INTRODUCTION
Chapter 1. The Role of Somatostatin
The Role of Somatostatin
“ Non c'è nulla interamente in nostro potere, se non i nostri pensieri. ”
René Descartes ( 1596 – 1650)
Chapter 1. The Role of Somatostatin
H2N
6 H O 3 H O H O H N 5 7 N NH H2N 2 N N N 4 N N 8 1 O H O H O H O O NH2 Ph S O S Ph OH 9 NH O H O H H O 11 13 N N N HO 12 N 10 N NH2 14 O H O O H OH HO
Figure 1. SRIF-14.
Advances in Human Growth Hormone Research 1973 74-612 J. Am. Chem. Soc. 1974 96 FEBS Letters. 1980 109 Metabolism. 1990 39 N. Engl. J. Med. 1983 309 N. Engl. J. Med. 1983 309 Chapter 1. The Role of Somatostatin
Figure 2. SRIF receptor-mediated modulation of signalling cascades leading to changes in hormone secretion, apoptosis and cell growth. 8
J. Endocrinol. Invest. 1997 20 Nat. Rev. 2003 2 Proc. Natl. Acad. Sci. 1992 89 Mol. Pharmacol. 1993 44 Rev. Physiol. Biochem. Pharmacol. 1998 133 J. of Neurochem. 2004 89 Chapter 1. The Role of Somatostatin
8 Figure 3. Structure of SRIF receptors exemplified by subtype sstr 2A receptor. et al.
Biopolymers Peptide Science 2002 66 Eur. J. Nucl. Med. 2001 28 Chapter 1. The Role of Somatostatin
Chapter 1. The Role of Somatostatin
1.1. Clinical use of Somatostatin analogues
Compound Sstr 1 Sstr 2 Sstr 3 Sstr 4 Sstr 5 SRIF-14 SRIF-28 Octreotide BIM 23014 RC-160
16 Table 1. Binding affinities (K i) nM of clinically useful Somatostatin analogues at
sst 1-5 receptors.
J. Clin. Endocrinol. Metab. 1974 39 Endocrinology 1994 135 Chapter 1. The Role of Somatostatin
in vivo in vivo
Lancet 1997 350 J. Clin. Endocrinol. Metab. 1997 82 Pituitary 1999 1 Life Sci. 1987 41 Endocrine Rev. 1991 12 Endocrinol Rev. 2003 24 J Nucl. Med. 2000 41 Lancet 1989 1 Life Sci. 1991 49 Chapter 1. The Role of Somatostatin
OH
O O HO N HOOC COOH N N N N N N OH HOOC COOH O O COOH
OH a b
Figure 4. Bifunctional chelating agents: a. DOTA and b. DTPA. in vivo
et. al Eur. J. Nucl. Med. 1993 20 Endocrinology 2000 141 Ann. Inter. Med. 1996 125 Lancet 1998 351 Chapter 1. The Role of Somatostatin
Eur. J. Nucl. Med. 2003 30 Cancer Res 1998 58 32 Clinical Cancer Research 2004 10 Int. J. Cancer. 2002 98 Chapter 1. The Role of Somatostatin
Radio- Physical Particle Energy Maximum nuclide half-life (MeV) tissue range 32 P β 67 Cu β γ 90 Y β 105 Rh β γ 109 Pd β 111 Ag β γ 125 I γ 131 I β γ 177 Lu β γ 186 Re β γ 188 Re β γ 198 Au β γ 211 At α µ 212 Bi α γ µ 99m Tc γ 64 Cu β γ µ
Table 2. Radio-nuclides physical half-life, decay and maximum penetration.
Chapter 2. Peptide Design
Peptide Design
“ Lo scienziato non è l'uomo che fornisce le vere risposte: è quello che pone le vere domande. “
Claude Levi Strauss (1908-2009)
Chapter 2. Peptide Design
silico silico
Curr. Opin. Drug. Disc. Dev. 1998 1 Chapter 2. Peptide Design
2.1 The Rational Approach
Science 1977 196 Metab. Clin. Exp. 1978 27 Nat. Rev. Drug Discov 2002 1 Chapter 2. Peptide Design
Scheme 1. Strategy for transforming peptides into small molecules.
α α,β β
Chapter 2. Peptide Design
in vitro in vivo
et al.
O O NH HN
H2N H NH
O S NH O S H O HO NH N H O OH NH2
Figure 5. Smallest peptide active to inhibit GH
n Vivo in Vitro Endocrinology 1983 37 Biochem Biophys Res Commun. 1975 65 PNAS 2000 97 1979 Chapter 2. Peptide Design
2.1.1 Somatostatin agonists et al. et al
O
H N N H N O O O N O NH H H N N H NH2 O OH
Figure 6. L-363, 301. β β
Int. J. Peptide Protein Res. 1983 21 et al Life Sci. 1984 34 Proc. Nat. Acad. Sci. USA. 1971 68 β Protein Sci. 1994 3 Chapter 2. Peptide Design
β β
et al. in vitro
O O O
NH HN
H2N HN H NH
O S NH O S H O H N NH HO N H O HO CH OH 3 NH2
Figure 7. Octreotide, SMS-201-995.
et al Life Sci. 1982 31 Metab.Clin. Exp. 1992 41 Chapter 2. Peptide Design
in vivo
in vitro, in vivo
OH
O O O NH HN
H2N HN H NH
O S NH O O S H O H N NH H2N N H O
NH2
N H
Figure 8. RC-160 Vapreotide .
New Engl. J. Med. 1996 334 Proc. Natl. Acad. Sci. USA 1987 84 Life Sci. 1987 40 Chapter 2. Peptide Design
OH
O O O NH HN
H2N HN H NH
O S NH O O S H O H N NH H2N N H O HO NH2
Figure 9. BIM 23014 lanreotide.
OH
O
H N N H HN O O O N O NH H H N N H NH2 O
Figure 10. MK-678 segletide.
et al. R S β cis
Life Sci. 1984 34 J. Med. Chem. 1998 41 Chapter 2. Peptide Design
In vitro
O H H N N N NH O H2N H N O O O
O O HN H N N H NH2 O
O
Figure 11. SOM230 first universal SRIF agonist.
Tetrahedron 2000 56 Chapter 2. Peptide Design
β
Figure 12. CH-275 sst 1 selective analogue.
J. Med. Chem. 2003 46 in vitro J. Clin. Endocrinol. Metab. 2004 89 J. Med. Chem. 2001 44 N- J. Med. Chem. 2001 44 Endocrinology 2001 142 Chapter 2. Peptide Design
NH O O H NH N N N H H O HN O O NH O H N N N NH2 O H O HO O H2N
Figure 13. PTR3173 cyclic- backbone analogue.
2.1.2 Somatostatin antagonists
Figure 14. CYN-154806 first SRIF antagonist.
Mol. Pharmacol. 1997 50 Chapter 2. Peptide Design
OH
O O H O H N N N H N N N 2 H H O O S O S O NH H O H N N HO N NH2 O H O HO
Figure 15. Sst 3-ODN-8 selective sst 3 antagonist.
Brain Res Bull. 2001 Br J Pharmacol. 2003 139 Mol Pharmacol. 2005 68 Chapter 2. Peptide Design
2.2 NMR conformational analysis approach
Table 3. 1H chemical shifts for the 20 common amino acid residues.
NMR of protein and nucleic acids 1986 Chapter 2. Peptide Design
Scheme 2. The NMR conformational analysis approach.
2.2.1 NMR Experiments
α
Chapter 2. Peptide Design
α via space
1996 Ann., Rev., Bioch 1989 Chapter 2. Peptide Design
• • 66 α α via d d
α ensamble
1971 Eur. J. Bioc. 1981 114 2002 Chapter 2. Peptide Design
2.2.2 Conformational Analysis
Mol Biol 1971 5 J. Mol. Biol., 1963 Chapter 2. Peptide Design
φ
∆δ ∆
Chapter 2. Peptide Design
φ ψ
2.2.3 Matching the Secondary Structure motif
ψ ϕ
Parameter α-helix 310 -helix γ-turn β-turn II β-turn II’ ϕϕϕ (°) ψψψ (°) n
Table 4. Average Parameters for Peptide Helices Based on α-Amino Acids.
ϕ
ψ
nm n m ← ← ←
m γ-turn β-turn α-turn
PNAS 1971 68 Biopolymers 1965 3 Chapter 2. Peptide Design
γ β α π Helix
γ-helix 310 helix α-helix α α
α ϕ ψ α
φ ψ
Biopolymers 2006 84 Crit. Rev. Biochem. 1980 . Trends. Biochem Sci. 1991; 16 Biochemistry 1990 29 Chapter 2. Peptide Design
a a Distance α-helix 310 -helix β βp Turn I Turn II dαN 3.2 3.2 dαN (i, i+2) dαN (i, i+3) dαN (i, i+4) dNN dNN (i, i+2) dβN
dαβ a For the turns, the first of two numbers applies to the distance between residues 2 and 3, the second to that between residues 3 and 4.
Table 5. Short (≤ 4.5 Å) sequential and Medium-range 1H-1H distances in Polypeptide secondary structures. β-bend ribbon spiral β β
γ-Turn γ γ
PNAS 1987 84 78 J. AM. Chem. Soc. 1992 ; 114 . γ Macromolecules. 1972 5 Chapter 2. Peptide Design
ϕ ≅ ψ ≅ γ ϕ ≅ ψ ≅ γ γ
γ γ β-Turn β β- β
β
short range . long range β β-Turn II ϕ
γ Macromolecules 1972 5 Biopolymers 1968 6 β J. Mol. Biol 1988 203 Chapter 2. Peptide Design
β-Turn II’ β α β
β Protein Science 1994 3 Chapter 2. Peptide Design
Figure 16. Types of β-turns. β-turns differ from each other based on the orientation the ϕ and ψ torsional angles of the peptide bond between residues in "i+1" and "i+2" (indicated with curved arrows). Additional differences based on the properties of the residues in the position "i+1" are also observed (in circles). Chains were colored according to the physiochemical properties of amino acids. Hydrophobic residues are colored grey, acidic residues and relatives are yellow, and basic residues are in blue.
α β
α β
Chapter 2. Peptide Design
Chapter 2. Peptide Design
2.3 In Silico approach
• • • Chapter 2. Peptide Design
ensemble ensemble ensemble
2.3.1 On the question of GPCR
Figure 17. Trans-membrane domains of GPCR.
Chapter 2. Peptide Design
Curr. Opin. Cell Biol. 1994 6 FASEB J 1995 9 EMBO J. 1995 14 DNA and Cell Biol. 1995 14 Biochem. Biophys. Res. Commun 1995 216 Chapter 2. Peptide Design
2.3.2 The pharmacophore model
Chapter 3. Aim of the Project
AIM OF THE PROJECT
"... Non vogliate negar l'esperienza di retro al sol, del mondo sanza gente. Considerate la vostra semenza fatti non foste a viver come bruti ma per seguir virtute e conoscenza."
Dante Alighieri, (1265-1321) (Divina Commedia, Inferno canto XXVI, 116-120
Chapter 3. Aim of the Project
in silico
β Letters in Organic Chemistry 2005 2 Journal Medicinal Chemistry 2008 51 Chapter 3. Aim of the Project
in silico sera c Peptides Xaa Yaa Zaa Kaa
27
28
45
29
48
30
46
31
32
33
34
. The Journal of Clinical Endocrinology & Metabolis. 2001 86 Chapter 3. Aim of the Project
Peptides Xaa Kaa Yaa Jaa Zaa
36
Peptides Xaa Kaa Yaa Jaa Zaa
37
47
Peptides Xaa Kaa Yaa Jaa Zaa
38
39
40
41
42
43
Chapter 3. Aim of the Project
Compounds Chelating Agents Unsaturated-Peptide 49 50 51 52
53
54
Table 6. Target compounds synthesized in this work.
Chapter 4. Results and Discussion RESULTS AND DISCUSSION
“ When a multistep process, such as the preparation of a long polypeptide or protein is contemplated, the saving in time and effort and materials could be very large. The fact that all of the steps just described are heterogeneous reactions between a soluble reagent in the liquid phase and the growing peptide chain in the insoluble solid phase led to the introduction of the name “solid phase peptide synthesis”.
Robert Bruce Merrifield (1921 – 2006) Nobel Prize in Chemistry , December 8 th 1984
Chapter 4. Results and Discussion
Nature 1965 207 Curr. Opin. Chem. Biol. 1997 1 Chapter 4. Results and Discussion
4.1 Chemical Synthesis
• • • • • •
•
Chapter 4. Results and Discussion
4.1.1 Linear peptides synthesis
t- t t • C • • • • • •
Anal. Biochem 1970 34 Chapter 4. Results and Discussion
Scheme 3. Chemical steps of the SPPS.
Figure 18. a: H–L-Thr(t-Bu)–ol–2-chlorotrityl resin; b: Wang Resin; c: Rink amide resin.
t-
Chapter 4. Results and Discussion
Scheme 4. Synthesis of linear 1-8 peptides on H–l-Thr(t-Bu)–ol–2-chlorotrityl resin. a. (i) Fmoc- L-Hag, HATU/NMM, 40 min r.t.; (ii) 20% piperidine in DMF (2 x 10 min); (iii) Coupling with the amino acid.
Fmoc NH X O
HN O K HN Y O NH-Boc HN O J HN OH O N H O NH O Z a O NH OH O O
O
9 X= L-Hag K= D-Phe Y= L-Phe J= D-2-Nal Z= D-Phe
Scheme 5. Synthesis of 9 on Wang resin. a.(i) Fmoc- L-Hag, HATU/NMM, 40 min r.t.; (ii) 20% piperidine in DMF (2 x 10 min); (iii) Coupling with the amino acid.
Chapter 4. Results and Discussion
Fmoc NH X O
HN O K HN Y O NH-Boc HN O J HN
O N W O H NH O Z MeO HN O b O NH
MeO O O MeO HN
MeO O 10 X= L-Hag K= D-Phe Y= L-Phe J= D-Trp W= L-Phe Z= D-Phe 11 X= L-Hag K= D-Phe Y= L-Phe J= D-Trp W= L-Thr Z= D-Phe 12 X= L-Hag K= L-Phe Y= L-Phe J= D-Trp W= L-Thr Z= L-Phe 13 X=D-Hag K=L-Phe Y=L-Phe J=D-Trp W=L-Thr Z=L-Phe 14 X= L-Hag K= L-Phe Y= L-Phe J= D-2-Nal W= L-Thr Z= L-Phe 15 X= D-Hag K= L-Phe Y= L-Phe J= D-2-Nal W= L-Thr Z= L-Phe 16 X= D-Hag K= L-Phe Y= L-Tyr J= D-2-Nal W= L-Thr Z= L-Phe
Scheme 6. Synthesis of linear 10-16 peptides on Rink amide resin. b. (i) 20% piperidine in DMF (2 x 10 min ; (ii) Coupling with the amino acid.
t-
t- 3 5
Chapter 4. Results and Discussion
Compound Linear peptides sequences 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Table 7. Linear epta-peptides( 1-8) and octa-peptides ( 9-16 ) synthesized in this work.
Chapter 4. Results and Discussion
4.1.2 Synthesis of unsaturated cyclic peptides
Ring formation
H2C CH2
[(Cy)3P]2RuCl2 CHR Catalyst
Ru Ru
H2C CH2 H2C Ru
Scheme 7. Olefin metathesis.
2006 45 Angew. Chem. Int. Ed. 2006 45 2003 Makromol. Chem. 1971 141 J. Am. Chem. Soc. 1988 110 Chapter 4. Results and Discussion
Figure 19. 1 st generation Grubbs catalyst ( 17 ). 2 nd generation Grubbs catalyst ( 18 ). 17 17 17 18
J. Am. Chem. Soc. 1993 115 Org. Lett 1999 1 Org. Lett 2000 2 J. Am. Chem. Soc. 1996 118 Chapter 4. Results and Discussion
Scheme 8. c. Catalyst 18 , 45°C, 24 h ( 20-24 ) and 48 h ( 19 ; 25-26 ); (i) 20% piperidine in DMF, Fmoc-D-Phe/HATU/NMM 40 min, r.t. d.(ii) Cleavage of 19, 20, 22, 24, 25, 26 TFA/DCM/EDT/Phenol (94:2:2:2); 21 and 23 TFA/DCM/EDT/Phenol (70:26:2:2).
Chapter 4. Results and Discussion
Scheme 9. c. Catalyst 18, 45 °C, 48h; d. (i) 20% piperidine in DMF, Fmoc-GLY /HATU/NMM 40 min, r.t. (ii). Cleavage of 36 TFA/DCM/EDT/Phenol (94:2:2:2).
Chapter 4. Results and Discussion
Scheme 10. c. Catalyst 18, 45 °C, 48h; cleavage: TFA/DCM/EDT/Phenol (94:2:2:2). N
27-34 36 27 29 18
Org. Lett 2003 5 Org. Lett 2002 4 J. Org. Chem. 2005 70, Chapter 4. Results and Discussion
Dicarba- Catalyst cyclic/linear % yield a analogues (hours of reflux) ratio b Six-mer-ring peptides 27 28 29 30 31 32 33 34 Eight-mer-ring-peptides
36 37 38 39 40 41 42 43
Table 8. Cyclization of the linear six-mer-ring peptides ( 27-34 ) and eight-mer-ring peptides (36-43 ) with catalyst 18. a Overall yield of isolated pure compounds(Z/E mixture), calculated on the basis of an average peptide loading of 0.5 mmol/g of resin. b Calculated on the basis of peaks area in analytical RP-HPLC. 39-43 36-38 via π
J. Chem Comm. 2009 Chapter 4. Results and Discussion
44
Figure 20. Hoveyda-Grubbs 1 st generation catalyst ( 44 ).
44 44 39-43 ≈ 29 32 ≈ 29 32
Org. Lett. 2003, 5, . Tetrahedron Lett. 1999 40 Org. Lett. 2000 2 Chapter 4. Results and Discussion
Compound [M+H] + calcd. [M+H] + found [M+2H] 2+ [M+Na] + 27 28 29 E 29 Z 30 31 32 E 32 Z 33 34 36 37 38 39 40 41 42 43
Table 9. Mass spectrometry data of pure unsaturated cyclic compounds.
Chapter 4. Results and Discussion
a Compound HPLC method Rt (min) 27 28 29 E 29 Z 30 31 32 E 32 Z 33 34 36 37 38 39 40 41 42 43
Table 10. RP-HPLC analytical data of the pure cyclic compounds with the alkenyl bridge. a A: H2O 0.1% TFA; B: CH 3CN 0.1% TFA.
4.1.3 Microwave-assisted RCM
Tetrahedron, 2001 57 2002 . Angew. Chem. Int. Ed. 2004 43 Chapter 4. Results and Discussion
Figure 21. Electromagnetic spectrum.
Tetrahedron 1995 51 Drug Discov. Today. 2001 6 Curr. Opin. Chem. Biol. 2002 6 Microwave Engineering 1993 Chapter 4. Results and Discussion
Figure 22. Heating by microwave irradiation.
Chapter 4. Results and Discussion
via
Angew Chem Int Ed Engl. 2004 43 Chapter 4. Results and Discussion
Figure 23. Inverted temperature gradients in microwave versus oil-bath heating: Difference in the temperature profile: after 1 min of microwave irradiation (left) and treatment in oil-bath (right).
Org. Lett. 2006 8 J. Pept. Sci. 2007 13 Chapter 4. Results and Discussion
4.1.3.1 Six-mer-ring peptides 27 32 33-34 1-6 18 27 29 29
Microwaves in Organic Synthesis 2006 2005 Microwaves in organic and Medicinal Chemistry 2005 Tetrahedron Lett 2003 Tetrahedron Lett. 2003 2005 2005 2006 2006 2006 Chapter 4. Results and Discussion
Cyclic/linear Compounds Microwaves methods ratio a 27
28
29
30
31
32
Table 11. Microwave-assisted RCM on peptides 27-32 ; a Calculated on the basis of peaks area in analytical RP-HPLC. b. 75% of 29 and 15% of 29 without benzyl moiety.
4.1.3.2 Eight-mer-ring peptides 39- 43, 44 12-16 , Chapter 4. Results and Discussion
Graphic 1. Temperature, pressure and power for each of the four employed methods. Mean values monitored during 30 minutes of reaction.
12-16 18 39-43 39-43
J. Pept. Sci 2007 13 Chapter 4. Results and Discussion
Graphic 2. Cyclic peptides ( 39-43) yields after 30 minutes of MW-assisted RCM performed with four different methods. 44 40 42 39 41
Microwaves-assisted ring-closing Metathesis revisited. On the question of non thermal effect 2003 68 Chapter 4. Results and Discussion
39 40 41 42 43 39-43 12-16 39 14 41
Chapter 4. Results and Discussion
Graphic 3. Time-dependent yield. Values are referred as a HPLC cyclic/linear ratio of the E/Z isomers mixture.
4.1.4 Synthesis of saturated cyclic peptides
28 29 30 37
− − − 29 45-47
Chapter 4. Results and Discussion
O O O Y NH HN H2N HN H NH O NH 28;30 e O H H O N NH HO N Z H O HO CH 3 NH2
45 Y= Phe Z= Thr 46 Y= 1-Nal Z= Thr
O O H H N K N 2 X N Y NH H O J O 37 e NH O H H2N N W N Z N NH2 H H O O O
47 X=L-Hag K=D-Phe Y=L-Phe J=D-Trp W=L-Phe Z=D-Phe Scheme 11. Synthesis of saturated dicarba-analogues 45-47. e: 20% Pd(OH) 2/C, H 2, 24 h, 30 °C.
29 48 29
J. Org. Chem 2002 67 1995 2 Can. J. Chem. 2005 83 Chapter 4. Results and Discussion
Scheme 12. Synthesis of the saturated analog 48 ; f. (i) MeOH/DCM, Wilkinson’s catalyst 2.5%, H 2 (60 psi); (ii) 20% piperidine in DMF, Fmoc- D-Phe/HATU/NMM, 40 min, r.t.; (iii) 20% piperidine in DMF, cleavage.
Compounds [M+H]+ calcd. [M+H]+ found [M+2H]2+ [M+Na]+ 45 46 47 48
Table 12. Mass spectrometry data of pure saturated cyclic compounds . Compound HPLC method a Rt (min) 45 46 47 48
Table 13. RP-HPLC analytical data of pure saturated cyclic compounds. a A: H 2O 0.1% TFA; B: CH 3CN 0.1% TFA.
Chapter 4. Results and Discussion
4.1.5 Synthesis of radiolabeled unsaturated peptides
J. Nucl. Med. 1990 17 Anticancer Drugs 1991 2 Eur. J. Nucl. Med. 1998 25 J. Labeled Cmpd. Radiopharm. 1993 32 Chapter 4. Results and Discussion
in vivo 29 31 32 28 30
4.1.5.1 Synthesis of DOTA conjugated peptides 29-32 t 49 51
Figure 24. General formula of unsaturated DOTA-dicarba-analogues 49-52 .
Chapter 4. Results and Discussion
Compound [M+H] + calcd. [M+H] + found [M+2H] 2+ [M+Na] + 49 50 51 52
Table 14. Mass spectrometry data of the pure conjugated peptides.
a Compound HPLC method Rt (min) 49 50 51 52 Table 15. RP-HPLC analytical data of the pure conjugated peptides. a A: H 2O 0.1% TFA;B: CH 3CN 0.1% TFA.
Chapter 4. Results and Discussion
4.1.5.2 Synthesis of PN 2S peptide conjugates
28 30
53 54
Scheme 13. Synthesis of PN 2S conjugated peptides 53 and 54 . h: (i) 20% piperidine in DMF; (ii) coupling with -Cys-(Trt), -Gly/HATU/NMM 40 min, r.t.; 3-diphenylphosphinopropionic acid succinimidic ester/NMM 40 min, r.t.(iii) Cleavage TFA/DCM/EDT/Phenol (94:2:2:2).
Inorg. Chem. 2003 42 Chapter 4. Results and Discussion
Compounds [M+H] + calcd. [M+H] + found [M+2H] 2+ [M+Na] + 53 54
Table 16. Mass spectrometry data of the pure conjugated peptides. a Compounds HPLC method Rt (min) 53 54 Table 17. RP-HPLC analytical data of the pure conjugated peptides. a A: H 2O 0.1% TFA;B: CH 3CN 0.1% TFA.
Chapter 4. Results and Discussion
4.2 NMR Analysis
28 45 29 E trans 48
29 Z 30 31 32 E 32 Z
Chem. Soc. Rev. 1992 21 Acta Biochimica Polonica 2001 48 J. Med. Chem. 2006 49 J. Med. Chem 2003 46 J. Mol. Biol. 1997 273 Chapter 4. Results and Discussion
4.2.1 Water/DMSO NMR spectra
4.2.1.1 NMR analysis of 28 28 J Z E 28
α α
α β αΝ
α α α
Protein Sci. 1996 5 Biochemistry 1997 36 Chapter 4. Results and Discussion
NH ( 3J , - Residue αααN CαααH CβββH ( 3J ) Others ∆δ∆δ∆δ /∆∆∆T) b αβαβαβ δ D-Phe 2 ε dhDsa-Nc γ δ Phe 7 ε δ ε D-Trp 8 ζ η Lys 9 δ ε ε Thr 10 γ dhDsa-Cc γ γ Thr(ol) 15 ω) Table 18. NMR Data a of Peptide 28 in Water/DMSO 8:2 solution. a Obtained at 277.1 K, with TSP ( δ 0.00 ppm) as reference shift. Chemical shifts are accurate to b 3 3 ±0.02 resonances. JαΝ and Jαβ coupling constants are in Hz. -∆δ /∆T = temperature coefficients (ppb/K). c Overlapped signals.
β β
α β β β
Chapter 4. Results and Discussion
Figure 25. Superposition of the 10 lowest energy conformers of 28 . Structures were superimposed using the backbone heavy atoms of residues 3-14. Heavy atoms are shown with different colours (carbon, green; nitrogen, blue; oxygen, red). Hydrogen atoms are not shown for clarity.
28
Chapter 4. Results and Discussion
Figure 26. Superposition of the most representative structure of 28 (blue) with octreotide structure (PDB entry 1SOC, red). Structures were superimposed using the backbone heavy atoms of residues 3-14. Hydrogen atoms are not shown for clarity. 4.2.1.2 NMR analysis of 45 45
− 28 J α 45 β γ δ δ
Bioorg. Chem. 1978 7 Int. J. Pept. Prot. Res. 1985 25, Chapter 4. Results and Discussion
NH ( 3J , - Residue αααN CαααH CβββH ( 3J ) Others ∆δ∆δ∆δ /∆∆∆T) b αβαβαβ δ 2 D-Phe ε ζ) Dsa-Nc γ δ Phe 7 ε δ 8 D-Trp ε ζ η γ Lys 9 δ ε Thr 10 γ Dsa-Cc γ γ Thr(ol) 15 ω) Table 19. NMR Data a of Peptide 45 in Water/DMSO 8:2 solution. a Obtained at 277.1 K, with TSP ( δ 0.00 ppm) as reference shift. Chemical shifts are accurate to b 3 3 ±0.02 resonances. JαΝ and Jαβ coupling constants are in Hz. -∆δ /∆T = temperature coefficients (ppb/K). c Overlapped signals. 45 45 β
Chapter 4. Results and Discussion
Figure 27. Superposition of the 10 lowest energy conformers of 45 . Structures were superimposed using the backbone heavy atoms of residues 3-14. Heavy atoms are shown with different colours (carbon, green; nitrogen, blue; oxygen, red). Hydrogen atoms are not shown for clarity.
β β β 45 45
Chapter 4. Results and Discussion
Figure 28. Superposition of the most representative structure of 45 (blue) with octreotide structure (PDB entry 1SOC, red). Structures were superimposed using the backbone heavy atoms of residues 3-14. Hydrogen atoms are not shown for clarity.
4.2.1.3 NMR analysis of 29E 29 E J
γ β E Z 29 E β 29 E
Chapter 4. Results and Discussion
Figure 29. Superposition of the 10 lowest energy conformers of 29 E . Structures were superimposed using the backbone heavy atoms of residues 3-14. Heavy atoms are shown with different colours (carbon, green; nitrogen, blue; oxygen, red). Hydrogen atoms are not shown for clarity.
29 E
Chapter 4. Results and Discussion
NH ( 3J , - Residue αααN CαααH CβββH ( 3J ) Others ∆δ∆δ∆δ /∆∆∆T) b αβαβαβ δ D-Phe 2 ε dhDsa-Nc γ δ Phe 7 ε δ ε D-Trp 8 ζ η γ Lys 9 δ ε δ ε Tyr(Bzl) 10 ω) dhDsa-Cc γ γ Thr(ol) 15 ω) Table 20. NMR Data a of Peptide 29 E in Water/DMSO 8:2 solution. a Obtained at 277.1 K, with TSP ( δ 0.00 ppm) as reference shift. Chemical shifts are accurate to b 3 3 ±0.02 resonances. JαΝ and Jαβ coupling constants are in Hz. -∆δ /∆T = temperature coefficients (ppb/K). c Overlapped signals.
29 E trans gauche - 4.2.1.4 NMR analysis of 48 48 45 − 48 45
Chapter 4. Results and Discussion
β 48 NH ( 3J , - Residue αααN CαααH CβββH ( 3J ) Others ∆δ∆δ∆δ /∆∆∆T) b αβαβαβ δ D-Phe 2 ε ζ Dsa-Nc γ δ Phe 7 ε δ ε D-Trp 8 ζ η γ Lys 9 δ ε δ ε Tyr(Bzl) 10 ω) Dsa-Cc γ γ Thr(ol) 15 ω) Table 21. NMR Data of Peptide 48 in Water/DMSO 8:2 solution. a Obtained at 277.1 K, with TSP ( δ 0.00 ppm) as reference shift. Chemical shifts are accurate to b 3 3 ±0.02 resonances. JαΝ and Jαβ coupling constants are in Hz. -∆δ /∆T = temperature coefficients (ppb/K). c Overlapped signals.
Chapter 4. Results and Discussion
Figure 30. Superpositionof the 10 lowest energy conformers of 48 . Structures were superimposed using the backbone heavy atoms of residues 3-14. Heavy atoms are shown with different colours (carbon, green; nitrogen, blue; oxygen, red). Hydrogen atoms are not shown for clarity.
β
α α α ∆δ ∆ χ trans gauche trans gauche gauche gauche
χ JΗαΗβ Chapter 4. Results and Discussion
χ
4.2.2 SDS NMR spectra
4.2.2.1 NMR analysis of 29 E 29 E trans E β β 29 E β
Chapter 4. Results and Discussion
β Residue CαααH CβββH Others
δ D-Phe 2 ε dhDsa-Nc γ δ Phe 7 ε δ D-Trp 8 ε ζ η γ Lys 9 δ ε δ Tyr(Bzl) 10 ε dhDsa-Cc δ Thr(ol) 15 γ Table 22. NMR Data of Peptide 29 E in SDS solution. Obtained at 277.1 K, with TSP ( δ 0.00 ppm) as reference shift. Chemical shifts are accurate to ±0.02 resonances.
χ trans trans gauche- gauche- gauche+ χ α Chapter 4. Results and Discussion
4.2.2.2 NMR analysis of 29 Z 29 Z Z 29 E γ 29 E 29 E β trans γ δ 29 E
Chapter 4. Results and Discussion
Residue CαααH CβββH Others
δ D-Phe 2 ε dhDsa-Nc γ δ Phe 7 ε δ D-Trp 8 ε ζ η γ Lys 9 δ ε ζ δ Tyr(Bzl) 10 ε dhDsa-Cc δ Thr(ol) 15 γ Table 23. NMR Data of Peptide 29 Z in SDS solution. Obtained at 277.1 K, with TSP ( δ 0.00 ppm) as reference shift. Chemical shifts are accurate to ±0.02 resonances. 4.2.2.3 NMR analysis of 30 30 29 Z β 29 E 29 Z Chapter 4. Results and Discussion
Residue CαααH CβββH Others δ D-Phe 2 dhDsa-Nc γ ζ 1-Nal 7 δ ε δ D-Trp 8 ε ζ η γ δ Lys 9 ε ζ Thr 10 dhDsa-Cc δ Thr(ol) 15 γ Table 24. NMR Data of Peptide 30 in SDS solution. Obtained at 277.1 K, with TSP ( δ 0.00 ppm) as reference shift. Chemical shifts are accurate to ±0.02 resonances.
29 Z 4.2.2.4 NMR analysis of 31 31 29 Z 30 31
Chapter 4. Results and Discussion
29 Z 30 γ β 30 trans gauche Residue CαααH CβββH Others δ D-Phe 2 ε dhDsa-Nc γ ζ 1-Nal 7 δ ε δ D-Trp 8 ε ζ η γ Lys 9 δ ε δ Tyr(Bzl) 10 ε dhDsa-Cc δ Thr(ol) 15 γ Table 25. NMR Dat a of Peptide 31 in SDS solution. Obtained at 277.1 K, with TSP ( δ 0.00 ppm) as reference shift. Chemical shifts are accurate to ±0.02 resonances.
Chapter 4. Results and Discussion
4.2.2.5 NMR analysis of 32 E 32 E 29 E Z 30 31 29 32 E 32 E 29 E Residue CαααH CβββH Others δ D-Phe 2 ε dhDsa-Nc γ δ Phe 7 ε δ D-Trp 8 ε ζ η γ Lys 9 δ ε ζ ε Tyr 10 dhDsa-Cc δ Thr(ol) 15 γ Table 26. NMR Data of Peptide 32 E in SDS solution. Obtained at 277.1 K, with TSP ( δ 0.00 ppm) as reference shift. Chemical shifts are accurate to ±0.02 resonances.
Chapter 4. Results and Discussion
4.2.2.6 NMR analysis of 32 Z 32 Z 29 Z 32 E 32 Z 29 Z Residue CαααH CβββH Others δ D-Phe 2 ε dhDsa-Nc γ δ Phe 7 ζ ε δ D-Trp 8 ε ζ η γ Lys 9 δ ε ζ δ Tyr 10 ε dhDsa-Cc δ Thr(ol) 15 γ Table 27. NMR Data of Peptide 32 Z in SDS solution. Obtained at 277.1 K, with TSP ( δ 0.00 ppm) as reference shift. Chemical shifts are accurate to ±0.02 resonances.
Chapter 4. Results and Discussion
4.3 Molecular Modeling
de novo
4.3.1 Peptide library realization
ensemble
J. Med. Chem. 1996 38 J. Med. Chem. 1994 ,37, J. Med. Chem. 2003 46, J. Med. Chem. 2008 51, Proc. Nat. Am. Sci 2000 97 Chapter 4. Results and Discussion
• •
• • • •
4.3.2 PPS: the algorithm
2008 Chapter 4. Results and Discussion
• • • • •
4.3.3 Pharmacophore model for SRIF receptors
4.3.3.1 SST1 model β
Chapter 4. Results and Discussion
4.3.3.2 SST2 model
β
4.3.3.3 SST3 model β 36 41 42 43 36 41 42 43
4.3.3.4 SST4 model β
4.3.3.5 SST5 model
Chapter 4. Results and Discussion
4.4 Biological Part
4.4.1 Stability essays in human serum
serum 27 28 29 27 28 29 27 28 29
27 28 29 27 28 29
4.4.2 Binding affinity to sstr 1-5
27 32 37 45 48
Chapter 4. Results and Discussion
IC 50 (nM) a
Compound sstr 1 sstr 2 sstr 3 sstr 4 sstr 5
SRIF-28 27 28 29 E 29 Z 30 31 32 E 418 ± 56 32 Z 37 45 46 47 48 Table 28. Receptor affinities of the somatostatin analogues. aThe number of independent repetitions to obtain the mean values ± SEM are indicated between brackets. SRIF-28 is used as internal control.
29 Z
45 28
vs 27 37 47 28 29 E 48
29 E Chapter 4. Results and Discussion
29 Z 29 Z
30 46
28 29 E 29 Z 30 31,
32 Z 48
β
29 Z 30 31, 32 Z 48
J. Med. Chem. 1998 41 Science 1998 282 . Am. Chem. Soc. 1999 121 ´ J. Med. Chem. 2007 50 Chapter 4. Results and Discussion
4.4.3 Radiolabeling of peptides with radioactive metals
4.4.3.1 Radiolabeling of 49 , 50 , 51 and 52 with 111 In
49-52 49-52 4.4.3.2 Radioalbeling of 53 and 54 with 99m Tc
53 54 53 54 53
53 54
Chapter 4. Results and Discussion
Chapter 5. Conclusions
CONCLUSIONS
“ In ogni attività la passione toglie gran parte della difficoltà ”
Erasmo Da Rotterdam (1466-1536)
Chapter 5. Conclusions
5.1 Peptide Design
31 32 33 34 29 Z 31 32 Z 30
5.2 Synthesis
silico 19-24 39-43
5.3 Conformation-affinity relationships and pharmacophore model for sst 5 selective analogues
Chapter 5. Results and Discussion
β - trans gauche
28 45 β 45 28
28 45
β 29 Z 48 45
29 E 48
β gauche - 48 29 E
29 E 48
29 E 48
Chem. Rev. 2005 105 Tetrahedron 1993 49 Chapter 5. Results and Discussion
29 E 48
29 E 48
31
a Compound 29 Z 31 48 sst 2/3/5 Ar 2-Ar 7 Ar 2-Ar 8 Ar 2-Lys 9 Ar 2-Ar 10 Ar 7-Ar 8 Ar 7-Lys 9 Ar 8-Lys 9 Ar 8-Ar 10 Lys 9-Ar 10 Table 29. C γ-Cγ distances (Å) between putative pharmacophoric residues. a 140 b Pharmacophore for the sst 2, sst 3, sst 5 selective SRIF analogues. Average distance and standard deviation calculated from the ensemble of ten structures. γ γ
29 Z 31
J. Med. Chem. 2005 48 . J. Pept. Sci. 2006 12 Chapter 5. Results and Discussion
29 Z β 29 Z
30 trans 31 29 Z 31
31 trans Gauche
32 E 29 E 32 Z 29 Z
Chapter 6. Experimental Part
EXPERIMENTAL PART
Chapter 6. Experimental Part
6.1 Instruments and methods
− −
t t
Chapter 6. Experimental Part
Chapter 6. Experimental Part
6.2 Synthesis
6.2.1 Synthesis of linear peptides
6.2.2 Synthesis of unsaturated cyclic peptides
J. Org. Chem. 1998 63 J. Org. Chem. 1998 63 Chapter 6. Experimental Part
6.2.3 Microwaves-assisted RCM
6.2.3.1 Six-mer-ring peptides
Chapter 6. Experimental Part
Temperature Power Pressure Method (°C) (W) (Psi)
Standard Fixed Variable Variable
Dynamic Variable Variable Variable
Power Fixed Fixed Variable Cycling Fixed Variable Fixed Variable Power Variable in a SPS Fixed Variable range
Table 30. MW-methods available in a CEM Discover MW instrument. 6.2.3.2 Eight-mer-ring peptides Chapter 6. Experimental Part
≈
6.2.4 Synthesis of saturated cyclic peptides
Chapter 6. Experimental Part
6.2.5 Synthesis of radiolabeled unsaturated peptides
6.2.5.1 Synthesis of DOTA conjugated peptides
6.2.5.2 Synthesis of PN 2S peptide conjugates
Chapter 6. Experimental Part
6.3 NMR Studies
6.3.1 NMR spectroscopy calculations
β
J. Am. Chem. Soc. 1982 104 Biochem. Biophys. Res. Commun. 1983 113 J. Magn. Reson. 1983 53 J. Chem. Phys. 1979 71 J. Magn. Reson. 1982 48 J. Biomol. NMR. 1995 6 J. Magn. Res. 1987 72 Chapter 6. Experimental Part
6.3.2 Structural determination and computational modeling
Proc. Natl. Acad. Sci. 1988 85 Chapter 6. Experimental Part
6.4 Molecular Modeling
6.4.1 Peptide virtual library construction
6.4.2 PPS algorithm
T1
R' R' R' 110° C O RO 120° RN 120° T R" N 1.8 A 1.8 A 1.8 A 1.8 A T2 T T R Chapter 6. Experimental Part
T O H
2.8 A
6.4.3 Pharmacophore model for SRIF receptors
Chapter 6. Experimental Part
6.5 Biological Part
6.5.1 In serum stability
6.5.2 Binding essays
µ
Eur. J. Nucl. Med. 2000 27 Chapter 6. Experimental Part
6.5.3 Radiolabeling procedure
∼
∼
Abbreviations
BFCA Boc Tert BSA c-AMP COSY CVFF DCM dhDsa-C dhDsa-N DMF DMSO DMSO-d6 DNA DOTA N,N’,N’’,N’’’ DQF-COSY Dsa-C Dsa-N DTCM DTPA EDT ESI Fmoc GEP GH GPCR Hag HATU I-Amp IGF MD MS NHC NMM NMR
Abbreviations NOE NOESY PE COSY PET PRL QSAR RCM
Rf RP-HPLC SDS SPE SPPS SRIF SRIF-28 Sst TOCSY Trt TSP UPLC VIP
Supplementary Information
SUPPLEMENTARY INFORMATION
Supplementary Information
Figure-S1. Peptide 27 .
Figure-S2. Peptide 28 .
Supplementary Information
O
O O (S)
NH HN (R) (R) NH H2N HN H
O NH
O (S)
H O H N (S) NH (S) HO (R) N H (R) O
HO CH3 NH2
O
Figure-S3. Peptides 29 ( E and Z).
Figure-S4. Peptide 30 .
Supplementary Information
Figure-S5. Peptide 31 .
Figure-S6. Peptides 32 (E and Z). Supplementary Information
OH
O
O O (S)
NH HN (R) (S) (R) NH H2N HN H
O NH
(Z) O (S)
H O H N (S) NH (S) HO (S) N H
(R) O
HO CH3 NH2
Figure-S7. Peptide 33 .
O
O O (S) NH HN (R) (R) (S) NH H2N HN H
O NH
O (S)
H O H N (S) NH (S) HO (R) N H (R) O
HO CH3 NH2
O
H3C
Figure-S8. Peptide 34 .
Supplementary Information
Figure-S9. Peptide 36 .
Figure-S10. Peptide 37 .
Supplementary Information
Figure-S11. Peptide 38.
Figure-S12. Peptide 39 .
Supplementary Information
Figure-S13. Peptide 40 .
Figure-S14. Peptide 41 .
Supplementary Information
Figure-S15. Peptide 42 .
Figure-S16. Peptide 43 .
Supplementary Information
Figure-S17. Peptide 45 .
Figure-S18. Peptide 46 .
Supplementary Information
Figure-S19. Peptide 47 .
Figure-S20. Peptide 48. Supplementary Information
Figure-S21. Peptide 49 .
Figure-S22. Peptide 50 . Supplementary Information
(S) (R) (R) (S)
(Z) (S)
(S) (S) (R)
(R)
Figure-S23. Peptide 51 .
Figure-S24. Peptide 52. Supplementary Information
(S)
(R) (S) (R) (R) (Z) (S) (S) (S) (R) (R) (R)
Figure-S 25. Peptide 53 .
Figure-S 26. Peptide 54 .
Aknowledgements
Peptide Synthesis
Laboratory of Peptides & Proteins, Chemistry & Biology, Department of Organic Chemistry, Prof. Mauro Ginanneschi University of Firenze Prof. Anna Maria Papini
Conformational Analysis
Department of Pharmaceutical Chemistry and Toxicology, University Federico II, Prof. Alfonso Carotenuto Napoli
Binding Essays
Institute of Pathology, Division of Cell Biology and Experimental
Cancer Research.,
University of Bern, Bern, Switzerland Prof. Jean Claude Reubi
99m Tc Radiolabeling
Department of Pharmaceutical Sciences, Prof. Ulderico Mazzi University of Padova Dr. Elena Zangoni
111 In Radiolabeling
Regional Center of Nuclear Medicine, Prof. Giuliano Mariani Dr.Paola Erba University of Pisa Dr. Chiara Manfredi
Molecular Modeling
Prof. Fabrizio Melani