Transcript 1. dia - RĂŠnyi Institute

Chemical ligation procedures
in peptide and protein chemistry
Gábor Mező
Research Group of Peptide Chemistry
Hungarian Academy of Sciences
Eötvös L. University
Budapest, Hungary
ERASMUS Teaching Mobility Grant
Konstanz
30th October 2008
a) Stepwise synthesis:
Z-NH-CHR2-CO-X
+
NH2-CHR1-CO-OY(NH2)
- HX
Z-NH-CHR2-CO-NH-CHR1-CO-OY
Cleavage of amino protecting group
NH2-CHR2-CO-NH-CHR1-CO-OY
+ Z-NH-CHR3-CO-X
- HX
Z-NH-CHR3-CO-NH-CHR2-CO-NH-CHR1-CO-OY
cleavage
coupling repetition till the end of the peptide sequence
Z-NH-CHRn-CO----------------NH-CHR3-CO-NH-CHR2-CO-NH-CHR1-CO-OY
final cleavage step
NH2-CHRn-CO----------------NH-CHR3-CO-NH-CHR2-CO-NH-CHR1-CO-OH
b) Fragment condensation
Z-NH-CHR3-CO-NH-CHR2-CO-NH-CHR1-CO-OY
cleavage of the carboxyl
protecting group
cleavage of the amino
protecting group
Z-NH-CHR3-CO-NH-CHR2-CO-NH-CHR1-CO-OH
NH2-CHR3-CO-NH-CHR2-CO-NH-CHR1-CO-OY
+ coupling agents
- H2 O
Z-NH-CHR3-CO-NH-CHR2-CO-NH-CHR1-CO-NH-CHR3-CO-NH-CHR2-CO-NH-CHR1-CO-OY
Final cleavage step
NH2-CHR3-CO-NH-CHR2-CO-NH-CHR1-CO-NH-CHR3-CO-NH-CHR2-CO-NH-CHR1-CO-OH
Limitations of stepwise synthesis
Consider a 100 amino acid residue synthesis in one run
Coupling yield (%)
Final yield (%)
99.99
99.00
99.00
36.60
1.4
81mer
13.26
Low yield;
VERY complex crude product;
Purification problems.
AUFS (214nm)
98.00
90mer
64mer
1.2
1
0.8
0.6
0.4
0.2
0
20
22
24
26
28
30
32
34
36
Time (min)
Chromatograms from Hubert Gaertner and Matteo Villain
Solutions:
a) higher coupling efficiency
b) capping after each coupling step
c) preparation of several smaller segments, followed by their ligation
Chemical ligation
a set of techniques used for creating long peptide or protein chains
Fragment condensation
Synthesis of (semi) protected peptides
(Convergent solid phase peptide synthesis; CSPPS)






Chemical ligation
Synthesis of unprotected peptides
by conventional SPPS
 selective coupling in aqueous solution
special resins (e.g. 2-ClTrt)
 different types of bonds
solubility problems
 up to 200-300 amino acid residues
difficult purification
fragment condensation in organic solvents
amide bond formation
up to 100-150 amino acid residues
P. Lloyd-Williams et al.: Tetrahedron 49, 11065 (1993)
Chemical ligation methods
 Chemoselective ligation
 Prior thiol capture
 Native chemical ligation
 Expressed protein ligation
 Acyl initiated capture
 Staudinger ligation
other than amide bond
amide bond
Chemoselective ligation
Component B
bromo-acetamido
O
NH
NH2
hydrazide
NH
HN
H2NO
hydroxyl-amine
SH
O
aldehyde
NH2
N-term. cysteine
O
O
N
hydrazone
aldehyde
O
aldehyde
O
O
O
O
O
thioether
O
O
NO
oxime
O
S
NH
thiazolidine
O
O
S
NH
HN
C-term. cysteine
NH2
O
NH
Br
thioester
O
O
SH
bromo-acetamido
HN
NH2
S
NH
HN
O
Br
O
thiocarboxylic acid
HN
SH
NH
O
O
O
Resulting bond
HN
Component A
Efficient chemical ligation via thioether bond formation
S. Futaki et al.: Bioorg. Med. Chem. 5, 1883 (1997)
Synthesis of a four-helix-bundle protein: SPPS, Fmoc/tBu, TMSOBr cleavage
SH
Ad
Cys(Ad)-
CH3CO-CGG-ELEELLKKLKELLK-GGCY-NH2
Acm
BrCH2CO-GG-ELEELLKKLKELLK-GGCY-NH2
linker
Cys(Ad)-
-Cys(SH)
Br-
=
-Cys(Acm)
linker
helix
Cys(Ad)-
-Cys(Acm)
-Cys(Acm)
-Cys(Acm)
0.1M Tris (pH 8)
100 eq AgOTf/TFA
6M GnHCl
RT, 18h
0oC, 1.5h
( then DTT)
-Cys(SH) Br-
+
S
Cys(Ad)-
-Cys(Acm)
S
BrCys(Ad)-
S
100 eq AgOTf/TFA
0oC, 1.5h
( then DTT)
S
S
Br
Cys(Acm)
S
-Cys(Acm)
Cys(Ad)-
Cys(SH)-
-Cys(Acm)
S
-Cys(SH)
S
1. 1M TFMSA-thioanisole/TFA
2. AgOTf/TFA
(both on 0oC, 1.5h)
S
S
S
=
S
S
S
Cys(SH)-
S
S
-Cys(SH)
air oxidation
0.1M NH4HCO3
S S
cpeptide= 0.05mg/mL
20oC, 16h
S S
Decomposition of bromoacetylated compound was observed under acidic cleavage or
storage in the freezer. (Yield 18% according to this method)
Application of chloroacetylated version gave better results (Yield 30%).
More stable derivative. But it needs longer reaction time.
ClAc-Peptide + KI
IAc-Peptide
J.A. Wilce et al.: J. Biol. Chem. 276, 25997 (2001)
Synthesis of chloroacetylated oligotuftsin (OT20) carrier
and its ligation with Ab(4-10)Cys epitope peptide
M. Manea et al. (2008) Biopolymers, 90, 94-104.
Ac-[Thr(Bzl)-Lys(ClZ)-Pro-Lys(Fmoc)-Gly]4-MBHA
Fmoc-cleavage (2% piperidine-2%DBU in DMF)
Boc-synthesis
Ac-[Thr(Bzl)-Lys(ClZ)-Pro-Lys(H-Gly-Phe-Leu-Gly)-Gly]4-MBHA
Chloroacetylation ( 5equiv ClAc-OPcp)
Ac-[Thr(Bzl)-Lys(ClZ)-Pro-Lys(ClAc-Gly-Phe-Leu-Gly)-Gly]4-MBHA
HF-cleavage
(10 mL HF – 0.5g p-cresol – 0.5g p-thiocresol)
Ac-[Thr-Lys-Pro-Lys(ClAc-Gly-Phe-Leu-Gly)-Gly]4-NH2
H-Phe-Arg-His-Asp-Ser-Gly-Tyr-Cys-NH2
0.1M Tris buffer (pH 8) (HCl elimination)
Ac-[Thr-Lys-Pro-Lys(CH2CO-Gly-Phe-Leu-Gly)-Gly]4-NH2
S
H-Phe-Arg-His-Asp-Ser-Gly-Tyr-Cys-NH2
3h
10.2
22.8
Ab4-10C
monomer
23.5
0,6
3910.1
Ligation of Ab(4-10)Cys with OT20(ClAc) followed by HPLC and MS
21.2
0,4
25.4
Ab4-10C
dimer
14.9
A220 nm
0,5
2000
3000
4000
5000
m/z
4856.8
27.9
0,3
0,2
5
10
15
20
25
30
Time(min)
6749.9
3373.0
2000
3000
3000
4000
5000
6000
7000
4000
5000
6000
5801.8
2000
m/z
2000
3000
4000
5000
6000
7000
m/z
m/z
Ac-[TKPK(S-CH2CO-GFLG)G]4-NH2
1,0
3842.8
0,9
0,8
7696.5
H-FRHDSGYC-NH2
21.2
24h
0,5
15.0
0,6
10.2
A 220 nm
0,7
0,4
0,3
0,2
10
15
20
25
30
35
3000
900
4000
983.6
983.7
Time(min)
1100
1300
1500
1700
1900
2100
m/z
1100
5000
6000
7000
1964.8
5
1600
2100
2600
m/z
8000
m/z
Thioether bond formation by the aid of
a bifunctional coupling reagent
NH2
O
O
NH2
+
NH2
N O
O
KLH
N
MBS
N-(3-maleimido-benzoyloxy)succinimide
O
T. Kitagawa, et al.: J. Biochem.
79, 233 (1976)
O
PBS-DMF, 30 min, RT
Sephadex G25, 10mM PBS (pH 6)
H-9LKNleADPNRFRGKDL22C-NH2
NH2
H
O
O
NH2
N
O NH
O
NH2
S
O
O
H-9LKNleADPNRFRGKDL22C-NH2
PBS solution, pH 7.5
NH2
N
O NH
O
Synthesis of peptide dendrimers with oxime, hydrazone,
and thiazolidine linkages
J Shao, JP Tam
JACS 117, 3893 (1995)
O
O
NH2OCH2C- VA20
CHOCO
CHOCO Lys
Lys-Ala-OH
CHOCO Lys
CHOCO
VA20 -CCH2ON=C-C)4-TLCP
(OxmVA20)4-TLCP
O
O
NH2NHC(CH2)2C- VA20
O
1. NaOH
O
O
VA20 -C(CH2)2CNHN=C-C)4-TLCP
(HydVA20)4-TLCP
O
SH
NH2CH-C- VA20
O
VA20 -C-
OO
(HC-C)4-TLCP
O
VA20 = VMEYKARRKRAAIHVMLALA
model peptide with helical structure
S
N
O
-C)4-TLCP
(ThzVA20)4-TLCP
2. HCl
(MeO)2CHCO
(MeO)2CHCO Lys
Boc
Lys-Ala-OMe
(MeO)2CHCO Lys
(MeO)2CHCO
1. TFA
2. (MeO)2CH2COONa+BOP/DIEA
(dimethyl acetal form)
Boc
Lys
Lys-Ala-OMe
Boc Lys
Boc
Rate of (OxmVA20)4-TLCP formation
reaction conditions (22oC)
(37oC)
pH: 4.7
4.2
5.2
5.7*
50% of AcN
DMF
DMSO
52h 64h 38h 32h
(1.0) (0.8) (1.4) (1.6)
35h
(1.5)
18h
(2.9)
8h
(6.5)
23h (2.3)
4.5h (12)
Rate of (HydVA20)4-TLCP formation
reaction conditions (22oC)
pH: 4.7
5.2
44h
(0.9)
(37oC)
5.7*
50% of AcN
DMF
DMSO
40h 34h
(1.0) (1.2)
76h
(0.5)
16h
(2.5)
2h
(20)
26h (1.5)
1.5h (27)
Rate of (ThzVA20)4-TLCP formation
reaction conditions (22oC)
pH: 4.0
30h
(0.8)
(37oC)
4.5*
5.0
50% of AcN
24h 16h
(1.0) (1.5)
18h
(1.3)
8h (3)
DMF
5h
(4.8)
30h
(0.8)
2h (12)
in case of 2.5 equiv peptide to a coupling site (10 equiv to lysine dendrimer)
* This buffer was mixed with the organic solvents.
TFE
Influence of different chemical linkages and
epitope orientations on biological activity
W. Zeng et al.: Vaccine 19, 3843 (2001)
B-cell epitope: EHWSYGLRPG
(LH-RH)
T-cell epitope: GALNNRFQIKGVELKS
(HA2 of influenza virus hemagglutinin)
-NH-CH2-CO(CH2)4
T-B; amide
NH2
Tandem linear SPPS
-NH-CH2-CONH2
T-S-B; thioether
CH2
S-CH2-CO-
T-cell (Cys, N- or C-terminus) and B-cell (BrAc, N-terminus)
-NH-CH2-CONH2
(CH2)4
T-oxm-B; oxime
NH-COCH=NOCH2CO-
T-K-B; amide
NH2-CH2-COCH2
S-CH2-CO-
B-S-T; thioether
CH-CONOCH2-COB-oxm-T; oxime
T-cell (Ser at N-terminus or at eNH2 of Lys to aldehyde) and B-cell (aminooxyacetyl group at N-terminus)
-NH-CH2-CONH2
NH2-CH2-CO-
CH2
CH2
S
S
NH2-CH2-CO-
T-S-S-B; disulfide
CH2
T-cell (Cys(Pys) either at N- or C-terminus)
and B-cell (Cys at N-terminus)
S
CH2
NH2-CH2-CO-
O
O
H2N
H2N
OH
OH
B-S-S-T; disulfide
Immunogen
NaIO4
HN
O
NH2
OH
HN
O
S
O
T-B
T-K-B
T-S-B
B-S-T
T-oxm-B
B-oxm-T
T-S-S-B
B-S-S-T
Total yield (%)
45
45
47
43
26
23
21
20
Vaccine
Average anti-LHRH titres (log10)
2weeks
9 weeks
20 weeks
after the second dose
Pregnancy
T-B
5.28
4.72
4.26
0/5
T-K-B
5.26
4.96
4.66
0/5
T-S-B
5.14
4.90
4.47
0/5
B-S-T
5.08
4.52
4.16
1/5
T-oxm-B
4.32
4.16
3.86
0/5
B-oxm-T
4.74
4.36
4.14
1/5
T-S-S-B
3.66
3.24
3.22
5/5
B-S-S-T
3.74
3.36
3.33
4/5
Saline control
1
1
1
5/5
Disulfide bonds are less stable in vivo than thioether or oxime bonds
Stability study of disulfide bond
Z. Bánóczi et al.: Bioconjugate Chemistry 18, 130 (2007)
Hca-RQIKIWFQQNRRMKWKKC-NH2
S
penetratin
S
Ac-CSK(Flu)PIIGPDDAIDALSSDFTS-NH2
calpastatin (calpain enzyme inhibitor )
COOH
O
O
O
HO
HO
O
O
4-(7-hydroxycumaril) acetic acid
(Hca)
lex = 360 nm
lem= 480 nm
HO
O
OH
5(6)-carboxyfluoresceine
(Flu)
lex = 492 nm
lem= 517 nm
Flu
Hca
COS-7 cells after 4h incubation with conjugates: measurement by fluorescence microscopy
Highly enantioselective amide ligation by prior thiol capture
D.S. Kemp, D.R. Buckler: Tetrahedron Letters 32, 3013 (1991)
 First ligation procedure that results in amide bond between unprotected
peptide fragments.
 The incorporated template helps the intramolecular acyl transfer.
O
Cys(Scm)---
+
O
SH
NH3-CH-COCH2
O
S
S
Cys(Acm)---
Scm: methoxycarbonylsulphenyl
HCO2H-HOAc-DMF (1:8:1, v/v)
OMe
 Fmoc synthesis on S-S linked dibenzofurane linker
 Cleavage of the peptide with Bu3P
 Cleavage of the protecting groups with TFA
ScmCl
HFIP-water (4:1, v/v)
30 min
O
O
+
NH3-CH-COCH2
S
S
+
O
NH3-CH-COCH2
O
S
S
DMSO solution
+ AgNO3
+ DIEA
O
O
O-N acyltransfer
2h, RT
OH
NH2-CH-COCH2
S
S
O
NH-CH-COCH2
S
S
Et3P
dioxan-water
O
NH-CH-COCH2
HS
Yield of the ligation step is ca. 80%
Native chemical ligation
(thioester based orthogonal ligation)
P.E. Dawson et al.: Science 266, 776 (1994)
The amide bond is formed without the aid of a template (difference from the „prior thiol
capture“ procedure)
N-terminal fragment in thioester form, C-terminal fragment with Cys at its N-terminus
+
O
SR
NH3-CH-COCH2
-S
 C-terminal amino acid should not be
Val, Ile, Thr (branching on b-C atom)
 R good leaving group
e.g. red. Ellman`s reagent
5-thio-2-nitrobenzoic acid
or thiophenol
(thiol ester exchange)
in water at pH 7
Chemoselective reaction
(reversible)
Other Cys without H N
2
any protection
CH-COO
S
CH2
Spontaneous rearrangment
S,N-acyl transfer
(irreversible)
Available for Cys-containing peptides
O
or de novo peptides with an incorporated
NH-CH-COCys, that can be used for another ligation
CH2
step
HS
Native chemical ligation resulting in a methionine in the sequence
J.P. Tam and Q. Yu: Biopolymers 46, 319 (1998)
+
O
NH3-CH-CO(CH2)2
-S
SR
Hcy instead of Cys
Chemoselective reaction
(reversible)
H2N
O
S
Spontaneous rearrangment
S-N acyl transfer
(irreversible)
O
NH-CH-CO(CH2)2
HS
CH-CO(CH2)2
methyl p-nitrobenzensulphonate
Selective methylation
O
No other Cys should be in the sequence !
in water at pH 7
NH-CH-CO(CH2)2
CH3S
Met
Native chemical ligation with Asp or Glu
as C-terminal residues
M. Villain et al.: Eur. J. Org. Chem. 3267 (2003)
 After native chemical ligation 20-30% isomer (b- (Asp), g (Glu) peptide bond)
 Migration of thioester moiety
 The side chain of Asp or Glu must be protected
 Boc-chemistry is prefered for the synthesis of thioester
 Side chain protecting groups must be stable under acidic conditions
 Clevable by base or reduction (hydrogenation)
In case of Glu:
Cleavage of BrZ from Tyr
OFm alkylation can occur!
O
-CO-O
O
S
O-CO9-fluorenylmethyl ester (OFm)
20% piperidine-20% DMF-10% 2-mercaptoethanol
(pH 13) for 15 min, RT
(phenylsulphonyl)ethyl ester
0.1M Na2CO3-10% 2-mercaptoethanol
(pH 9) for 2h, 37oC
Alkaline solutions can not be used in case of Asp(X) containing peptides because
of aspartimide formation:
H 2C
NH
CH
O
O
C
C
OX
C
CH2-SH
NH CH
O
C
H2C
- XOH
NH
CH2-SH
N
CH
CH C
O
C
O
O
-Asp-Cys-
-Asu-Cys-
+H2O
+H2O
O
H 2C
NH
CH
C
C
CH2-SH
O
OH
CH2-SH
NH CH
O
~30%
a-Asp-peptide
C
O
H2C
NH
C
N
CH C
O
CH
C
O
OH
~70%
b-Asp-peptide
In case of Asp:
CH3
-CO-O
O
Stability of esters under ligation conditions
phenacyl ester (OPac)
2,2,2-trichloroethyl ester (OTce)
(2“,4“-dimethoxy)phenacyl ester (OdiMePac)
1-methyl-2-oxo-2-phenyethyl ester (OMop)
1.2 h
1.5 h
3.8 h
9.9 h
1-methyl-2-oxo-2-phenylethyl ester
Zn/30% AcOH
reduction for 30 min, RT
Synthesis: Boc-strategy, on MPAL (b-mercaptopropionic acid-Leu) modified resin
in situ neutralization (sensitive thioester bond), HBTU activation.
Boc-Asp(OMop)-OH were activated by PyBOP and attached to SH-group.
HBTU: O-(Benzotriazol-1-yl)-N,N,N´N´-tetramethyluronium-hexafluorophosphat
PyBOP: (Benzotryazol-1-yloxy)-tripyrrolidinophosphonium-hexafluorophosphat
Ligation: 1 equiv N-terminal and 1.5 equiv C-terminal fragments dissolved
in 0.2 M phosphate buffer containing 6 M Gn.HCl (pH 7.5);
Add 1% thiophenol (catalyst) and 1% benzylmercaptan,
then adjust the pH to 6.5; ligation is ready in 1h at RT.
H-LYRAD(OMop)-SR + H-CSYRFL-OH
H-LYRAD(OMop)CSRFL-OH
H-LYRADCSRFL-OH
Experiments with Na-(1-phenyl-2-mercaptoethyl) auxiliary group
P. Botti et al.: Tetrahedron Letters 42, 1831 (2001)
The drawback of the mentioned native chemical ligation is that a Cys is needed in the sequence.
New procedure:
O
Br + HS
CH3
H3C
O
CH3
S
H3 C
SYRFL- R
O
HN
SYRFL- R
O
O
S
O CH3
CH3
S
BH3 *THF
H3C
O
R : PAM
HN
HF
SYRFL-OH
O
H3C
CH3
NH2-O-CH3
N
O
1-(4-methoxyphenyl)-2-(4´-methylbenzylthio)ethylamine
Br
CH3
S
DMF
H3C
O
NH2
H3C
O
DIEA
O
SH
Na-(1-(4-methoxyphenyl)-2-mercaptoethyl-peptide
PEPTIDE -COSR
HN
ligation
PEPTIDE –CO-N
O
SH
SYRFL-OH
O
SH
R
O
SYRFL-OH
CH 3
R
O
CH3
HF (R = H)
or
TFA (R = OMe)
PEPTIDE –CO-NH-GSYRFL-OH
PEPTIDE –COSR
Phe-Gly-Gly
Phe-Gly-Gly
TBRA 1-67 (His)
Mouse Larc 1-31 (Ala)
N-auxiliary
I
II
II
I
II
MCP1 1-35 (Lys)
I
reaction time
ligation yield
removal
16
16
16
16
40
16
40
16
40
>98
>98
87
81
92
73
85
69
76
HF
TFA
TFA
HF
HF
TFA
TFA
TFA-TMSBr
TFA-TMSBr
Ligation: 6M Gn.HCl in phosphate buffer (pH 7), 25oC
1.5 equiv thioester part, 1 equiv N-auxiliary part
Native Chemical Ligation at DBSB
size
synthetized
failed
41-70
45
1
71-100
37
5
101-130
24
5
131-164
9
1
unpublished data from Hubert Gaertner and Paolo Botti
Expressed protein ligation
Native chemical ligation: from unprotected linear peptides
Can be prepared by recombinant-DNA-based expression methods
Preparation of a C-terminal fragment with Cys at its N-terminus
Production of a thioester of the N-terminal fragment
1. Express in E. coli ;
2. Affinity purification
RECOMBINANT POLYPEPTIDE -CO-NH-Cys- INTEIN
CBD
Chitin
beads
CBD
Chitin
beads
HS
NH2-Cys- INTEIN
RECOMBINANT POLYPEPTIDE –CO- S
transthioesterification
HSR
RECOMBINANT POLYPEPTIDE –CO-SR
Acyl initiated capture
Similar to native chemical ligation, but not from a thioester derivative
R
SH
O
thioacid
R
Br
+
H2N
O
Br-Ala
alkaline
conditions
S
OH N
2
O
S-N acyl shift
HS
C SH
Thiol trityl resin
Substitution at the highly hindered
trityl thiol can be very difficult.
Long reaction time and highly activated
reagents may be required for acylation.
R
HN
O
O
Cys containing peptide
Bidirectional tandem pseudoproline ligation
Z. Miao, J.P. Tam: JACS 122, 4253 (2000)
Available for the synthesis of Pro rich peptides and proteins
H
O
O
O
HX
+
H2N
HX
H
R
O
R
X
N
O
O
X = S and R = H (S Pro)
X = O and R = H (O Pro)
X = O and R = CH3 (O ProMe)
O
O
O
X = S and R = H (Cys)
X = O and R = H (Ser)
X = O and R = CH3 (Thr)
HO
N
R
O,N-acyl
transfer
Ring chain
tautomerization
H X
O
R
N
H
O
O
Thiaproline formation runs in aqueous solution
Oxaproline formation runs in anhydrous pyridine- AcOH
H
O
Ser/Thr-
Cys-
+
O
O
1.
2.
Ser/ThrO
pH 5.3, 10 h, RT thiazolidine formation
pH 6.6, 20 h, RT O,N-acyl shift
Yield 86%
-S ProH
O
Yield 78%
Pyr/AcOH (1:1, v/v), 35 h, RT
O
-O Pro-
-S Pro-
Peptide fragments: Cys-Phe-Lys-Ile (C-terminal fragment)
Ser-Leu-Ile-Leu-Asn-Gly (middle fragment)
Asp-Ser-Phe-Gly (N-terminal fragment)
Final product:
Synthesis on normal (PAM) resin
Synthesis on cyclic acetal resin
Asp-Ser-Phe-Gly-O Pro-Leu-Ile-Leu-Asn-Gly-S Pro-Phe-Lys-Ile
Synthesis of Bac 7 antimicrobial peptide (59 amino acids)
from fragments 1-24, 25-38, 39-59 by the same procedure
resulted in peptide(s) that has a little change in the secondary
structure (determined by CD) and biological activity.
HO
O
O
Staudinger ligation
H. Staudinger, J. Meyer: Helv. Chim. Acta 2, 635 (1919)
P:
+ N=N-N
triphenylphosphane
+ -
azide
P
N
P+ N-
iminophosphorane
aza-ylide
- N2
P =N-N=N
N
P
N
phosphazide
N
Reactions of aza-ylide
H2O
H2N-R´
-R3PO
O
R´´
R
R-P-N
+ R
R´
-R3PO
R´´
H
R´´
O
NH-R´´´
-R3PO
R´´-N=C=O
-R3PO
R´´
N R´
H
Less reactive carbonyl electrophiles
(e.g. amides, esters) can undergo
this reaction, especially if the
electrophilic attack proceeds in
an intramolecular fashion
N R´
NH-R´´´
R´´-N=C=N-R´
(phosphane oxide derivative)
Advantages of Staudinger ligation:
 The azide moiety is absent in almost all naturally occurring compounds („bioorthogonal“)
 Despite their high intrinsic reactivity, azides undergo a selective ligation with a very
limited number of reaction partners
 The azide group is small and can be introduced into biological samples without altering the
molecular size significantly
M. Köhn and R. Breinbauer: Bioorganic Chemistry 43, 3106 (2004) minireview
Staudinger ligation as peptide ligation procedure
B.L. Nielsen et al.: Org. Lett. 3, 9 (2001)
O
H3C
N
H
HS-CH2-PPh2
DCC/HOBt
OH
S
O
O
N3
R
+
+
PPh2
PPh2
N
H
O
amidophosphonium salt
-
HS
O
H
N
O
R
N
H
Ph
Other than Gly
Much lower efficiency
Ph
N-peptide2
peptide1
O
Ph
N
H
H3C
- N-peptide2
H2O
PPh2
THF/H2O (3:1)
RT, 12 h
90-99%
O
iminophosphorane
-S
S
O
PPh2
N3-peptide2 - N2
S
N
H
phosphinothioester
O
peptide1
H3C
in DMF, RT
95%
N-acetyl-glycine
peptide1
O
P=O
Ph
NH-peptide2
peptide1
O
Presence of Lys
(eNH2-group)
might cause
problems
Preparation of azido derivatives from amino acids
by diazo transfer
J. Zaloom and D. Roberts: J. Org. Chem. 46, 5173 (1981)
O
H2N
CH
C
X
R
+ CF3SO2N=N=N
trifluormethanesulfonyl azide
(triflyl azide)
Explosive!
X = OH, OR´, NHR´
- +
N=N=N
O
CH
C
X
+
CF3SO2-NH2
triflamide
R
Azido-amino acid derivative
 Amino acid is dissolved in 1N NaOH (pH 9.5)
 Add 1 equiv triflyl azide dissolved in DCM (~ 1M solution)
 Stirring overnight (keep the pH between 9-9.5)
 Phase separation
 Add 1 equiv of 1M aqueous HgOAc to the aqueous phase
 Filter off the mercuric salt of triflamide
 The pH is adjusted to 3.5 with 2N H2SO4 then evaporation
 The product can be solidified by adding DCHA (salt formation)
Leu
Phe
Glu
Met
Trp
Asn
Gly-Phe
Tyr-Gly
Leu-OEt
Ala-OEt
Phe-OEt
67%
23%
24%
31%
24%
19%
37%
33%
40%
62%
44%
Azido-amino acids are stable under alkaline conditions but sensitive in aqueous acidic
conditions especially under pH 2. Azido-peptides can undergo racemization in alkaline
solutions (e.g. Ala-Phe resulted in 30 % epimerization in 1N NaOH at RT for 5 min).
Synthesis of RNAse A, a 124 amino acids containing protein
B.L. Nielsen et al.: JACS 125, 5268 (2003)
SPPS
RNAse A
(110-111)
cleavage
RNAse A
(110-111)
Kenner-type safety catch resin
SPPS
N3
S-CH2-PPh2
RNAse A
(110-124)
RNAse A
(112-124)
cleavage
PEGA resin
mRNA translation
RNAse A
(1-109)
S-R
+
RNAse A
(110-124)
Cys
61%
RNAse A
(1-124)
RNAse A
(110-111)
= Fmoc-Cys(Trt)-Glu(OtBu)-
RNAse A = N CH CO-Asn(Trt)-Pro-Tyr(tBu)-Val-Pro-Val-His(Trt)-Phe-Asp(OtBu)-Ala-Ser(tBu)-Val3
2
(112-124)
RNAse A
(110-124)
= Fmoc-Cys(Trt)-Glu(OtBu)-Gly-Asn(Trt)-Pro-Tyr(tBu)-Val-Pro-Val-His(Trt)-Phe-Asp(OtBu)-Ala-Ser(tBu)-Val-
Kinetically controlled ligation for the
convergent chemical synthesis of proteins
D. Bang et al.: Angew. Chem. Int. Ed. 45, 1 (2006)
Based on the different reactivity of thioesters and on thiazolidine form of
N-terminal Cys as a protection.
S
N
H
O
+
S
peptide1
HS
O
H2N
peptide2
S
R
Native chemical ligation
S
N
H
O HS
O
HN
peptide1
peptide2
S
R
Thiophenol catalyst
0.2M HCl.NH2OCH3
S
methoxyamine hydrochloride
2h, RT
HS
H2N
N
H
O HS
peptide1
O HS
HN
peptide1
O
peptide2
S
R
HN
O
peptide2
S
The synthesis can be further carried out
either at N- or C-terminus, either with
(Thz)-peptide thio-phenylester or Cyspeptide (thioester).