1. Occurrence and Functions of Peptides in Nature and Every Day Life …hormones, neurotransmitters, therapeutics, artificial sweetener, … 2. Peptide Synthesis a) Aspartam: Properties of amino acids; nomenclature; solution phase synthesis b) Glucagon: Solid phase peptide synthesis c) Fuzeon, an anti HIV drug: Solution and solid phase peptide synthesis 3. Peptide Structures – Nanostructured Materials – Self-assembly a) Fuzeon: α-helices and coiled-coil structures b) Amyloids: β-sheets c) Collagen: PPII helices 4. Applications of Peptides in Chemistry, Biology and Material Sciences a) Peptide-based materials b) Therapeutically active peptides (Cetrorelix, Fuzeon) b) Cyclic peptides and cancer imaging c) Asymmetric Catalysis with Peptides – Combinatorial chemistry

Syllabus

2 Prof. H. Wennemers

http://courses.cm.utexas.edu/emarcotte/ch339k/fall2005/Lecture-Ch4.html

• • •

• • •

3 Prof. H. Wennemers

Polyproline II (PPII) helix

Polyproline II Helixleft handed helix (Φ = -75°, Ψ = 145°)

side view view along helix axis

9.4 Å 3 residues

4 Prof. H. Wennemers

Common but NOT limited to proline-rich peptides!

Which factors stabilize PPII helices?

PPII helix (all-trans amide bonds)

PPI helix (all-cis amide bonds)

in H2O

in n-PrOH

Case study: Oligoprolines

Giralt: Biopolymers 1993, 33, 1019. Ochsenfeld, Wennemers: J. Am. Chem. Soc. 2009, 131, 15474.

cis/trans Isomers of Proline

N CO2CH3

O

N CO2CH3

O

N CO2CH3

OH

N CO2CH3

HO

N

OH

N

HO

trans cis

4.8 : 1

1.5 : 1

1 : 2

steric effect

stereoelectronic effect – n->π* interaction

6

M. P. Hinderacker; Raines, R. T. Prot. Sci. 2003, 12, 1188.

DeRider, Wilkens, Waddell, Bretscher, Weinhold, Raines, Markley, J. Am. Chem. Soc. 2002, 124, 2497.

all K values in D2O

Zusammenhang ΔG° und K bei Raumtemperatur (ΔG = -RTlnK)

ΔG° (kcal/mol) K % stabileres Isomer

-0.1 1.2 54.5

-0.5 2.4 69.7

-1 5.4 84.4

-2 29.3 96.7

-5 4631 99.98

-10 2.1x107 99.999999

7 Prof. H. Wennemers

Oligoprolines, the Progenitors of the PPII Helixleft handed helix (Φ = -75°, Ψ = 145°)

side view view along helix axis

9.4 Å 3 residues

8

Original structure (Powder diffraction): Cowan, McGavin, Nature 1955, 176, 501 Reviews Rabanal, Ludevid, Giralt, Biopolymers 1993, 33, 1019 . Rath, Davidson, Deber, Biopolymers (Pept. Sci.) 2005, 80, 179 Crystal Structure (x-ray): Wilhelm, Lewandowski, Wennemers, JACS 2014, 136, 15829. Prof. H. Wennemers

Power diffraction structure

Crystal Structure of a PPII-helical Hexaproline – A First

P. Wilhelm, B. Lewandowski, N. Trapp, H.W. JACS 2014, 136, 15829.

No water in crystal structure

N N N N NN

O O O O O

OH

O

p-Br-C6H4

O

crystallized from aqueous CH3CN

8.98±0.14 Å

Pro1 Pro2 Pro3 Pro4 Pro5 Pro6

Power Diffraction: Cowan, McGavin, Nature 1955.

N

Oi-1

Ci

Oi

N"

Ciα" Ci"

Ci#1" Oi#1""

Δ"

θBD

d"

Oi"n π*

interaction

Patrick  Wilhelm

d = 1.9–3.1 Å Θ = ~100°

Δ = 0.01–0.04 Å

Collagen – Roles in Nature and Medicine

Tyrannosaurus rex Science 2007, 316, 277.

Most abundant protein in mammals skin, bones, connective tissue,

cellular communication

Medical applications, e.g., wound dressings, implant coatings Use of collagen isolated from biological source – allergic response

Stability of Amide Bonds

11 Wolfenden: Annu. Rev. Biochem. 2002, 71, 847

PEP = carboxylpeptidase

t1/2: ~600 years (water, neutral pH, 25°C)

N

ONCH2

ON

CH2

NO

HO

N

NO

ONCH2

ON

NO

ONCH2

O

NO

ONCH2

O

H

N

NO

O

ONCH2

NO

H

HO

HO

HO

HO

N

O

HO

N

O

O H

H

H

H

Collagen – Basic Structure

PPII-like helices

Triple helix Common repeat unit: Xaa-Yaa-Gly

Pro

Hyp

Gly

Reviews Fields, G. B.; Prockop, D. J. Biopolymers 1996, 40, 345. Engel, J.; Bachinger, H. P. Top. Curr. Chem. 2005, 247, 7. Greenspan, D. S. Top. Curr. Chem. 2005, 247, 149. Shoulders, M. D.; Raines, R. T. Annu. Rev. Biochem. 2009, 78, 929.

Collagen

13

Fields, G. B.; Prockop, D. J. Biopolymers 1996, 40, 345. Engel, J.; Bachinger, H. P. Top. Curr. Chem. 2005, 247, 7. Greenspan, D. S. Top. Curr. Chem. 2005, 247, 149. Shoulders; Raines, Annu. Rev. Biochem. 2009, 78, 929.

Prof. H. Wennemers

Stabilizing Effect of Hyp

N CO2CH3

O

N CO2CH3

O

N CO2CH3

OH

N CO2CH3

HO

N

OH

N

HO

trans cis

4.8 : 1

1.5 : 1

1 : 2

steric effect

stereoelectronic effect – n->π* interaction 98°

2.9 Å• gauche effect leads to C(4)-exo ring pucker • favorable geometry for n->π* interaction

N CO2CH3

O

N3

N CO2CH3

O

N3

N CO2CH3

O

HO

N CO2CH3

O

HO

6.1 : 1

6.1 : 1

trans cis

14

M. P. Hinderacker; Raines, R. T. Prot. Sci. 2003, 12, 1188.

DeRider, Wilkens, Waddell, Bretscher, Weinhold, Raines, Markley, J. Am. Chem. Soc. 2002, 124, 2497.

Sonntag, Schweizer, Ochsenfeld, Wennemers, J. Am. Chem. Soc. 2006, 128, 14697.

Sonntag, Schweizer, Ochsenfeld, H.W. JACS 2006, 14697

1 : 2.6 (D2O)

1 : 6.1 (D2O)

cis trans

(R) (R)

cis trans

(S) (S)

N

N3

CO2CH3

O

N

N3

CO2CH3

O

N

N3

CO2CH3

O

N

N3

CO2CH3

O

C(4)-endo pucker C(4)-exo pucker

(4S)- and (4R)-Azidoprolines – “Azido Gauche Effect“

For Fluoroproline, see: Raines JACS 2001, 777; Moroder ACIE 2001, 923.

Sonntag, Schweizer, Ochsenfeld, H.W. JACS 2006, 14697

1 : 2.6 (D2O)

1 : 6.1 (D2O)

cis trans

(R) (R)

cis trans

(S) (S)

N

N3

CO2CH3

O

N

N3

CO2CH3

O

N

N3

CO2CH3

O

N

N3

CO2CH3

O

C(4)-endo pucker C(4)-exo pucker

(4S)- and (4R)-Azidoprolines – “Azido Gauche Effect“

For Fluoroproline, see: Raines JACS 2001, 777; Moroder ACIE 2001, 923.

98°

2.9 Åstabilizing n π* interaction

Functionalizable Collagen Model Peptides

N NHN

N3

O O Om

N NHN

Ac

OH

O O O n

N NHN

OH

O O O

NH2

N NHN

O O Om

N NHN

Ac

OH

O O O n

N NHN

OH

O O O

NH2

NN

N

R

“Click Chemistry”

N NHN

NH

O O Om

N NHN

Ac

OH

O O O n

N NHN

OH

O O O

NH2

ROAmidation

Erdmann, H.W. JACS 2010, 13957. ACIE 2011, 6835. OBC 2012, 1982. JACS 2012, 17117. BMC 2013, 3565.

Functionalized Collagen Model Peptides

N

O

N

O

HN

O

*N3

N

O

N

O

HN

O

*N3 * = (4R) or (4S)Xaa Yaa

N

O

N

O

HN

O

NH

R1

O

*

N

O

N

O

HN

O

NH

R1

O

*N

O

N

O

HN

O

NNN

R2

*

* = (4R) or (4S)

Reduction/ Acylation

“Click Chemistry”

R2 = CO2Me CH2OH

or

OOH

HOOH

O

OH

OHOHO

OHO

OH

N

O

N

O

HN

O

NNN

R2

*

R1 = Me or tBu Xaa Xaa

Yaa Yaa

3

N NHN

Ac

OH

O O O 3

N NHN

OH

O O O

NH2middle repeat unit

Thermal Stability of Collagen Triple Helices

(4R)Hyp

N NHN

Ac

OH

O O

NH2

O 7

N NHN

Ac

N3

O O

NH2

O 7

N NHN

Ac

N3

O O

NH2

O 7

(4R)Azp

(4S)Azp

(R)

(R)

(S)

-1.0

0.0

1.0

2.0

3.0

4.0

10 20 30 40 50 60 70

Temperature [°C]

ΘM

RW

, 225

nm

[10

3 deg

cm

2 dm

ol-1

]

Tm = 43°C

Tm = 44°C

Tm = <5°C

For work on Fluoroproline, see: Raines, et al. Nature 1998, 392, 666. Erdmann, H.W. JACS 2010, 13957.

Functionalized Triple Helices – Thermal Stabilities

Triazoles Amides

Tm

Xaa Yaa(R) (S) (R) (S) Xaa Yaa(R) (S) (R) (S)

Pro-Hyp

Pro-Pro

Erdmann, H.W. JACS 2010, 13957. ACIE 2011, 6835. OBC 2012, 1982. JACS 2012, 17117. BMC 2013, 3565.

• Comparable stabilities of triazolylated collagen triple helices. • Significant differences in stability of amidated collagen triple helices.

References

C(4)-endo

M. Kümin, Y. Nagel, F.W. Monnard, S. Schweizer, C. Ochsenfeld, H.W., Angew. Chem. Int. Ed. 2010, 49, 6324.

C(4)-exo

(R) (R) (S) (S)

Functionalized Collagen Model Peptides – Amidation

trans cis trans cisN

O

CO2CH3

NH

O

N

O

CO2CH3

NH

O

N

O

N

CO2CH3

H

O

N

O

CO2CH3

NH

O

4.3 : 1 (D2O) 5.8 : 1 (CDCl3) 5.8 : 1 (D2O) 5.2 : 1 (CDCl3)

Comparable to N3, OH, F (Kt:c ~6:1) Different from N3, OH, F (Kt:c ~2.5:1)

2.0 Å

2.8 Å

104°

transannular H-bond

n π* interaction

2.8 Å

102°

n π* interaction

• Transannular H-bond triggers high trans:cis ratio in C(4)-endo pucker.

R. Erdmann, H.W., Angew. Chem. Int. Ed. 2011, 50, 6835 and J. Am. Chem. Soc. 2012, 134, 17117.

No Steric repulsion

•

Crystal structure from Okuyama, Biopolymers 2004, 76, 367.

Residue points to the outside of the triple helix

(4R)Amidoproline in the Xaa Position of Collagen

Tm (R = Me): 43°C Tm (R = tBu): 44°C

Tm (Ac[POG]7NH2): 43°C

!A C(4)-exo pucker is tolerated in the Xaa position of Collagen

Ac[Pro-Hyp-Gly]3N

O

N

O

HN

O

[Pro-Hyp-Gly]3NH2

NH

R

O(R)

R. Erdmann, H.W., Angew. Chem. Int. Ed. 2011, 50, 6835 and J. Am. Chem. Soc. 2012, 134, 17117.

•

Crystal structure from Okuyama, Biopolymers 2004, 76, 367.

Competition of transannular with interstrand H-bond

(4S)Amidoproline in the Xaa Position of Collagen

Tm (R = Me): 32°C Tm (R = tBu): 26°C

Tm (Ac[POG]7NH2): 43°C

Interference with interstrand H bonding and steric effects destabilize collagen triple helices.

Ac[Pro-Hyp-Gly]3N

O

N

O

HN

O

[Pro-Hyp-Gly]3NH2

NH

R

O(S)

• Steric repulsion

Aminoproline – pH-Responsive Amino Acid

C(4)-endo pucker

(S)

N

O

H3N

CO2CH3

- H+

+ H+

Ktrans/cis= 5.7 (D2O)

n π* interaction

H bonding and stereoelectronic effects

N

HγN

O OCH3H

Ψ ~145°

HH

O

Comparable with (4S)Acp: Kt/c = 4.3 ACIE, 2010 49, 6324.

Siebler, Erdmann, H.W., Angew. Chem. Int. Ed. 2014, 53, 10340.

(S)

N

O

H2N

CO2CH3

Ktrans/cis= 7.1 (D2O)

OCH3Ψ ~145°O

N OHγ

H2N

C(4)-exo pucker

steric effect

Comparable with (4S)Mep: Kt/c = 7.4 Raines JACS 2006, 128, 8112.

Chris0ane  Siebler

Acidic pH Basic pH

Hγ

H2N

O

N O

N

HγN

O

O

HHH

competition with interstrand H bonds no constraint with triple helix

N NHN

O O OProHypGly

3ProHypGly

NH3

Ac NH23

+ H+

- H+ N N

HN

O O OProHypGly

3ProHypGly

NH2

Ac NH23

pH-Responsive Collagen Triple Helices

Applications: e.g. drug delivery

pH-Responsive Collagen Triple Helices

N NHN

O O OProHypGly

3ProHypGly

NH2

Ac NH23N N

HN

O O OProHypGly

3ProHypGly

NH3

Ac NH23

+ H+

- H+

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

10 20 30 40 50 60

Temperature [°C]

ΘM

RW

, 224

nm

[103

deg

cm

2 dm

ol-1

]

pH 10: Tm = 33°C

pH 3: Tm ~13°C

Siebler, Erdmann, H.W., Angew. Chem. Int. Ed. 2014, 53, 10340.

1.5

1.0

2.0

50 0 100 200 150 250 300 Time [min]

ΘM

RW

, 224

nm

[103

deg

cm

2 dm

ol-1

]

Reversibility

J. Chmielewski (Angew. Chem. 2009)

R. T. Raines (PNAS 2006)

V. P. Conticello/J.D. Hartgerink (JACS 2007/JACS 2013)

L. Moroder (Chem. Eur. J. 2007)

reviews: J. A. Fallas, L. E. R. O’Leary, J. D. Hartgerink, Chem. Soc. Rev., 2010, 39, 3510. S. Chattopadhyay, R.T. Raines, Biopolymers 2014, 101, 821. 27

Towards Synthetic Collagen – Biocompatible Materials

Crosslinked Synthetic Collagen

F. W. Kotch, R. T. Raines Proc. Natl. Acad. Sci. 2006, 103, 3028.

AFM images and TEM images of 1 (top) and 2 (bottom)

200 nm

200 nm

Prof. H. Wennemers 28

Synthetic Collagen with Complementary Charges

Raines Nature Chem. 2016, 8, 1008.see also: J.D. Hartgerink et al., JACS 2014, 136, 14417; JACS 2014, 136, 7535.