N-terminal dual protein functionalization by strain-promoted alkyne-nitrone cycloaddition

Article abstract of DOI:10.1039/c3ob00043e

Strain-promoted alkyne-nitrone cycloadditon (SPANC) was optimized as a versatile strategy for dual functionalization of peptides and proteins. The usefulness of the dual labeling protocol is first exemplified by the simultaneous introduction of a chloroquine and a stearyl moiety, two endosomal escape-improving functional groups, into the cell-penetrating peptide hLF (human lactoferrin). Additionally, we demonstrate that dual labeling of proteins is feasible by combining metal-free and copper-catalyzed click chemistry. First, SPANC is applied to enhanced green fluorescent protein to introduce both biotin and a terminal alkyne. The terminal acetylene then serves as a convenient anchor point for the CuAAC reaction with azido-containing fluorescein, thereby demonstrating the potential of combined SPANC and CuAAC for the straightforward, dual functionalization of proteins.

Full text of DOI:10.1039/c3ob00043e

Organic &
Biomolecular Chemistry
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PAPER
N-terminal dual protein functionalization by strain-
promoted alkyne–nitrone cycloaddition†
Cite this: Org. Biomol. Chem., 2013, 11,
2772
Rinske P. Temming,a Loek Eggermont,a Mark B. van Eldijk,b Jan C. M. van Hestb and
Floris L. van Delft*a
Strain-promoted alkyne–nitrone cycloadditon (SPANC) was optimized as a versatile strategy for dual func-
tionalization of peptides and proteins. The usefulness of the dual labeling protocol is first exemplified by
the simultaneous introduction of a chloroquine and a stearyl moiety, two endosomal escape-improving
functional groups, into the cell-penetrating peptide hLF (human lactoferrin). Additionally, we demon-
strate that dual labeling of proteins is feasible by combining metal-free and copper-catalyzed click chem-
istry. First, SPANC is applied to enhanced green fluorescent protein to introduce both biotin and a
terminal alkyne. The terminal acetylene then serves as a convenient anchor point for the CuAAC reaction
with azido-containing fluorescein, thereby demonstrating the potential of combined SPANC and CuAAC
for the straightforward, dual functionalization of proteins.
Received 9th January 2013,
Accepted 20th February 2013
DOI: 10.1039/c3ob00043e
www.rsc.org/obc
bifunctional linker to the protein, appears most attractive but
requires the potentially complex synthesis of a branched struc-
Introduction
Chemical conjugation of a functional tag, e.g. a polyethylene ture before conjugation. Approach B is more straightforward in
glycol (PEG) tail, a small molecule toxin, or a reporter mole- design and is based on combining two existing tools for
cule, is a well-established technique to modulate behavior or protein conjugation, which traditionally rely on the selective
study the biological function of proteins. As a further exten- targeting of canonical amino acids such as cysteine, lysine,
sion of the strategy, interest is growing in the dual conjugation tyrosine or tryptophan.2 However, such a process typically pro-
of two orthogonal functionalities to a single protein. For ceeds with low site-specificity and poor control over the
example, multimodality imaging is offering increased accuracy number of reactions. As a consequence, protein function may
for the diagnosis and surgical treatment of human disease. To be attenuated in case conjugation takes place in close proxi-
this end, an antibody (or another targeting protein) with high mity to for example the active site or a binding region. Some
affinity for a site of disease is labeled with two different approaches for selective N-terminal labeling have been devel-
imaging reporters to enable the simultaneous visualization of oped in recent years, however these require specific enzymes3,4
a single target with two diagnostic techniques, e.g. positron or careful fine-tuning of the reaction conditions to reach full
emission tomography (PET), computated tomography (CT), conversion to a singly modified product.5
fluorescence imaging, or magnetic resonance imaging (MRI).1
To overcome the limitations associated with conjugation to
Synthesis of the requisite dual-labeled targeting agents, natural amino acids, labeling techniques based on non-native
however, is more challenging than attachment of a single functional handles have been developed, for example hydr-
reporter molecule, as schematically depicted for three different azone6 or oxime ligation7 to an aldehyde. An aldehyde can be
strategies in Fig. 1. Approach A, involving conjugation of a chemically introduced into a protein by oxidation of the
N-terminal amino acid upon the action of sodium periodate8
or pyridoxal 5-phosphate,9 or at an internal site by genetically
aSynthetic Organic Chemistry, Institute for Molecules and Materials, Radboud
encoding a cysteine-containing pentapeptide tag for enzymatic
University Nijmegen, Nijmegen, The Netherlands. E-mail: f.vandelft@science.ru.nl;
oxidation.10 Another often applied non-natural functional
Fax: +31-(0)24-3653393; Tel: +31-(0)24-3652373
handle is azide,11 for reaction with a phosphine probe by Stau-
bBioorganic Chemistry, Institute for Molecules and Materials, Radboud University
dinger ligation,12 with a terminal acetylene by the copper-cata-
Nijmegen, Nijmegen, The Netherlands
†Electronic supplementary information (ESI) available: Results from SPANC con-
lyzed click reaction (CuAAC),13,14 or with a strained alkyne by
strain-promoted azide–alkyne cycloaddition (SPAAC).15 Several
dition optimization on SKYRAG and HspB2, mass spectra of S-hLF after functio-
nalization with BCN-CH2OH and N-methyl hydroxylamine, mass spectra of
methods have been developed for the site-selective introduc-
HspB2 after functionalization with BCN-CH2OH and N-propargyl hydroxylamine,
synthesis of SKYRAG, expression and purification of S-eGFP and 1H and 13C
NMR spectra of described compounds. See DOI: 10.1039/c3ob00043e
tion of an azide into a protein, such as post-translational di-
azotransfer,16,17 by protein expression in auxotrophic E. coli,18
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Fig. 1 Different approaches for the introduction of two functional labels to a peptide or protein. A. Via a pre-synthesized complex containing the two labels; B. via
two separate functional handles; C. via the N-terminal, one-pot SPANC reaction.
or by genetic encoding of azide-containing amino acids in the concentration-dependent manner.27 It was reported that at a
presence of a heterologous suppressor tRNA/aminoacyl tRNA concentration of 20 μM nearly all cells showed a homogeneous
synthetase pair with altered specificity.19 The latter technique cytoplasmic and nuclear peptide distribution next to vesicular
has also been found suitable for site-specific incorporation of staining. However, at lower concentrations (up to 10 μM), the
cyclooctyne-charged lysine into proteins,20,21 thereby enabling peptide was localized predominantly in vesicles, thus hamper-
the alternative SPAAC route for protein conjugation by reaction ing cytosolic delivery. In order to facilitate escape from the
with an azide-containing probe.
Despite the existence of
acidic endosomal compartment and enabling cytoplasmic tar-
a
multitude of conjugation geting, Andaloussi et al.28 introduced a chloroquine and a
methods, it requires great effort to introduce two functional stearyl moiety into the cell-penetrating peptide TP10 by coup-
handles into a protein, and to our knowledge only a few ling of stearic acid to the N-terminus, and a chloroquinoline
groups have successfully adapted this approach.22–24 In analog to a lysine residue of resin-bound TP10. We envisioned
summary, there is great need for a straightforward and practi- that the SPANC procedure could be employed to simul-
cal method for the simultaneous introduction of two imaging taneously introduce both the chloroquine analog and the
modalities or other functional labels into a peptide or protein.
stearyl group post-synthetically to hLF, providing a method for
In 2010, we reported that cycloaddition between cyclo- the improvement of the cell-penetrating properties of commer-
octynes and nitrones, the so-called strain-promoted alkyne– cially available CPPs. To this end, screening of SPANC con-
nitrone cycloaddition (SPANC),25 is exceptionally suitable for ditions was undertaken first, in order to determine the
the site-specific functionalization of peptides and proteins at optimized stoichiometry of reagents, because relatively high
the N-terminus. It was postulated by us, but not yet demon- concentrations of the peptide or protein (up to 8 mM) and/or a
strated, that the SPANC procedure would also enable dual large excess of reagents (60–80 equiv.)26 were earlier required.
protein functionalization. In addition, our earlier protocol As depicted in Scheme 1, the SPANC protocol involves the
required relatively high concentrations of the peptide or following sequence of events: first oxidative cleavage of an
protein and a large excess of reagents.26 We now report dual N-terminal serine (or threonine) with sodium periodate,
functionalization of peptides and proteins using an optimized followed by a benzenethiol quench to remove excess (per)
SPANC procedure (Fig. 1, approach C). The usefulness of the iodate. Then, without work-up or intermediate purification, an
dual labeling protocol is exemplified by the simultaneous alkylated hydroxylamine is added to the reaction mixture,
introduction of two endosomal escape-improving functional preferably in the presence of p-anisidine, leading to in situ
groups into the cell-penetrating peptide hLF (human lacto- nitrone formation and cycloaddition with a strained alkyne. As
ferrin). Additionally, we demonstrate that dual labeling of the strained alkyne, we selected bicyclo[6.1.0]nonyne (BCN)26
proteins is feasible by combining metal-free and copper-catalyzed over the more lipophilic cyclooctyne DIBAC,29 or DIBO.30 After
click chemistry, enabling the introduction of both biotin and a a first round of optimization on a model hexapeptide (ESI,
fluorophore into enhanced green fluorescent protein (eGFP).
Table S1†), it was found that, at a peptide concentration of
1 mM, the SPANC procedure was best executed with 10 equi-
valents of both MeNHOH·HCl and BCN-CH2OH. In the next
step, an hLF analog containing the requisite N-terminal serine
residue (S-hLF, Scheme 1) was oxidized under carefully con-
trolled conditions (0 °C, 1.0 equiv. NaIO4, 10 min) in order to
Results and discussion
Optimization of SPANC on S-hLF
Optimization of the one-pot three-step protocol for the intro- avoid oxidation of the disulfide linkage. Under these con-
duction of two functional labels into a biologically relevant ditions, mass spectrometric analysis (ESI, Fig. S1b†) showed
system was performed on hLF, a recently established cell-pene- the formation of the desired aldehyde 2 along with only a
trating peptide (CPP). hLF is a 20-mer peptide derived from minor amount of a p-methoxybenzenethiol adduct, presum-
the N-terminal domain of human lactoferrin and it was ably by opening of the disulfide linkage. Then, the optimized
recently found to transport cargo across cell membranes in a SPANC conditions were applied to the resulting glyoxyl-hLF
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Scheme 1 Procedure for the one-pot N-terminal dual SPANC labeling of the peptide S-hLF.
derivative, by addition of N-methyl hydroxylamine and incubation gave low conversion (20%) to the desired isoxazo-
BCN-CH2OH 5, leading to full conversion of the desired isox- line product, probably caused by the increased steric hin-
azoline product 4.
drance of both BCN-QN and stearylhydroxylamine compared
to their simpler analogs. Much to our satisfaction, by partially
increasing the stoichiometry of BCN-QN (40 equiv.) and by
prolonging the reaction time (3 days), full conversion to the
desired dual functionalized product was observed (Fig. 2C).
Dual functionalization of S-hLF
Now, the stage was set for the introduction of two functional
labels onto hLF via the SPANC procedure, i.e. a chloroquine
analog and a stearyl moiety for endosomal escape. Thus, we
synthesized the required BCN-trifluoromethylquinoline analog
Optimization of SPANC on the protein HspB2
8 (BCN-QN, Scheme 2) from commercially available BCN The next stage of SPANC labeling of a biomolecule involved
succinimidyl carbonate and amine-functionalized trifluoro- the application to a model medium-sized protein (20 kDa). To
methylquinoline 7.28 Similarly straightforward, N-stearyl- this end, we selected heat shock protein HspB2, a protein with
hydroxylamine 10 was synthesized by nucleophilic substitution an N-terminal serine that was conveniently expressed in-house
of stearyl bromide with N,O-diBoc-protected hydroxylamine31 (along with 25% of HspB3).32 Not unexpectedly, it was found
followed by Boc deprotection (TFA in DCM) to give the desired that periodate cleavage of the N-terminal aminoalcohol of
product 10 in 80% overall yield. These reagents were then HspB2 was accompanied by significant concomitant oxidation
applied in the SPANC reaction on glyoxyl-hLF 2 in the stoichio- of the free cysteine. Gratifyingly, alkylation of the free cysteine
metry that was also applied before (10 equiv. of N-stearyl- with iodoacetamide33 prior to periodate oxidation led to clean
hydroxylamine 10, 10 equiv. of BCN-QN 8). However, overnight conversion of the alkylated HspB2 to the desired glyoxyl
Scheme 2 Synthesis of BCN-trifluoromethylquinoline 8 and N-stearylhydroxylamine 10.
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Fig. 2 Deconvoluted mass spectra of S-hLF. A. Native peptide; B. after oxidation with NaIO4; C. after dual SPANC labeling with BCN-trifluoromethylquinoline and
N-stearylhydroxylamine.
product (ESI†). Given the compromised introduction of the propargyl isoxazoline derivative, as indicated by mass spectro-
sterically hindered stearyl hydroxylamine, we now opted to metric analysis (ESI, Fig. S2†).
introduce propargyl hydroxylamine instead. Propargyl
Dual functionalization of S-eGFP
hydroxylamine is small, readily synthesized and may serve as a
convenient handle for the subsequent further introduction of
azide-substituted functional handles by means of copper-cata-
lyzed click chemistry.13,14 Thus, varying amounts of reagents
were applied to HspB2 (ESI, Table S2†) for p-anisidine (10
equiv.–100 mM), BCN-CH2OH (10–100 equiv.) and N-propargyl-
hydroxylamine (10–100 equiv.), while opting to keep the
BCN-CH2OH stoichiometry to a minimum. From these experi-
ments it was concluded that optimal conditions involve 50
equivalents of N-propargylhydroxylamine and 20 equivalents
of BCN-CH2OH, leading to efficient formation of the desired
Finally, the 28 kDa protein enhanced green fluorescent protein
(eGFP)34 was selected to explore the generality of the optimized
SPANC reaction conditions, as well as to investigate the envi-
sioned further functionalization of the protein by a second,
copper-catalyzed derivatization. To this end, eGFP with an
N-terminal serine residue was expressed in E. coli. The result-
ing S-eGFP was subjected to oxidation with sodium periodate
(1.5 equiv., MW = 28 313, Fig. 3B), followed by quenching
with p-methoxybenzenethiol (10 equiv.), leading to the near
quantitative formation of the aldehyde derivative of eGFP as
Fig. 3 Deconvoluted mass spectra of S-eGFP. A. Native protein; B. after NaIO4 oxidation (hydrate form); C. after dual SPANC labeling with BCN-biotin and
N-propargylhydroxylamine; D. after CuAAC reaction with azido-fluorescein 12.
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Scheme 3 CuAAC of alkyne-functionalized eGFP with azido-fluorescein 12.
expected. To our delight, in this particular case no competitive indicated. TBTA was synthesized according to the literature
oxidation of the cysteine residues in eGFP was observed. Then, procedure,35 azido fluorescein 1236 was synthesized according
p-anisidine (100 mM final concentration), N-propargylhydroxyl- to the literature procedure with DCM as the solvent. All reac-
amine (50 equiv.) and BCN-biotin (20 equiv.) were added. After tions were monitored by TLC on Kieselgel F254 (Merck).
4 h, mass spectrometric analysis showed full conversion of Detection was by examination under UV light (254 nm) and
glyoxyl-eGFP to the dual labeled product (MW = 28 898 Da, by charring with aqueous KMnO4. Silica gel (Acros
Fig. 3C). The minor peak in the MS spectrum belongs to 0.035–0.070 mm) was used for chromatography.
residual (unoxidized) native S-eGFP (MW = 28 326 Da).
Dual labeling of S-hLF with BCN-CH2OH 5 and
N-methylhydroxylamine
CuAAC on alkyne-functionalized eGFP
To show the full extent of this technology for dual functional
labeling, a CuAAC reaction was next performed between the
N-terminal propargyl function and an azido-containing fluore-
scein derivative 12 (Scheme 3). To this end, isoxazoline 11 was
S-hLF-K-Fluo (EMC microcollections, SKCFQWQRNMRKV-
RGPPVSCIKRK(ε-carboxyfluorescein)-NH2 1, 1.6 mg, 486 nmol)
was dissolved in 1.1 mL of 0.1 M NH4OAc buffer (adjusted to
pH 8.34 using NaOH) and shaken (300 rpm) at 40 °C for 2 h to
dissolved in 20 mM phosphate buffer (pH 7.6) and subjected
form the disulphide bridge. 0.1 M HCl (5.5 μL) and 0.1 M
to typical CuAAC conditions (CuSO4, TBTA, sodium ascorbate
NH4OAc buffer pH 6.8 (1.1 mL) were added to adjust to pH 6.8.
The reaction mixture was cooled to 0 °C and NaIO4 (0.093 μg,
and azido-fluorescein 12). After overnight incubation at room
temperature, mass spectrometry analysis showed the near
486 nmol in water) was added. The reaction mixture was incu-
complete conversion of the starting alkyne to the desired tria-
bated for 10 min at 0 °C and immediately quenched with
zole product 13 (MW = 29 506, Fig. 3D), thereby demonstrating
p-methoxybenzenethiol (1.22 mL, 9.8 μmol dissolved in THF).
the potential of combined SPANC and CuAAC for the dual
After incubation at 0 °C for 1 h, the reaction mixture was
functionalization of proteins.
warmed to rt and diluted with THF (2.2 mL) to get a mixture of
THF–water 1 : 1. Then, p-anisidine (1.20 mg, 9.8 μmol dis-
solved in THF), N-methylhydroxylamine (0.82 mg, 9.8 μmol in
water) and BCN-CH2OH 5 (1.5 mg, 9.8 μmol in THF–water 1 : 1)
Experimental procedures
General procedures
were added. The reaction was incubated at rt for 16 h, until
mass spectrometry showed full conversion to isoxazoline 4.
ESI: calculated 3440.1 Da, found 3438.2 Da.
1H NMR spectra were recorded in CDCl3, DMSO or D2O on
Bruker DMX 300 or Varian Inova-400 spectrometers at 300 K.
TMS (δH 0.00), DMSO (δH 2.50) or D2O (δH 4.79) was used as
the internal reference. 13C NMR spectra were recorded in
CDCl3, DMSO or MeOD at 75 MHz on a Bruker DMX 300
Synthesis of N1-methyl-N1-(2-((7-trifluoromethyl)quinolin-4-yl)-
aminoethyl)propane-1,3-diamine (QN, 7)
spectrometer, using the central resonance of CDCl3 (δC 77.2), 4-Chloro-7-(trifluoromethyl)quinoline
(ABCR,
1.02
g,
DMSO (δC 39.5) or MeOD (δC 49.0) as the reference. Mass 4.4 mmol) and 2,2′-diamino-N-methyldiethylamine (TCI,
spectra were obtained on a JEOL AccuToF or a Thermo LCQ 6.6 mL, 50.9 mmol) were heated neat to 140 °C for 2 hours to
Fleet. Deconvoluted mass spectra were obtained with MagTran give a red solution. After cooling to rt, DCM (20 mL) was
1.03b2. Chemicals were purchased from Sigma-Aldrich or added and the solution was washed with a 5% aqueous
Acros and used without further purification, unless otherwise NaHCO3 solution (2 × 20 mL) and water (2 × 20 mL). The
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organic phase was dried (Na2SO4), filtered and the solvent was Synthesis of N-stearylhydroxylamine TFA salt (10)
removed under reduced pressure to give QN 7 (1.14 g, 83%). RF
N,O-diBoc-N-stearylhydroxylamine 9 (84.3 mg, 0.17 mmol) was
dissolved in DCM (1 mL) and TFA (1 mL) was added dropwise.
After stirring at rt for 1 h, solvents were removed in vacuo and
the product was co-evaporated with toluene (3 × 60 mL) to give
the TFA salt of N-stearylhydroxylamine 10 (67 mg, quant.). RF =
not determined; 1H NMR (300 MHz, DMSO) δ 3.10–3.05 (m,
2H), 1.60–1.52 (m, 2H), 1.24 (s, 30H), 0.89–0.82 (m, 3H); 13C
NMR (75 MHz, DMSO) δ = 50.2, 31.3, 30.7, 29.0, 28.9, 28.8,
28.7, 28.5, 25.7, 23.0, 22.1, 13.9; FT-IR νmax (cm−1): 3386, 2906,
2859, 1653, 1467, 1200, 1018, 616; HRMS (ESI+) m/z calcd for
C18H40NO (M + H)+: 286.3110, found: 286.3102.
= 0.10 (MeOH–DCM 1 : 2); 1H NMR (300 MHz, CDCl3) δ 8.61
(d, J = 5.3 Hz, 1H), 8.25 (s, 1H), 7.97 (d, J = 8.8 Hz, 1H), 7.57
(dd, J = 8.8, 1.9 Hz, 1H), 6.46 (d, J = 5.4 Hz, 1H), 6.25 (bs, 1H),
3.33 (m, 2H), 2.90 (t, J = 6.1 Hz, 2H), 2.81 (dd, J = 6.4, 5.1 Hz,
2H), 2.57 (t, J = 6.1 Hz, 2H), 2.33 (s, 3H); 13C NMR (75 MHz,
CDCl3) δ 152.4, 149.9, 147.7, 131.0, 130.6, 127.5, 125.9, 121.6,
120.0, 100.2, 59.7, 55.1, 41.9, 40.2, 39.5; FT-IR νmax (cm−1):
3250–2794, 1584, 1537, 1324, 1161, 1122, 811; HRMS (ESI+)
m/z calcd for C15H20F3N4 (M + H)+: 313.1640, found: 313.1631.
Synthesis of (1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-ylmethyl
2-(methyl(2-((7-(trifluoromethyl)quinolin-4-yl)amino)-
ethylamino)ethyl)carbamate (BCN-QN, 8)
Dual labeling of S-hLF with BCN-QN 8 and
N-stearylhydroxylamine 10
BCN succinimidyl carbamate (Synaffix B.V., 50.6 mg,
0.17 mmol) was dissolved in DCM (2 mL), QN 7 (82 mg,
0.26 mmol) and triethylamine (75 μL, 0.54 mmol) were added
and the reaction mixture was stirred at rt for 90 min. Saturated
NH4Cl solution (15 mL) was added and extracted with dichloro-
methane (2 × 15 mL). The DCM layer was dried using Na2SO4,
filtered and solvents were removed in vacuo. Products were sep-
arated by silica gel column chromatography (DCM–MeOH
19 : 1) to give BCN-QN 8 (79 mg, 93%). RF = 0.20 (DCM–MeOH
19 : 1); 1H NMR (300 MHz, CDCl3) δ 8.56 (d, J = 5.4 Hz, 1H),
8.26 (s, 1H), 8.07 (d, J = 8.9 Hz, 1H), 7.57 (d, J = 9.2 Hz, 1H),
6.44 (d, J = 5.4 Hz, 1H), 5.13 (bs, 1H), 4.07 (d, J = 7.9 Hz, 2H),
3.36–3.33 (m, 4H), 3.13 (bs, 1H), 2.83 (t, J = 5.7 Hz, 2H), 2.61 (t,
J = 5.9 Hz, 2H), 2.33 (s, 3H), 2.29–2.08 (m, 6H), 1.55–1.40 (m,
2H), 1.29–1.16 (m, 1H), 0.92–0.81 (m, 2H); 13C NMR (75 MHz,
CDCl3) δ 157.2, 151.5, 150.2, 146.8, 131.4, 131.0, 126.7, 125.8,
121.9, 120.6, 100.1, 98.8, 63.1, 57.3, 55.1, 42.1, 40.2, 38.7, 29.0,
21.5, 20.2, 17.8; FT-IR νmax (cm−1): 3334, 2924, 2846, 1701,
1588, 1571, 1537, 1372, 1325, 1268, 1156, 1126, 1070; HRMS
(ESI+) m/z calcd for C26H32N4O2F3 (M + H)+: 313.1640, found:
313.1631.
S-hLF-K-Fluo
(SKCFQWQRNMRKVRGPPVSCIKRK(carboxy-
fluorescein)-NH2, 1.6 mg, 486 nmol) was dissolved in 1.1 mL
of 0.1 M NH4OAc buffer (adjusted to pH 8.34 using NaOH) and
shaken (300 rpm) at 40 °C for 2 h to form the disulphide
bridge. 0.1 M HCl (5.5 μL) and 0.1 M NH4OAc buffer pH 6.8
(1.1 mL) were added to adjust to pH 6.8. The reaction mixture
was cooled to 0 °C and NaIO4 (0.093 μg, 486 nmol in water)
was added. The reaction mixture was incubated for 10 min at
0 °C and immediately quenched with p-methoxybenzenethiol
(1.22 mL, 9.8 μmol dissolved in THF). After incubation at 0 °C
for 1 h, the reaction mixture was warmed to rt and diluted
with THF (2.2 mL) to get a mixture of THF–water 1 : 1. Then,
p-anisidine (1.20 mg, 9.8 μmol dissolved in THF), N-stearyl-
hydroxylamine 10 (3.92 mg, 9.8 μmol in water) and BCN-QN 8
(4.8 mg, 9.8 μmol in THF–water 1 : 1) were added. The reaction
was incubated at rt for 3 days, until mass spectrometry showed
full conversion to the product. ESI: calculated 4016.9, found
4015.4 Da.
Synthesis of N,O-diBoc-N-propargylhydroxylamine
N,O-diBoc hydroxylamine (435 mg, 1.86 mmol) was dissolved
in dry DMF (15 mL) under an argon atmosphere and K2CO3
(343 mg, 2.48 mmol) was added. Propargyl bromide (80% solu-
tion in toluene, 230 μL, 2.07 mmol) was added dropwise, and
the reaction mixture was stirred at rt for 20 h. Water (150 mL)
was added and extracted with DCM (2 × 200 mL). The organic
layer was dried over Na2SO4, filtered and evaporated under
reduced pressure. The product was purified by silica gel
column chromatography (heptane–EtOAc 5 : 1) to give the title
compound as a colourless oil (488 mg, 93%). RF = 0.29
(heptane–EtOAc 5 : 1); 1H NMR (400 MHz, CDCl3) δ 4.31 (bs,
2H), 2.25 (t, J = 2.4 Hz, 1H), 1.51 (s, 9H), 1.47 (s, 9H); 13C NMR
(75 MHz, CDCl3) δ 154.7, 152.1, 85.1, 83.5, 77.1, 72.7, 40.6,
28.2, 28.1, 27.7, 27.7; FT-IR νmax (cm−1): 3296, 2980, 1792,
1718, 1372, 1230, 1148, 1091, 832; HRMS (CI+) m/z calcd for
C13H22NO5 (M + H)+: 272.1498, found: 272.1487.
Synthesis of N,O-diBoc-N-stearylhydroxylamine (9)
Stearyl bromide (1.7 g, 5.1 mmol) was dissolved in DMF
(20 mL) before addition of N,O-diBoc hydroxylamine (1.2 g,
5.1 mmol) and K2CO3 (861 mg, 6.2 mmol). The reaction
mixture was stirred for 3 days at room temperature. Water
(40 mL) was added and the product was extracted using EtOAc
(3 × 30 mL). The organic layer was dried over Na2SO4, filtered
and the solvent was removed under reduced pressure. The
product was purified by silica gel column chromatography
(heptane–EtOAc 15 : 1) to give N,O-diBoc-N-stearylhydroxyl-
amine 9 (1.98 g, 80%). RF = 0.58 (heptane–EtOAc 9 : 1); 1H
NMR (400 MHz, CDCl3) δ 3.56 (bs, 2H), 1.62–1.56 (m, 2H), 1.53
(s, 9H), 1.48 (s, 9H), 1.25 (s, 30H), 0.90–0.86 (m, 3H); 13C NMR
(75 MHz, CDCl3) δ 155.1, 152.5, 84.7, 82.2, 50.4, 32.1, 29.9,
29.5, 29.4, 28.4, 28.3, 27.9, 27.7, 26.7, 22.8, 14.3; FT-IR νmax
(cm−1): 2920, 2855, 1783, 1718, 1476, 1394, 1372, 1247, 1130,
Synthesis of N-propargylhydroxylamine HCl salt
841; HRMS (ESI+) m/z calcd for C28H56NO5 (M + H)+: 486.4159, N,O-diBoc-N-propargylhydroxylamine (563 mg, 2.075 mmol)
found: 486.4181.
was dissolved in EtOAc (10 mL) and H2O (5 mL) was added. To
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this mixture was added conc. HCl (5 mL) and the reaction was Cu/ascorbate mixture to give a 5 mM solution of all reagents.
stirred at rt for 2 h. The solvents were evaporated in vacuo to 20 μL of the previously prepared 31 μM solution of 11 in
give N-propargylhydroxylamine HCl salt as an off-white solid 20 mM phosphate buffer (pH 7.6) was taken (0.6 nmol protein)
(223 mg, quant.). RF = not determined; 1H NMR (400 MHz, and to this mixture was added 30 equivalents of Cu/ascorbate/
D2O) δ 4.14 (d, J = 2.6 Hz, 2H), 2.98 (t, J = 2.6 Hz, 1H); 13C NMR ligand and 20 equivalents of azido-fluorescein 12 (12 nmol,
(75 MHz, MeOD) δ 79.4, 72.8, 42.1; FT-IR νmax (cm−1): 3378, 7.3 μg in MeCN–water 1 : 1). After incubation at rt for 19 h,
3291, 2915, 2747, 2613, 1632, 664; HRMS (ESI+) m/z calcd for mass spectrometry analysis showed approximately 90% conver-
C3H6NO (M + H)+: 72.0449, found: 72.0436.
sion to triazole product 13. ESI-TOF: calculated 29 505 Da,
found 29 507 Da.
Dual labeling of HspB2/3 with N-propargylhydroxylamine and
BCN-CH2OH 5
A 245 μM stock solution of rHspB2/3 complex 3 : 1 in PBS Conclusions and outlook
buffer (150 μL) was diluted with 0.1 M NH4OAc buffer pH 6.8
For the first time, the SPANC protocol was employed for the
(600 μL) to give a 50 μM solution. An equal amount of a
10 mM stock solution of iodoacetamide in water (750 μM) was
added, and the mixture was incubated for 3.5 h at rt. The reac-
tion mixture was purified with a Millipore Amicon ultra cen-
trifugal filter (10 kDa cut-off). After each spin cycle the spinfilter
was refilled with 0.1 M NH4OAc buffer pH = 6.8, finally resulting
in a 100 μM solution. 1.1 equivalent NaIO4 was added
(40.6 nmol, 7.8 μg in water) and the reaction mixture was incu-
bated for 1 h at rt. 10 Equivalents of p-methoxybenzenethiol
(370 nmol, 53 μg in MeCN) was added and allowed to react for
16 h at rt. The reaction mixture was divided in 7 and to each
tube was added the required amount of p-anisidine (0 equiv.–
100 mM in MeCN), N-propargylhydroxylamine (10–100 equiv. in
water) and BCN-CH2OH 5 (10–100 equiv. in MeCN–water 1 : 1).
After 16 h at rt, the conversion was analysed with mass spectro-
metry. ESI-TOF: calculated 20 444, found 20 448.
site-selective one-pot introduction of two functional groups to
a peptide and a protein. First, a chloroquine analog and a
stearyl moiety were introduced into the CPP hLF, thus poten-
tially providing a method for the improvement of the cell-pene-
trating properties of commercially available CPPs. Currently,
investigations on the effectiveness of the dual labeled hLF
derivatives are ongoing. Further optimization of the SPANC
protocol for proteins led to conditions highly suitable for intro-
duction of N-propargyl hydroxylamine and BCN-biotin into
S-eGFP within a few hours. The terminal alkyne served as a
convenient anchor point for a CuAAC reaction with azido-
containing fluorescein, thereby demonstrating the potential of
combined SPANC and CuAAC for the dual functionalization of
proteins. In this respect, it is of relevance to note that the
applicability of combined SPANC and CuAAC is particularly
facilitated by the commercial availability of a wide range of
functionalized azides and cyclooctynes, in contrast to for
example N-functionalized hydroxylamines. We are currently
investigating various alternative synthetic routes towards such
functionalized N-hydroxylamines. We anticipate that the
SPANC protocol will find in particular application in the simul-
taneous visualization of a single target with two diagnostic
techniques, i.e. multimodality imaging. However, numerous
other (combinations of) functional groups can be envisioned,
e.g. polyethylene glycol (PEG) chains, imaging modalities,
drugs, targeting elements, nanoparticles and solid surfaces,
thereby further enhancing the prospect of SPANC for dual
labeling of peptides and proteins.
Dual labeling of S-eGFP with N-propargylhydroxylamine and
BCN-biotin
A 92 μM solution of S-eGFP in 0.1 M NH4OAc buffer pH 6.8
(65 μL, 6.0 nmol S-eGFP) was diluted with the same buffer
(130 μL), to give a 30 μM solution. NaIO4 was added (14.3 nmol,
2.7 μg in water) and the reaction mixture was incubated for
65 min at rt. p-Methoxybenzenethiol (60 nmol, 8.4 μg in MeCN)
was added and allowed to react for 16 h at rt. p-Anisidine was
added to give a final concentration of 100 mM (2.9 μmol,
0.36 mg in MeCN) and N-propargylhydroxylamine HCl salt
(285 nmol, 30.6 μg in water) and BCN-biotin (Synaffix B.V.,
114 nmol, 62.8 μg in MeCN–water 1 : 1) were added. After incu-
bation for 4 h at rt, mass spectrometry analysis showed full con-
version to isoxazoline product 11, along with a minor amount
of unoxidized, native S-eGFP. The product was purified using a
Millipore Amicon Ultra-0.5 mL centrifugal filter (10 kDa cut-
off). After each spin cycle the spinfilter was refilled with 20 mM
phosphate buffer (pH 7.6) to finally give 187 μL of a 31 μM solu-
tion. ESI-TOF: calculated 28 898 Da, found 28 899 Da.
Acknowledgements
Marjoke Debets is kindly acknowledged for the synthesis of
azido-fluorescein. This research was supported financially by
the Council for Chemical Sciences of The Netherlands Organ-
ization for Scientific Research (NMW-CW, F.L.v.D.)
CuAAC on propargyl-isoxazoline 11 with azido-fluorescein 12
A 20 mM solution of CuSO4·5H2O in water was prepared and
mixed in a 1 : 1 ratio with a 20 mM solution of sodium ascor-
bate in water. A 10 mM solution of the Cu(I) ligand Tris-
(benzyltriazolylmethyl)amine (TBTA) in tBuOH–DMSO 4 : 1
was prepared, and this was mixed in a 1 : 1 ratio with the
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