Solid phase peptide synthesis

1
Solid phase peptide synthesis Part
I Applications of Boc/Bzl strategy Gábor
Mezo Research Group of Peptide
Chemistry Hungarian Academy of Sciences Eötvös L.
University Budapest, Hungary
2
(No Transcript)
3
Hormones oxytocin vasopressin insulin
somatostatin GnRH etc.
Neuropeptides substance P cholecystokinin neurote
nsin
Immune peptides synthetic antigens vaccines diag
nostic tools immunostimulator peptides muramyl
dipeptide tuftsin derivatives
Antibiotics tachikinin gramicidine S
Applications of synthetic peptides
Transporter peptides penetratin
oligoarginine HIV-Tat protein
Toxins conotoxins spider toxins snake
toxins ionchanel blockers
Carriers templates miniproteins
Peptides for structural studies turn mimicking
cyclic peptides
Enzymes and enzyme inhibitors Ribonuclease A
4
(No Transcript)
5
Peptidek mint gyógyszerek ?

• A peptidekhez, fehérjékhez számos biológiai és
  élettani funkció kapcsolható.
• Ezért a '60-as évektol a jövo gyógyszereinek
  gondolták.
• Elonyök nagy specifitás, magas aktivitás,
  viszonylag kis dózis, kicsi toxicitás,
• kevés mellékhatás.
• Hátrány gyors lebomlás, magas költségek.

2000-ben a világ gyógyszeriparának kb. 265
milliárd USD bevételébol 28 milliárd USD a
peptidek és fehérjék bevételébol származott.
Évente 35-40 új vegyület kerül gyógyszerként
bevezetésre. Ezek között a peptidek száma egyre
növekszik.
6
(No Transcript)
7
(No Transcript)
8
(No Transcript)
9
(No Transcript)
10
(No Transcript)
11
(No Transcript)
12
(No Transcript)
13
(No Transcript)
14
(No Transcript)
15
(No Transcript)
16
The final cleavage results in peptides
with carboxamide (CONH2) group at the C-terminus.
17
(No Transcript)
18
(No Transcript)
19
(No Transcript)
20
(No Transcript)
21
(No Transcript)
22
(No Transcript)
23
(No Transcript)
24
(No Transcript)
25
(No Transcript)
26
(No Transcript)
27
Side chain functional group protecting group
name (abbreviation)
benzyloxymethyl (Bom) (p)

• Cleavage of Bom results in Bzl and CH2O
• Formaldehyde can react with nucleophiles
• H2CO eNH2-Q H2CN-Q (Schiff
  base)

H

• H2CO OH-R

hemiacetale acetale
Work up the peptide as soon as possible !
However, Bom must not be used in case of peptides
containing Cys at the N-terminus
MMcalc12
thiazolidine-4-charboxylic acid (thioproline)
Use Cys as scavanger under the cleavage
condition to catch the formaldehyde !
28
(No Transcript)
29
2,3,6-trimethyl- benzenesulfonyl
or mesitelenesulfonyl (Mts)
30
(No Transcript)
31
(No Transcript)
32
(No Transcript)
33
When might be double coupling necessary

• Incorporation of the 10-15th amino acids
• Attachment to Pro
• Coupling of amino acids containing a branch on
  b-C atom (Val, Ile, Thr)
• Attachment to these amino acids
• Coupling of Arg or attachment to Arg
• Attachment to e-amino group of Lys (synthesis of
  branched peptides).

• Influence on the efficacy of the coupling
• Solvent change DCM-DMF to NMP
  (N-methyl-pyrolidone)
• Coupling reagent change DCC/HOBt to BOP, HBTU
  or HATU
• Application of these expensive reagents is
  suggested for the third coupling.

If the nynhidrine test is still blue make
acetylation to block the unreacted amino groups
(acetic anhydride and DIEA in DMF).
34
(No Transcript)
35
(No Transcript)
36
Diketopiperazine formation
37
(No Transcript)
38
M Mcalc- 47.0
39
(No Transcript)
40
C
H
O
3

• Cresol is prefered in case of Glu

O
M Mcalc 90.05
O
N
H
N
H
O

• Dont use indole as scavanger for Trp in strong
  acids

M Mcalc 117.1
Indole dimerisation can occur also in case of
peptides containing Trp at the N-terminus
resulting in dimer peptide connected through
indole rings.

• Asp-Pro bond might be cleaved under acidic
  condition

Use dried materials and equipments !
41

• 2-mercaptopyridine (10 equiv.) was suggested to
  prevent Met(O)
• formation or Met(O) reduction under HF
  cleavage. However it
• decreases the acidity of HF, so some
  protecting groups (eg. Tos)
• cant be removed effectively. Add Met and
  DTT to eliminate
• Met(O) formation under HF cleavage.

• N-O acyl shift in case of Ser or Thr

This reaction can be reversed by either
neutralizing with NH4OH or relyophilisation from
5 NH4HCO3
42
Pull-push mechanism in the presence of
thioanisole
SiMe3-O3S-CF3
SiMe3

S
C
H
3

CF3SO3-
m-cresol
H2O (NH4F)

C
H
HO-SiMe3
3
CF3SO3H

HO
Thioanisole reversible scavanger Cresol
irreversible scavanger
Dont use reversible scavanger alone !
43
(No Transcript)
44
(No Transcript)
45
(No Transcript)
46
(No Transcript)
47
(No Transcript)
48
(No Transcript)