Click-enabled heterotrifunctional template for sequential bioconjugations

Article abstract of DOI:10.1039/c1ob06398g

A heterotrifunctional template was developed that utilizes thiol-maleimide and click chemistries (both copper-free and copper-mediated) to effect sequential biomolecule conjugations in a one-pot process. The breadth of compatible substrates was illustrated through highly efficient conjugations of protein, peptide, sugar, lipid, fluoroalkane, biotin and fluorophore molecules. This template should be useful for the creation of chemically-enhanced/enabled biotherapeutics, especially through the expression of discontinuous (and heterogeneous) epitopes.

Full text of DOI:10.1039/c1ob06398g

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PAPER
Click-enabled heterotrifunctional template for sequential bioconjugations†
David M. Beal,*‡ Victoria E. Albrow, George Burslem, Louisa Hitchen, Carla Fernandes, Cris Lapthorn,
Lee R. Roberts, Matthew D. Selby and Lyn H. Jones§
Received 16th August 2011, Accepted 10th October 2011
DOI: 10.1039/c1ob06398g
A heterotrifunctional template was developed that utilizes thiol–maleimide and click chemistries
(both copper-free and copper-mediated) to effect sequential biomolecule conjugations in a one-pot
process. The breadth of compatible substrates was illustrated through highly efficient conjugations of
protein, peptide, sugar, lipid, fluoroalkane, biotin and fluorophore molecules. This template should be
useful for the creation of chemically-enhanced/enabled biotherapeutics, especially through the
expression of discontinuous (and heterogeneous) epitopes.
using amide coupling/thiol-mediated chemistry to attach multiple
Introduction
copies of single peptides to proteins, such as Bovine Serum
The ability to label biological molecules such as proteins/peptides,
Albumin (BSA). The utilization of a multifunctional template to
mimic a greater area of the target protein, a discontinuous epitope,6
could greatly enhance the potency and selectivity of the antibodies
produced.
sugars and lipids with multiple functional groups is becoming
a key focus for applications in biotherapeutics and chemical
biology.1–3 The modification of these biological systems relies
on a ‘toolbox’ of suitably reactive reagents that can be effi-
ciently conjugated to protein molecules. The increase in breadth
of bioconjugation chemistries available for incorporation into
these types of system augments this toolbox of multifunctional
templates. For example Renard, Romieu and co-workers have
developed a heterotrifunctional cross-linking template for the
synthesis of bioconjugates which uses Copper-catalysed Azide–
Alkyne Cycloaddition (CuAAC, Fig. 1 upper), oxime and thiol-
based chemistries.4,5
Our initial goal to present discontinuous epitopes on protein
surfaces, whilst keeping the two units physically linked, required
a template which would satisfy specific requirements, including:
the formation of stable, irreversible bonds between the coupled
biomolecules and the template; the ability to derive a diverse
array of constructs from simple azide monomers, ideally al-
lowing a combinatorial one-pot methodology; incorporation of
a thiol to enable attachment to proteins using reliable thiol–
maleimide chemistry. In addition to this function, the ability to
utilize this reactivity to build up ‘tagging’ reagents for proteins
which had been labeled selectively with a suitably functionalized
molecule was also of interest as a potential tool for chemical
biology.7–10
To satisfy these stringent requirements for the linker, the work
of Aucagne,11 on sequential CuAAc reactions using silyl-protected
alkynes, and also that of Kele,12 on the incorporation of the
functional groups required for both Strain-Promoted Azide–
Alkyne Cycloaddition (SPAAC, Fig. 1 lower) and CuAAc onto
Bovine Serum Albumin (BSA), were encouraging. These works
suggest that by combining CuAAc and SPAAC into one template
it is possible to incorporate orthogonality whilst maintaining the
simplicity of the azide monomer set and avoiding the need for a
protecting group strategy. This therefore indicates that sequential
addition of diverse monomers to this template will allow for
expedient synthesis of compound arrays.
CuAAC13–16 and, more recently, SPAAC17 have become popular
methods for bioorthogonal labeling of biological systems. Both
methods are based on the Huisgen cyclization,18 the thermal
reaction of an alkyne and an azide to give a 1,2,3-triazole as
a mixture of regioisomers. The use of copper to catalyze this
process allows the reaction to proceed at room temperature giving
Fig. 1 CuAAC vs. SPAAC.
The synthesis of immunogens used to generate antibodies by the
stimulation of an immune response has classically been performed
Chemical Biology Group, Worldwide Medicinal Chemistry, Sandwich
Laboratories, Pfizer Global Research and Development, Ramsgate Road,
Sandwich, Kent, CT13 9NJ, UK. E-mail: dmb38@kent.ac.uk; Tel: +44 (0)
1304 824209
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c1ob06398g
‡ Current address: School of Biosciences, University of Kent, Canterbury,
Kent, CT2 7NZ
§ Current address: WorldWide Medicinal Chemistry, Pfizer, 200 Cam-
bridge Park Drive, Cambridge, MA 02140, USA.
548 | Org. Biomol. Chem., 2012, 10, 548–554
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a single regioisomer (CuAAC). The introduction of ring strain to
the alkyne-reacting partner lowers the required energy to affect
cycloaddition so that reaction occurs at ambient temperature,
giving a mixture of regioisomers (SPAAC).
TEG-azide 1524 as a reporter model system (Scheme 2). Initial
experiments showed that, at a reaction concentration of 1 mg ml-1
template and 1.1 equivalents of azide, conversion was clean but
slow, reaching 89% conversion after 24 h, but with both starting
materials remaining. By increasing the reaction concentration to
10 mg ml-1, complete conversion was achieved within 6 h.25 As
observed by the HPLC traces in Scheme 2, significant conversion
was achieved after one hour, suggesting either that the reaction
would be complete within six hours, or that there was scope to
lower the initial concentration of the reaction.
The subsequent CuAAC step (Scheme 3) was then explored us-
ing the fluorous tag 16,26 which is becoming increasingly common
for enrichment of samples, employing classic ‘click’ conditions of
CuSO4·5H2O and sodium ascorbate. The efficiency of the CuAAC
between 7 and 16 was such that complete conversion of the
intermediate template was achieved within 2 h and required no
optimization. To exemplify the compatibility of the template with
complex biomolecules, an array of click reactions with suitably
functionalized azides was undertaken (Scheme 4).
The investigated monomer set included peptides with a variety
of functional groups (aromatic, acidic and basic: the cyclic RGD
peptide 1027 and two random peptides DAVE-N3 11 and FAKL-
N3 12), to benchmark the template’s use for the display of
discontinuous epitopes.
To illustrate the breadth of chemistry, representative sugar
and lipid biomolecules 13 and 1428 were also conjugated. Other
conjugated molecules included several tags common in chemical
biology for enrichment, and the analysis of biomolecules from
complex mixtures 1524 (biotin), 1626 (fluorous tag) and 1729
(fluorophore).
The results of this array show that various functional groups
are tolerated by the template and the conditions required to
produce the transformation. In some cases, such as 7a and 7e,
a broadening of the LC-MS and a change in the peak shape
was observed. This peak shape can be attributed to the four
template components formed in the reaction, although there was
not adequate separation to delineate the ratio of isomers created.
To demonstrate the potential of this system for the synthesis
of immunogens, these molecules were incorporated onto com-
mercial maleimide-activated BSA,30 a standard starting material
for the development of immunogens to be used for the genera-
tion of poly- and mono-clonal antibodies. Maleimide activation
of BSA is achieved using 4-(N-maleimidomethyl)cyclohexane-
1-carboxylate, SMCC, and typically incorporates between 15
and 25 maleimides on each BSA molecule. The method of
choice for analyzing protein conjugates is via mass spectrometry
and, in particular, Matrix Assisted Laser Desorption Ionization
(MALDI). Conversion of the parent BSA to the maleimide-
activated species caused significant difficulties with the analysis of
the system by MALDI-analysis. The suspected reason for this was
the heterofunctional nature of the BSA activation giving different
points of attachment, as well as the presence of numerous dimers,
trimers and ‘polymeric’ species meaning that the sample contains
many sub species rather than a single component (see gel Fig.
2a). This therefore makes analysis by mass spectroscopy unfea-
sible. The incorporation of the thiol-reactive template with the
maleimide-activated species adds an extra layer of variation to the
system and further deteriorates the mass spectroscopy data. For
these reasons it was necessary to analyze the protein conjugates
The inherent trifunctionality of amino acids is such that the
core of the template can be derived from many of the abundant
amino acids, in particular lysine, cysteine, serine, aspartic and
glutamic acid. Since thiol–maleimide chemistry was to be used
for the attachment to proteins, the sulfur contained in cysteine
immediately suggested itself for use as a core. In order to enable
this chemistry, advantage was taken of the ability to deprotect
thioacetates in the presence of molecules containing maleimides,
using hydroxylamine, thus affecting a one-pot bioconjugation.19
Due to the lack of suitable commercial starting points and
concerns over transfer of the acyl group from sulfur to the b-
amino functionality, as observed in native chemical ligation,20 it
was decided to use alternatives to cysteine. The ideal method for
building the functionality onto the central core of the molecule
is by way of amide coupling, and therefore the use of aspartic
acid, glutamic acid and lysine offers more flexibility than serine.
The size, reactivity and ease of synthesis of the fluoro-cyclooctyne
acid, as described by Pigge,21 showed promise as a reagent for
incorporating the SPAAC functionality into the template. The
presence of the carboxylic acid in the cyclooctyne enabled further
refinement of the selection of amino acid core to lysine. An issue
associated with this choice of cyclooctyne is the incorporation of
a racemic centre into the template, resulting in two diastereomeric
products. Additionally, the SPAAC step provides a mixture of
regioisomers and therefore the final constructs are a mixture of
four compounds. However, for our initial purposes (the colocation
of two peptide epitopes on a single protein carrier) we decided
to continue with this approach for our proof of concept studies.
One could also argue that the four isomers formed by the
template after the SPAAC and CuAAC step allow the two
epitope fragments to sample a greater conformational space and
therefore may be more likely to adopt the native protein conforma-
tion.
Results and discussion
The synthesis of the template was achieved by way of solution-
phase chemistry using the readily available Fmoc-Lys(Boc)-
OH (Scheme 1). The order of incorporation of the reactive
functionalities onto the starting material was dictated by a need
to avoid subjecting the cyclooctyne to strong acid, due to its
instability in such conditions (unpublished observation). Initial
amide formation to incorporate the terminal alkyne using 2-
(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexaflu-
orophosphate methanaminium (HATU) gave 222 Fmoc depro-
tection of 2 using 4-methylpiperidine to give 322 was followed
by amide coupling with S-acetylthioacetic acid (SATA) using
HATU, providing 4. Subsequent Boc deprotection of 4 using HCl
in 1,4-dioxane furnished the amine scaffold, 5. 5 was coupled
to 1-fluorocyclooct-2-yne carboxylic acid using HATU yielding
the final template 6.23 This short and effective route enabled the
preparation of the desired template in 5 steps and gave 6.3% overall
yield as a 1 : 1 mixture of diastereomers.
The reactivity of the activated cyclooctyne towards azides in the
initial SPAAC step was investigated and optimized using biotin-
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Scheme 1 Synthesis of heterotrifunctional template.
Table 1 Description of the template composition added to BSA-
maleimide. Assay method/treatment of gel: A = Coomassie stain, B =
Coomassie stain and fluorescence scan, C = Coomassie stain, fluorescence
scan and western blot, D = Coomassie stain and western blot (see
supplemental)
which were synthesized with a qualitative method such as gel
electrophoresis and subsequent imaging of the gels. Following gel
electrophoresis, conjugates (Scheme 4, 9a–j) were then analyzed
using Western Blot (to assay for biotin incorporation, Fig. 2c),
fluorescence scanning (to assay for dye incorporation, Fig. 2b)
and Coomassie staining (to assay for molecular weight increase,
Fig. 2a) to confirm addition of the templates to the protein
(Table 1).
Conjugates 9b, 9c and 9d all show appreciable movement to
a higher molecular weight following Coomassie staining of the
gel (Fig. 2a, line shows mid-point of the commercial maleimide-
activated BSA band), displaying the successful reaction of the
templates with BSA. Estimation of the construct ‘loading’ onto the
BSA from these molecular weight shifts suggests that incorpora-
tion of up to five copies of the template onto the BSA was achieved.
Confirmation of the attachment of 9c and 9f to the protein was
achieved by scanning for the fluorescence on the gels at 488 nm
(Fig. 2b), showing appendage of the desired templates to BSA. For
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
R1N3
R2N3
Assay
14
10
A
10
12
A
10
17
B
10
11
A
12
11
A
15
17
C
15
10
D
15
14
D
15
16
D
15
13
D
the biotinylated samples 9f, 9g, 9i, 9j, 9h, Western Blot analysis
using an antibiotin antibody (Fig. 2c) demonstrates successful
conjugation to the carrier protein. Unfortunately samples 9e and
9a do not display a movement to a higher molecular weight.
Comparison with the line marking the mid-point of the maleimide
activated BSA (Fig. 2a), shows a shift to a greater electrophoretic
mobility for these conjugates.
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Fig. 2 Gel images of conjugates 9a–9j: (a) Coomassie stain, (b) Fluorescence scan of gel of dye labeled samples, (c) Western blot analysis of biotinylated
samples.
biomolecules could be synthesized in under two days. This
approach appears to augment discontinuous epitope expressions
for synthetic vaccine creation and antibody elicitation significantly
and is under further investigation.
Conclusions
This newly-developed template is an effective method for com-
bining various classes of molecule in a simple, robust and quick
procedure which does not require purification. Moreover, the
diversity available through this route offers great flexibility for
the formation of multiple functions, including discontinuous
epitope libraries, glycan arrays for tissue targetting and therapeutic
delivery and the synthesis of multifunctional ‘tags’ for use in
chemical biology. Further work in our group will explore templates
that avoid diastereomeric mixtures and display multiple epitopes
in rigidified architectures.
Experimental procedures
General methods
All reagents were purchased from Sigma-Aldrich, Fluka, VWR,
Expedion, Berry and Associates, Fluorous Technologies, Invitro-
gen, Pierce, and Charnwood Molecular, and used without further
Scheme 2 SPAAC reactivity at 10 mg mL-1.
purification, unless otherwise noted. Dry solvents were used as
The functionality contained in these templates did not allow for
visualization by alternative methods, and therefore no definitive
evidence for conjugation can be taken from the gels of these
samples (although through inference, it is highly likely these
conjugations were also successful). Although it was possible to
choose a simpler protein substrate to illustrate proof of concept
for our template that would aid characterization, it was decided to
pursue the more relevant BSA-maleimide system for our synthetic
immunology programs.
The results of the array display the robustness and efficiency
of both the SPAAC and CuAAC steps. Complete conversion
was achieved for both steps, as assessed by LC-MS, irrespective
of the azide-coupling partner utilized. The rate of both steps,
combined with the avoidance of the need to purify at any point
in the sequence, demonstrated that an array of trifunctional
purchased from Sigma-Aldrich. Chromatographic purifications
were performed on ISCO Companion Combiflash using Redisep
cartridges (normal phase 69-2203-304 etc. and reverse phase 69-
2203-410 etc. available from Teledyne ISCO) or using Merck
silica gel 60 (0.040 mm–0.63 mm) 23-400 MESH, catalogue no.
1.09385 and the indicated solvent eluents. Analytical thin layer
chromatography (TLC) was performed on Merck silica gel 60 F254
TLC glass plates. Infrared (IR) spectra were obtained using a
Nicolet Avatar 360 FTIR. Proton, carbon and fluorine nuclear
magnetic resonance spectra (1H NMR, 13C NMR and 19F NMR)
were recorded on a Varian Mercury 400BB spectrometer with
1
solvent resonance as the internal standard. H NMR data are
reported as follows: chemical shift, multiplicity (s = singlet, BS =
broad singlet, d = doublet, dd = doublet of doublets, dt = doublet of
triplets, t = triplet, q = quartet, m = multiplet), coupling constants
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Scheme 3 CuAAC reactivity – ELSD profile after 2 h.
Scheme 4 Synthesis of array of conjugates 9 from azide monomers 10–17.
552 | Org. Biomol. Chem., 2012, 10, 548–554
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(Hz), and integration. LC-MS acquired on a Waters ZQ ESCI
single quadrupole LC-MS using method: A: 0.1% formic acid in
water; B: 0.1% formic acid in acetonitrile; Column: Agilent Extend
C18 phase 50 ¥ 3 mm with 3 micron particle size; Gradient: 95–0%
over 3.5 min. Accurate mass data was acquired on a Thermo LTQ
Orbitrap ESI LC-MS or Bruker microTOF.
For details regarding the synthesis of azide monomers and
known compounds synthesized by alternative methods, 2 and 3,
please see the supplemental information†.
evaporated in vacuo to leave a yellow gum, which was washed with
diethyl ether and then acetonitrile to give the product, HCl salt, as
a white solid which was dried under vacuum (350 mg, 92%) m.p.
72–74 ◦C.
IR:
(amide), 2855 (NH2), 1634 (C O), 1535 (C O). 1H NMR
(400 MHz, CD3OD) (ppm) 1.34–1.55 (m, H, HN-
CH(CO)-CH2CH2CH2CH2NH3Cl), 1.59–1.75 (m, H,
CH2CH2CH2NH3Cl NH-CH(CO)CH2CH2CH2), 1.81–
1.93 (m, H, NH-CH(CO)CH2CH2CH2), 2.37 (s, H,
n
max/cm-1 3264 (C C–H), 3043 (amide), 2929
d
2
3
+
1
3
SCOCH3), 2.58 (t, J = 2.45 Hz, 1 H, CH2C CH), 2.92
(t, J = 7.6 Hz, 2 H, CH2CH2NH3Cl), 3.63–3.74 (m, 2 H,
CH3COSCH2CONH), 3.96 (t, J = 2.45 Hz, 2 H, NHCH2C CH),
4.30–4.38 (m, 1 H, NHCH(CO)CH2). 13C NMR (101 MHz,
CDCl3) d (ppm) 23.7 (HN-CH(CO)-CH2CH2CH2CH2NH3Cl),
28.1 (CH2CH2CH2NH3Cl), 29.7 (CONHCH2CCH), 30.2
(CH3COSCH2), 32.6 (NH-CH(CO)CH2CH2CH2), 34.0 (CH3-
COSCH2CONH), 40.7 (CH2CH2NH3Cl), 54.6 (NHCH(CO)-
CH2), 72.4 (CH2C CH), 80.5 (CH2C CH), 170.9
(SCH2CONH), 173.5 (CHCONHCH2CC, 197.1 (CH3COSCH2).
HRMS (ESI) calc. For C13H22N3O3S [M + H]+: 300.1382; found
300.1381, error = -1.70 ppm.
(R)-S-2-(6-(tert-Butoxycarbonylamino)-1-oxo-1-(prop-2-
ynylamino)hexan-2-ylamino)-2-oxoethyl ethanethioate (4)
SATA (592 mg/4.41 mmol) was dissolved in dichloromethane
(10 ml) and to this solution the (COCl)2 (339 ml/3.88 mmol)
was added, followed by treatment with dimethylformamide
(14 ml/0.176 mmol). The reaction was stirred at ambient tem-
perature under N2 for one hour. On stirring of the reaction
effervescence was observed, caused by the liberation of CO2. 3
(1 g/3.529 mmol) was dissolved in dichloromethane (5 ml) and
treated with TEA (1.48 ml/10.6 mmol) and cooled to 0 ◦C. To this
solution the acid chloride solution was added over one minute. The
reaction was allowed to warm to ambient temperature and then
stirred for one hour. The reaction was quenched by the addition of
2 M HCl (10 ml). The organics were extracted, dried over MgSO4
and evaporated in vacuo to leave an oil. Acetonitrile was added
to this oil causing precipitation and the resulting material was
collected by filtration to give a white solid (450 mg, 32%) m.p.
135–137 ◦C. (The mother liquors contained a large amount of
relatively pure product which was not isolated.)
S-(2-(((2R)-6-(1-Fluorocyclooct-2-ynecarboxamido)-1-oxo-1-
(prop-2-yn-1-ylamino)hexan-2-yl)amino-2-oxoethyl) ethanethioate
(6)
To a stirred solution, under N2, of amine (5) (189 mg,
0.56 mmol) in dichloromethane (5 ml) was added DIPEA (330 ml,
1.89 mmol). A solution of cyclooctyne (19) (204 mg, 1.2 mmol)
in dichloromethane (4 ml) was then added to the amine solution
followed by HATU (456 mg, 1.2 mmol). The solution was stirred
overnight at ambient temperature. The solvent was evaporated
in vacuo and the crude mixture suspended in ethyl acetate. The
organic phase was washed with water (¥2) and brine (¥1), dried
over (MgSO4), filtered and concentrated under reduced pressure.
The crude mixture was purified by flash column chromatography
(silica gel, mesh, 20 to 100% ethyl acetate in heptane) to give
the product as an off-white solid with a lower running impurity.
The material was dissolved in ethyl acetate and washed with water
(¥3), dried over (MgSO4), filtered and concentrated under reduced
pressure to give the product as a white solid (94 mg, 39%) as a 1 : 1
mixture of diastereomers, m.p. 59–61 ◦C.
IR: nmax/cm-1 3280 (C C–H), 2974 (amide), 2917 (amide),
1687 (C O), 1629 (C O), 1531 (C O), 1249 (C–O), 792
1
(C C–H). H NMR (400 MHz, DMSO-d6) d (ppm) 1.14–1.28
(m, 2 H, HN-CH(CO)-CH2CH2CH2CH2NH), 1.28–1.40 (m,
11 H, NHCO-OC(CH3)3
1.40–1.54 (m, H, NH-CH(CO)CH2CH2CH2), 1.54–1.68
(m, H, NH-CH(CO)CH2CH2CH2), 2.30–2.38 (m, H,
+ CH2CH2CH2NHCO-O(CH3)3),
1
1
3
SCOCH3), 2.86 (m, 2 H, CH2CH2NHCO-OC(CH3)3), 3.08
(t, J = 2.4 Hz, 1 H, CH2C CH), 3.65 (d, J = 1.2 Hz, 2 H,
CH3COSCH2CONH), 3.81–3.86 (m, 2 H, NHCH2C CH),
4.17 (td, J = 8.4, 5.2 Hz, 1 H, NHCH(CO)CH2), 6.60–6.79
(m, 1 H, CH2CH2NHCO-OC(CH3)3), 8.22 (d, J = 8.0 Hz,
1 H, CH3COSCH2CONHCH), 8.34 (t, J = 5.6 Hz, 1 H,
HCCCH2NHCOCH). 13C NMR (100 MHz, DMSO-d6): d (ppm)
22.5 (NH-CH(CO)CH2CH2CH2), 27.9 (NHCH2C CH), 28.2
IR: nmax/cm-1 3272 (C CH), 3064 (C C in ring), 2929
(amide), 2871 (amide), 1662 (C O), 1634 (C O), 1531 (C O),
1120 (C–O), 959 (C C–H). 1H NMR (400 MHz, CDCl3
mixture of diastereomers) d (ppm) 1.22–1.80 (m, 8 H), 1.81–
2.16 (m, 5 H), 2.17–2.52 (m, 7 H), 3.15–3.44 (m, 2 H,
CH2CH2NHCOCF), 3.53–3.69 (m, 2 H, CH3COSCH2CONH),
(NHCO-OC(CH3)3),
29.1
(CH2CH2CH2NHCO-O(CH3)3),
30.1 (SCOCH3), 31.7 (NH-CH(CO)CH2CH2CH2), 32.5
(CH3COSCH2CONH), 40.5 (CH2CH2NHCO-O(CH3)3), 52.6
(NHCH(CO)CH2), 72.9 (CH2C CH), 77.3 (OC(CH3)3), 80.9
(CH2C CH), 155.5 (CO), 166.7 (CO), 171.1 (CO), 194.5 (CO)
HRMS (ESI) calc. For C18H29N3O5SNa [M + Na]+: 422.1726;
found 422.1733, error = -3.1 ppm.
3.97–4.08 (m,
2 H, NHCH2CCH), 4.31–4.44 (m, 1 H,
NHCH(CO)CH2), 6.52 (m, 1 H, HC CCH2NHCOCH), 6.75
(m, 1 H, CH3COSCH2CONHCH), 6.88 (d, J = 7.6 Hz, 0.5 H,
CH2NHCOCF), 6.94 (d, J = 7.4 Hz, 0.5 H, CH2NHCOCF).
13C NMR (101 MHz, CDCl3 mixture of diastereomers) d (ppm)
20.6, 22.1, 22.3, 25.7, 28.9, 28.9, 29.2, 30.3, 30.3, 30.9, 31.1, 33.2,
(R)-S-2-(6-Amino-1-oxo-1-(prop-2-ynylamino)hexan-2-ylamino)-
2-oxoethyl ethanethioate·HCl (5)
2
33.3, 33.9, 33.9, 38.6, 38.8, 46.3 (d, JC–F = 24.3 Hz), 46.4 (d,
2
Boc protected amine (4) (450 mg, 1.13 mmol) was treated with HCl
in 1,4-dioxane (4 M, 10 ml, 40 mmol) and the reaction was stirred
at ambient temperature under N2 for one hour. The reaction was
2JC–F = 24.3 Hz), 53.2, 53.3, 71.5, 79.3, 79.3, 87.3 (d, JC–F
=
2
31.7 Hz), 87.3 (d, JC–F = 31.7 Hz), 94.4 (d, JC–F = 185.8 Hz),
94.5 (d, JC–F = 185.8 Hz), 109.4 (d, JC–F = 10.3 Hz), 109.5
3
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(d, 3JC–F = 10.3 Hz), 168.3, 168.4, 168.7 (d, 2JC–F = 19.3 Hz), 168.9
Notes and references
2
(d, JC–F = 19.3 Hz), 170.8, 170.9, 195.6. 19F NMR (376 MHz,
1 M. E. Bunnage, D. E. Thurston and D. Fox, New Frontiers in Chemical
Biology: Enabling Drug Discovery, Royal Society of Chemistry, Cam-
bridge, 2010, ISBN-10: 184973125X.
2 S. L. Schreiber, T. M. Kapoor and G. Wess, Chemical Biology: From
Small Molecules to Systems Biology and Drug Design, Wiley-VCH,
Weinheim, 2007, ISBN-10: 3527311505.
CDCl3 mixture of diastereomers) d (ppm) -145.89–-145.53 (m).
HRMS (ESI) calc. For C22H30FN3O4SNa [M + Na]+: 474.1839;
found 474.1840, error = -1.40 ppm.
SPAAC general method
3 T. P. Begley, Wiley Encyclopedia of Chemical Biology, Wiley on-line
library, Online ISBN: 978047004867210.1002/9780470048672.
4 G. Clave´, H. Volland, M. Flaender, D. Gasparutto, A. Romieu and
P.-Y. Renard, Org. Biomol. Chem., 2010, 8, 4329–4345.
5 G. Clave´, H. Boutal, A. Hoang, F. Perraut, H. Volland, P.-Y. Renard
and A. Romieu, Org. Biomol. Chem., 2008, 6, 3065–3078.
6 C. Chamorro, J. A. W. Krujitzer, M. Farsaraki, J. Balzarini and R.
Liskamp, Chem. Commun., 2009, 821–823.
A 10 mg ml-1 solution of 6 in DMSO was treated with 1.1 eq of an
azide DMSO stock solution. The reaction was rolled for six hours
at which point LC-MS analysis showed complete consumption of
6 and the formation of a single product, 7. No purification was
carried out before the CuAAC step was undertaken. For more
information please see the supplemental information†.
7 G. C. Adam, E. J. Sorensen and B. F. Cravatt, Mol. Cell. Proteomics,
2002, 10, 828–835.
8 A. F. H. Berry, W. P. Heal, A. K. Taradfer, T. Tolmachova, R. A. Baron,
M. C. Seabra and E. W. Tate, ChemBioChem, 2010, 11, 771–773.
9 M. Trester-Zedlitz, K. Kamada, S. K. Burley, D. Fenyo¨, B. T. Chait
and T. W. Muir, J. Am. Chem. Soc., 2003, 125, 2416–2425.
10 D. S. Wilbur and B. E. B Sandberg, US pat 0023288 A1, 2001.
11 I. E. Valverde, A. F. Delmas and V. Aucagne, Tetrahedron, 2009, 65,
7597–7602.
CUAAC general method
The DMSO solution of compound 7, from the SPAAC reaction,
was treated with 2 eq of an azide DMSO stock solution, followed
by the addition of 0.5 M sodium ascorbate (7 eq) and 0.25 M
CuSO4.5H2O (5 eq). The reaction was rolled for two hours before
LC-MS analysis of the reaction showed complete conversion
from 7 to 8. For further information please see the supplemental
information†.
12 P. Kele, G. Mezo, D. Achatz and O. Wolfbeis, Angew. Chem., Int. Ed.,
2009, 121, 344–347.
13 C. W. Tornøe, C. Christensen and M. Meldal, J. Org. Chem., 2002, 67,
3057–3064.
14 V. V. Rostovtsev, L. G. Green, V. V. Fokin and B. K. Sharpless, Angew.
Chem., Int. Ed., 2002, 41, 2596–2599.
15 V. Hong, S. I. Presolski, C. Ma and M. G. Finn, Angew. Chem., Int.
Ed., 2009, 48, 9879–9883.
16 M. Meldal and C. W. Tornøe, Chem. Rev., 2008, 108, 2952–3015.
17 J. C. Jewett and C. R. Bertozzi, Chem. Soc. Rev., 2010, 39, 1272–
1279.
18 R. Huisgen, Angew. Chem., Int. Ed. Engl., 1963, 2, 565–598.
19 F. Morhard, J. Pipper, R. Dahint and M. Grunze, Sens. Actuators,
2000, B 70, 232–242.
20 P. E. Dawson, T. W. Muir, I. Clark-Lewis and S. B. Kent, Science, 1994,
266, 776–779.
21 M. K. Schultz, S. G. Parameswarappa and F. G. Pigge, Org. Lett., 2010,
12, 2398–2401.
22 M. Van Dijk, M. L. Nollet, P. Weijers, A. C. Dechesne, C. F. Van
Nostrum, W. E. Hennink, D. T. S. Rijkers and R. M. J. Liskamp,
Biomacromolecules, 2008, 9, 2834–2843.
23 Template 6 is isolated as a mixture of two diastereomers due to the use
of racemic cyclooctyne acid.
24 The Biotin-TEG-Azide is available from Berry and Associates, BT 1085.
25 HPLC separation of regioisomeric products formed in SPAAC is
dependent on azide used. 7a and 7e show evidence of two unresolved
peaks.
Conjugation to BSA-maleimide
BSA-maleimide (Pierce – 77115) 1 mg was reconstituted in 100 ml
distilled water and then diluted to 1.5 ml with dPBS. Aliquots
of 75 ml (74.5 nmol) were treated with 8.3 ml of PBS + 5 M
NH2OH·HCl +50 mM EDTA·2Na (pH had been adjusted to
pH4). To an aliquot 10 ml of DMSO template solution 8 was
added and then the reaction was incubated on a roller for four
hours. These samples were then subjected to purification by
size-exclusion chromatography, using a GE Healthcare illustra
Nap 5 col. eluting with dPBS. The resulting solutions were then
concentrated to approximately 95 ml using a Millipore Amicon
ultra centrifugal filter device with a 3 KDa cut off (cat. No.
UFC800324). Confirmation of conjugation to the protein was
achieved by gel electrophoresis, using conditions described in the
supplemental information.
26 The perfluorinated azide is available from Flourous technologies,
BR017190.
Acknowledgements
27 I. Dijkgraaf, A. Y. Rijnders, A. Soede, A. C. Dechesne, G. W. van Esse,
A. J. Brouwer, F. H. M. Corstens, O. C. Boerman, D. T. S. Rijkers and
R. M. J. Liskamp, Org. Biomol. Chem., 2007, 5, 935–944.
28 A. K. Sanki and L. K. Mahal, Synlett, 2006, 3, 455–459.
29 The Alexafluor 488 azide is available from Invitrogen, A10266.
30 The Imject Maleimide-Activated BSA is available from Pierce, 77116,
http://www.piercenet.com.
We thank Nichola Davies and Torren Peakman (SASS, Pfizer
Ltd) for help with NMR assignment, Hannah Vuong (Sandwich
Chemistry, Pfizer Ltd) for the supply of reagents and Kirsty
Corrigan for proof-reading of the manuscript.
554 | Org. Biomol. Chem., 2012, 10, 548–554
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The Royal Society of Chemistry 2012
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