Synthesis of peptide-protein conjugates using N-succinimidyl carbamate chemistry

Article abstract of DOI:10.1021/bc900154r

Peptide-protein conjugates are useful tools in different fields of research as, for instance, the development of vaccines and drugs or for studying biological mechanisms, to cite only few applications. N-Succinimidyl carbamate (NSC) chemistry has been scarcely used in this area. We show that unprotected peptides, featuring one lysine residue within their sequences, can be converted in good yield into NSC derivatives by reaction with disuccinimidylcarbonate (DSC). No hydrolysis of the NSC group was observed during RP-HPLC purification, lyophilization, or storage. NSC peptides reacted efficiently within minutes with lysozyme used as model protein. To illustrate usefulness of the method consisting of the synthesis of a peptide-protein conjugate of biological interest, a NSC peptide derived from a peptide substrate for tyrosylprotein sulfotransferase (TS) was synthesized and ligated to receptor-binding nontoxic B-subunit of Shiga toxin (STxB). Immunofluorescence studies showed the intracellular delivery of the TS-STxB conjugate and its ability to circulate to the Golgi as the native STxB protein. Moreover, we demonstrate that the TS label could be sulfated by tyrosylprotein sulfotransferases present in the Golgi. Thus, NSC chemistry permitted rapid synthesis of a peptide-protein conjugate worthwhile for studying the transport of proteins from the plasma membrane to the Golgi. The second part of this article describes a more general method for synthesizing peptide-protein conjugates without any limitation of the peptide sequence. The conjugates were assembled by combining NSC chemistry and α-oxo semicarbazone ligation. To this end, a glyoxylyl NSC peptide was synthesized and reacted with lysozyme. The glyoxylyl groups on the protein were then reacted with a semicarbazide peptide to produce the target peptide-protein conjugate. Both reactions, namely, urea bond formation and α-oxo semicarbazone ligation, were carried at pH 8.0 using a one-pot procedure.

Full text of DOI:10.1021/bc900154r

Bioconjugate Chem. 2010, 21, 219–228
219
Synthesis of Peptide-Protein Conjugates Using N-Succinimidyl Carbamate
Chemistry
Reda Mhidia,† Aure´lie Vallin,† Nathalie Ollivier,† Annick Blanpain,† Getao Shi,‡,§ Romain Christiano,‡,§
Ludger Johannes,‡ and Oleg Melnyk*,†
UMR CNRS 8161 Universite´ de Lille Nord de France, Institut Pasteur de Lille, IFR 142, 1 rue du Pr Calmette
59021 Lille Cedex, France, Institut Curie s Centre de Recherche, Trafic, Signaling and Delivery Laboratory, 26 rue d’Ulm,
75248 Paris Cedex 05, France, and UMR CNRS 144, France. Received April 8, 2009; Revised Manuscript Received December 10, 2009
Peptide-protein conjugates are useful tools in different fields of research as, for instance, the development of
vaccines and drugs or for studying biological mechanisms, to cite only few applications. N-Succinimidyl carbamate
(NSC) chemistry has been scarcely used in this area. We show that unprotected peptides, featuring one lysine
residue within their sequences, can be converted in good yield into NSC derivatives by reaction with
disuccinimidylcarbonate (DSC). No hydrolysis of the NSC group was observed during RP-HPLC purification,
lyophilization, or storage. NSC peptides reacted efficiently within minutes with lysozyme used as model protein.
To illustrate usefulness of the method consisting of the synthesis of a peptide-protein conjugate of biological
interest, a NSC peptide derived from a peptide substrate for tyrosylprotein sulfotransferase (TS) was synthesized
and ligated to receptor-binding nontoxic B-subunit of Shiga toxin (STxB). Immunofluorescence studies showed
the intracellular delivery of the TS-STxB conjugate and its ability to circulate to the Golgi as the native STxB
protein. Moreover, we demonstrate that the TS label could be sulfated by tyrosylprotein sulfotransferases present
in the Golgi. Thus, NSC chemistry permitted rapid synthesis of a peptide-protein conjugate worthwhile for
studying the transport of proteins from the plasma membrane to the Golgi. The second part of this article describes
a more general method for synthesizing peptide-protein conjugates without any limitation of the peptide sequence.
The conjugates were assembled by combining NSC chemistry and R-oxo semicarbazone ligation. To this end, a
glyoxylyl NSC peptide was synthesized and reacted with lysozyme. The glyoxylyl groups on the protein were
then reacted with a semicarbazide peptide to produce the target peptide-protein conjugate. Both reactions, namely,
urea bond formation and R-oxo semicarbazone ligation, were carried at pH 8.0 using a one-pot procedure.
acid N-succinimidyl ester (11). However, the sensitivity of Tat-
Cys-NHSE peptide toward hydrolysis precluded its purification
by RP-HPLC. Consequently, the crude Tat-Cys-NSCE peptide
was stored at -80 °C and directly used in the conjugation
experiments. A similar approach was reported by Inglese et al.
for the attachment of a protein kinase A substrate to proteins
(12).
N-Succinimidyl carbamate derivatives (NSC), whose chem-
istry is related to NSCE, have been scarcely used for the
synthesis of conjugates. Morpurgo et al. reported the synthesis
of an NSC derivative of biotin for protein labeling (13). Later
on, Clave´ et al. described the synthesis of a fully protected NSC
dipeptide scaffold (14). This reagent was coupled to the
N-terminal amino group of substance P. Pasut et al. described
the synthesis of a PEG-ꢀ-alanine NSC derivative for protein
pegylation (15). In these studies, besides the NSC group, no
other functional moiety was present on the molecules. To the
best of our knowledge, the synthesis of NSC peptides incor-
porating functional side chains, their purification, and coupling
to proteins was not reported in the literature.
We describe here the synthesis of NSC peptides incorporating
functional side chains (phenol group of tyrosine, hydroxyl group
of serine, imidazole group of histidine, carboxylic acid group
of glutamic acid, and/or indole group of tryptophan). These
peptide derivatives were successfully purified by RP-HPLC and
coupled to proteins. Lysozyme was used as a model protein to
optimize preparation of conjugates. Obviously, the partial
conversion of NSC group into an isocyanate group during the
conjugation experiment is a fact. To illustrate the value of the
method consisting of the synthesis of a peptide-protein
INTRODUCTION
Nowadays, various methods are available for the synthesis
of peptide-protein conjugates. Site-specific ligation methods,
by exploiting a unique functional group present within the
protein, lead to well-characterized conjugates. Reaction of a
cysteine residue with haloacetyl (1) or maleimidyl (2) peptides
is often used in this field. Alternatively, unnatural functional
groups helpful for site-specific ligation such as keto or aldehyde
groups can be introduced into protein-specific sites by using
chemical methods (3, 4), enzymatic techniques (5), or living
organisms (6).
Random peptide-protein conjugates are also useful tools in
different fields of research as, for example, the development of
vaccines (7) or drugs (8, 9) or for studying biological mecha-
nisms (1). Their preparation is often carried out using N-
succinimidyl carboxylic ester (NSCE)-based bifunctional re-
agents (10). Usually, the bifunctional reagent is reacted first
with the protein leading to formation of amide bonds between
surface-exposed ε-amino groups and NSCE moiety. Then, the
second functional group is used to attach peptide molecules (7).
The synthesis of peptide-protein conjugates by direct reaction
of NSCE peptides with proteins is less common. For instance,
Kida et al. have recently described the synthesis of a Tat-Cys-
NSCE peptide by reacting Tat-Cys with 6-maleimidohexanoic
* Corresponding author. Phone: 333 20 87 12 14, Fax: 333 20 87
12 35, E-mail: oleg.melnyk@ibl.fr.
† UMR CNRS 8161.
‡ Institut Curie s Centre de Recherche.
§ UMR CNRS 144.
10.1021/bc900154r  2010 American Chemical Society
Published on Web 01/07/2010

220 Bioconjugate Chem., Vol. 21, No. 2, 2010
Table 1. Yields for Peptides 1-4a,b and MS Data
Mhidia et al.
0.05% TFA by vol (6 mL/min, detection 215 nm, 0-40% B in
60 min). Fractions were collected and lyophilized to produce
1-4b (yields are indicated in Table 1).
The purity of peptides 1-4b was >95% as determined by
analytical RP-HPLC and CZE. MS data are in accordance with
the proposed structures (see Table 1 and Supporting Informa-
tion).
peptide
m/z calcd
m/z found
yield (%)
1a
2a
3a
4a
1b
2b
3b
4b
1601.7a
2183.1a
2339.2a
2495.3a
871.9b
1601.5a
2182.8a
2339.0a
2495.2a
872.2b
73
34
42
47
59
56
56
61
1162.6b
1240.6b
1318.7b
1163.1b
1241.2b
1319.5b
Synthesis of NSCE Peptide 1c. A solution of suberic acid
bis(N-succinimidyl ester) DSS (64 mg, 175 µmol) in dry DMF
(700 µL) at rt under nitrogen was added to a stirred solution of
peptide 1a (30 mg, 17.5 µmol) and triethylamine (2.4 µL, 17.5
µmol) in dry DMF (340 µL). The mixture was stirred at rt for
1 h. The crude mixture was then diluted with 20 mL of 1% by
vol aqueous TFA and purified by RP-HPLC on a 100 Å 5 µm
C18 Nucleosil column using a linear water/acetonitrile gradient
containing 0.05%TFA by vol (6 mL/min, detection 215 nm,
0-40% B in 60 min). Fractions were collected and lyophilized
to produce 15 mg of pure peptide 1c (46%). The purity of
peptide 1c was >95% as determined by analytical RP-HPLC.
MS data are in accordance with the proposed structure (see
Supporting Information).
a [M+H]+ MALDI-TOF analysis in a reflector mode using R-cyano-
b
4-hydroxycinnamic acid as matrix. [M+2H]2+ LC-MS analysis (source
temperature 120 °C, desolvation temperature 350 °C, cone 30 V, capillary
3000 V).
conjugate of biological interest, a NSC peptide derived from a
peptide substrate for tyrosylprotein sulfotransferase (TS) (10)
was synthesized and ligated to receptor-binding nontoxic
B-subunit of Shiga toxin (STxB). STxB binds to glycosphin-
golipid Gb3 in the plasma membrane of target cells. After
binding, STxB is transported to the endoplasmic reticulum, via
endosomes and the Golgi. Due to this property, STxB can
potentially be used for vectorization of drugs (16). The
colocalization of TS-STxB conjugate with a Golgi marker was
studied by confocal immunofluorescence microscopy. The
ability of the TS label within the TS-STxB conjugate to be
sulfated by Golgi tyrosylprotein sulfotransferase was also
examined.
In the second part of this article, we describe a more general
method for synthesizing peptide-protein conjugates, which is
not limited by the peptide sequence. The conjugates are
assembled using a one-pot procedure combining NSC chemistry
and R-oxo semicarbazone ligation. To this end, a glyoxylyl NSC
peptide was synthesized and reacted with lysozyme at pH 8.0
to produce glyoxylyl lysozyme. The crude reaction mixture was
then reacted with a semicarbazide peptide. Ligation between
the glyoxylyl groups on the protein and the semicarbazide group
on the peptide resulted in one-pot formation of the target
peptide-protein conjugate.
Labeling of Lysozyme, General Procedure. Lysozyme (Ap-
pligene ref 130172, 0.41 mg, 0.028 µmol, 0.2 mM final
concentration) in 10 mM pH ) 7.4 PBS buffer (140 µL) was
added to NSC peptide 1-4b (0.2 µmol). The mixture was stirred
at 4 °C for 1 h. Analysis of the reaction mixtures by RP-HPLC
and CZE showed that ∼50% of lysozyme molecules were
modified. MALDI-TOF analysis of the mixtures revealed the
presence of up to 4 peptide chains on lysozyme.
Labeling of STxB. STxB was produced and purified as
described elsewhere (17). STxB (1.11 mg, 0.14 µmol, 0.32 mM
final concentration) in pH ) 8.0 sodium phosphate buffer
(450 µL) was added to NSC peptide 1b (2.24 µmol). The
mixture was stirred at rt for 1 h. The conversion was ∼70% as
determined by LC-MS. MALDI-TOF analysis of the mixture
revealed grafting of up to 3 peptide chains on STxB. The
conjugate was purified by filtration to produce TS-STxB-a or
purified by RP-HPLC to produce TS-STxB-b.
Purification by Filtration, TS-STxB-a. 300 µL of the above
reaction mixture was filtered and washed with PBS (3 × 20
mL) on a 3 kD membrane (3000 MWCO PES Vivascience,
UK). The total volume after this step was 750 µL. The solution
concentration (0.92 µg/µL) was determined using the bicincho-
ninic BCA assay (18).
EXPERIMENTAL PROCEDURES
Solid-Phase Peptide Synthesis of Peptides 1-4a. Solid-
phase synthesis was performed using the Fmoc/tert-butyl
strategy on a Novasyn TGR resin (0.1 mmol scale) in a
microwave peptide synthesizer (CEM µ WAVES, Saclay,
France). Couplings were performed using 5-fold molar excess
of each Fmoc L- or D-amino acid, 4.5-fold molar excess of
HBTU, and 10-fold molar excess of DIEA. Final deprotection
and cleavage from the resin was carried out with TFA/thioanisol/
TIS/H2O 92.5/2.5/2.5/2.5 by vol. The resin beads were filtered,
and the filtrate was precipitated in diethyl ether/heptane 1/1 by
vol, dissolved in water, and lyophilized. The crude peptide was
purified by RP-HPLC on a 100 Å 5 µm C18 Nucleosil column
using a linear water/acetonitrile gradient containing 0.05%TFA
by vol (6 mL/min, detection 215 nm, 0-40% B in 60 min).
Fractions were collected and lyophilized to produce 1-4a
(yields are indicated in Table 1). The purity of peptides 1-4a
was >95% as determined by analytical RP-HPLC and CZE. MS
data are in accordance with the proposed structures (see Table
1 and Supporting Information).
Synthesis of NSC Peptides 1-4b. DSC (16.6 mg, 65 µmol)
was dissolved in dry DMF (340 µL) at rt under N2. Peptide
1-4a (6.5 µmol) and triethylamine (0.45 µL, 3.2 µmol) were
solubilized in dry DMF (85 µL) and then added in one portion
to DSC solution. The mixture was stirred at rt for 1 h. The
crude mixture was diluted with 20 mL of 1% by vol aqueous
TFA and purified by RP-HPLC on a 100 Å 5 µm C18 Nucleosil
column using a linear water/acetonitrile gradient containing
RP-HPLC Purification, TS-STxB-b. 100 µL of the above
reaction mixture was purified by RP-HPLC on a 300 Å 5 µm
C18 XBridge column (4.6 × 250 mm) using a linear water/
acetonitrile gradient (1 mL/min, eluent A is 0.05% TFA in water,
eluent B is 0.05% TFA in CH3CN/water 4/1 by vol, 0-100%
B in 30 min, detection at 215 nm). Fractions were collected
and lyophilized to produce TS-STxB-b. The conjugate was
dissolved in 100 µL of PBS. The solution concentration
(0.5 µg/µL) was determined using the bicinchoninic BCA assay
(18).
Synthesis of Peptide 5 and Glyoxylyl Peptide 6. Solid-phase
synthesis of peptide 5 H-SAAFGGK-NH2 was performed using
the Fmoc/tert-butyl strategy on a Novasyn TGR resin (0.1 mmol
scale) in a microwave peptide synthesizer (CEM µ WAVES,
Saclay, France). Couplings were performed using 5-fold molar
excess of each Fmoc L-amino acid, 4.5-fold molar excess of
HBTU, and 10-fold molar excess of DIEA. Final deprotection
and cleavage from the resin was carried out with TFA/TIS/
H2O 95/2.5/2.5 by vol. The resin beads were filtered and the
filtrate was precipitated in diethylether/heptane 1/1 by vol,
dissolved in water, and lyophilized to produce 71 mg of peptide
5 (82%). The purity of peptide 5 was >90% as determined by

Synthesis of Peptide-Protein Conjugates
Bioconjugate Chem., Vol. 21, No. 2, 2010 221
analytical RP-HPLC and CZE. MS data are in accordance with
the proposed structure (see Supporting Information).
glycerol, and 59% Tris-HCl 0.2 M pH 8.5). Cells were analyzed
by confocal microscopy (Leica Microsystems, Mannheim,
Germany).
A solution of peptide 5 (21.3 mg, 0.024 mmol) in 0.1 M pH
7.0 sodium phosphate buffer (6.4 mL) was reacted for 10 min
with sodium periodate (10.5 mg, 0.049 mmol) at rt. The reaction
mixture was quenched with ethanolamine (3.5 µL, 0.037 mmol)
and purified immediately by RP-HPLC on a 100 Å 5 µm C18
Nucleosil column using a linear water/acetonitrile gradient
containing 0.05% TFA by vol (6 mL/min, detection 215 nm,
0-40% B in 60 min). Fractions were collected and lyophilized
to produce peptide 6 (11 mg, 64%).
Sulfation Assay. Sulfation assay was performed as described
elsewhere (21) using STxB, TS-STxB-a (conjugate purified by
filtration), TS-STxB-b (conjugate purified by RP-HPLC), and
TS-STxB-c. TS-STxB-c is a recombinant STxB protein modified
at the C-terminus by two tandem sulfation sites. It is used as a
positive control; see ref 22. Proteins were incubated with
300 000 HeLa cells at 4 °C in sulfate-free Dulbecco’s Modified
Eagle Medium (DMEM, Invitrogen) for 30 min. Cells were
washed with sulfate-free DMEM. 250 µL of radioactive sulfate
(35SO42-, Perkin-Elmer) in sulfate-free DMEM (final activity
480 µCi/mL) were added to cells (120 µCi/300 000 cells). Cells
were incubated for 2 h at 37 °C and then lysed in 900 µL of
RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid,
0.1% SDS, 50 mM Tris pH 8.0) and 9 µL of protease-inhibitor
cocktail (1 mg/mL aprotinin, 1 mg/mL leupeptin, 1 mg/mL
antipain, 1 mg/mL pepstatin, 1 M benzamidine, 40 mg/mL
phenylmethanesulfonyl fluoride in DMSO). The cell lysate was
precipitated with protein G-sepharose (GE Healthcare) loaded
with anti-STxB antibody 13C4. Precipitated proteins were
analyzed by tris-tricine gel (5 mL gel contains 1.7 mL 30%
acrylamide,1.9% bis-acrylamide; 1.7 mL 3 M Tris-HCl with
0.3%SDS pH 8.45; 1.1 mL water; 0.7 mL glycerol; 50 µL 10%
ammonium persulfate; 5 µL N,N,N′,N′-tetramethylethylenedi-
amine) electrophoresis. Sulfated proteins were detected by
autoradiography (STORM860, Molecular Dynamics).
The purity of peptide 6 was 90% by analytical RP-HPLC
and 92% by CZE. MS data are in accordance with the proposed
structure (see Supporting Information).
Synthesis of Glyoxylyl NSC Peptide 7. A solution of glyoxylyl
peptide 6 (11 mg, 0.015 mmol) and triethylamine (1.07 µL,
0.007 mmol) in dry DMF (153 µL) was reacted with DSC (58.7
mg, 0.229 mmol). The mixture was stirred at rt for 1 h. The
crude mixture was diluted with 10 mL of 0.3% by vol aqueous
TFA and purified immediately by RP-HPLC on a 100 Å 5 µm
C18 Nucleosil column using a linear water/acetonitrile gradient
containing 0.05% TFA by vol (6 mL/min, detection 215 nm,
0-40% B in 60 min). Fractions were collected and lyophilized
to produce glyoxylyl NSC peptide 7 (6 mg, 53%).
The purity of peptide was 87% by analytical RP-HPLC. MS
data are in accordance with the proposed structure (see Sup-
porting Information).
Preparation of Peptide-Protein Conjugates. Reaction of
Lysozyme with Peptide 7 and then r-Oxo Semicarbazone
Ligation with Peptide 8. The synthesis of semicarbazide peptide
8 was described in detail elsewhere (19). This peptide sequence
is derived from HIV1 POL 476-484 protein.
Labeling with Peptide 7. A solution of lysozyme (0.85 mg,
0.06 µmol, 0.2 mM) in 0.1 M pH 8.0 sodium phosphate buffer
(300 µL) was added to peptide 7 (0.30 mg, 0.42 µmol). The
mixture was stirred at rt. The reaction was monitored by
MALDI-TOF analysis. The reaction was complete after 1 h as
visualized by MALDI-TOF mass spectrometry. The MS spectra
showed grafting of up to three peptide 7 molecules to lysozyme.
RESULTS AND DISCUSSION
Molecules exposed at the plasma membrane can be circulated
to early endosomes for recycling or targeted to lysosomes for
degradation. Some molecules are directed to other cell compart-
ments such as the Golgi apparatus or the endoplasmic reticulum
(ER). This pathway, termed the retrograde route (21), plays a
key role in important cellular functions such as antigen
presentation (16) and cell signaling (23).
One strategy used to prove that a protein is transported from
the plasma membrane to the Golgi is to modify it with a peptide
sequence (TS) which can be enzymatically modified in the
Golgi. Peptides presented in Table 1 are derived from the
tyrosine sulfation site of rat chromogranin B (24). The tyrosine
residue within the EEPEYGE motif is sulfated by tyrosylprotein
sulfotransferase, an enzyme which is exclusively located in the
trans Golgi network. Previous studies have shown TS-protein
conjugates utility for studying the retrograde transport of
proteins. Sulfation of the TS label was detected by incubating
cells with radioactive sulfate followed by immunoprecipitation
of the conjugate (1).
The work described in the first part of this article was
motivated by the need to have an easy access to various
TS-protein conjugates. TS peptides were modified at the
C-terminus by a Lys(biotin) residue, thereby allowing capture
of the TS-protein conjugates using streptavidin-coated beads.
TS peptide sequence features four glutamic acid residues, with
a negative charge at physiological pH. Thus, the labeling of
proteins with TS peptide may significantly lower the isoelectric
point (pI) of proteins. The solubility, binding, and biological
properties of proteins heavily depend on their pI (25-29). The
interactions of proteins with cell membranes (28, 30) are also
often dictated by charge effects. Finally, pI control of labeled
proteins by varying the label charge has been demonstrated to
facilitate purification of labeled proteins by isoelectric focusing
techniques (31). In this context, synthesis of a series of TS
peptides with a range of pI was worth considering. To this end,
2 to 5 arg residues were incorporated at the N-terminus of
peptides 1-4. D-Arg residues were chosen to minimize degrada-
tion of the peptide label in biological media.
Ligation between Glyoxylyl Lysozyme and Semicarbazide
Peptide 8. The above mixture was then added to semicarbazide
peptide 8 (1.00 mg, 0.84 µmol). The mixture was stirred at 37
°C for 2 h and then at rt for 72 h. MALDI-TOF analysis showed
ligation of up to two semicarbazide peptide 8 molecules to
lysozyme.
Retrograde Transport of TS-STxB-a,b Conjugates from
the Plasma Membrane to the Trans Golgi Network. Immuno-
fluorescence Analysis. 200 000 HeLa cells were placed on
coverslip in 24-well test plate (TPP, Switzerland) and incubated
with 2 µM TS-STxB-a,b conjugate or STxB for 40 min at 4 °C
in PBS++ (PBS containing 0.9 mM CaCl2, 0.52 mM MgCl2
and 0.16 mM MgSO4) and then shifted to 37 °C for 1 h (water-
jacketed incubator, Forma Scientific). Cells were fixed with
paraformaldehyde (300 µL of a 4% w/v solution in PBS),
washed with PBS++, and then permeabilized with saponin
solution (300 µL, 0.2% w/v saponin, 2% w/v BSA in PBS
buffer). Each coverslip was incubated with 30 µL saponin
solution containing 0.2% by vol of 13C4 murine anti-STxB
antibody (hybridoma from ATCC #CRL 1794; see ref 20) and
0.5% by vol of goat antibody against GM130 (C-19, Santa
Cruz). Cells were washed with saponin solution and then
incubated with 30 µL saponin solution containing 0.5% by vol
antimurine Cy3-labeled secondary antibody (Beckman Coulter)
and 0.5% by vol antigoat Alexa 488-labeled secondary antibody
(Molecular Probes). Coverslips were mounted using 10%
aqueous polyvinyl alcohol (Mowiol 4-88 from Fluka), 25%

222 Bioconjugate Chem., Vol. 21, No. 2, 2010
Mhidia et al.
Figure 1. TS-peptides 1-4 synthesized in this study. Two to five D-Arg residues were incorporated at the N-terminus of the TS substrate to
modulate the pI of peptides.
Figure 3. Effect of base stoichiometry on the RP-HPLC yield. Peptide
3a was reacted at rt in anhydrous DMF with DSC (10 equiv) in the
presence of triethylamine. Peptide and base were mixed and added in
one portion to DSC. (A) TEA 0.5 equiv, 5 min; (B) TEA 0.5 equiv, 30
min; (C) TEA 10 equiv, 5 min; (D) TEA 10 equiv, 30 min; (E) TEA
10 equiv, 2 h.
Figure 2. Effect of the DSC mode of addition and stoichiometry on
the RP-HPLC yield. Peptide 1a was reacted at rt in anhydrous DMF
with DSC in the presence of triethylamine (2 equiv). (A) Peptide and
base were mixed and added over 1 h period to DSC (2 equiv). (B)
Peptide and base were mixed and added in one portion to DSC (2
equiv). (C) Same as B using 10 equiv of DSC.
was not detected. Alternately, the use of 0.5 equiv of base
(Figure 3A,B) permitted the clean conversion of peptide 3a into
NSC peptide 3b. After 30 min, 3b was formed nearly quanti-
tatively (Figure 3B). The use of an excess of DSC minimized
the formation of urea side-products by reaction of the formed
NSC peptide 1-4b with starting peptide 1-4a. The critical role
played by base stoichiometry can be explained by the potential
conversion of NSC group into the highly reactive isocyanate
moiety in basic conditions. Evidence for this degradation is
introduced later on. Isocyanate groups react efficiently with
amino groups as well as with the phenol group of tyrosine, and
thus can be responsible for the multiple side-products observed
when two or more equivalents of triethylamine were used. DSC
high reactivity allows formation of NSC groups using substo-
ichiometric amounts of base. In these experimental conditions,
NSC group is stable and TS-peptides 1-4b could be isolated
NSC Peptide Synthesis and Stability. Peptides 1-4b were
obtained by reacting the corresponding precursors 1-4a with
DSC in anhydrous DMF. In a first approach, formation of the
N-succimidylcarbamate moiety was examined using peptide 1a
and 2 equiv of both base and DSC. Slow addition of peptide
1a to DSC led to a complex mixture (Figure 2A) in which target
peptide 1b represented only 37% of the total detected area.
Alternately, addition of peptide 1a in one portion (Figure 2B)
led to a 67% RP-HPLC yield. The use of 10 equiv of DSC
slightly improved the yield up to 73% (Figure 2C).
Next, the amount of DSC was fixed to 10 equiv, and we
examined the influence of the base amount on the reaction yield
using peptide 3a (Figure 3). The use of an excess of base (Figure
3C-E) produced complex mixtures in which target peptide 3b

Synthesis of Peptide-Protein Conjugates
Bioconjugate Chem., Vol. 21, No. 2, 2010 223
2 h. Peptide 1d (Figure 1) was the sole species detected by
RP-HPLC. The half-life of peptide 1b was around 10 min. In
this case, LC-MS analysis of the mixture revealed, besides
peptide 1a, formation of a novel compound corresponding to
isocyanate 1e (Figure 1). Reaction of this intermediate with
benzylamine led to formation of urea 1f, which displayed the
expected fragment ions by matrix-assisted laser desorption
ionization-time-of-flight postsource decay analysis.
In situ conversion of NSC peptide 1b into isocyanate 1e is
probably at the origin of this peptide short half-life. Indeed,
carbamates, which cannot be converted into isocyanates such
as those derived from secondary amines, are known to be less
reactive toward nucleophilic attack than parent carboxylic esters.
For example, Gassman et al. have shown that a p-nitrophenyl-
carbamate derived from a secondary amine is less reactive with
nucleophiles than p-nitrophenyl acetate (32). As demonstrated
later on, in situ formation of isocyanate moiety during the
conjugation step might also explain the rapid formation of
conjugates relative to NSCE derivatives.
Conjugation of Peptides 1-4b to Lysozyme. Lysozyme
contains seven Lys residues within its sequence. Thus, 7 equiv
of NSC peptide was used in the conjugation experiment.
Lysozyme (0.2 mM final concentration) was reacted with NSC
peptides 1-4b at pH 7.4 in PBS buffer at 4 °C. A combination
of RP-HPLC and CZE analyses showed a conversion of 50%
for all tested NSC peptides. Lysozyme labeled with peptide 1b
coeluted by RP-HPLC with the native protein, whereas CZE
permitted separation of the labeled protein from the native one
due to the low pI of peptide 1b. Alternately, lysozyme labeled
with peptide 4b could be separated from lysozyme by RP-HPLC,
with the labeled material eluting earlier than the unlabeled one,
whereas no separation was observed by CZE. The pI of peptide
4b is close to the pI of lysozyme, explaining why the conjugate
between peptide 4b and lysozyme was not separated from the
native protein by CZE. These experiments highlight the interest
of varying the NSC peptide pI to match the physicochemical
properties.
Figure 4 shows the MALDI-TOF spectra of the reaction
mixtures. Up to four peptide chains were linked to lysozyme
showing the efficiency of the conjugation reaction. Several
studies have proven that NSCE derivatives react with ε-amino
groups of proteins and sometimes with hydroxyl-containing
amino acids too (33). Likewise, reaction of isocyanates with
amino, thiol, hydroxyl, and/or imidazole groups within proteins
is also possible (34). Thus, it cannot be excluded that such
reactions might also occur with NSC peptides, which are at least
partially converted in situ into isocyanate peptides.
RP-HPLC analysis of the crude reaction mixtures showed
also, after 1 h, the absence of NSC peptides 1-4b and two
novel peaks in the TS peptide region. The latest eluting peak
corresponded to peptides 1-4a, formed by the NSC group
hydrolysis. The other peak was attributed to cyclic peptide 1-4g
(Figure 5), resulting from the intramolecular reaction between
the phenol group of tyrosine and the N-succinimidyl carbamate
or isocyanate. Indeed, peptide 4g showed a band at 263.5 nm,
typical of an O-acetyl tyrosine, whereas the tyrosine residue
within peptide 4a displayed a band at 275.3 nm as expected.
Peptide 4g was isolated by RP-HPLC and treated with 0.1 M
NaOH. These experimental conditions led to conversion of 4g
into a compound that coeluted with peptide 4a. MALDI-TOF
and UV spectra of 4a and of the compound obtained from 4g
were also identical. Overall, these data support the structure
proposed for cyclic peptides 1-4g.
Figure 4. Reaction of lysozyme with peptides 1-4b. MALDI-TOF
analysis of the reaction mixtures after 1 h (pH 7.4, 4 °C). (A) without
peptide, (B) peptide 1b, (C) peptide 2b, (D) peptide 3b, (E) peptide
4b.
successfully with a 59-61% yield after RP-HPLC purification.
Lyophilized peptides 1-4b were homogeneous by LC-MS and
CZE; they displayed the expected molecular ions and were stable
for months at -20 °C. 1H and 13C NMR analysis of peptide 1b
confirmed the NSC group presence on the ε-amino group of
the C-terminal Lys residue. This procedure was successfully
used for converting Ac-YLFSVHWPPLNKA-OH into corre-
sponding NSC derivative Ac-YLFSVHWPPLNK(COOSu)A-
OH, showing its compatibility with histidine, serine, tryptophan,
and asparagine residues also (see Supporting Information).
For comparison, the same experimental conditions were used
to convert peptide 1a into 1c in the presence of DSS (Figure
1). The RP-HPLC yield reached only 50% after 90 min of
reaction. Peptide 1a could efficiently be converted into peptide
1c by using 1 equiv of base (46% isolated yield). Thus, synthesis
of NSC peptides requires lower amounts of base compared to
NSCE peptides, probably due to the higher reactivity of DSC
compared to DSS.
Afterward, we examined the stability of peptides 1b,c in
water. Peptides 1b,c were stable enough at room temperature
in water containing TFA (pH 2) allowing their purification by
RP-HPLC. Typically, hydrolysis of 1b amounted to 3% after
3 h and only 19% after 18 h. The stability of peptides 1b,c was
also studied at pH 6.0. The half-life of NSCE peptide 1c was
Finally, we have compared the reaction rate of peptides 1b,c
with lysozyme. LC-MS analysis of the reaction mixtures
revealed that reaction of NSC peptide 1b with lysozyme
occurred in less than 5 min (50% conversion). Alternately, the

224 Bioconjugate Chem., Vol. 21, No. 2, 2010
Mhidia et al.
Figure 5. Cyclic peptides 1-4c formed during the labeling reaction by reaction between the phenol group of tyrosine and the NSC group on
C-terminal Lys residue.
Figure 6. Immunofluorescence study of the retrograde transport of STxB and TS-STxB-a,b conjugates from the plasma membrane to the trans
Golgi network. HeLa cells were incubated with 2 µM STxB or TS-STxB-a,b conjugates. Cells were fixed with paraformaldehyde, permeabilized
with saponin, and then incubated with 13C4 murine anti-STxB antibody (hybridoma from ATCC #CRL 1794, see ref 20) and C-19 goat antibody
against GM130, a marker of the Golgi. Cells were washed with saponin solution and then incubated with secondary antibodies (Cy3-labeled antimurine,
Alexa 488-labeled antigoat). Cells were analyzed by confocal microscopy (Leica Microsystems, Mannheim, Germany).
reaction of lysozyme with peptide 1c led to only 20% conversion
after 5 min and required about 1 h to go to completion (80%
conversion). Thus, NSC peptides allow rapid conversion of
proteins into peptide-protein conjugates, a property that can
be an advantage for the labeling of unstable proteins.
Synthesis of TS-STxB Conjugates and Biological Studies. The
retrograde route is used by Shiga toxin (35) to exert its toxic
effects. This toxin has an AB5 structure. The A domain is
responsible for the toxic effects of Shiga toxin. The B-subunit
STxB (7.7 kD) forms a pentamer which preferentially binds
the eukaryotic receptor globotriaosylceramide (Gb3). The B
pentamer has the capacity of targeting the toxin to the retrograde
route, allowing transfer of the A-subunit to the cytosol by retro-
translocation from the endoplasmic reticulum. STxB is a

Synthesis of Peptide-Protein Conjugates
Bioconjugate Chem., Vol. 21, No. 2, 2010 225
Scheme 2. Synthesis of Glyoxylyl NSC Peptide 7
peptide 1b did not alter the capacity of the protein to target the
retrograde route. We next studied the TS label capacity within
the conjugates to be sulfated by tyrosylprotein sulfotransferase,
an enzyme which is associated with the Golgi membrane (Figure
7) (37, 38). STxB was used as a negative control (lane A, Figure
7), whereas TS-STxB-c, a recombinant STxB protein modified
at the C-terminus by two tandem sulfation sites, was used as a
positive control (21). Cells were first incubated with the different
Figure 7. Sulfation assay using STxB (lane A), TS-STxB-a (lane B),
TS-STxB-b (lane C), and TS-STxB-c (lane D). Proteins were incubated
with HeLa cells at 4 °C in sulfate-free DMEM, and then incubated
with 35SO4 in sulfate free DMEM at 37 °C for 2 h. Cells were
2-
lysed and STxB proteins were captured using protein G-sepharose
loaded with anti-STxB antibody 13C4. Precipitated proteins were
separated by tris-tricine gel electrophoresis, and gels were analyzed
by autoradiography. The arrow points to the different STxB proteins.
2-
STxB proteins and then in the presence of 35SO4 in sulfate-
free DMEM to allow incorporation of radioactive sulfate within
the TS labels. Cells were lysed and STxB proteins were
immunoprecipitated using anti-STxB monoclonal antibody.
Radioactive bands were detected by autoradiography. The arrow
in Figure 7 points to STxB proteins. This experiment shows a
radioactive band for the positive control (TS-STxB-c, lane D)
but not for the negative control (STxB, lane A). A band is also
seen for TS-STxB a or b, showing that the TS label introduced
using NSC chemistry is sulfated by tyrosylprotein sulfotrans-
ferase present in the Golgi. Overall, these data show that NSC
chemistry allows the synthesis of conjugates that preserve the
capacity of STxB protein to target the retrograde route and the
ability of TS peptide to be sulfated in the cell.
valuable marker for the retrograde route and was used here to
prove the usefulness of TS conjugates prepared using NSC
chemistry.
STxB labeling was performed using peptide 1b as described
above. LC-MS analysis of the product revealed that 80% of
STxB was modified. MALDI-TOF analysis of the product
showed the presence of up to three TS peptides per STxB
monomer. The conjugate was purified either by ultrafiltration
on a 3 kD membrane (TS-STxB-a) or by RP-HPLC (TS-STxB-
b). TS-STxB-a,b were identical by LC-MS. Both conjugates
were tested to evaluate the impact of RP-HPLC and lyophiliza-
tion steps on the TS-STxB-b biological activity.
The retrograde transport of STxB and of TS-STxB-a,b from
the plasma membrane to the trans Golgi network was first
studied by immunofluorescence confocal microscopy using
HeLa cells (Figure 6). The three proteins colocalized with
GM130, a protein which is bound to Golgi membranes (36).
This experiment shows that modification of STxB by NSC
One-Pot Synthesis of Peptide-Protein Conjugates. The
method described above can only be used with peptides lacking
Lys residues within their sequences such as the TS peptide
described above. The two-step strategy depicted in Scheme 1
allows overcoming this limitation. The first step involves
reaction of bifunctionnal peptide A with the protein using NSC
Scheme 1. One-Pot Synthesis of Peptide Protein Conjugates Can Be Performed by Combining NSC Chemistry and r-Oxo
Semicarbazone Ligation

226 Bioconjugate Chem., Vol. 21, No. 2, 2010
Mhidia et al.
Scheme 3. Synthesis of Semicarbazide Peptide 8
Peptide A synthesized in this work is used as spacer. A Phe
residue was inserted into the sequence to facilitate UV detection.
However, peptide A can potentially be used for introducing a
bioactive sequence or a label depending on the final application.
The strategy described in Scheme 1 requires glyoxylyl NSC
peptide 7 and semicarbazide peptide 8 synthesis. Peptide 7 was
prepared starting from peptide 5 H-SAAFGGK-NH2 (Scheme
2). Oxidation of peptide 5 into peptide 6 COCHO-AAFGG-
NH2 by sodium periodate was performed using standard
experimental conditions (4). Glyoxylyl NSC peptide 7 was then
synthesized by treating peptide 6 with 0.5 equiv of triethylamine
and an excess of DSC, as described above for NSC TS peptides
1-4b. Attempts to convert peptide 6 into the corresponding
NSCE ester with DSS, as described above, failed probably due
to imine formation between R-oxo aldehyde and ε-amino
moieties in basic conditions.
Table 2. MS Data for the One-Pot Reaction of Lysozyme with
Glyoxylyl NSC Peptide 7 and Semicarbazide Peptide 8a
entries
lysozyme L(n,m)
calcd
found
1
2
3
4
5
6
7
0,0
1,0
2,0
3,0
1,1
2,1
2,2
14.306
14.936
15.567
16.198
15.996
16.626
17.684
14.306
14.937
15.571
16.198
15.995
16.626
17.684
a MALDI MS spectra were acquired in the linear mode by using a
PerSeptive Biosystems Voyager DE-STR instrument fitted with a 337
nm nitrogen laser (25 kV, grid 92%, delay time 750 ns). Matrix:
3,5-Dimethoxy-4-hydroxycinnamic acid (20 mg/mL). Entry 1: lysozyme.
Entries 2-4: reaction with peptide 7. Entries 5-7: reaction with peptide
7 and peptide 8.
Model peptide 8 synthesis was performed by coupling Boc-
NHNHCO-imidazole to peptidyl resin assembled using Fmoc/
t-Bu strategy (Scheme 3) (19). Semicarbazide peptide 8 was
stable during TFA deprotection and cleavage and was purified
by RP-HPLC in a pH 6.5 triethylamonium acetate buffer.
Lysozyme was used again as a model protein. Reaction
between lysozyme and glyoxylyl NSC peptide 7 was performed
in a pH 8.0 sodium phosphate buffer. MALDI-TOF analysis of
the reaction mixture revealed grafting of up to three glyoxylyl
NSC peptide 7 molecules (L1-3,0 see entries 2-4, Table 2;
see also Figure 8). After 1 h, the reaction mixture was reacted
with semicarbazide peptide 8 at same pH. Up to two semicar-
bazide peptide 8 molecules were linked to lysozyme (entries
chemistry. This allows introducing an R-oxo aldehyde group
on the surface of the protein, which can be subsequently ligated
chemoselectively with peptide B featuring a semicarbazide group
at the N-terminus (Scheme 1). To date, semicarbazone chemistry
has been mainly used for linking peptides to surfaces (39-42).
To the best of our knowledge, however, ligation between
semicarbazide and glyoxylyl peptides was not reported before.
This may be due to the difficulty synthesizing peptides featuring
a semicarbazide group at the N-terminus, because this moiety
is rapidly hydrolyzed in the acid eluents (pH 2) often used for
peptide RP-HPLC analysis or purification. A solution to this
problem has recently been described by the use of a nearly
neutral buffer instead for the purification step (19).
Figure 8. MALDI-TOF analysis of the one-pot conversion of lysozyme L into L(n,m) peptide-lysozyme conjugates. (A) after reaction with glyoxylyl
NSC peptide 7, (B) after reaction with peptide 7 and semicarbazide peptide 8. See Table 2 for experimental conditions. The asterisk corresponds
to protein-matrix adducts.

Synthesis of Peptide-Protein Conjugates
Bioconjugate Chem., Vol. 21, No. 2, 2010 227
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5-7, Table 2). Usually, Schiff-base formation between glyoxylyl
peptides and peptide hydrazides is carried out at pH 5.5. In this
study, successful formation of the R-oxo semicarbazone bond
at pH 8.0 permitted the one-pot synthesis of peptide-protein
conjugates in a straightforward manner.
In conclusion, we have shown that NSC peptides can easily
be synthesized in high yield by reacting unprotected peptide
precursors with DSC. The method requires the presence of only
one reactive ε-amino group within the peptide sequence.
Importantly, NSC peptides were stable during standard RP-
HPLC purification at pH 2 and in the lyophilized form for
months. Their reaction with ε-amino groups of proteins occurred
efficiently and rapidly (<5 min), probably due to in situ partial
conversion of NSC group into isocyanate moiety. The method
was used for grafting a peptide substrate for tyrosylprotein
sulfotransferase to lysozyme, a model protein, or STxB the
receptor-binding nontoxic B-subunit of Shiga toxin, a valuable
marker for the retrograde route. The biological activity of the
STxB conjugate was verified by immunofluorescence confocal
microscopy or using a sulfation assay. We have also described
a novel method for synthesizing peptide-protein conjugates,
which is compatible with all proteinogenic amino acids. The
conjugates were assembled by combining NSC chemistry and
R-oxo semicarbazone ligation. To this end, a glyoxylyl NSC
peptide was synthesized and reacted with lysozyme to produce
glyoxylyl lysozyme. The crude reaction mixture was then
reacted with a semicarbazide peptide. Ligation between the
glyoxylyl groups on the protein and the semicarbazide group
on the peptide resulted in formation of the target peptide-protein
conjugate. Both reactions were carried out at pH 8.0, thus
allowing the one-pot synthesis of the conjugate without the need
to adjust pH in between.
Overall, NSC chemistry described here is an interesting tool
for preparation of conjugates of biological interest and comple-
ments the chemical toolbox of the peptide chemist.
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ACKNOWLEDGMENT
We are grateful to the French Ministry of Research for its
financial support, to the EEC, CNRS, Re´gion Nord Pas-de-Calais
and Cance´ropoˆle Nord-Ouest for their grants. The work was
performed in Chemistry Systems Biology platform facility of
the Institute of Biology (Lille, France, http://csb.ibl.fr). GS was
supported by a PhD fellowship from Fondation pour la
Recherche Me´dicale (FRM).
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Supporting Information Available: RP-HPLC, CZE and
MS data for all compounds. 1H NMR spectra for peptides 1a,b.
This material is available free of charge via the Internet at http://
pubs.acs.org.
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