha helics are amphipathic. (e.g. alcohol dehydrogenase)• Alpha helical sequences of ≤15 amino acids are estimated to accountfor ~30% of protein structure and frequently mediate biologicalprocesses through interactions with proteins, DNA or RNA.
• The folded states of most proteins are only ≤10 kcal/mol lower inenergy than their unfolded states. (PNAS, 2006, 103, 16623)
But short peptide sequences are usually unfolded in aqueous solution,
awy from the helix stabilizing environments of proteins.

SerAspGlyLeu

AsnLeuGlu

ArgGlyPheIle1

If short peptide alpha helics could be stabilized or mimicked by small molecules, such compunds
might be valuable chemical or biological probes and lead to development of novel pharmaceuticals,
vaccines, diagnostics, biopolymers and industrial agents.

• Circular Dichroism (CD) used to provide a quantitative measure of helical content.
2pz
π∗210~220 nm (n→π*)
2pz
190 nm (π→π*)N

π1C2pzOO 2pypeptide bondπpeptide bond[θ(λ)] = χα[θα(λ)] + χβ[θβ(λ)] + χrc[θrc(λ)][θα(λ)], [θβ(λ)],[θrc(λ)]; 2次構造が既知のポリペプチド鎖�����������������������������������のα, β, rc構造に対する[θ]χα,χβ,χrc; それぞれの存在割合[θ]208nm+4000

alpha helix(%) =x 100-33000+4000

AlanineLysineAspartic acid
Glutamic acid
Asparagine
Ariginine
Isoleucine
glycineGlutamine
Cystein
SerineAla
LysAsp
Glu
Asn
Arg
Ile
GlyGln
Cys
SerA
K
DE
N
R
I
GQ
CS

TyrosineTyr
Tryptophan
Trp
Threonine
Thr
ValineVal
Histidine
His
Phenylalanine Phe
ProlinePro
Methionine
Met
Leucine
Leu

YW
TV
HFPM
LAla; CH3Glu; (CH2)2CO2H
Lys; (CH2)4NH2
Asp; CH2CO2H

2. some strategies to stabilize alpha helics conformation
2-1 Metal chelation
i+42-2-Template

iTemplate
M. Ghandiri JACS. 1990, 112, 9633
Rev) Tetrahedron 1999, 55, 11711

2-3 Hydrogen-Bond Surrogate

D. S. Kemp et al. JOC, 1991, 56, 6672
cross-linkingii+4
large entropy loss
covalent bondParamijit S. Arora et al. JACS. 2004, 126, 12252
less entropy loss

21 residues

3 cross-linking with lactam bridge
Taylor, J. W. JACS, 1990, 112, 6046
Taylor, J. W, JACS, 1992, 114, 69663-1-1 design
peptide1-1-1i
i+4-Lys-Glu-Leu-Lys-Glu-

peptide2-2-2i
i+4-Lys-Glu-Leu-Lys-AspH
NOHNO

3-1-2 synthesis of peptide 1-1-1i
i+4 i+7
i+11 i+14
i+18
K-L-K-E-L-K-E-K-L-K-E-L-K-E-K-L-K-E-L-K-E
1

1

K-L-K-E-L-K-E
1i
i+4
Lys-Leu-Lys-Glu-Leu-Lys-Glu

1i
i+4 i+7i+11 i+14i+18
K-L-K-E-L-K-E-K-L-K-E-L-K-E-K-L-K-E-L-K-E

1, TFA

3DCC

Boc-Lys(2Cl-Z)-Glu-OPacoxime resin(74%)

O
COOPacBoc-Lys(2Cl-Z)-NH-CH-CH2-CH2-C-O-N
(74%)
Resin

1, TFA

Boc-Glu(OBzl)-Leu-Lys(2Cl-Z)-Glu(O-resin)-OPac Pac =2, BOP, DIEA
C(E-L-K-E)(99.3%)
H2
Boc-Glu(OBzl)-OH

2, BOP, DIEA
Boc-Leu-OH

NEt2 PO

HN

Synthesis of 1 (K-L-K-E-L-K-E)
H-Glu-OPac
NaHCO3+BocN-Lys(2Cl-Z)-Su

K-L-K-E-L-K-E

O

Ph
Trt =
Ph ,Ph
Ph

O
N
N

O
N
N
N+ Boc-Leu-COO

Boc-Leu-COO-P(NEt2)3N

BOP

H-Lys(Trt)-OMe

H2N NH2

Boc-Lys(2Cl-Z)-Leu-N2H3(88%)
NaOH
Boc-Lys(2Cl-Z)-Leu-Lys(Trt)-OMe(42%)

Boc-Lys(2Cl-Z)-Leu-OMe

Boc-Glu(OBzl)-Leu-Lys(2Cl-Z)-Glu(O-resin)-OPac(E-L-K-E)
1) TFA
2) HOBt, DIC, DIEA
Boc-Lys(2Cl-Z)-Leu-Lys(Trt)-OH(K-L-K)

NaNO2/HClO
O N
Nactivated ester N
Boc-Leu-C

Boc-Lys(2Cl-Z)-Leu-N3

Boc-Lys(2Cl-Z)-Leu-Lys(Trt)-OH(94%)(K-L-K)O
COOPacLeu-Lys(2Cl-Z)-NH-CH-CH2-CH2-C-O-N
Resin
Glu(OBzl)-OC-CH-(CH2)4-NH-Trt
Boc-Lys(Cl-Z)-Leu-NH

Boc-Lys(2Cl-Z)-Leu-Lys-Glu(OBzl)-Leu-Lys(2Cl-Z)-Glu-OPac(K-L-K-E-L-K-E) (2)(61%)

5% TFA (deprotection of Trt)

(64%)

peptide-peptide coupling (DIC/HOBt)
O

H
N

R1OOH

DIC

H
N

R1

R2NO

NH

O
R2O

iPr

O

R2iPr

O

NOH

R2N

R1NOH
O
R1R1R3 NH2O
R2O

N
HOBt

O

H
NN

N

O

NN

R1O

R2

R3 NH2NO

H
N

R1ON
H

R2R3O

H
N

R1O

R2N R3Hsynthesis of 1-1-1
Boc-Lys(2Cl-Z)-Leu-Lys-Glu(OBzl)-Leu-Lys(2Cl-Z)-Glu-OPacsynthesis of 3-3-3(3 = Lys-Leu-Lys-Glu-Leu-Lys-Gln)

(84%)

(66%)

(27%)

(17%)

11→13
O

3-1-3 CD spectra

ROAr
NO

HO-PipAr

R

Zn/AcOH
ON

ROH
ref. JOC, 1983,48,678

Effect of TFE on helix formation

CD spectra of 1-1-1, 2-2-2, 3-3-3
Cross-linking through Lys-Asp
could stabilize alpha-helics in 21
residues peptide.
Cross-linking length is important?

O3-2 single turn peptide alpha helices
Nicholas E. Shepherd and David P. Fairlie JACS, 2005, 127, 2974

3-2-1 design

single turn alpha helics ??
X = Lys or Orn (Orn side chain; -CH2-CH2-CH2-NH2)
Y = Asp or Glu
Z = any amno acidsii+4
Y/X-Z-Z-Z-X/Y

3-2-2 solid phase peptide synthesis (Fmoc protocol)
OMe

OMe
OMe

H2N

FmocN-Xxx-OH
BOP reagent / DIPEA
resin
Fmoc-Xxx-NHOMe1) piperidine
resin2) Fmoc-N-Xxx-OH
BOP reagent / DIPEA

OMe

OMe
Fmoc-(Xxx)n-NH

1) piperidine

nNH2-(Xxx)n-NH2

resin 2) TFA• Some Resins are used. Such as
HOH2C

O (CH2)nresin• Acids are not used except final step (cleavage from resin).
Side chains are protected with OtBu (for Asp, Glu), Boc (for Lys, Ser and His),Mtr, Pmc (for Arg) and so on which
can be deprotected with TFA.
Mtr; 4-methoxy-2,3,6-trimethylbenzenesulfonyl
OSO

Pmc; 2,2,5,7,8-pentamethylchroman-6-sulfonyl
OS
O

OMeO

O
Alloc;
amine group

OAllyl;HBTU (BOP reagent)
N
N NMe2NNMe2O

O
Fmoc HNOH

DIPEA

O NNHPGNNNNO
NHPG

carboxy lateNOH

N NMe2O

NHPG

O

N
Fmoc-Ala-Lys-NH

NO
activated ester

AllocO
H2N

N
4 H
NHOO

NMe2O

NMe2O

NMe2

path a; Fmoc-K/O-A-R-A-D/E-NH-resin
path b; Fmoc-D/E-A-R-A-K/O-NH-resin

R = Fmoc

When Fmoc-D/E-A-R-A-K/O-NH- was cyclized
through path a, some side reactions occurred.
Fmoc-D-A-R-A-K/O-NH-resin Fmoc-E-A-R-A-K/O-NH-resinO
OOO
piperidine
H2N

piperidine
OON
H
O

H2N

A-R-A-K/O-NH-resn

OOO
H2NO

NOpath apath bNHNuHNOON
HThanks Dr. Nich.

3-2-3 result
CD-spectra-1 Ac-(cyclo-1,5)-[X/Y-ARA-Y/X]

HN

35,7,8,96 2,44356O

HNOOONHNH

CD-spectra-2 Ac-(cyclo-2,6)-R[K-ZZZ-D] (helics dependence on sequences)

Rank order of helical propencities(rev. Teterahedron, 1999,55,11711)

21
18,19,20
13,14,15,16
17

ref. CD spectra-1

9,10,11,123

3-2-4 NMR analysis of short peptides
A low temperature dependence for amide NH
chemical shifts (values ≤4ppb/K) are indicativeof hydrogen bonds.
13 Ac-(cyclo-2,6)-R[KAAAD]
Δδ/T of each residue NH O H
N
Arg1 8ppb/K
O
Lys2 6ppb/K
6
Ala3 2ppb/K
5
H N
Ala4 3ppb/K
O
N
Ala5 1ppb/K
O O
Asp6 4ppb/K
H
4
NH1 3ppb/K
N
NH2 8ppb/K
3
H 2N
O
H
O HN
Ac

The NMR analysis indicated the presence of
three hydrogen bonds within the pentapeptide.

542Ac

11
Ac-(cyclo-1,5)-K-A-R-A-D-NH2 (3)NH
Ac-(cyclo-2,6)-R[KAAAD] (13)Gua. HCl (protein denaturant) effect to the stability of 3

Temperature effect to the stability of 3bottom line at 5 ºC
middle line at 45 ºC
top line at 85 ºC

Gua. HCl
NH2 Cl
H2NNH2

3-3 biological activity of lactam linking peptides
cross-linking

ideal; parent peptidealpha helicity↑, biological activity↑

example 1 (�A. M. Felix et al. Biopolymers, 1995, 37,67)
GRF; growth hormone-releasing factor (Nature, 1982, 300, 276)
human-GRF

hGRF1-29; 0.8
ref. I. J.Pept. Protein. Res(A)(B)(C)

CD measurements of [Ala15]-hGRF29 show a slight increase in alpha helicity relative to the parent peptide .
ref. Proc. 10th American Peptide Symposium (G. Marshall ed. p.465)

example 2 Bcl-2-BakBH3 (regulation of apoptosis)
B. Yang et al. Bioorg.Chem. Lett. 2004, 14, 1403

1

5

10

BakBH315

none of theselactam cross-linking peptides
showed any binding to Bcl-2.
(Data were not shown in this paper.)Bcl-2

4 cross-linking with an all hydrocarbon staple

Gregory L. Verdine et al. JACS, 2000, 122, 5891

4-1 design
Fmoc

OHN

Fmoc

OHornOHNRCMnOH introduction into peptides

nnnn

S-1n
R-1n
They used α,α-disubstituted amino acids to capitalize on
the helix-stabilizing effect of them.
α helics; φ ≈ -60º, ψ ≈ -40º
α,α-disubstituted amino acids; φ ≈ -60±20º, ψ ≈ -30±20º

cross-linking withan all hydrocarbon staple

4-2 synthesis
The actual structure of cross-links is dependent synthesis of S-1 and R-1
on the stereochemistry at the attachment points.
PhPh 516 O KN(SiMe3)2, R X
t-BocN
THF, -80 ºCO3PhPhOt-BocN

KN(SiMe3)2, R2X
OTHF, -80 ºC

R1Ph
Ph

Li, NH3Ot-BocN

OTHF, EtOH

R1 R2
FmocHN
R1t-BocHN

CO2H 1) CF3CO2H

R1 R22) Fmoc-N-hydroxysuccinimide, Na2CO3

intermediate
CO2H
R2• The wild-type peptide (RNAse A) has the sequence:
Ac-EWAETAAAKFLAAHA-NH2• The peptides synthesized in the Ri,i+7S(x) series have the sequences:
Ac-EWAEyAAAKFLzAHA-NH2 where (y,z) were substituted with (R-13,S-13), (R-13,S-14), (R-14,S-14), (R13,S-16), (R-14,S-16) for the peptides Ri,i+7S(8), Ri,i+7S(9), Ri,i+7S(10), Ri,i+7S(11), andRi,i+7S(12), respectively.
• The peptides synthesized in the Si,i+4S(x), Ri,i+4R(x) and Ri,i+4S(x) series have the sequences:
Ac-EWAETAAyKFLzAHA-NH2 where (y,z) were substituted with (S-11,S-13), (S-11,S-14), (S-13,S-13), (R11,R-13), (R-11,S-12), (R-11,S-13) and (R-11,S-14)for the peptides Si,i+4S(6), Si,i+4S(7), Si,i+4S(8), Ri,i+4R(6), Ri,i+4R(7),Ri,i+4R(8), Ri,i+4S(5), Ri,i+4S(6), andRi,i+4S(7), respectively.
Metathesis reaction

PCy3PhCl
Ac-EWAETAAyKFLzAHA-NH2
Ac-EWAETAAyKFLzAHA-NH
Ar resin1) Cl Ru
H (20 mol%)
DCE,2hPCy
3Arn'nnn'O
N2) TFA

percent conversion product / product+starting material (determined by HPLC)OO

i+4ONH

65N
4 5.4 ÅHNO 3HO HN 21iNH

4-3 result

un-cross-linkcross-link

Percent helix

cleavage by trypsin (protease resistance in vitro)

Ri,i+7S(x) = Ac-EWAEyAAAKFLzAHA-NH2ZcisLK FAy A A

trans4-4 biological activity of α helics peptide stabilized by a hydrocarbon staple
Gregory L. Verdine et al. Science, 2004, 305, 1466
Bcl-2 (B-cell lymphoma 2) proteins constitute a critical control point for the regulation of apoptosis.
BID is a pro-apptotic protein that, in response to death receptor signaling, interconnects the extrinsec and core
intrinsic apoptotic pathways. Activated BID can bind to BCL-2 and trigger activation of pro-apoptotic proteins,
resulting in cytochrome c release.
BIDcytochrome c release

apoptosis

BCL-2
BID-BH3 (23 amino acid residues)
15

10

1520

Si,i+4S(8)Si,i+4S(8)Si,i+4S(8)Si,i+4S(8)Ri,i+7S(11)Serum(血清) stability

CD spectra

BIDBH3SAHBD

SAHBC
SAHBA(G→E)

Serum includes all proteins not used in blood clotting and all theelectrolytes, antibodies, antigens, hormones, and any exogenous
substances (e.g., drugs and microorganisms).

SAHBB
SAHBA

KD (fluorescence pilarization binding assay)

Percent cytochrome c release
SAHBA, wt.mito

SAHBA, Bak-/-. mito
BIDBH3, wt.mito

intravenous injection (静注) leukemia cells that stably
expressed luciferase

VehicleSAHBA

SAHBA(G→E)

4-5 the physical forces that underlie stapled peptide stabilization
Gregory L. Verdine and Eugene I. Shakhnovich et al. JACS, 2009, 131, 4622
Why some cases (RNAse A) an i, i+7 staple can be more stabilizing than an i,i+4 staple, while in other cases (BIDBH3)
the opposite is true?
What factors are most important?
Four variables in Ebbtor
H = Econ+ whbEhb + wbbtorEbbtor + wsctEsct + wlinktorElinktor
function are φΑ+1,ψA+1,Econ; intermolecular potencial energy
the angle between bAi
Ehb; hydrogen-bonding potential
and bAi+2 and the angle
Ebbtor; sequence-dependent
between PAi and PAi+2.
backbone torsional potential
Esct; side-chain torsional angle potential
Conformations that were obtained during MC simulations
Elinktor; torsional potential for hydrocarbon linker
Exp
S = configurational entropy of the peptide + that of the staple
ExpSimulation
Simulation
Ala

Their simulation was able to predict which staple wouldbe more stabilizing for each peptide.
In all cases, the stapled peptides are more helical thanthe corresponding alanine-substituted versions,suggsting that the physical constraint conferred bymacrocyclic ring formation contributes to increased
helical propensity.

For all stapled peptides ∆∆H makes a larger contribution to ∆∆G than -T∆∆S.

5 cross-linking with acetylenic cross-linking agent

M. Inouye et al. Chem Comm, 2004, 1280
M. Inouye et al. Chem. Eur. J. 2008, 14, 857
G. A Wooley et al. JACS. 2007, 129, 14154

5-1 design
Lyscross-linking agentsLys

cross-linking agentsLys

5-2 synthesis

solid phase peptide synthesis using the standard Fmoc chemistry
NH-resin

Fmoc-Ala-OH
BOP reagent

Fmoc-Ala-NH-resin

piperidine

H2N-Ala-NH-resin

Fmoc-Glu(PG)-OH
BOP reagent

H2N-Glu(PG)-Ala-resin

Fmoc-Ala-Ala-Glu(PG)-Ala-Trp-Ala-Lys(PG)-Ala-Glu(PG)-Ala-Ala-Glu(PG)-Ala-Lys(PG)-Glu(PG)-Ala-resin
1) piperidine
Ac-Ala-Ala-Glu(PG)-Ala-Trp-Ala-Lys(PG)-Ala-Glu(PG)-Ala-Ala-Glu(PG)-Ala-Lys(PG)-Glu(PG)-Ala-resin
2) Ac2O
TFA

Ac-Ala-Ala-Glu-Ala-Trp-Ala-Lys-Ala-Glu-Ala-Ala-Glu-Ala-Lys-Glu-Ala-NH2

cross-linking agent
buffer

Ac-Ala-Ala-Glu-Ala-Trp-Ala-Lys-Ala-Glu-Ala-Ala-Glu-Ala-Lys-Glu-Ala-NH2
cross-linking yieldcross-linking agent

38%

5-3 result
peptideA

peptide B

21b
1a1b2
1a

3a
4a, 3b, 3c

3a, 3b, 3cnativenative(ºC)(ºC)

peptide B (Lys→X); Ac-Ala-Ala-Glu-Ala-Trp-Ala-X-Ala-Glu-Ala-Ala-Glu-Ala-X-Glu-Ala-NH2
NH2

X residues

Other cross-linking agents

NH2

NH2NH6ÅODap5Å

NH2NHODabNHO

OrnN
HO

Lys

helical content
CL-agent
10 Å

9ÅReversible photoregulation of helical structure inshort peptides (M. Inouye et al. Org. Lett. 2006, 8, 285)
Isomerization of the spiropyran to the merocyanine

darkN OR
1

NO2 indoorlighting
RN O
RNO2
R~35
1~65
2
3
4
5
6
78
9101112
H = 40-60%, M = 20-40%, L = 0-20%
O
R = (CH2)2CO2NO
photochromic features of spiropyran skeltons
1) only merocyanine forms strongly absorbvisible light2) mericyanines are more stable than the
spiropyrans in aqueous media under dark
conditions3) [merocyanine]/[spiropyran] = 1.13 (dark)= 0.01 (light)

peptide A

native peptide A (12% helicity)
dark condition (48% helicity)indoor lighting (62% helicity)