Supporting Information
© Wiley-VCH 2005
69451 Weinheim, Germany
Multivalent Peptide and Protein Dendrimers using Native
Chemical Ligation
Ingrid van Baal, Hinke Malda, Silvia A. Synowsky, Joost L.J. van
Dongen, Tilman M. Hackeng, Maarten Merkx, and E.W. Meijer
1
General methods.
Unless stated otherwise, all solvents (p.a. quality) and other chemicals were obtained
from commercial sources and used as received. Water was demineralized prior to use.
Dichloromethane was obtained by distillation from P2O5. Amine-terminated poly
(propylene imine) dendrimers were kindly provided by DSM (Geleen, The Netherlands)
and dried prior to use. Trityl-protected cysteine (Trt-Cys(Trt)-OH) was obtained from
Bachem (Bubendorf, Switserland). Standard 1H NMR and 13C NMR experiments were
performed on a Varian Gemini-2000 300 MHz spectrometer, a Varian Mercury Vx 400
MHz spectrometer, and a Varian Unity Inova 500 MHz spectrometer at 298K. Chemical
shifts are reported in parts per million relative to tetramethylsilane (TMS).
Reversed phase high pressure liquid chromatography (RP HPLC) was performed on a
Varian Pro Star HPLC system coupled to a UV-Vis detector probing at 214 nm using a
VydacTM protein & peptide C18 column. MALDI-TOF spectra were obtained on a
Perspective Biosystems Voyager DE-Pro mass spectrometer using α-cyano-4hydroxycinnamic acid as matrix. ESI-MS spectra were measured on either an Applied
Biosystems ESI Mass Spectrometer API-150EX, a Micromass Q-TOF Ultima Global
Mass Spectrometer, or a Thermo Finnigan LCQ Deca XP MAX, all in positive mode.
Native mass spectrometry was performed in positive mode using a Micromass LCT. The
protein was injected onto a Superdex 200 H/R size-exclusion column (3.2 mm x 300 mm;
Amersham Biosciences) using a mobile phase of 50 mM ammonium acetate, pH 6.7 at a
flow rate of 50 µl/min. The post-column eluent was guided into the electrospray source
using a fused silica emitter. The electrospray source was optimized for transmission of 1(GFP)x complexes. Mass determinations were performed under conditions of increased
pressure in the source and intermediate pressure regions in the mass spectrometer.[1]
Micromass MaxEnt 1 software was used for deconvolution.
2
Succimide activation of trityl-protected cysteine (Trt-Cys(Trt)-OSuc)
Trt-Cys(Trt)-OH (1 eqv., 4.1 mmol, 2.5 g) was dissolved in 20 mL acetonitrile while
pyridine (1.1 eq., 4.5 mmol, 0.37 mL) and disuccinimidyl carbonate (2 eq., 8.2 mmol, 2.1
g) were added. The mixture was stirred overnight and acetonitrile was evaporated using
reduced pressure. The crude product was dissolved in ethylacetate and filtered to remove
the precipitate (pyridine salt). Subsequently, the clear solution was washed with a sodium
bicarbonate solution. The organic layer was dried over magnesium sulfate and, after
filtration, the solvent was evaporated using reduced pressure. Column chromatography
(silica, gradient EtOAc:heptane 1:3 – 1:1) yielded a white fluffy solid in 77% yield as the
product.
1
H NMR (400MHz, CDCl3); δ (ppm); 7.12-7.52 (m, 30H, Trt), 3.72 (dt, 1H, CH), 2.67 (s,
4H, CH2, Suc), 2.6 (d, 1H, NH), 2.5 (dd, 1H, CH2), 2.4 (dd, 1H, CH2)
13
C NMR (CDCl3, 100 MHz); δ (ppm); 168.1, (2xCq, Suc), 167.7 (C=O), 144.8 (3xCq,
NHTrt), 144 (3xCq, NHTrt), 129.4 (3xCq, STrt), 129.3 (3xCq, STrt), 128 (3xCH, STrt),
127.7 (3xCH, NHTrt), 127.4 (3xCH, NHTrt), 126.9 (3xCH, STrt), 126.2 (3xCH, STrt),
126.1 (3xCH, NHTrt), 70.7 (Cq, NHTrt), 66.4 (Cq, STrt), 53.4 (CH), 36.6 (CH2), 25
(CH2 (Suc)).
Mass (ESI): calculated: 702.8, found: 725.1 (M+Na+) and 1426.9 (2M+Na+)
Functionalization of PPI dendrimers with Trt-Cys(Trt)-OSuc and deprotection to
yield dendrimers 1-3
PPI dendrimer (G1, G2, or G3) was dissolved in 5 mL dichloromethane and 5 (G1), 10
(G2) or 15 (G3) equivalents of triethylamine were added. 4, 8, or 16 equivalents,
respectively, of Trt-Cys(Trt)-OSuc were added and the reaction mixture was stirred for 2
hours at room temperature. Subsequently the reaction mixture was washed twice with a
potassium carbonate solution (pH 10) and once with a saturated solution of potassium
bisulfate solution (pH 0.2). The organic layer was dried over magnesium sulfate and after
filtration the solvent was evaporated using reduced pressure. The obtained compound was
kept at 0° C using ice while a mixture of TFA with 2.5% of Et3SiH and 2.5% of
demiwater was added to remove the trityl protective groups. After stirring for 1 hour,
demiwater was added and the solution was washed three times with diethyl ether. The
water layer was lyophilized to obtain the product.
Dendrimer 1:
1
H NMR (300MHz, CD3OD); δ (ppm); 4.01 (t, 4H, CH), 3.48 (q, 8H, CH2NH), 3.26-3.17
(m, 12H, CH2N), 3.09-2.94 (m, 8H, CH2S), 1.97 (m, 8H, NCH2CH2CH2NH), 1.81 (m,
4H, NCH2CH2CH2CH2N).Mass (ESI): calculated: 725.1 (oxidized), found: 724.4
Dendrimer 2:
Mass (ESI): calculated: 1590.4 (oxidized), found: 1589.7
Dendrimer 3:
Mass (ESI): calculated: 3321.1 (oxidized), found: 3321.1
3
Synthesis of MPAL-activated peptides
Standard tBoc-mediated solid phase peptide synthesis was used to make the peptides. For
the formation of the thioester, first a leucine was coupled to an MBHA (4-methylbenzhydrylamine) resin.[2] Subsequently, 1.1 mmol of trityl-mercaptopropionic acid (TrtMPA) was activated with HBTU in DMF, added to the resin and allowed to couple for 30
minutes. After washing the resin with DMF, the trityl group was removed by washing
with a mixture of TFA with 2.5% of Et3SiH and 2.5% of demiwater, followed by washing
with DMF. Then, the first amino acid of the desired peptide was activated with HBTU in
DMF, added to the resin and allowed to couple for 10 minutes, resulting in thioester
formation. The remaining peptide sequence was made using normal tBoc-mediated solid
phase peptide synthesis. After cleavage of the peptide from the resin with HF, the product
was further purified using RP HPLC.
Ac-LYRAG-MPAL
Mass (ESI): calculated: 821.0, found: 821.3
Ac-GRGDSGG-MPAL
Mass (ESI): calculated: 846.9, found: 847.3
Recombinant expression and purification of GFP thioester
Cloning of expression plasmid for GFP-intein-CBD fusion protein
The expression vector pBAD-GFPuv (Clontech) was used as the source of the GFP gene
in this study. GFPuv is a GFP variant that is optimized for high level expression in E.coli
and high fluorescent intensity when illuminated by UV light. It contains 3 amino acid
mutations relative to wt GFP: F99S, M153T, and V163A. Site-directed mutagenesis
(QuickChange site-directed mutagenis kit, Stratagene) was used to delete the NdeI
restiction site within the GFP gene using the primers 5’CCCGTTATCCGGATCATGAAACGGCATGAC-3’and 5’GTCATGCCGTTTCATGTGATCCGGATA ACGGG-3’. The mutant plasmid was
digested with NdeI and EcoR1, and the GFP fragment was ligated into the NdeI and
EcoR1 sites of pET28a yielding pET28GFP. Site-directed mutagenesis using primers
5’CGGAGCTCGAATTCATTTTTGTAGAGCTCATCC-‘3 and 5’CGATGAGCTCTACAAAAATGAATTCGAGCTCCG-3’ was used to delete the first
nucleotide of the TAA stop codon. The mutated plasmid was again digested with NdeI
and EcoR1 and the GFP fragment was inserted into the MCS of pTXB1 (New England
Biolabs) yielding pGFPX1. This cloning strategy inserts a stretch of 8 amino acids
(NEFLEGSS) between the original C-terminus of GFP and the intein cleavage site. DNA
sequencing using T7 promoter and intein specific reversed primers (New England
Biolabs) confirmed the correct in-frame fusion of GFP with the intein sequence.
Protein expression and purification
pGFPX1 was transformed into chemically competent E. coli BL21(DE3) (Novagen) and
plated on LB agar plates containing 100 mg/L ampicilin. Single colonies were used to
inoculate 2 ml LB medium containing 100 mg/L ampicilin. Cultures were incubated
overnight at 37°C and subsequently used to start 200 ml culture containing 100 mg/L
4
ampicilin. At OD600 = 0.5 the temperature was lowered to 15 °C and 0.3 mM IPTG was
added to induce expression of the target protein. Cells were collected after overnight
expression at 15 °C and 250 rpm by centrifugation, resuspended in BugBuster (Novagen)
lysis buffer that contained 1 µL/mL Benzonase and incubated for 20 minutes at 20 °C. A
clarified cell extract was obtained by centrifugation at 40000 × g for 45 minutes. The
supernatant was loaded onto a 10 mL chitin column (New England Biolabs) that was
equilibrated with 20 mM sodium phosphate, 0.5 M NaCl, 0.1 mM EDTA, pH 8.0
(column buffer). The column was washed with 10 volumes (100 ml) of column buffer to
remove non- and weak binding proteins. Subsequently, 3 volumes (30 ml) of cleavage
buffer (200 mM sodium phosphate, 0.5 M NaCl, 0.1 mM EDTA, 50 mM MESNA, pH
6.0) were flushed quickly through the column. After overnight incubation of the column
at 20 °C, the MESNA thioester of GFP was eluted from the column using 1 volume of
cleavage buffer. SDS-PAGE analyis of the eluted protein showed a single band at ~27
kDa. Electro Spray Ionization Mass Spectrometry (ESI-MS) showed a single protein peak
with a mass of 27705 Da that is consistent with the MESNA thioester of GFP (theoretical
mass: 27703.9 Da). This procedure typically yields 40 mg of pure GFP thioester from 1 L
of E. coli culture.
References
[1]
[2]
N. Tahalla, M. Pinkse, C. S. Maier, A. J. R. Heck, Rapid Commun. Mass Spectr.
2001, 15, 596.
T. M. Hackeng, J. H. Griffin, P. E. Dawson, Proc. Natl. Acad. Sci. USA 1999, 96,
10068.
5
Figure S1. ESI mass spectra (m/z and deconvoluted mass spectra) of cysteine dendrimers
1-3 after incubation with 1% (v/v) β-mercaptoethanol (reduced) and 1% (v/v) hydrogen
peroxide (oxidized).
6
Figure S2. ESI mass spectra (m/z and deconvoluted mass spectra) of various peptide
dendrimers.
7
H2N
NH2
O
H2N
H2N
HN
O
NH
HN
NH
SH HN
O
3-(LYRAG) 15
(calc.: 11746)
N
H
N
H
N
N
O
HN
HN
NH
O
NH
HN
HN
O
NH
O
NH
O
HN
N
H
NH 2
O
N
H
NH2
NH
O
NH
HN
O
HN
NH2
NH2
HN
O
OH
NH2
NH
NH
NH
O
O
O
HN
OH
HN
H2N
H
N
HN
OH
HN
O
NH
O
HN
N
H
O
NH
O
O
NH2
O
O
NH
HN
HN
H2N
H
N
HO
O
O
NH2
N
H
O
HO HN
HN
O
O
HN
NH
NH
OH
O
HS
O
SH
O
O
O
HN
NH
SH
O
O
SH
HN
N
H
H2N
NH
NH
NH
O
O
H
N
NH
HN
SH
O
O
O
N
N
N
NH
HN
OH
NH
H2N
N
N
NH2
HN
O
N
H
HS
O
O
O
HO
H
N
N
H
O
N
O
HN
O
O
H
N
SH
NH
NH
O
HS
N
H
HN
O
NH
HN
N
H
HN
N
N
O
OH
NH
H2N
11698
N
N
N
H
SH
O
O
O
H2N
O
NH2
NH
HN
O
HS
SH
H
N
O
H
N
O
O
O
N
H
O
HN
H
N
H2 N
NH2
HN
O
NH
HN
N
N
H
N
HO
HS
O
H
N
O
OH
HN
O
O
HN
SH
O
NH
NH
HS
O
HN
NH
N
H
O
NH
H2N
NH
O
O
HS
HN
O
NH
HN
O
NH
O
O
O
OH
H
N
N
H
O
NH2
O
HO
O
HN
O
HN
O
H
N
H2N
NH2
NH
NH
O
O
HN
NH
H
N
O
NH
NH
O
OH
O
O
HN
HO
O
HO
HN
NH
HN
NH
NH
HN
O
O
NH
O
NH2
HN
O
NH
HN
NH
O
H2N
NH
H2N
H2N
O
O
HN
HN
O
NH
NH2
HN
O
NH2
NH2
12262
3-(LYRAG) 16
(calc.: 12307)
5000
10000
15000
Mass (m/z)
Figure S3. MALDI-TOF spectrum obtained after ligation of 3rd generation cysteine
dendrimer 3 with LYRAG-MPAL
8
A
B
Mw (kDa) GFP
75
50
37
800
1000
1200
1400
m/z
25
27705
26000
27000
28000
29000
30000
Mass (Da)
Figure S4. Characterization of MESNA thioester of GFP. Panel A: SDS-PAGE (12%
v/v) of GFP obtained after overnight cleavage of GFP-intein-CBD fusion protein bound
to chitine column with 50 mM MESNA and subsequent elution. Panel B: ESI mass
spectrum (m/z and deconvoluted spectrum) of GFP thioester (calculated mass: 27703.9).
9