A disulfide intercalator toolbox for the site-directed modification of polypeptides

Article abstract of DOI:10.1002/chem.201403965

A disulfide intercalator toolbox was developed for site-specific attachment of a broad variety of functional groups to proteins or peptides under mild, physiological conditions. The peptide hormone somatostatin (SST) served as model compound for intercalation into the available disulfide functionalization schemes starting from the intercalator or the reactive SST precursor before or after bioconjugation. A tetrazole-SST derivative was obtained that undergoes photoinduced cycloaddition in mammalian cells, which was monitored by live-cell imaging.

Full text of DOI:10.1002/chem.201403965

DOI: 10.1002/chem.201403965
Full Paper
&
Peptide Modification |Hot Paper|
A Disulfide Intercalator Toolbox for the Site-Directed Modification
of Polypeptides
Tao Wang,[a] Yuzhou Wu,[a] Seah Ling Kuan,[a] Oliver Dumele,[b] Markus Lamla,[a]
David Y. W. Ng,[a] Matthias Arzt,[a] Jessica Thomas,[a] Jan O. Mueller,[c, d] Christopher Barner-
Kowollik,*[c, d] and Tanja Weil*[a]
Abstract: A disulfide intercalator toolbox was developed for
site-specific attachment of a broad variety of functional
groups to proteins or peptides under mild, physiological
conditions. The peptide hormone somatostatin (SST) served
as model compound for intercalation into the available di-
sulfide functionalization schemes starting from the intercala-
tor or the reactive SST precursor before or after bioconju-
gation. A tetrazole–SST derivative was obtained that under-
goes photoinduced cycloaddition in mammalian cells, which
was monitored by live-cell imaging.
Introduction
polypeptides containing additional substituents at predefined
positions.[7] In this way, the aforementioned limitations could
be addressed and, in addition, novel features that, for example,
allow bioactivity or self-assembly to be controlled could be in-
troduced.[8] For most applications, formation of a homogeneous
product is crucial. Thus, chemical modification must be per-
formed at a distinct site in high yield, which also facilitates pu-
rification. Most approaches are based on the attachment of
a bioorthogonal anchor group that can react in the presence
of abundant functional groups such as amino, carboxyl, and
hydroxyl. Often, a single accessible cysteine residue is intro-
duced by genetic engineering since its thiol group selectively
reacts with maleimide moieties under pH control.[9] Probably
one of the most elaborate strategies is based on the incorpora-
tion of reactive, noncanonical amino acids at distinct positions
in the polypeptide sequence.[10] In this way, halogen,[11] ethyn-
yl,[12] and azido[13] groups have been attached that allow fur-
ther post-modifications. Since only very few native proteins
contain unpaired and accessible cysteine residues, recombi-
nant techniques are often the only possibility to attach an ap-
propriate bioorthogonal anchor to the polypeptide surface.
A less common approach involves the functionalization of
disulfide groups. Disulfides are abundant in proteins and cyclic
polypeptides, and theoretical evaluation of protein databases
revealed that most therapeutically relevant proteins offer at
least one disulfide bond close to the surface.[14] Previously,
Brocchini et al. demonstrated that disulfide bridges of proteins
can be addressed with high site-specificity.[15] Bis-alkylation
conjugation reagents, often referred to as intercalators, have
been introduced that react with accessible disulfide bonds in
a two-step reaction mechanism (Scheme 1).[14] First, the disul-
fide bond is reduced, and then disulfide rebridging occurs
through two successive Michael addition reactions with reten-
tion of the tertiary structure of the protein.[14,15] The broad ap-
plicability of disulfide intercalators has been demonstrated by
the successful modification of peptide hormones, antibodies,
Peptides and proteins are versatile biomacromolecules that are
of emerging interest in medicine and materials science.[1] Due
to their unique chemical, physical, and biological properties,
polypeptides have been used as, for example, drug mole-
cules,[2] tissue-engineering scaffolds,[3] drug-delivery matrices,[4]
and detectors and transducers in biosensor technology.[5] They
have many distinct advantages, such as high target affinities,
specificity, and biocompatibility, but their applications are
often still limited by low chemical and metabolic stability or
other unfavorable pharmacokinetic parameters.[6] Therefore,
there has been increasing interest in the development of syn-
thetic procedures that open access to chemically modified
[a] T. Wang, Dr. Y. Wu, Dr. S. L. Kuan, Dr. M. Lamla, Dr. D. Y. W. Ng, M. Arzt,
J. Thomas, Prof. Dr. T. Weil
Institute of Organic Chemistry III, University of Ulm
Albert-Einstein-Allee 11, 89081 Ulm (Germany)
Fax: (+49)731-5022883
E-mail: tanja.weil@uni-ulm.de
[b] O. Dumele
Max Planck Institute for Polymer Research
Ackermannweg 10, 55128 Mainz (Germany)
[c] J. O. Mueller, Prof. Dr. C. Barner-Kowollik
Preparative Macromolecular Chemistry
Institute of Technical Chemistry and Polymer Chemistry
Karlsruhe Institute of Technology (KIT)
Engesserstrasse 18, 76131 Karlsruhe (Germany)
E-mail: christopher.barner-kowollik@kit.edu
[d] J. O. Mueller, Prof. Dr. C. Barner-Kowollik
Institute for Biological Interfaces, Karlsruhe Institute of Technology (KIT)
Hermann-von-Helmholtz-Platz 1
76344 Eggenstein-Leopoldshafen (Germany)
Fax: (+49)721-60845740
E-mail: christopher.barner-kowollik@kit.edu
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201403965.
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Scheme 1. A) Mechanism of disulfide intercalation. B,C) Synthesis of various intercalators with different functionalities. a) HBTU, DIEA, propargylamine, 78%;
b) 33, EDC, DMAP, 68%; c) DOX, HBTU, DIEA DMF, 68%; d) 36, HBTU, DIEA, DMF, 82%; e) TFA, CH2Cl2, 95%; f) Sulforhodamine B acid chloride, DIEA, DMF, 45%;
g) Palmitic acid, HBTU, DIEA, DMF, 78%; h) 4-Carboxyphenylboronic acid, HBTU, DIEA, DMF, 60%; i) 34, HBTU, DIEA, DMF, 63%; j) TZ, HBTU, DIEA, DMF, 62%;
k) HBTU, DIEA, DMF, 70%; l) Oxone, MeOH/H2O (1:1), 90%; m) 35, CuSO4, sodium ascorbate, 84%; n) Boc2O, Et3N, MeOH, 238C, quant.; o) [Cu(CH3CN)4]PF6,
Et3N, THF, 708C, 1 h, then 238C, 10 h, 56%; p) TFA, CH2Cl2, quant.
and therapeutic proteins.[16] We have further investigated the
reaction mechanism of disulfide intercalation[16c] by NMR spec-
troscopy and have reported the introduction of a single iodo
or ethynyl group by applying the appropriately functionalized
bis-alkylation reagents. In addition, the combination of disul-
fide intercalation and maleimide chemistry even allowed the
cross-conjugation of biomolecules,[17] which has been shown
to be particularly attractive for the design of tumor-responsive
therapeutics.[18] However, little to no solubility of most interca-
lators in aqueous solution as well as a lack of more versatile
and easily adaptable reaction schemes currently limits broader
usage of this promising approach.
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Herein, we report the synthesis of a versatile toolbox of bis-
alkylation reagents 3, 4, 6–11, 14, 15, 18a, and 18b
(Scheme 1) offering improved solubility and multiple function-
alities that “reanneal” the original disulfide sites and thus intro-
duce the desired functionality at a predefined position of pep-
tides or proteins (Figure 1A). The burgeoning interest in bioor-
thogonal chemistry that responds to environmental stimuli
motivated us to consider phenyl boronic acid (PBA) and tetra-
zole (TZ) units. PBA reacts with salicylhydroxamic acid (SHA)
with fast kinetics and high binding affinity in response to pH
changes. The photoinduced, bioorthogonal nitrile imine-medi-
ated tetrazole–ene cycloaddition reaction (NITEC) takes place
under UV irradiation.[19] The great potential of the NITEC ap-
proach has been demonstrated, for instance, in site-specific
(bio)polymer labeling, patterning of various biosubstrates for
controlled cell attachment,[20] light-responsive materials,[21]
probing of protein dynamics and function, and investigation of
biological processes inside living cells.[22]
before intercalation, but they are not advisable thereafter due
to the ubiquitous presence of amino and carboxyl groups in
proteins and peptides.
Amine bis-sulfone 6 is an attractive building block for gener-
ating further intercalator derivatives by simple condensation of
the desired functionalities. It was synthesized in 78% yield by
treating bis-sulfone 1 with N-Boc-4,7,10-trioxa-1,13-tridecanedi-
amine (36, Supporting Information) in the presence of 2-(1H-
benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophos-
phate (HBTU) and N,N-diisopropylethylamine (DIEA), followed
by removal of the Boc protecting group with trifluoroacetic
acid (TFA). Exemplarily, the widely used chromophore sulforho-
damine B acid chloride, TZ, palmitic acid, 4-carboxyphenylbor-
onic acid, and Boc-protected thiol 34 (Supporting Information)
were attached to give the corresponding intercalators 7–11
after purification by column chromatography in moderate to
good yields in a single reaction step (Scheme 1B).
For the reaction of compounds that contain many reactive
primary amino or carboxyl acid groups with the intercalator,
cycloaddition reactions involving azide or ethynyl groups are
a better alternative, since they are absent from almost all natu-
rally occurring compounds and therefore react with high
chemical selectivity in high yields.[25] The fastest approach to
ethynyl bis-sulfones is the condensation of propargylamine
with bis-sulfone 2 in the presence of HBTU and DIEA providing
alkyne bis-sulfone 2 in 78% yield. However, 2 is limited by
poor labeling efficiency, most likely due to the high lipophilici-
ty of the respective monosulfone (Supporting Information, Fig-
ure S10B).
Disulfide intercalation was studied by applying the polypep-
tide hormone somatostatin (SST) with emphasis on steric re-
strictions of the intercalation reaction. SST was selected as
model polypeptide due to its important biological activity, the
presence of only one well-accessible disulfide bond, as well as
its efficient uptake into cells and transport into the cytosol,
which facilitates investigating chemical reactions even inside
living cells. The biological effects of SST are mediated through
five G protein-coupled membrane-bound receptors SSTR1–
SSTR5, which are aberrantly expressed in high levels in various
tumors (e.g., breast, lung, renal, pancreatic, prostate, and neu-
roendocrine cancers) in comparison to normal tissues.[23] SST
conjugates are of emerging interest for radiotherapy and
tumor imaging by labeling SST with radionuclides and for che-
motherapy by conjugating SST with antineoplastic agents.[24]
To address this limitation, bifunctional linker 38 (Supporting
Information) was designed to improve the water solubility of
the intercalator. A single amino group of 1,13-diamino-4,7,10-
trioxatridecane was Boc-protected, whereas the other was con-
verted to an azide. The most common method for converting
amines to azides involves diazo transfer reagents such as tri-
fluoromethanesulfonyl azide (TfN3) serving as diazo donors.[26]
However, due to the explosive nature and relatively poor shelf
life of TfN3, imidazole-1-sulfonyl azide 32 (Supporting Informa-
tion) was applied instead.[27] Compound 36 (Supporting Infor-
mation) was treated with 32 in the presence of copper sulfate
and potassium carbonate to give the corresponding azido de-
rivative 37 (Supporting Information). After Boc deprotection,
the bifunctional linker 38 (Supporting Information) was ob-
tained, which was condensed with bis-sulfide 12 in the pres-
ence of HBTU and DIEA in 70% yield. Subsequent oxidation of
bis-sulfide 13 with Oxone yielded the corresponding azido bis-
sulfone 14 in 90% yield. The analogous intercalator ethynyl
bis-sulfone 3 with improved water solubility was synthesized
accordingly (Supporting Information, Scheme S1). Azido bis-
sulfone 14 was then treated with ethynyl biotin 35 (Support-
ing Information), which was synthesized from ethynyl triethy-
lene glycol 33 (Supporting Information) by using 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide (EDC) as activator and 4-di-
methylaminopyridine (DMAP) as catalyst, as shown in
Scheme S1 of the Supporting Information.
Results and Discussion
Synthesis of the intercalator toolbox
The intercalation reaction with accessible disulfide bonds of
proteins and peptides is based on two consecutive Michael ad-
ditions. Reagents containing bis-sulfone groups undergo an
elimination reaction with formation of the reactive monosul-
fone derivative and p-toluenesulfinic acid as byproduct
(Scheme 1A). This reaction usually proceeds in situ under
weakly basic conditions. The respective monosulfone subse-
quently reacts with a free thiol group, which eliminates the
second p-toluenesulfinic acid group with formation of
a second Michael system. In the presence of a second thiol
group in close vicinity, the second Michael addition occurs
with concomitant formation of a C3-rebridged bis-sulfide. One
refers to an intercalation reaction in the case that both consec-
utive reactions occur. There are several advantages associated
with the introduction of functional groups already in the inter-
calator. A large variety of substituents can be attached, purifi-
cation can be carried out by chromatography, and removal of
traces of catalyst is usually less challenging. Condensation reac-
tions that provide intercalator libraries proceed smoothly
Since the alkyne is electron-deficient, it readily undergoes
CuI-catalyzed cycloaddition with azido bis-sulfone 14 in THF/
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Figure 1. A) Site-specific functionalization of SST. 1) Direct intercalation of SST with functionalized intercalators. 2) Introduction of bioorthogonal groups onto
SST and post-modification by bioorthogonal reactions. B) HR-ESI mass spectrum of PBA-SST 25 (calcd exact mass: 714.99514 [MÀ2H2O+3H]3+, formula:
C104H137BN20O25S2; 1080.99436 [MÀH2O+2H]2+, formula: C104H139BN20O26S2); C) HR-ESI mass spectrum of tetrazole-SST 22 (calcd exact mass: 760.34745
[M+3H]3+, 1140.01753 [M+2H]2+, formula: C111H144N24O25S2; D) HR-MALDI mass spectrum of PBA-SST 25 with DHB as matrix (calcd exact mass: 2296.99563
[M+DHBÀ2H2O+H]+, formula: C111H143BN20O29S2).
H2O to afford biotin bis-sulfone 15 in 84% yield. In addition,
more bulky substituents such as the anticancer drug doxorubi-
cin (DOX) and a bulky polyamidoamine (PAMAM) dendron
were attached to afford 4 and 18a,b, respectively. Compound
18b was synthesized by CuI-catalyzed cycloaddition in CH2Cl2/
MeCN in 56% yield. Boc deprotection in TFA/CH2Cl2 (1:1) yield-
ed the dendritic intercalator G2-PAMAM(NH2)4·TFA salt 18a in
quantitative yield. DOX was attached to bis-sulfone 1 to afford
DOX bis-sulfone 4 by HBTU coupling in 68% yield. In total, 13
novel bis-sulfone intercalators were prepared for the introduc-
tion of, for example, fluorescence labels, bioorthogonal reac-
tive groups, affinity tags, and polymer or drug substituents.
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Synthesis of a SST library
the intercalator. If the substituent is too bulky, it might not be
able to reach the reduced disulfide groups anymore, since no
addition products were observed by LC-MS. Therefore, bulky
substituents must be introduced into the polypeptide after the
intercalation reaction. Both azido-SST 20 and ethynyl-SST 19
offer improved water solubility and enable further bioorthogo-
nal modifications. Therefore, azido-SST 20 was conjugated to
G2 PAMAM dendron 44 (Supporting Information) and azido-
DOX[18] by CuI-catalyzed cycloaddition in water. G2 PAMAM-SST
2 and DOX-SST 28[18] were isolated in 30 and 47% yield, re-
spectively, and characterized by MALDI MS (Supporting Infor-
mation, Figure S18B) with no CuI catalyst remaining in the
sample.
CuI-catalyzed cycloaddition reactions have the inherent dis-
advantage that the CuI catalyst must be removed entirely due
to its cytotoxicity. Even though removal of the catalyst could
be accomplished easily at the level of the intercalator as well
as for peptides such as SST by column chromatography or
HPLC, purification of proteins can be tedious, and product loss
or reduced bioactivity can occur. Therefore, there is an increas-
ing interest in the synthesis of alternative, catalyst-free reaction
schemes that can be accomplished at the level of the bioma-
cromolecule. In principle, thiol, SHA, PBA, and tetrazole sub-
stituents that were introduced by intercalation offer site-direct-
ed reactions at the level of the protein. Proteins or peptides
containing these functional groups such as SST derivatives 21,
22, 25, and 27 would offer bioconjugation without application
of cytotoxic metal catalysts.
SST was derivatized by applying intercalators that contained af-
finity tags, reactive bioorthogonal groups, a fluorescent probe,
or a fatty acid (Figure 1A) in moderate yields. First, the disul-
fide of SST was reduced by tris(2-carboxyethyl)phosphine
(TCEP) and then allowed to react with the respective intercala-
tor under slightly basic conditions (pH 7.8) in two successive
Michael addition reactions. Azido and ethynyl bis-sulfones (14,
3) were incubated in 50 mm phosphate buffer (pH 7.8) contain-
ing 40% MeCN to allow elimination of p-toluenesulfinic acid
and thus yield the corresponding monosulfones in situ. Reduc-
tion by TCEP and intercalation by the respective monosulfones
gave the respective azido or ethynyl SST derivatives (Fig-
ure 1A).
SH-SST 21 was produced in a similar fashion after intercala-
tion of the Boc-protected SBoc-bis-sulfone 10 followed by Boc
deprotection in 80% TFA solution for 5 h. Thiol–maleimide
chemistry is widely used in bioconjugation reactions due to
fast reaction kinetics in aqueous media, the lack of byproducts,
and the improved stability of the thioether addition product.
Therefore, 21 is an attractive SST building block that gives
access to more sophisticated SST derivatives and which was
prepared by purely chemical approaches without the necessity
to express a SST cysteine mutant.
PBA-SST 25 containing a single boronic acid group was pre-
pared as depicted in Figure 1A and characterized by HRMS.
HR-ESI-MS revealed dehydration products with mass signals at
714.99575 [MÀ2H2O+3H]3+ and 1080.99517 [MÀH2O+2H]2+
(Figure 1B) due to thermally induced dehydration of boronic
acid or cyclization to boroxines, as reported before.[28] The iso-
topic pattern of the dehydration products matched the calcu-
lated pattern (Supporting Information, Figure S16). In addition,
MALDI HRMS with a-cyano-4-hydroxycinnamic acid (CHCA) as
matrix gave the dehydration product with mass signal of
2142.96879 [MÀ2H2O+H]+ (Supporting Information, Fig-
ure S15), the isotopic pattern of which fits the theoretical pat-
tern. By using 2,5-dihydroxybenzoic acid (DHB) both as matrix
and derivatizing reagent due to its complexation to boronic
Phototriggered ligation reaction of tetrazole-SST 22 inside
live cells
The photoinduced 1,3-dipolar cycloaddition of tetrazole deriva-
tives with maleimides usually proceeds within 5 min at ambi-
ent temperature and is one of the contemporary tools for
rapid modular ligation conjugation.[35] It occurs with formation
of a photoluminescent product that also allows the reaction ki-
netics to be monitored.[22b,36] Tetrazole-SST 22 was obtained by
sequential reduction and intercalation of SST with TCEP and
11, respectively, and purified by HPLC. Tetrazole-SST 22 was
obtained in 41% yield and was characterized by HR-ESI-MS
(Figure 1C). Bioconjugation of 22 and 6-maleimideocaproic
acid in tenfold excess proceeded by tetrazole–ene cycloaddi-
tion in aqueous medium under UV irradiation for 2 h (Fig-
ure 2A). No formation of side products was observed (Fig-
ure S20A). Compound 30 was isolated by HPLC (Supporting In-
formation, Figure S20B) in moderate yield (50%) and charac-
terized by HR-ESI-MS (Figure 2B). For proof of concept, we in-
vestigated whether this reaction could also be accomplished in
a biological environment. Since SST readily penetrates cellular
membranes expressing SST2 receptors via receptor-mediated
endocytosis, a model NITEC reaction was performed inside
living cells.
acids,
only
the
final
mass
signal
2296.99563
[M+DHBÀ2H2O+H]+ was detected owing to formation of the
borate ester with DHB (Figure 1D), which supports that
a single boronic acid group was successfully introduced.[28] The
attachment of boronic acids to polypeptides is of great interest
for biosensing,[29] therapy,[30] and pH-responsive materials,[31]
since boronic acids efficiently and reversibly react with bifunc-
tional alcohols,[32] amines,[33] and other nucleophiles[34] under
physiological conditions. They offer great opportunities to con-
struct stimulus-responsive materials based on their bioorthog-
onal click chemistry.
Interestingly, direct intercalation of 18a, 18b, and 4 bearing
the sterically demanding G2 PAMAM dendron or the drug DOX
was not successful. In both cases, complex product mixtures
that still contained unconsumed starting materials were ob-
tained repetitively (Supporting Information, Scheme S2 and
Figures S7, S8). We presume that the intercalation reaction is
limited by the steric demand of the substituent attached to
Tetrazole-SST 22 (0.1 mm) and the membrane-permeable
compound methoxypolyethylene glycol maleimide (MI-
PEG5000, 2 mm, Mn =5000) were incubated with A549 cells for
4 h. Thereafter, cells were washed thoroughly to remove any
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Figure 2. A) Synthetic scheme of the tetrazole–ene cycloaddition of tetrazole-SST 22 with 6-maleimideocaproic acid or MI-PEG5000 under UV irradiation at
254 nm. B) HR-MALDI mass spectrum of click product 30 with CHCA as matrix (calcd mass: 2461.09888, formula: C121H157N23O29S2). C) Confocal image of A549
cells incubated with 2 mm MI-PEG5000 and 0.1 mm tetrazole-SST before UV irradiation. D) Incubated with 2 mm MI-PEG5000 and 0.1 mm tetrazole-SST under
UV irradiation for 10 min. E) The normalized single-cell fluorescence intensity (single-cell fluorescence intensity/area of cell) was quantified by ImageJ software
based on the confocal images (Supporting Information, Figure S22); data were plotted as meansÆstandard errors of the means (SEM) by using GraphPad
Prism5 Software (n=10).
Conclusion
remaining tetrazole-SST and MI-PEG5000 that were not taken
up. The tetrazole–ene cycloaddition inside cells was initiated
by 254 nm UV irradiation and investigated by confocal micros-
copy after 10 min. The emission inside A549 cells incubated
with both tetrazole-SST 22 and MI-PEG5000 under UV irradia-
tion for 10 min was significantly enhanced as compared to
A549 cells incubated with both reagents but without UV irradi-
ation (Figure 2C, D). The emission intensity inside the cells
under UV irradiation for 10 min was quantified by the normal-
ized single-cell fluorescence intensity (single-cell fluorescence
intensity/area of cell) by using the ImageJ software (Support-
ing Information, Figure S22, Table S1).[37] It was at least seven
times higher than that of the control experiments (Figure 2E).
The weak emission of A549 after incubation with tetrazole-SST
(Figure 2E) was most likely due to the weak emission of the ni-
trile imine intermediate. These results clearly support that the
photoinduced click reaction was accomplished inside living
cells. With the tetrazole intercalator, a chemically addressable,
bioorthogonal group can be readily introduced into polypep-
tides or proteins of interest in a site-directed fashion. The tetra-
zole group can undergo photoinduced 1,3-dipolar cycloaddi-
tion reactions whereby a new chromophore is formed. This re-
action is of potential interest to probe protein function and dy-
namics inside living cells or to control chemical reactions both
spatially and temporally in real time inside a living system by
light irradiation.
We have designed a versatile intercalator library that rebridges
disulfide bonds and introduces various functionalities into
polypeptides in a site-specific fashion. In total, 13 novel inter-
calators were prepared containing bioorthogonal groups such
as ethynyl, azido, PBA, and TZ, bioaffinity tags such as biotin,
and imaging probes such as rhodamine. They were connected
by triethylene oxide linkers that impart attractive water solubil-
ity, which is particularly suitable for peptide and protein
chemistry. The successful functionalization of the peptide hor-
mone SST containing a single disulfide bridge was demonstrat-
ed by applying the intercalator toolbox, and various new SST
conjugates were obtained, which could be of great signifi-
cance for biomedical research. Tetrazole-SST allowed photoin-
duced reaction under UV irradiation for bioconjugation via
photoinduced, bioorthogonal tetrazole–ene cycloaddition reac-
tion (NITEC). Since SST penetrates into cells by receptor-medi-
ated uptake, photoinduced NITEC reaction was accomplished
in living cells and was monitored by live-cell imaging.
Thus, we have presented a versatile and robust synthetic
strategy for introducing desired functional groups into poly-
peptides that contain accessible disulfide bonds. Due to the
improved water solubility of these intercalators, site-directed
functionalization of proteins could be envisaged, since most
proteins have at least one accessible disulfide bridge. There-
fore, our approach complements the introduction of reactive
functionalities such as free thio, ethyny, or azido groups
through recombinant techniques offering the advantage that
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Synthesis of DOX intercalator 4: Bis-sulfone
1
(50 mg,
the reaction can be accomplished at the native protein and no
protein mutant need to be expressed.
0.1019 mmol, 1 equiv) was dissolved in 1 mL of anhydrous DMF.
Then HBTU (63 mg, 0.1657 mmol, 1.9 equiv) and DIEA (46 mL,
0.2786 mmol, 3.2 equiv) were added to this solution at 08C under
argon. The reaction mixture was stirred for 10 min before doxoru-
bicin hydrochloride (384 mg, 1.2 mmol, 1.2 equiv) was added. The
resulting mixture was stirred overnight at RT. The solvent was re-
moved under high vacuum, and the product was dissolved in
CH2Cl2. The organic phase was washed with NaHCO3 and brine and
dried over anhydrous Na2SO4. The solvent was evaporated under
vacuum and the crude product was purified by column chroma-
tography with 5% methanol in chloroform to afford 60 mg of 4 in
68% yield. The product was characterized by LC-MS, which re-
vealed that the monosulfone had already partially formed during
column chromatography due to elimination of p-toluenesulfinic
acid; LC-MS: m/z=868 [monosulfoneÀH]À, 1024 [MÀH]À (Support-
ing Information, Figure S2).
Experimental Section
General
Unless otherwise noted, all operations were performed without
taking precautions to exclude air and moisture. All solvents and re-
agents were purchased from commercial sources and were used
without further purification. Reaction progress was monitored by
TLC on Merck 60 F254 precoated silica gel plates with visualization
by UV illumination (254 nm) or by using appropriate stains. Flash
column chromatography was carried out on Merck silica gel 70–
230 mesh. NMR spectra were measured on a Bruker 400 or
500 MHz NMR spectrometer and the chemical shifts were refer-
enced to residual solvent shifts in the respective deuterated sol-
vents. Chemical shifts are reported in parts per million relative to
the residual solvent peaks. MALDI-TOF mass spectra were acquired
on a Bruker Reflex III. HR-MALDI-MS and HR-ESI mass spectra were
recorded on a Solarix (Bruker) FTICR-MS. LC-MS analysis was per-
formed on a Shimadzu LC-MS 2020 equipped with an electrospray
ionization source and a SPD-20A UV/Vis detector (Shimadzu, Duis-
burg, Germany). FD-MS (8 V) spectra were obtained on a VG Instru-
ments. ZAB 2 SE-FPD. The absorbance and emission were mea-
sured on Microplate Readers (Tecan Infinite M1000 PRO). Azido-G2
dendron 45 and 2-hydroxy-4-pent-4-ynamido-N-(trityloxy)benza-
mide were synthesized according to previous literature.[38]
Synthesis of tert-butyl (1-oxo-1-{4-[3-tosyl-2-(tosylmethyl)propa-
noyl]phenyl}-6,9,12-trioxa-2-azapentadecan-15-yl)carbamate (5):
Bis-sulfone 1 (500 mg, 1 mmol, 1 equiv) was dissolved in 5 mL of
anhydrous DMF. Then HBTU (606 mg, 1.6 mmol, 1.6 equiv) and
DIEA (330 mL, 2 mmol, 2 equiv) were added to this solution at 08C
under argon. The reaction mixture was stirred for 10 min before
Boc-ethylene glycol (36, 384 mg, 1.2 mmol, 1.2 equiv) was added.
The resulting mixture was stirred overnight at RT. The solvent was
removed under high vacuum, and the product was dissolved in
CH2Cl2. The organic phase was washed with NaHCO3 and brine and
dried over anhydrous Na2SO4. The solvent was evaporated under
vacuum and the crude product was purified by column chroma-
tography with 10% methanol in chloroform to afford 658 mg of 5
1
Synthesis of intercalators with different functionalities
in 82% yield. H NMR (400 MHz, CDCl3): d=1.36 (s, 9H), 1.61–1.67
(m, 2H), 1.82–1.88 (m, 2H), 2.74 (s, 6H), 3.11–3.13 (m, 2H), 3.40 (t,
J=5.95 Hz, 2H), 3.44–3.45 (m, 2H), 3.54–3.63 (m, 11H), 4.29 (s, 4H),
7.28–7.31 (m, 2H), 7.57–7.66 (m, 4H), 7.71–7.78 (m, 4H), 7.82–
7.84 ppm (m, 2H); 13C NMR (100 MHz, CDCl3): d=21.6/21.7, 28.4,
28.7, 29.6, 38.3, 39.0, 57.7, 69.4, 70.0, 70.2, 70.4, 70.6, 78.8, 127.1,
128.3, 129.6, 129.9/130.2, 135.2/135.9, 135.7/135.8, 138.3, 138.4,
145.1/145.5, 156.0, 166.2, 194.2 ppm; LC-MS: m/z=669 [monosul-
fone+H]+, 825 [M+Na]+.
Synthesis of N-(prop-2-yn-1-yl)-4-[3-tosyl-2-(tosylmethyl)propa-
noyl]benzamide (2): Bis-sulfone 1 (20mg, 0.04581mmol, 1 equiv)
was dissolved in 1mL of DMF. Then HBTU (24mg, 0.06392mmol,
1.6 equiv) and DIEA (13 mL, 0.0799mmol, 2 equiv) were added to
this solution at 08C under argon. The reaction mixture was stirred
for 10min before propargylamine (3 mL, 0.04794mmol, 1.2 equiv)
was added. The resulting mixture was stirred overnight at RT. The
solvent was removed under high vacuum, and the product was
dissolved in CH2Cl2. The organic phase was washed with aqueous
NaHCO3 and brine and dried over anhydrous Na2SO4. The solvent
was evaporated under vacuum and the product was purified by
column chromatography to afford 19mg of 2 in 78% yield.
1H NMR (500 MHz, CDCl3): d=2.41 (s, 6H), 3.49 (q, J=6.9 Hz, 2H),
3.62 (q, J=6.9 Hz, 2H), 4.27 (q, J=2.5 Hz, 2H), 4.37 (m, 1H),7.36 (d,
J=8.2 Hz, 4H), 7.69 (d, J=8.2 Hz, 6H), 7.77 ppm (d, J=8.2 Hz, 2H);
LC-MS: m/z=538 [M+H]+, 382 [monosulfone+H]+ (Supporting In-
formation, Figure S1).
Synthesis of N-(3-{2-[2-(3-aminopropoxy)ethoxy]ethoxy}propyl)-
4-[3-tosyl-2-(tosylmethyl)propanoyl] benzamide (6): Compound
5 (500 mg, 0.623 mmol, 1 equiv) was dissolved in 3 mL of CH2Cl2,
and TFA (476 mL, 10 equiv) was added. The CH2Cl2 and TFA were re-
moved under vacuum at 308C to afford 416 mg of amine bis-sul-
1
fone 6 as a yellow oil in 95% yield. H NMR (400 MHz, CDCl3): d=
1.84–1.90 (m, 2H), 1.94–1.99 (m, 2H), 2.41 (s, 6H), 3.20–3.25
(m,2H), 3.47–3.55 (m, 4H), 3.57–3.62 (m, 8H), 3.72 (t, J=5.22 Hz,
2H), 4.33 (s, 4H), 7.33–7.35 (m, 2H), 7.59–7.67 (m, 4H), 7.69–7.77
(m, 4H), 7.83–7.85 ppm (m, 2H); 13C NMR (100 MHz, CDCl3): d=
21.74, 26.15, 29.30, 38.01, 40.97, 57.91, 69.04, 69.42, 69.74, 69.80,
70.28, 71.01, 127.33, 128.43, 129.78, 130.12, 135.01, 135.66/135.82,
137.53, 138.98, 145.48, 167.68, 194.50 ppm; LC-MS: m/z=547
[monosulfone+H]+, 703 [M+H]+.
Synthesis of 2-{2-[2-(prop-2-yn-1-yloxy)ethoxy]ethoxy}ethyl 4-[3-
tosyl-2-(tosylmethyl)
propanoyl]benzoate
(3):
Bis-sulfone
1 (200 mg, 0.4 mmol, 1 equiv), DMAP (6 mg, 48 mmol, 0.12 equiv),
EDC (128 mg, 0.48 mmol, 1.2 equiv) and ethynyl triethylene glycol
33 (90 mg, 0.48 mmol, 1.2 equiv) were dissolved in 4 mL of anhy-
drous CH2Cl2 under argon at 08C. The reaction mixture was stirred
at RT overnight. The mixture was purified by column chromatogra-
phy with hexane/ethyl acetate (1:2) to afford 182 mg of product 3
Synthesis of rhodamine bis-sulfone 7: Sulforhodamine B acid
chloride (60 mg, 0.102 mmol, 1 equiv) was dissolved in 2 mL of an-
hydrous DMF at 08C under argon. Then, amine bis-sulfone 6
(100 mg, 0.142 mmol, 1.4 equiv) and DIEA (50 mL, 0.306 mmol,
3 equiv) were added sequentially. The reaction mixture was stirred
at RT overnight. The solvent was removed under vacuum and the
mixture was purified by column chromatography with 10% metha-
nol in chloroform to afford 57 mg of the product 7 as pink solid in
45% yield. The product was characterized by LC-MS, which re-
vealed that monosulfone had already partially formed during
1
as pale yellow oil in 68% yield. H NMR (400 MHz, CDCl3): d=2.39
(s, 6H), 3.44 (m, 1H), 3.65 (m, 8H), 3.83 (m, 4H), 4.07 (m, 2H), 4.32
(m, 2H), 4.49 (m, 2H), 7.34 (d, J=8.1 Hz, 4H), 7.66 (m, 6H),
8.09 ppm (d, J=8.4 Hz, 2H); 13C NMR (400 MHz, CDCl3): d=21.4,
30.5, 60.1, 63.6, 64.4, 69.3, 69.4, 69.5, 77.0, 78.5, 128.5, 128.9, 129.9,
130.0, 134.6, 139.6, 141.1, 145.2, 165.7, 201.9 ppm; MALDI-TOF-MS:
m/z=671.39 [M+H]+.
Chem. Eur. J. 2014, 20, 1 – 12
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column chromatography due to elimination of p-toluenesulfinic
acid; LC-MS: m/z=1087 [monosulfone+H]+, 1243 [bis-sulfo-
ne+H]+ (Supporting Information, Figure S3).
uct was purified by column chromatography with 4% MeOH in
CHCl3. The solvent was removed under vacuum to afford 44 mg of
the product in 62% yield. The product was characterized by LC-
MS, which revealed that the monosulfone had already partially
formed during column chromatography due to elimination of p-
toluenesulfinic acid; LC-MS: m/z=795 [monosulfone+H]+, 951
[M+H]+ (Supporting Information, Figure S6).
Synthesis of palmityl bis-sulfone 8: Palmitic acid (134 mg,
0.5122 mmol) was dissolved in 3 mL of anhydrous DMF. Then
HBTU (312 mg, 0.82 mmol) and DIEA (169 mL, 1.024 mmol) were
added to this solution at 08C under argon. The reaction mixture
was stirred for 10 min before 6 (300 mg, 0.4268 mmol) was added.
The resulting mixture was stirred overnight at RT. The solvent was
removed under high vacuum and the product was dissolved in
CH2Cl2. The organic phase was washed with saturated NaHCO3
twice and with brine and dried over anhydrous Na2SO4. The crude
product was purified by column chromatography with 2% metha-
nol in chloroform to afford 313 mg of the product in 78% yield.
The product was characterized by LC-MS, which revealed that the
monosulfone had already partially formed during column chroma-
tography due to elimination of p-toluenesulfinic acid; LC-MS: m/
z=941 [M+H]+, 963 [M+Na]+, 785 [monosulfone+H]+, 807 [mon-
osulfone+Na]+ (Figure S4).
Synthesis of N-(3-{2-[2-(3-azidopropoxy)ethoxy]ethoxy}propyl)-4-
{3-(p-tolylthio)-2-[(p-tolylthio)methyl]propanoyl}benzamide (13):
Bis-sulfide 12 (380 mg, 0.866 mmol, 1 equiv) was dissolved in 4 mL
of anhydrous DMF. Then HBTU (530 mg, 1.386 mmol, 1.6 equiv)
and DIEA (290 mL, 1.732 mmol, 2 equiv) were added at 08C under
argon. The reaction mixture was stirred for 10 min before 38
(210 mg, 0.866 mmol, 1.2 equiv) was added and the resulting mix-
ture stirred overnight at RT. The solvent was evaporated under
high vacuum and the crude product was dissolved in CH2Cl2. The
organic phase was washed with saturated NaHCO3 twice and with
brine and dried over anhydrous Na2SO4. The solvent was evaporat-
ed under vacuum and crude product was purified by flash chroma-
1
tography to afford 403 mg of 13 in 70% yield. H NMR (400 MHz,
Synthesis of PBA bis-sulfone 9: 4-Carboxyphenylboronic acid
(58 mg, 0.3415 mmol) was dissolved in 2 mL of anhydrous DMF.
Then HBTU (208 mg, 0.5464 mmol) and DIEA (113 mL, 0.683 mmol)
were added to this solution at 08C under argon. The reaction mix-
ture was stirred for 10 min before 6 (200 mg, 0.2845 mmol,
1.2 equiv) was added. The resulting mixture was stirred overnight
at RT. The solvent was removed under high vacuum and the prod-
uct was dissolved in CHCl3. The organic phase was washed with sa-
turated NaHCO3 twice and with brine and dried over anhydrous
Na2SO4. The crude product was purified by column chromatogra-
phy with 4% MeOH in CHCl3. The solvent was removed under
vacuum to afford 145 mg of 9 in 60% yield; LC-MS: m/z=695
[monosulfone+H]+, 717 [monosulfone+Na]+, 851 [M+H]+, 875
[M+Na]+ (Supporting Information, Figure S5).
CDCl3): d=1.72–1.97 (m, 2H), 1.85–1.90 (m 2H), 2.32 (s, 6H), 3.09–
3.24 (m, 2H), 3.30 (t, J=6.7 Hz, 2H), 3.41–3.47 (m, 4H), 3.54–3.66
(m, 10H), 3.73–3.80 (m, 1H), 7.03 (d, J=8.0 Hz, 4H), 7.10 (d, J=
8.1 Hz, 4H), 7.56 (d, J=8.4 Hz, 2H), 7.73 ppm (d, J=8.4 Hz, 2H);
13C NMR (125 MHz, CDCl3): d=21.1, 28.6, 29.0, 36.4, 39.4, 45.6, 48.4,
67.9, 70.2, 70.3, 70.4, 70.4, 127.2, 128.5, 129.8, 131.2, 131.5, 137.2,
138.5, 138.9, 166.03, 200.33 ppm; ESI-MS: m/z=665 [M+H]+, 687
[M+Na]+.
Synthesis of N-(3-{2-[2-(3-azidopropoxy)ethoxy]ethoxy}propyl)-4-
[3-tosyl-2-(tosylmethyl)propanoyl]benzamide (14): Compound 13
(250 mg, 0.376 mmol, 1 equiv) and Oxone (1.39 g, 2.256 mmol,
6 equiv) were added to methanol/deionized water (1 mL, 1:1). The
reaction mixture was allowed to stir at RT for 48 h. The mixture
was diluted with deionized water (60 mL) and extracted with
chloroform (4ꢁ50 mL). More water was added to dissolve all the
inorganic solids and the aqueous layer was extracted once with
chloroform (50 mL). The chloroform extracts were combined and
washed once with brine. Then the organic layer was dried over an-
hydrous sodium sulfate and filtered. The solvent was removed
under vacuum to afford 247 mg of 14 in 90% yield. 1H NMR
(400 MHz, CDCl3): d=1.72–1.79 (m, 2H), 1.87–1.92 (m, 2H), 2.47 (s,
6H), 3.28–3.32 (t, J=6.7 Hz, 2H), 3.42–3.49 (m, 6H), 3.57–3.69 (m,
12H), 4.30–4.35 (m, 1H), 7.34 (d, J=8.0 Hz, 4H), 7.62–7.67 (m, 6H),
7.78 ppm (d, J=6.6 Hz, 2H); 13C NMR (125 MHz, CDCl3): d=21.8,
28.5, 29.0, 35.5, 48.4, 55.5, 67.9, 70.2, 70.3, 70.5, 71.0, 127.6, 128.3,
128.7, 130.2, 132.9, 135.3, 136.0, 145.6, 165.8, 202.5 ppm; MALDI-
TOF-MS: m/z=729.65 [M+H]+.
Synthesis of SBoc bis-sulfone 10: Compound 34 (98 mg,
0.4268 mmol) was dissolved in 2 mL of anhydrous DMF. Then
HBTU (312 mg, 0.82 mmol) and DIEA (169 mL, 1.024 mmol) were
added to this solution at 08C under argon. The reaction mixture
was stirred for 10 min before amine bis-sulfone
6 (300 mg,
0.4268 mmol) in 1 mL of anhydrous DMF was added. The resulting
mixture was stirred overnight at RT and the solvent was removed
under high vacuum. The product was dissolved in CHCl3 and
washed with NaHCO3 (3ꢁ) and brine and dried over anhydrous
Na2SO4. The solvent was evaporated under vacuum and the crude
product was purified by column chromatography with 5% metha-
nol in CHCl3 to afford 236 mg of 10 in 63% yield. 1H NMR
(400 MHz, CDCl3): d=1.47 (s, 9H), 1.70–1.74 (m, 2H), 1.90–1.93 (m,
2H), 2.40/2.47 (s, 6H), 3.29–3.34 (m, 2H), 3.41–3.44 (m, 2H), 3.45–
3.50 (m, 4H), 3.57–3.67 (m, 10H), 4.32 (s, 2H), 7.30–7.42 (m, 4H),
7.62–7.72 (m, 4H), 7.76–7.82 (m, 2H), 7.87–7.89 ppm (m, 2H);
13C NMR (100 MHz, CDCl3): d=21.8/21.9, 28.3, 28.9, 29.1, 34.8, 38.0,
39.3, 57.8, 69.6, 70.2, 70.52, 70.5, 70.4, 70.8, 86.1, 127.2, 128.4,
129.8, 130.0/130.3, 135.4/136.0, 135.9, 138.5, 139.8, 145.2/145.7,
166.3, 168.6, 194.4 ppm; MALDI-TOF-MS: m/z=899.42 [M+Na]+.
Synthesis of 2-[2-(2-{[1-(1-oxo-1-{4-[3-tosyl-2-(tosylmethyl)propa-
noyl]phenyl}-6,9,12-trioxa-2-azapentadecan-15-yl)-1H-1,2,3-tria-
zol-4-yl]methoxy}ethoxy)ethoxy]ethyl 5-[(3aS,4S,6aR)-2-oxohexa-
hydro-1H-thieno(3,4-d)imidazol-4-yl]pentanoate (15): Compound
14 (200 mg, 0.276 mmol, 1 equiv) and ethynyl biotin 35 (136 mg,
0.328 mmol, 1.2 equiv) were mixed in 2 mL of THF/H2O (1:1).
Sodium ascorbate (56 mg, 0.276 mmol, 1 equiv) and copper sulfate
(4.4 mg, 27.6 mmol, 0.1 equiv) were added to the mixture sequen-
tially. The reaction mixture was stirred at RT for 24 h. The solvent
was removed under reduced pressure by rotary evaporator. The
residue was dissolved in CHCl3 and the precipitate was removed
by filtration. The crude product was purified by column chroma-
tography with 10% methanol in chloroform to afford 265 mg of
the product 15 as a slightly yellow oil in 84% yield. 1H NMR
(400 MHz, CDCl3): d=1.86–1.93 (m, 4H), 2.06–2.12 (m, 2H), 2.33 (t,
2H), 2.71 (d, 1H), 2.47 (s, 6H), 2.86–2.91 (m, 1H), 3.11–3.15 (m, 1H),
Synthesis of tetrazole bis-sulfone 11: 4-(2-Phenyl-2H-tetrazol-5-yl)-
benzoic acid (TZ, 20 mg, 0.07512 mmol, 1 equiv) was dissolved in
1 mL of anhydrous DMF. Then HBTU (46 mg, 0.1202 mmol,
1.6 equiv) and DIEA (25 mL, 0.1502 mmol, 2 equiv) were added at
08C under argon. The reaction mixture was stirred for 10 min
before 6 (63 mg, 0.09014 mmol, 1.2 equiv) was added. The result-
ing mixture was stirred overnight at RT. The solvent was evaporat-
ed under high vacuum and the product was dissolved in ethyl ace-
tate. The organic phase was washed with saturated NaHCO3 twice
and with brine and dried over anhydrous Na2SO4. The crude prod-
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3.39 (t, 2H), 3.46–3.51 (m, 4H), 3.56–3.68 (m, 24H), 4.19–4.21 (m,
2H), 4.26–4.30 (m, 1H), 4.35–4.42 (m, 3H), 4.46–4.49 (m, 1H), 4.64
(s, 2H), 5.03 (s, 1H), 5.50 (s, 1H), 7.35 (d, 4H), 7.49 (t, 1H), 7.61 (s,
1H), 7.67 (d, 6H), 7.82 ppm (d, 2H); 13C NMR (400 MHz, CDCl3): d=
195.0, 173.5, 165.7, 163.5, 145.4, 144.5, 139.4, 135.8, 135.0, 130.0,
128.4, 128.0, 127.5, 123.1, 70.3, 70.3, 70.2, 70.2, 70.0, 69.9, 69.4,
68.9, 67.0, 59.9, 55.4, 55.3, 46.9, 40.3, 38.8, 35.3, 33.6, 30.0, 28.6,
28.1, 28.0, 24.5, 21.6 ppm; MALDI-TOF-MS: m/z=1143.61 [M+H]+.
Tetrazole-SST 22: HR-ESI-MS: m/z=760.34593 [M+3H]3+
,
1140.02032 [M+2H]2+
;
LC-MS: m/z=760 [M+3H]3+
,
1140
[M+2H]2+; calcd: 2278.02052 (Figure 1C and Supporting Informa-
tion, Figure S12).
Rhodamine-SST 23: MALDI-TOF-MS (CHCA matrix): m/z=2570.80
[M+H]+; calcd: 2569.09 (Supporting Information, Figure S13).
Biotin-SST 24: MALDI-TOF-MS (CHCA matrix): m/z=2470.73
[M+H]+; calcd: 2469.12 (Supporting Information, Figure S14).
Synthesis of dendritic intercalator G2-PAMAM(NHBoc)4 18b: A
degassed solution of 17b (142 mg, 121 mmol) and intercalator 16
(44 mg, 136 mmol) in 4.5 mL of MeCN/CH2Cl2 (2:1) was treated with
2,6-lutidine (5 mL, 4.3 mg, 36 mmol) and [Cu(MeCN)4]PF6 (4.2 mg,
11 mmol) at 238C under argon atmosphere. After 10 min the reac-
tion mixture turned green and the solution was heated to 708C for
1 h. After stirring for further 10 h at 238C, the reaction mixture was
diluted with 30 mL of CH2Cl2, washed with NaHCO3 (0.7m, 3ꢁ
10 mL), water (1ꢁ20 mL), and brine (1ꢁ20 mL), dried over MgSO4,
filtered, and concentrated. Flash chromatography (SiO2, gradient
13 to 23% MeOH in CH2Cl2) yielded 102 mg of dendritic intercala-
tor 18b as a colorless solid in 56% yield. Rf =0.21 (18% MeOH in
PBA-SST 25: HR-ESI-MS: m/z=714.99575 [MÀ2H2O+3H]3+
,
1080.99517 [MÀH2O+2H]2+; HR-MALDI-MS (DHB matrix): m/z=
2296.99563 [M+DHBÀ2H2O+H]+; m/z=2142.96879 (CHCA matrix):
[MÀ2H2O+H]+; calcd: 2177.98477 (Figure 1B, D and Supporting
Information, Figures S15, S16).
Palmityl-SST 26: MALDI-TOF-MS (CHCA matrix): m/z=2268.75
[M+H]+, 2290.82 [M+Na]+; calcd: 2267.18 (Supporting Informa-
tion, Figure S17).
Synthesis of SST conjugates by click chemistry
Synthesis of G2-SST 29: N3-G2 45 (4 mg, 5.71 mmol) and ethynyl-
SST 19 (5 mg, 2.50 mmol) were dissolved in 2 mL of H2O. To this so-
lution, sodium ascorbate (4 mg, 20.2 mm) and CuSO4 (2 mg,
12.5 mm) were added sequentially. The resulting reaction mixture
was stirred for 24 h at RT. The mixture was concentrated and puri-
fied by preparative HPLC on an Atlantis Prep OBD T3 Column (19ꢁ
100 mm, 5 mm) with the mobile phase starting from 100% solven-
t A (0.1% TFA in water) and 0% solvent B (0.1% TFA in acetonitrile)
(0–5 min), raising to 23% B in 2 min, 23% B for 10 min, raising to
26% B in 0.5 min, 26% B for 10 min, and finally reaching 100% B
in 5 min with a flow rate of 10 mLminÀ1. The absorbance was
monitored at 280 and 254 nm. The retention time for 29 was
37.3 min. 2 mg of the product 29 was obtained from lyophilization
in 30% yield. The product was characterized by MALDI-TOF-MS
with CHCA as matrix: m/z=2697.41 [M+H]+, 2719.33 [M+Na]+;
calcd: 2697.33 (Supporting Information, Figure S18).
1
CH2Cl2); H NMR (300 MHz, CD2Cl2): d=8.38 (s, 1H, Htriazole), 7.98 (d,
J=8.0 Hz, 2H), 7.79 (m, 4H), 7.47 (broad, 6H, NHamide), 7.39 (d, J=
8.0 Hz, 2H), 6.08 (s, 1H, Holefin), 5.99 (s, 1H, Holefin), 5.71 (s, 4H,
NHBoc), 4.52 (s, 2H, triazole CH2), 4.34 (s, 2H, CH2SO2), 3.26 (m, 12H,
CH2NHamide), 3.18 (d, J=4.7 Hz, 10H, CH2NHcarbamate
, triazole
CH2CH2N), 2.96 (s, 4H, NHCH2CH2N), 2.75 (s, 12H, CH2CO), 2.44 (s,
3H, PhCH3), 2.36 (s, 8H, NCH2CH2COouter), 2.27 (s, 4H,
NCH2CH2COinner), 1.40 ppm (s, 36H, CH3,Boc); FTIR: n˜ =544, 667, 710
(s), 796, 860, 955 (s), 1039, 1167 (vs), 1250, 1365, 1454, 1525 (s),
1645 (vs), 1685, 2330, 2362, 2827, 2978, 3084, 3300 cmÀ1 (vbr); FD-
MS: m/z=1518.6 [M+Na]+; MALDI-TOF-MS: m/z=1497 [M+H]+,
1535 [M+K]+; HR-ESI-MS: m/z (%): 1517.8171 (100), 1518.8258
(90), 1519.8469 (31), 1520.8556 (11); calcd for [M+Na]+: 1517.8166
(100), 1518,8200 (76), 1519, 8233 (29), 1520,8267 (7).
Synthesis of dendritic intercalator G2-PAMAM(NH2)4 TFA salt
(18a): 18b (27 mg, 18 mmol) was dissolved in 1 mL of TFA/CH2Cl2
(1:1) and the solution stirred for 2 h at 238C and concentrated to
obtain 44 mg of 18b in quantitative yield as a colorless solid
which is soluble in MeOH or water, but not in CH2Cl2. 1H NMR
(300 MHz, CD3OD): d=8.67 (s, 1H, Htriazole), 8.04 (d, J=8.3 Hz, 2H),
7.86 (m, 4H), 7.48 (d, J=8.2 Hz, 2H), 6.09 (s, 1H, Holefin), 6.00 (s, 1H,
Holefin), 5.05 (NHCO), 4.50 (s, 2H, SO2CH2), 3.96 (NHCO), 3.59 (s, 8H),
3.49 (t, J=5.9 Hz, 16H), 3.36 (4H), 3.09 (t, J=5.8 Hz, 8H), 2.91–2.67
(m, 12H), 2.47 ppm (s, 3H, PhCH3); MALDI-TOF-MS: m/z=1095
[M+H]+ (calcd: 1094.62), 1117 [M+Na]+.
Synthesis of SHA-SST (27): 2-Hydroxy-4-pent-4-ynamido-N-(trity-
loxy)benzamide (2.4 mg, 4.89 mmol, 2 equiv) was dissolved in 40 mL
of DMSO and the solution further diluted in 1 mL of CH2Cl2. To this
solution, azido-SST 20 (5 mg, 2.43 mmol, 1 equiv) in 1 mL of H2O
was added followed by addition of sodium ascorbate (1 mg,
5.05 mmol, 2 equiv) and CuSO4 (0.4 mg, 2.51 mmol, 1 equiv) sequen-
tially. The resulting mixture was shaken for 24 h at RT. The mixture
was concentrated and purified by preparative HPLC using an Atlan-
tis Prep OBD T3 Column (19ꢁ100 mm, 5 mm) with the mobile
phase starting from 100% solvent A (0.1% TFA in water) and 0%
solvent B (0.1% TFA in acetonitrile) (0–10 min), raising to 40% B in
5 min, 40% B for 9 min, raising to 45% B in 1 min, 45% B for
10 min, and finally reaching 100% B in 5 min with a flow rate of
10 mLminÀ1. The absorbance was monitored at 280 and 254 nm.
4 mg of trityl-protected SHA-SST was obtained from lyophilization
and its retention time was 29.789 min. The trityl-protected SHA-
SST was dissolved in 1 mL of MeCN/H2O (1:1), and 200 mL of TFA
and 120 mL of triisopropylsilane were added. After 12 h reaction at
RT, the mixture was concentrated and purified by preparative HPLC
on an Atlantis Prep OBD T3 Column (19ꢁ100 mm, 5 mm) with the
mobile phase starting from 100% solvent A (0.1% TFA in water)
and 0% solvent B (0.1% TFA in acetonitrile) (0–5 min), raising to
36% B in 5 min, 36% B for 8 min, and finally reaching 100% B in
5 min with a flow rate of 10 mLminÀ1. The absorbance was moni-
tored at 280 and 220 nm. 2 mg of product 27 was obtained from
lyophilization in 35% overall yield and its retention time was
13.12 min. The product was characterized by HR-ESI-MS and HR-
MALDI-MS with CHCA as matrix. HR-ESI-MS: m/z=577.01294 [M+
Functionalization of SST
Bis-sulfones with different functionalities were preincubated in
50 mm phosphate buffer (pH 7.8) overnight to allow the formation
of the corresponding monosulfone. SST was reduced by 2 equiv
TCEP for 30 min and subsequently treated with the monosulfone
to afford SST conjugates with corresponding functionalities (ethyn-
yl, azido, biotin, SBoc, rhodamine, tetrazole, palmityl, and PBA;
Figure 2). The SST derivatives were purified by HPLC and character-
ized by MALDI-MS, LC-MS, or HR-MS.
Ethynyl-SST 19: MALDI-TOF-MS (CHCA matrix): m/z=1998.22
[M+H]+; calcd: 1996.87 (Supporting Information, Figure S9).
Azido-SST 20: MALDI-TOF-MS (CHCA matrix): m/z=2056.40
[M+H]+; calcd: 2054.94 (Supporting Information, Figure S10).
SH-SST 21: MALDI-TOF-MS (CHCA matrix): m/z=2104.69 [M+H]+;
calcd: 2102.93 (Supporting Information, Figure S11).
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4H]4+, 769.01462 [M+3H]3+, 1153.01891 [M+2H]2+, HR-MALDI-
MS: m/z=2305.01095 [M+H]+; calcd: 2304.02091 (Supporting In-
formation, Figure S19).
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Synthesis of compound 30: Tetrazole-SST 22 (2 mg, 0.878 mmol,
1 equiv) was dissolved in 1 mL of H2O and 6-maleimideocaproic
acid (1.9 mg, 9 mmol, 10 equiv) in 200 mL of DMSO was added. The
resulting mixture was irradiated by UV lamp (254 nm) for 2 h. The
mixture was purified by analytical HPLC on an MerckChroCART
125-4 Column with the mobile phase starting from 95% solvent A
(0.1% TFA in water) and 0% solvent B (0.1% TFA in acetonitrile)
(0–2 min), raising to 35% solvent B in 3 min and then to 38% sol-
vent B in 13 min, kept at 38% solvent B for 4 min, decreasing to
5% solvent B in 2 min, and finally balancing the column with 5%
solvent B for 1 min with a flow rate of 1 mLminÀ1. The absorbance
was monitored at 220 nm. The retention time of conjugate 30 was
19.08 min (Supporting Information, Figure S20). The product was
characterized by HR-ESI-MS and HR-MALDI-MS. HR-ESI-MS: m/z=
821.37410 [M+3H]3+, 1231.55805 [M+2H]2+ (Supporting Informa-
tion, Figure S21); HR-MALDI-MS (CHCA matrix): m/z=2462.08742
[M+H]+ (calcd: 2461.09888, Figure 2B).
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Confocal microscopy study on phototriggered reaction in
live cells
Thirty thousand A549 cells were plated in a m-Slide 8-well cham-
bered cover slip (ibidi, Martinsried, Germany) in 300 mL of high glu-
cose DMEM medium fortified with 10% fetal bovine serum, 1%
penicillin/streptomycin. The cells were cultured overnight to allow
adhesion. The next day, 0.1 mm of tetrazole-SST, 2 mm of MI-
PEG5000, or both compounds were added with fresh medium. The
cells were then further incubated for 4 h in the incubator. Before
imaging, cells were washed with phosphate-buffered saline (3ꢁ).
Then cells were irradiated under a 254 nm UV lamp for 0, 10, or
20 min. Imaging was performed with an LSM 710 laser scanning
confocal microscope system (Zeiss, Germany) coupled to an XL-
LSM 710 S incubator and equipped with a 63ꢁ oil-immersion ob-
jective. The fluorescence of formed 31 was recorded by using
a 440–510 nm filter and a Diode 405-30 laser for excitation. The ac-
quired images were processed with Image-J software. The photo-
triggered reaction was monitored by means of the normalized
single-cell fluorescence intensity (single-cell fluorescence intensity/
area of cell) by using the ImageJ software to quantify the fluores-
cent conjugate 31 generated in situ. Data were plotted as meansÆ
standard errors of the means (SEM) by using GraphPadPrism5 Soft-
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C.B.K. acknowledges continued funding from the Karlsruhe In-
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Interfaces program. T.W. is grateful to the financial support
from the BMBF project Biotechnologie 2020+ and the ERC
Synergy Grant 319130-BioQ.
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Keywords: disulfides
peptides · somatostatin
· imaging agents · intercalation ·
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Published online on && &&, 0000
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FULL PAPER
Site-specific protein chemistry: A ver-
satile disulfide intercalator toolbox was
prepared for the site-specific modifica-
tion of proteins or peptides with various
functional groups (see figure) under
mild, physiological conditions. Function-
alization of the peptide hormone soma-
tostatin with a tetrazole substituent fa-
cilitated the photoinduced, bioorthogo-
nal tetrazole–ene cycloaddition reaction
(NITEC) in living cells, which was moni-
tored by live-cell imaging.
&
Peptide Modification
T. Wang, Y. Wu, S. L. Kuan, O. Dumele,
M. Lamla, D. Y. W. Ng, M. Arzt, J. Thomas,
J. O. Mueller, C. Barner-Kowollik,* T. Weil*
&& – &&
A Disulfide Intercalator Toolbox for
the Site-Directed Modification of
Polypeptides
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