Spectroscopic investigation of β-peptides: Ac-β3-Phe-NHMe, Ac-β3-Phe-β3-Ala-NHMe and Ac-β3-Ala-β3-Phe-NHMe.

Soo Hyuk Choi and Samuel H. Gellman Department of Chemistry, University of Wisconsin-Madison, Madison, WI

53706

Esteban E. Baquero, William H. James, III, , Timothy S. Zwier, Department of Chemistry, Purdue University, West

Lafayette, IN 47907

Peptide Containing Systems in the Gas Phase • α-peptides have been extensively studied in the jet by several groups.

• Gas phase studies are advantageous in probing the conformational preferences of isolated molecules, and give the best connection to theory.

Michel Mons

• β-peptides differ from α-peptides by an extra carbon linking the peptide groups.

• The extra linkage provides extra flexibility and a different set of conformational preferences.

• Conformational preferences of β-peptides are not as well known or understood.

Angew. Chem. Int. Ed. 2007, 46, 2463-2466Angew. Chem. Int. Ed. 2006, 45, 5166-5169

Mattanjah S. deVriesM. GerhardsMolecular Physics, Vol. 103, No. 11–12, 10–20 June 2005, 1521–1529

C

N1

C1

O1

C

C2

N2

O2

C

CC3

N3

O3

H1

H2 H3

NH

NH

NH

O OO

Ac-β3-Phe-NHMe

Ac-β3-Phe- β3-Ala-NHMe Ac-β3-Ala-β3-Phe-NHMe

Cß

HN1 C

C1

H3C

O1

C2

N2

CH3

O2

H1 H2

The conformational preferences and H-bonding properties of synthetic foldamers: Beta peptides

Collaboration with Samuel Gellman and Soo-Hyuk ChoiUniv. of Wisconsin-Madison

C6C8C10C12

ExperimentalExperimental

Resonant Two-Photon Ionization spectroscopy (R2PI)

Biomolecule* (S1)

Biomolecule (S0)

Biomolecule+ + e- - Heat to raise vapor pressure of molecules

- Molecules entrained in gas pulse (neon backing gas at 1-5 bar for these studies)

- Collisional cooling to zero point vibrational levels

- Heating (190 -250 °C) leads to concerns about thermal decomposition. The R2PI method gives mass analysis to confirm the spectrum is due to the molecule of interest.

UV-UV Hole-burning SpectroscopyUV-UV Hole-burning Spectroscopy

- Records the UV spectrum of a single conformation free from interference from others present in the expansion

Biomolecule* (S1)

Biomolecule (S0)

Biomolecule+ + e- H

ole-

burn

Pro

be

Conformer A

Hol

e-bu

rn

Pro

be

Conformer B

UV Hole-burn laser fixed: Provides selectivity UV probe laser tuned

Boltzmann distributionof conformers in the pre-expansion

Collisional cooling to zero-point vibrational level

B*

B*

B*CA

B*

C

CB

A

A

A

CA

ABC C

AAB BBB B

B B

UV

UV

C

Laser Timing

50-200nsec

UVHole-burn

UVprobe

Ion

In

ten

sity

(a

.u.)

3756037520374803744037400Wavenumbers (cm-1)

A

B

*

Resonant 2 Photon Ionization (R2PI) and Hole Burning Spectrum of Betapeptide

Ac--Phe-NHMe

Cluster BandMasses Observed:

Dimer and M+149 g/mol

Cluster BandMasses Observed:

Water 1 and Water 2

NH

O

NH

O

One dominant conformerMinor

Conformer

So Resonant Ion-dip Infrared Spectroscopy (RIDIRS)

Biomolecule *(S1)

Biomolecule+ + e-

Biomolecule (A) NH or OH stretch

(S0, v=1)

UV Source fixed: Provides selectivity IR Source tuned

Laser Timing

50-200nsec

IRHole-burn

UVprobe

Active Baseline Subtraction

3000 3200 3400 3600 3800Wavenumbers (cm-1)

SubtractedSignal

UV only

UV +IR

Difference

Cß

HN1 C

C1

H3C

O1

C2

N2

CH3

O2

H1 H2

C6C8

RIDIR Spectrum of Conformer A and B Ac-β3-Phe-NHMe

1.m

ol-1.c

m-1

Cß

H2N1 C

C1

H3C

O1

C2

N2

CH3

O2

H1 H2

A. Aubry, M.T. Cung and M. Marraud J. Am. Chem. Soc. 1985, 107, 7640-7647

Ion

in

te

ns

ity

(a

.u.)

3500345034003350 Wavenumber (cm

-1)

A

B

N1H1 …O2=C2

3400 cm-1

N2H2

3488 cm-1

N2H2…O1=C1

3416 cm-1

N1 H1

3454 cm-1

FT-IR Spectrum of beta-peptide chain L-Pro-L-Ala

N1H1 3437-3440 cm-1 N2H2 3466-3469 cm-1

C6

C8?

C6(c)

C6(b)

C6(a) C8(a)

C8(b)

C8(c)

12 3

Chromophore Position

φ1θ1

ψ1HN N

H

O O

Betatripeptide H-bonding architectures

•3 types of C6 rings•5 types of C8 rings•3 chromophore positions

C6(a)(1)

C6(b)(1)

C8(a)(3)

C6(c)(3)

C8(b)(1)

C8(c)(3)C8(b)(2)C8(d)(3)C8(a)(2)C6(b)(1)

C6(a)(2)

C8(a)(1)

C8(e)(3)

C6(a)(1)

C6(b)(1)

C8(a)(3)

C8(b)(1)

C8(b)(2)

C8(a)(2)

-2.00

3.00

8.00

13.00

18.00

23.00

28.00

Re

lati

ve

En

erg

y (

kJ

/mo

l)

DFT (OPT) MP2 (SP) RIMP2 (OPT)

C6(c)(1)

C6(b)(1)

C6(a)(1) C8(a)(3)

C8(b)(3)

C8(c)(3)

Calculated Relative Conformer Energies Ac-3-Phe-NHMe

RepresentativeC6 structures

RepresentativeC8 structures

•C6(a)(1) most stable•Many C8 structures in next tier

Ion

inte

nsi

ty (

a.u

.)

3500345034003350 Wavenumber (cm

-1)

C6(a)(1) 0.00 kJ/mol

C6(b)(1) 5.86 kJ/mol

C8(a)(2) 10.05 kJ/mol

C8(b)(3) 8.66 kJ/mol

C8(d)(3) 9.88 kJ/mol

RepresentativeC8 H-bonded NH

Unusual C8’s withweak H-bond

• Dominant conformer = C6• Minor conformer = unusual C8 (return to after consider tripeptides)

C6 (a)(1)

C6 (b)(1)

C8(b)(3)

C8(d)(3)

C8(a)(2)

Comparing Expt. and Calculated IR spectra Ac-3-Phe-NHMe

Acute angle

Long distance

Ion

Inte

sity

(a.

u.)

3760037550375003745037400Wavenumbers (cm

-1)

D

A B

C

E

A

B

C

D

E

*

*

*

*

*

Ion

In

tesi

ty (

a.u

.)

3480344034003360Wavenumbers (cm

-1)

A

B

C

D

E

C6C8C10C12

C

N1

C1

O1

C

C2

N2

O2

C

CC3

N3

O3

H1

H2 H3

RIDIR Spectrum of Conformer A - E ofAc-β3-Phe-β3-Ala-NHMe

R2PI and HB Spectra of Betapeptide Ac-β3-Phe-β3-Ala-NHMe

• 5 conformations, 2 major, 3 minor• Several unique H-bonding architectures

UV

HB

Spe

ctra

C6/C6

C6(a) C6(b) C8(a) C8(b)

C8/C8C10 C12

C8/C122→1. 3→1

Double ring / double acceptor

C6/C81→2, 3→2

C6C8

1→22→3

3→22→1

1→3 3→1

Single ring structures

12

3

Chromophore Position

Double ring structures

Betatripeptide H-bonding architectures

C6a/C6a

C6b/C6a

C6a/C6b

C8a/C8b

C6b/C6a

C10

C10

C6a/C8a

C8a/C8aC6b/C6b

C8a/C8bC8a/C12C10C8a/C8aC6b/C6b

C10/C6

C12

C12

-2.0

3.0

8.0

13.0

18.0

23.0

28.0

33.0

Rel

ativ

e E

nerg

y (k

J/m

ol)

C6a/C6a (1)

C6a/C8a (1)

C8a/C8a (2) C12 (1)

C10 (1)

C8/C12 (2)

DFT B3LYP/6-31+G* relative energies Ac-β3-Phe-β3-Ala-NHMe

C6a/C6a Double ring lowest

C6/C6C6/C8

C8/C8 Double rings

Double ring/Double acceptor

Single ring

Single ring

Double ring

Double ring

Double ring

348034403400336033203280Wavenumber (cm

-1)

C6(a)/C6(a) (1) 0.00 kJ/mol

C6(b)/C6(a) (1) 4.89 kJ/mol

C8(a)/C8(b) (3) 7.37kJ/mol

C8(a)/C8(a) (3) 11.05 kJ/mol

C6a/C6a (1)C6b/C6a (1)

C8a/C8b (3)C8a/C8a (3)

Conformer B

Conformer D

Conformer C

Comparing IR spectra with calculations

Ac-β3-Phe-β3-Ala-NHMe

•Conformers B,D = C6/C6 double rings (B=one of major conformers)•Conformer C = C8/C8 double ring

Assignments:

3480344034003360Wavenumber (cm

-1)

C10(b) (3) 28.44kJ/mol

C10 (a) (3) 14.23 kJ/mol

C6(a)/C8(a) (1) 8.65 kJ/mol

C8(a)/C12 (3) 14.02 kJ/mol

C8/C12 (2)

C10(a)(1)C10(b)(3)

C6a/C8a (1)

Conformer A

Conformer E

Comparing IR spectra with calculations Ac-β3-Phe-β3-Ala-NHMe

•C8 H-bonded NH now lower in frequency than C6

Ion

inte

nsity

(a.

u.)

3765037600375503750037450Wavenumbers (cm

-1)

A

B

CD

E

*

*

*

Ion

In

ten

sity

(a

.u.)

348034403400336033203280Wavenumber (cm

-1)

C6C8C10C12

A

NH

NH

NH

O OO

R2PI and HB Spectra of Betapeptide Ac-β3-Ala-β3-Phe-NHMe

RIDIR Spectrum of Conformer A - E ofAc-β3-Ala-β3-Phe-NHMe

B

C

A

B

C

D

E

•5 conformations: 2 major (A,B), 3 minor (C-E)

Ion

Inte

nsi

ty (

a.u

.)

3480344034003360Wavenumber (cm

-1)

Phe-AlaC6/C6double

ring

Phe-AlaC8/C8double

ring

Ion

Inte

nsity

(a.u

.)

3480344034003360Wavenumber (cm

-1)

Phe-AlaC10

Phe-AlaC6/C8

C8/C12

A B

D E

Comparing single-conformation IR spectra

Ac-β3-Ala-β3-Phe-NHMe

•A=C6/C6 double ring•B=C10 single ring

2 major conformers•D=C8/C8 double ring•E= Double ring/double acceptor•C= TBD (C8/C8 double ring???)

3 minor conformers

Same as Ac-β3-Phe-β3-Ala-NHMe Similar to Ac-β3-Ala-β3-Phe-NHMe

ConclusionsAc-β3-Phe-NHMe• 2 conformations 1 major (C6) and 1 minor (Unusual C8) •Unusual C8 creates a weaker than normal H-bonded structure •This has to be due to a long H--O distance or an acute angle between peptides.

CßH

N1 C

C1

H3C

O1

C2

N2CH3

O2

H1 H2

• 5 conformations, 2 major, 3 minor

• 2 major conformations are C6/C6 and C10

• 3 minor conformations are C8/C8, C6/C6 and the possibility of a double acceptor conformer C6/C8 or C8/C12. C

N1

C1

O1

C

C2

N2

O2

C

CC3

N3

O3

H1

H2 H3

Ac-β3-Phe- β3-Ala-NHMe

NH

NH

NH

O OO

Ac-β3-Ala-β3-Phe-NHMe• 5 conformations, 2 major, 3 minor

• Assignments can be done by comparison to Phe-Ala arrengement.

• 2 major conformations C10 and C6/C6

• 3 minor C8/C8 , C8/C12 or C6/C8 and one unusual conformer not yet assignned.

AcknowledgementsAcknowledgements

Professor Timothy S. Zwier

Current Group Members:Dr. Ching-Ping LiuDr. Christian MüllerWilliam H. James III *V. Alvin Shubert Tracy LeGreveNathan PillsburyJoshua Newby Chirantha Rodrigo Joshua Sebree

Former Group Members:Dr. Jaime StearnsDr. Talitha SelbyDr. Jasper Clarkson

Professor Samuel H. GellmanSoo-Hyuk Choi

Funding

Professor Kenneth JordanDr. Daniel Schofield