Transcript Bin Poster1(alkylation) - Indiana University Bloomington

A Novel Approach to Resin-based Cysteine Alkylation
Bin Yang and Richard DiMarchi
Department of Chemistry, Indiana University, Bloomington, Indiana, 47405 U.S.A.
Abstract
A novel method in the synthesis of S-alkylated cysteine peptides is described. The S-alkylation reaction was achieved “on resin” with unprotected cysteine containing peptides, in aqueous methanol/DMF buffered in Tris base (pH, 8.5). Alkylating
agents such such as bromoacetic acid, iodoacetic acid, 3-iodopropionic acid and 3-maleimidopropionic acid(MPPA), were used in a 2-5 molar excess to selectively S-alkylate cysteine peptides in yields that range between 50-90%. A model peptide
(GCSWARKHT) and a specific glucagon analog that each contained a thiol group and a set of additional nucleophilic functional groups (amino, hydroxy, indole, imidazolyl and guanidine) were utilized as alkylation substrates. Other aklylating agents that
introduce chemical substitutions of known biological importance, such as farnesyl bromide were also investigated to further test the generality of this methodology.
Introduction
Cysteine alkylation is a powerful and versatile chemical approach in peptide
and protein structure-function analysis. Modification with structurally diverse
alkylating agents has been successfully employed in protein identification,
assessment of protein folding, and localization of ligand binding sites.
S-alkylation of cysteine also provides a rapid means to optimize a specific
side chain structure starting from a single peptide. This minimizes the
requirement for total synthesis of individual peptides with a unique unnatural
amino acid. With pH control the thiol group (RSH) can be selectively alkylated
in the presence of other nucleophilic natural amino acid aide chains, such as
amino and hydroxy. Here we report a novel “on-resin” cysteine alkylation
method for unprotected peptides in aqueous methanol/DMF,
buffered with Tris base.
Table 1.
“On-resin” Alkylation Condition and Yields with Various Agents and Model Peptides
Rxn
Alkylating
agents
Template peptide
Rxn 1
Rxn 2
Rxn 7
Rxn 9
Molecular Weight
Alkylating solvent
and time
Alkylating
yeild (%)
90
3527.8
3528.0
90
3390.7
3391.4
Calculated/ESI-MS
1.
[Cys9]Glucagon
BrCH2COOH
MeOH/H2O/Tris(pH8.5), 4h
2.
[desHis1, Cys9]Glucagon
BrCH2COOH
MeOH/H2O/Tris(pH8.5), 4h
3.
[Cys9]Glucagon
ICH2CH2COOH
MeOH/H2O/Tris(pH8.5), 4h
55
3541.8
3542.0
4.
[desHis1, Cys9]Glucagon
ICH2CH2COOH
MeOH/H2O/Tris(pH8.5), 4h
65
3404.7
3404.0
5.
[desHis1, Cys9]Glucagon
ICH2CH2CH2COOH
MeOH/H2O/Tris(pH8.5), 16h
0
3555.8
3332.8
MeOH/H2O/Tris(pH8.5), 4h
90
3502.7
3500.0
80
4181.6
4180.3
75
4044.5
4043.2
O
6.
[desHis1, Cys9]Glucagon
N
COOH
O
Chemistry
Reaction Scheme 1 describes the general details of this on-resin peptide
cysteine alkylation procedure. Peptides were typically assembled on
4-methylbenzhyrylamine resin (MBHA) resin using traditional Fmoc/tBu
chemistry. All the side chain protection groups of the peptides were removed
by anhydrous TFA treatment to yield an unprotected peptidyl-resin. The
unprotected peptide still covalently attached to the resin was alkylated in
50% methanol or DMF aqueous that was buffered with Tris at pH 8.5, with 2-5
equiv excess of alkylating agent to susceptible thiol group. Each alkylated
peptide was cleaved from the resin with anhydrous HF and the extent and
selectivity in cysteine modification was assessed by MS and HPLC.
7.
[Cys9]Glucagon
m-dPEG12TM-MAL
MeOH/H2O/Tris(pH8.5), 4h
8.
[desHis1, Cys9]Glucagon
m-dPEG12TM-MAL
MeOH/H2O/Tris(pH8.5), 4h
9.
[Cys0, Ahx1, Glu9]Glucagon
m-dPEG12TM-MAL
MeOH/H2O/Tris(pH8.5), 4h
85
4285.6
4285.0
10.
[Cys0, Ahx1, Glu9]Glucagon
Br
DMF/H2O/Tris(pH8.5),16h
55
3780.1
3780.0
11.
[Cys0, Ahx1, Glu9]Glucagon
Br
DMF/H2O/Tris(pH8.5), 4h
90
3776.0
3774.0
5757.5
55
1102.1
1102.0
O
12.
GCSWARKHT
BrCH2COOH
13.
GCSWARKHT
ICH2CH2COOH
14.
15.
Br
GCSWARKHT
Br
GCSWARKHT
O
SH
NH 2
TFA
Fmoc/tBu chemistry
O
N
H
SPPS
MBHA
H
N
O
O
RX
N
H
MeOH /DMF / H 2 O / Tris (pH 8.5)
S
S
O
N
H
Glucagon
[Cys 0 , A hx 1 , G lu 9 ]G lucagon
R
R
H
N
O
O
HF cleavage
N
H
O
N
H
H
N
m-dPEG 12T M -MA L
O
S
NH 2
S
MeOH/H2O/Tris(pH8.5), 4h
MeOH/H2O/Tris(pH8.5), 4h
40
1116.1
1116.0
DMF/H2O/Tris(pH8.5),16h
50
1248.1
1248.0
DMF/H2O/Tris(pH8.5), 4h
85
1245.1
1244.8
H SQ GTFTSDY SKYLD SR R AQDFVQW LM NT
SH
H
N
H2N
SQ GTFTSEYSKYLD SRR AQDF VQW LM N T
O
O
O
H
N
N
O O
O
O
O
O
O
O
O
O
O O
O
O
O
Fig 1. RP-HPLC analysis of representative alkylated peptides
Rxn 1
Rxn 7
Rxn 3
Rxn 9
Rxn 2
Rxn 10
Scheme 1 Synthesis of cysteine S-alkylated peptide by on-resin alkylation
Result and Discussions
• BrCH2COOH & ICH2CH2COOH & ICH2CH2CH2COOH BrCH2COOH was highly
effective in alkylation of glucagon analogs (rxn 1, 2) and the model peptide
(rxn 12). Alkylation with ICH2CH2COOH (rxn 3, 4, 13) was slightly less reactive
than BrCH 2 COOH but still provided high yield and selectivity.
ICH2CH2CH2COOH was not reactive in this on-resin reaction (rxn 5).
• PEG-MAL Polyethylene glycol (PEG) maleimidopropionyimide was highly
reactive and selective using similar reaction conditions to the previously
described halo-acids (rxns 7-9).
• Farnesyl Bromide Modification of cysteine by addition of a farnesyl motif is
involved in many membrane signal transduction proteins, so alkylation of
cysteine by farnesyl bromide is a very desirable modification. The alkylation
with the farnesyl bromide was slow and thus required additional time (16hr)
but eventually yielded ~ 50% of the desired products (rxn 10, 14).
• 2-Bromo-1-(5-phenyl-2-thienyl)-ethanone is a selective and strong alkylating
agent. We observed it to alkylate the thiol to high degree (rxn 15) and resulted in a
dehydration product when alkylating an N-terminal cysteine thiol (rxn 11). The
dehydrated S-alkylation product provided additional evidence that the alkylation
reaction was selective to the thiol group. Our results demonstrate that 2-bromo-1(5-phenyl-2-thienyl)-ethanone can function as a new and highly effective cysteine
S-alkylating agent.
S
O
S
S
H N
2
H
N
O
-H O
2
O
N
H
H
N
N
S
O
S
O
N
H
-H O
2
H
N
N
S
O
HF
M -1 8
M -1 8
Scheme 2. Mechanism of formation of dehydration product when
2-bromo-1-(5-phenyl-2-thienyl)-ethanone to alkylate the N-terminal cysteine
Rxn 11
Fig 2. ESI-MS analysis of representative cysteine S-alkylated peptides after HF cleavage
Acknowledgements
O
NH
Rxn 6
2
We would like to thank David L. Smiley, Jay L. Levy and Jonathan A. Karty for their kind help in
peptide synthesis and mass spectrometry analysis.
References
1. Or, Y. S., Clark, R. F. and Luly J. R. J. Org. Chem. 56, 3146-3149(1991).
2. Perrey, D. A. and Uckun, F. M. Tetrahedron letters, 42, 1859-1861(2001).
3. Yang, C. C., Marlowe, C. K. and Kania, R. J. Am. Chem. Soc. 113, 3176-3177(1991).