CONFORMATIONAL ANALYSISCONFORMATIONAL ANALYSIS

Conformer Conformer BB

INTRODUCTIONINTRODUCTION

Conformationally constrained analogues of biologically important peptides can be useful mechanistic probes. Although much attention has been focused on peptidomimetics based on -turns,1 our search for peptidomimetic inhibitors of hydroxylase enzymes has led us to consider peptides based on -turns2 (Fig 1a).

A study of the three-dimensional requirements involved in the binding of hypoxia inducible factor (HIF) to FIH (Factor Inhibiting HIF) based on crystal structures3 (Fig 1b) revealed an inverse -turn in the HIF residues Val802-Asn803-Ala804 (Fig 1c), the design target for our peptidomimetics.

Design, Synthesis, and Conformational Analysis of Inverse Design, Synthesis, and Conformational Analysis of Inverse -Turn Cyclic Peptidomimetics of HIF-Turn Cyclic Peptidomimetics of HIFMorakot Kaewpet, Biswadip Banerji, Barbara Odell, Timothy D. Claridge, and Christopher J. Schofield

Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Mansfield Road, Oxford OX1 3TA, UK

SYNTHESISSYNTHESIS The synthesis of glycine- and proline-containing cyclic peptides 1-3 was achieved by standard amide coupling procedure and ring closure metathesis (RCM) reaction as shown in Scheme 1.

Scheme 1.Scheme 1. Synthesis of cyclic peptidesSynthesis of cyclic peptides

[2H]6-DMSO-CDCl3 ratio

NH

che

mic

al s

hift

/ppm

Figure 8.Figure 8. [2H]6-DMSO titration of 3 at 298K.

Temperature/K

NH

che

mic

al s

hift

/ppm

Figure 7.Figure 7. Temperature dependence of 3 in CDCl3.

Compound 3Compound 3

Figure 8.Figure 8. NOESY of 3 in CDCl3 at 298K – expanding the region

of strong NOE between H10’ H7’.

Figure 4.Figure 4. Tr-ROESY of 2 in [2H]5-pyridine at 238K – showing relevant NOEs and an expansion of the weak NOE of NH4B to H2’B, whereas NH1B has NOEs to H2’B (strong) and H2 (medium).

Results for 2

A:B (3:1) in [2H]5-pyridine at 238K.

In A, NH4 has the lowest temp coeff,

suggesting intramolecular H-bonding. In B, no intramolecular H-bonding. A has both trans-amide bonds, pointing

to the opposite faces due to strong NOEs

between NH1 H2’ and NH4 H2. B has one cis-amide bond due to strong

NOE between NH1 H2’, and weak NOEs

between NH4 H2 and NH4 H2’.

Figure 2.Figure 2. Tr-ROESY of 1 in [2H]5-pyridine at 238K – showing NOEs which define confermers A and B.

Conformer BConformer B

Conformer AConformer A

Figure 3.Figure 3. Temperature dependence of conformers A and B of 1 in CDCl3.

Temperature/K

NH

che

mic

al s

hift

/ppm

NH

che

mic

al s

hift

/ppm

Temperature/K

Conformer BConformer B

Conformer AConformer A

Figure 5.Figure 5. Temperature dependence of conformers A and B of 2 in CDCl3.

y = -0.0117x + 10.714

y = -0.0016x + 7.283

5.5

6

6.5

7

7.5

8

8.5

220 240 260 280 300 320

Temp/K

Am

ide C

hem

ical

sh

ift/

pp

m

NH1A

NH4A

y = -0.0032x + 7.7351

y = -0.0094x + 9.4097

5.5

6

6.5

7

7.5

300 305 310 315 320 325

Temp/K

Am

ide c

hem

ical shift/ppm

NH1B

NH4B

y = -0.0032x + 7.7351

y = -0.0094x + 9.4097

5.5

6

6.5

7

7.5

300 305 310 315 320 325

Temp/K

Am

ide c

hem

ical shift/ppm

NH1B

NH4B

Temperature/K

NH

che

mic

al s

hift

/ppm

NH

che

mic

al s

hift

/ppm

Temperature/K

Conformer BConformer B

Conformer AConformer A

CONCLUSIONSCONCLUSIONS REFERENCESREFERENCES1. Fink, B. E.; Kym, P. R.; Katzenellenbogen, J. A. Journal of

the American Chemical Society 1998, 120(18), 4334-4344.

2. Bystrov, V. F.; Portnova, S. L.; Tsetlin, V. I.; Ivanov, V. T.; Ovchinnikov, Y. A. Tetrahedron 1969, 25(3), 493-515.

3. Elkins, J. M.; Hewitson, K. S.; McNeill, L. A.; Seibel, J. F.; Schlemminger, I.; Pugh, C. W.; Ratcliffe, P. J.; Schofield, C. J. J Biol Chem 2003, 278(3), 1802-6.

Figure 1.Figure 1. (a) (a) The structure of The structure of -turn; -turn; (b)(b) The crystal structure of HIF (light pink) in The crystal structure of HIF (light pink) in the active site of FIH (sphere) together with Fe(II) (orange) and the active site of FIH (sphere) together with Fe(II) (orange) and 2-oxoglutarate (cyan); and 2-oxoglutarate (cyan); and (c)(c) Model of cyclic peptide design. Model of cyclic peptide design.

(a)

(c)

(b)

5

23 NH

O

NHO

NH2HO2C

H H

H H6

7 8

9

1011

4

1

3

13a1 N

O

NHO

NHBocMeO2C

H H4

5 6

7

89

2

H

1211

10

5

23 NH

O

NHO

NH2HO2C

H H6

7 8

9

1011

4

1

1

2

3

Compound 2Compound 2Compound 1Compound 1

Results for 1 A:B (2:1) in [2H]5-pyridine at 238K. Similar temp-dependence behaviour. -0.009 ppm/K for NH1 -0.005 ppm/K for NH4 NH4 has more of a propensity for H-bonding than NH1. Torsional rotations involving in the conversion of A to B.

NHO

NHOHO2C NH2

t1

t2

t4

t3

Results for 3Only one conformer in CDCl3 at rt. NH2 involved in intramolecular H-bonding. NH2 & H13a are on the same face, due to strong NOE. H8 has strong NOEs between both -proline (H10 slightly stronger), thus a trans-Pro. Strong NOE between H10’ H7’, together with weak NOEs between H10’ H5/H6, suggest the aliphatic chain is folded towards the -proline moiety.

Conformer AConformer A

This work has focused on peptidomimetics of HIF designed to form inverse -turns, initiated from the X-ray structure of HIF bound to FIH. Ring-closing olefin metathesis was shown to be an effective method for closing medium-ring amides with good yields, representing the first example of cyclisation to form eleven-membered peptides using RCM reaction The following conclusions were observed for this family of cyclic tripeptides; · 

There is a structural propensity of compounds 1-3 to form inverse -turns.   Restraints can either be introduced at the (i+1)th residue using proline as in 3 or on the alkyl chain side of the molecule employing unsaturation as in 1.

In 2, there is increased flexibility in the saturated system as noted by two conformers in which the predominant one is still an inverse -turn, but the minor conformer has a cis-amide bond and is therefore no longer an inverse -turn

3 is an interesting example where there is only one observable conformation containing a stable inverse -turn with the alkyl chain folded towards the proline side chain.

An inverse -turn might still be expected to play an important role for inhibition of FIH, but the core motif is not a sufficient requirement for activity. We conclude that appropriate functionality of the side chains must be involved. The next stage of this work will focus on designing proline analogues that contain extended functional groups which will be capable of binding to other strategic parts of the active site of FIH.

ACKNOWLEDGEMENTACKNOWLEDGEMENT : : The Royal Thai Government