United States Patent [19] Gellman et al.

US006060585A

[11] Patent Number: 6,060,585 [45] Date of Patent: May 9, 2000

[54] [3-POLYPEPTIDE FOLDAMERS OF WELL DEFINED SECONDARY STRUCTURE

[75] Inventors: Samuel H. Gellman; Daniel H. Appella; Laurie A. Christianson; Daniel A. Klein, all of Madison, Wis.; Susanne Krauthauser, Overath, Germany; Yong Jun Chung; Xifang Wang, both of Madison, Wis.

[73] Assignee: Wisconsin Alumni Research Foundation, Madison, Wis.

[21] Appl. No.: 09/034,509

[22] Filed: Mar. 4, 1998

Related US. Application Data [60] Provisional application No. 60/039,905, Mar. 4, 1997.

[51] Int. Cl.7 .. ....................... .. C07K 5/023

[52] US. Cl. ........................ .. 530/323; 530/324; 530/350; 530/333; 530/332; 530/331; 530/330

[58] Field of Search ................................... .. 530/323, 324,

530/330, 331, 332, 333, 350

[56] References Cited

PUBLICATIONS

Seebach et al. (1996), Helv. Chim. Acta. 79:913—941. Seebach et al. (1996), Helv. Chim. Acta. 79:2043—2066. Dado and Gellman (1994) J. Am. Chem. Soc. 116:1054—1062.

2.0 —

Methanol

1.5

1.0 -

Water 0.5 - [9) x16‘ (deg cm 2Ideclmol)

‘0.5 -

Suhara et al. (1996) Tetrahedron Lett. 37:(10):1575—1578. Ellman (1996) Acc. Chem. Res. 29:132—143. Nohira et al. (1970) Bull. Chem. Soc. Jpn. 43:2230. Lam et al. (1977) Chem. 97:411—448. Herradon and Seebach (1989) Helv. Chim. Acta. 72:690—714. Tilley et al. (1992) J. Med. Chem. 35:3774—3783. Kobayashi et al. (1990) Chem. Pharm. Bull. 38:350. Blake et al. (1964) Am. Chem. Soc. 86:5293. Cooper et al. (1993) J. Chem. Soc. Perkin Trans. 1:1313. Jefford and McNulty (1994) J. Helv. Chim. Acta. 7712142. Podlech and Seebach (1995) Liebigs Ann. 1217. BodansZky et al. (1984) The Practice of Peptide Synthesis; Springer Verlag, New York. Appella et al, J. Am. Chem. Soc. 1996, vol. 118, pp. 13071—72. Karle et al., Journal Am. Chem. Soc., Oct. 1, 1997, vol. 119, N0. 39, 9087—9095.

Primary Examiner—Keith D. MacMillian Assistant Examiner—T. D. Wessendorf Attorney, Agent, or Firm—DeWitt Ross & Stevens SC.

[57] ABSTRACT

Disclosed are [3-peptides containing cylcoalkyl, cycloalkenyl, and heterocylic substituents Which encompass the (X and [3 carbons of the peptide backbone. The [3-peptides adopt stable helical and sheet structures in both the solid state and in solution. Method of generating combinatorial libraries of peptides containing [3-peptide residues and the libraries formed thereby are disclosed.

20 Claims, 19 Drawing Sheets

l l I 200 210 220

I l l l 230 240 250 260

Wavelength (nm)

0 O O O O O O

H N H N H N 142* HQ‘ 2 H,+

U.S. Patent May 9, 2000 Sheet 1 0f 19 6,060,585

20-Helix

FIG. 1

18-Helix

U.S. Patent May 9, 2000 Sheet 2 0f 19 6,060,585

U.S. Patent May 9, 2000 Sheet 3 0f 19 6,060,585

FIG. 2B

U.S. Patent May 9, 2000 Sheet 4 0f 19 6,060,585

FIG. 2C

U.S. Patent May 9, 2000 Sheet 5 0f 19 6,060,585

65 hr 32 hr 19 hr 9 hr

6 min

I h * ‘ ‘? "I"""""?"""'I“f

8 00 7.00 6 00 PPM

U.S. Patent May 9, 2000 Sheet 6 0f 19 6,060,585

‘Wm :00 .w 12

2

‘m 0

..4

O 2

Id

0 0) I‘ I3 m

1m .n

_l - i& W

Km % %@1 ‘0 v

mm 0 I0 2 0

AW _ _ _ _ _ mm

0 O 0 O 0. m m. 0. m. w m m m

m

{9% NEG mmamg H2

FIG. 4

U.S. Patent May 9, 2000 Sheet 7 0f 19 6,060,585

40000- 3 A 2 = Dimer

'- s0ooo~ .

“g 3 = Tnmer u 2o0oo~ 4 = Tetramer N

E 0 10000~

8) 4 B 0' 5

91 ~1000o~ I I I I I

190 200 210 220 250 240 250

Wavelength (nm)

U.S. Patent May 9, 2000 Sheet 8 0f 19 6,060,585

maNo mauo 0

@AQ :ZILH OCH3 H H

50 hr

39 hr

15hr

40 min

8 min

8.50 8.00 7.50 7.00

FIG. 6

U.S. Patent May 9, 2000 Sheet 11 0f 19 6,060,585

4 z Totramer 6 a Hammer 8 :- Octamar

I I I l 200

O

5

1.0

_ w s. o. o 0 AEEQQENEQ 33 .2." § _ 5. 0.

0 6 2 O 5 2 O 4 2) m n 0( Sum 29 n .w 0% 2! 2W 0 1 2

o O

’§Z\ Q; NH: N H24"

i N H

FIG. 9

U.S. Patent May 9, 2000 Sheet 12 0f 19 6,060,585

1.5-—

5 0

r 1%

an“ 2

mmm

“KM 0 ‘QC IIS THo 2

a... 468

0 I4 2) m

. n

u (

“ lmm “ 2O . n

u h n 0

u 0v “ In... n W

I I

. O

“ I1 H 2

“ m

n 12

n _ 0. O

_ O. 1

:35 “.5 93 kc“ HQ

0 o 0 0 0

Nb ~¢~¢~~1 H N H H N H “2+ “2+

n

n = 0,1 or 2

NH NH )1

FIG. 10

U.S. Patent May 9, 2000 Sheet 13 0f 19 6,060,585

Methanol

I 260

l 250

I 240

I I I 210 220 230

Wavelength (am)

I 200

_ _ _

0 s. n 5 2 1 4| 0

205.82%“. 93 k m 0. O Fx

FIG. 11

U.S. Patent May 9, 2000 Sheet 14 0f 19 6,060,585

+ + 2 N22 3 0 n N22

N22 2

o o o 0 0mm _

ovm _

0mm _

com

2... 0007 I 8.9 l 88 I. ooom I ooov

+ +

5 n

o o o 0

A85 £32053 0mm .

O

U.S. Patent May 9, 2000 Sheet 16 0f 19 6,060,585

meg.

QwOO . mmOO . wUZGmEDWmE 3500 3000 3&00

NFWENUMESER 3400 @000.

FIG. 14A

U.S. Patent May 9, 2000 Sheet 17 0f 19 6,060,585

0 01m‘. 101m \mNHQ.

O U 1% m B M U UN OF 1|.TV an“ N

O O

_ _ _ arm wwoo. mmoo. 0000. wuzcmmommc

FIG. 14B

U.S. Patent

I Coupling Step:

II (9 dipeptides):

III (27 tripeptides):

6,060,585 May 9, 2000 Sheet 18 0f 19

-supp01t (split)

A G V

A-support G-support V-support

(Randomize, Split)

A G V

AA-support GA-support VA-support AG-support GG-support VG-support AV-support GV-support VV-support

(Randomize, Split)

A G V

AAA-support GAA-support VAA-support AAG-support GAG-support VAG-support AAV-support GAV-support VAV-support

AGA-support GGA-support VGA-support AGG-support GGG-support VGG-support AGV-support GGV-support VGV-support

AVA-support GVA-support VVA-support AVG-support GVG-support VVG-support AVV-support GVV-support VVV-support

FIG. 15