Triazole biotin: A tight-binding biotinidase-resistant conjugate

Article abstract of DOI:10.1039/c3ob41837e

The natural amide bond found in all biotinylated proteins has been replaced with a triazole through CuAAC reaction of an alkynyl biotin derivative. The resultant triazole-linked adducts are shown to be highly resistant to the ubiquitous hydrolytic enzyme biotinidase and to bind avidin with dissociation constants in the low pM range. Application of this strategy to the production of a series of biotinidase-resistant biotin-Gd-DOTA contrast agents is demonstrated. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3ob41837e

Organic &
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Triazole biotin: a tight-binding biotinidase-resistant
conjugate†‡
Cite this: Org. Biomol. Chem., 2013, 11,
7700
Anne I. Germeroth,a Jill R. Hanna,a Rehana Karim,a Franziska Kundel,a
Jonathan Lowther,a Peter G. N. Neate,a Elizabeth A. Blackburn,b Martin A. Wear,b
Dominic J. Campopianoa and Alison N. Hulme*a
The natural amide bond found in all biotinylated proteins has been replaced with a triazole through
CuAAC reaction of an alkynyl biotin derivative. The resultant triazole-linked adducts are shown to be
highly resistant to the ubiquitous hydrolytic enzyme biotinidase and to bind avidin with dissociation con-
stants in the low pM range. Application of this strategy to the production of a series of biotinidase-
resistant biotin-Gd-DOTA contrast agents is demonstrated.
Received 18th July 2013,
Accepted 30th September 2013
DOI: 10.1039/c3ob41837e
www.rsc.org/obc
shown to catalyze the hydrolysis of other esters and
amides,11–13 and engineering in biotinidase resistance is
Introduction
The use of the triazole as an amide bond isostere was high- important in the design of pre-targeted therapies and imaging
lighted by Sharpless in his seminal review of the impact of agents.14–18 Although not yet explored, hydrolysis-resistant bio-
“click” chemistry in 2003.1 However, the application of this tinylation agents for histone modification might allow recent
strategy to confer hydrolytic stability in amide linkages to the advances in site-specific histone labelling using near-native
essential cofactor biotin 1a, such as found in the biological labels to be exploited,19 enabling understanding of the epi-
carrier biocytin 2 (biotinyl-ε-lysine),2 and recently suggested as a genetic role of biotinylation. Additionally, the development of
rare epigenetic modification of histone lysines,3 has not been biotinylated, hydrolytically stable, drug conjugates which
explored. Functionalisation of (bio)molecules with biotin is exploit the over-expression of the sodium-dependent multi-
employed across a wide array of separation, immobilisation, pre- vitamin transporter,20 which mediates the cell uptake of
targeted therapeutic and medical imaging technologies due to its biotin, in several aggressive cancer cell lines is attractive.
extremely strong affinity for the proteins avidin4 and streptavidin.5
This “avidity” results from unusually strong hydrogen-bonding
interactions within the binding site for the ureido moiety of
Results and discussion
Design of biotinidase-resistant conjugates
biotin,6 and improved protein packing on binding the ligand.7
Regulation of histone biotinylation through formation and
Some resistance to cleavage by biotinidase has been achieved
by blocking,14,15 reversing the sense,16 or even removing the
carbonyl of the amide bond to biotin;15,17 however, each of
these approaches has an impact on binding to the target
protein avidin or streptavidin.17 We sought to design a biotin
label which would be resistant to cleavage by biotinidase, but
would retain a high binding affinity for avidin. Our hypothesis
was that triazole 3, prepared by copper(I)-catalysed azide
alkyne cycloaddition (CuAAC) coupling of alkyne 4 to an azide
(RN3), would provide an optimum non-hydrolysable isostere of
the amide bond (Fig. 1b). Correspondingly, triazole 5, derived
from coupling known azide 6 to a terminal alkyne (RCuCH),
would provide a non-hydrolysable isostere of the reversed
amide bond (Fig. 1b).§ Novel alkyne 4 was readily prepared
hydrolysis of the histone lysine-biotin amide bond is only
partly understood,3,8 but maintenance of levels of biotin from
dietary sources is known to be achieved through the biotini-
dase-induced cleavage of biotin from both biocytin 2, and
short biotinylated peptides.9 Indeed, biotinidase deficiency is
an inherited genetic disorder that if left untreated can result
in major disabilities.10 However, biotinidase has also been
aEaStCHEM School of Chemistry, University of Edinburgh, West Mains Road,
Edinburgh, EH9 3JJ, UK. E-mail: Alison.Hulme@ed.ac.uk; Fax: +44 (0)131 650 4743;
Tel: +44 (0)131 650 4711
bCentre for Translational and Chemical Biology, University of Edinburgh, Mayfield
Road, Edinburgh, EH9 3JR, UK
†Celebrating 300 years of Chemistry at Edinburgh.
‡Electronic supplementary information (ESI) available: Synthesis and character-
ization of azide 6, complexes 7–9, biotinidase activity assay, enrichment of bioti-
nidase from serum, biotinidase stability assay, HABA displacement and ITC
binding assays. See DOI: 10.1039/c3ob41837e
§For synthetic ease the azide 6 corresponding to the amide bond reversal of an
amide of homobiotin was chosen for this investigation.
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Fig. 1 (a) Biotin 1a and biocytin 2. (b) Non-hydrolysable triazoles (3, 5) arising
from CuAAC coupling of biotin-alkyne 4 and biotin-azide 6 (respectively).
from D-biotin methyl ester via reduction to the corresponding
aldehyde and reaction with the Ohira–Bestmann reagent (47%
overall yield);‡ whilst azide 6 was prepared in 3 steps (62%
overall yield) from the same starting material.21
Fig. 2 Biotin Gd-DOTA complexes for pre-targeted MRI.
To determine the efficacy of the triazole-linked biotin
species, in terms of biotinidase resistance and avidin binding,
a series of biotinylated Gd-DOTA complexes appropriate for
pre-targeted magnetic resonance imaging were generated.22
Synthesis of MR imaging agents 7–9 was achieved through the
CuAAC coupling of 4 and 6 with the Gd-complexes of three
known DOTA “click” ligands (Fig. 2).23–25 These complexes
allowed us to investigate both modes of CuAAC coupling (7 vs. 8),
as well as to probe the effects of linker length between the
triazole-linked biotin and bulky Gd-DOTA complex (8 vs. 9).
The precursor Gd-complexes were readily prepared from the
DOTA “click” ligands,¶ and were coupled (CuSO4, NaASc,
t
TBTA, 4 or 6, BuOH–H2O, rt, 16 h) to give triazoles 7–9 which
were purified by HPLC prior to biological assays.
Fig. 3 Stability of 7–9 to cleavage by partially-purified biotinidase contrasted
with cleavage of N-biotinyl-PABA under the same conditions. Samples were
incubated with biotinidase (0.57 AU) in phosphate buffer (0.05 M, pH 6) for up
to 126 h; the percentage of the complex remaining was determined by HPLC
against an internal standard (resorcinol).
Biotinidase resistance
Human biotinidase was fractionated from human serum,26
and its activity was assessed by measuring the rate of hydro-
lysis of the commercially available substrate, N-biotinyl-p-amino-
benzoic acid (N-biotinyl-PABA).27 HPLC analysis revealed
that all three triazole complexes (7–9) were resistant to cleavage
by biotinidase up to 126 h, in marked contrast to N-biotinyl-
PABA which was fully hydrolyzed under the same conditions
within 6 h (Fig. 3).
Binding affinity for avidin
The binding affinity of complexes 7–9 to avidin was probed
using spectrophotometric competition experiments against
the known ligand 4-hydroxy-azobenzene-2-carboxylic acid
(HABA, KD = 5.8 × 10−6 M).28 All of the triazoles 7–9 were
shown to inhibit the formation of the HABA:avidin complex in
a dose-dependent manner (Fig. 4), and comparison with three
additional ligands of known binding affinity (biotin 1a KD
=
10−15 M; desthiobiotin 1b KD = 10−13 M; and diaminobiotin 1c
KD = 1.2 × 10−7 M)4,29 suggested binding affinities for
¶Attempted coupling of DOTA ligands in the absence of the lanthanide metal
was found to be unsuccessful unless excess Cu(I) was employed.
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Fig. 4 Binding of MRI agents 7–9 to avidin assessed spectrophotometrically by
displacement of HABA from the HABA–avidin complex at pH 7.5. [Diaminobiotin
displacement assay conducted at pH 10.]
complexes 7–9 in the nM to pM range. Both the amide bond
and reversed amide bond triazole isosteres bound to avidin
strongly; but complex 9 bound least strongly, indicating that
longer linker lengths to the bulky DOTA complex were more
favourable.
Fig. 5 Calorimetric titration of 12 into avidin in competition with 10 in sodium
acetate buffer (100 mM; pH 5.5). Top panel: individual injections of a solution of
12 (and 10) into a solution of avidin (and 10). Bottom panel: titration curve,
enthalpy/(mole of 12 injected) versus 12/(avidin) molar ratio.
Comparison with other biotinidase-resistant conjugates
To compare this isostere-based strategy against other biotini-
dase-resistant approaches we took arguably the most studied
DOTA ligand, the reduced biotinamidohexylamine–DOTA con-
jugate referred to as r-BHD17b or ST221030 (Gd-complex 10,
measurement. To determine the binding affinities of amide-
linked complex 11 and the natural biotin carrier biocytin 2,
and compare them with the triazole-linked complex 12, the
amine-linked complex 10 was added to bring the apparent
Fig. 2); for which an avidin dissociation constant (KD
=
equilibrium dissociation constant (K′ ) closer to the range of
D
2.9 × 10−7 M) has recently been determined by isothermal
calorimetry (ITC).31 Ligand ST2210 was synthesised as pre-
viously described,17b and both it and the conjugate of biotin-
PEG3-amine with the mono-activated DOTA NHS ester
(DO3A-NHS) were treated with GdCl3 to effect the formation of
amine-linked complex 10 and amide-linked complex 11
respectively. Synthesis of the isostere-based, biotinidase-resist-
ant complex 12 was achieved through CuAAC coupling of
alkyne 4 with a commercial bi-functional PEG3-aminoazide;
followed by coupling to DO3A-NHS and Gd-complex formation
as for complex 11. Each of the complexes (10–12) was purified
the instrument (Fig. 5). The binding affinities of these amide-
and triazole-linked ligands were determined to be in the low
picomolar to femtomolar range (KD ≤ 10 × 10−12 M), and of
equivalent strength to each other suggesting that the triazole-
linked biotin mimics the natural biotin amide bond very
closely in its binding to avidin.
Conclusions
by HPLC prior to the determination of avidin dissociation con- We have demonstrated that a triazole-linked biotin conjugate
stants by ITC.32
derived either from alkynyl biotin 4, or from the related biotin
Since the binding of complexes 10–12 to avidin was pre- azide 6, provides an excellent biotinidase-resistant isostere of
dicted to be very strong, we determined KD values in a series of the biotin-amide bond. Derivatives of biotin alkyne 4 linked to
competition experiments using amine-linked complex 10 as a appropriate spacers have equilibrium dissociation constants
reference.33 Calorimetric titration of 10 was determined to be which are equivalent to their amide counterparts, in contrast
close to the limit of the experimental range (KD = 4.1 1.5 × to other biotinidase-resistant linkages. Hydrolase-resistant
10−9 M); the binding was assumed to be non-cooperative biotin conjugates based on the triazole isostere will lead to
across the avidin tetramer and the ligand was shown to satu- efficient pre-targeting agents for therapeutic and imaging
rate each of the four binding sites. This measured binding applications, and an enhanced understanding of the role of
affinity is ∼102 fold greater than previously reported,31 but we protein biotinylation where hydrolytic cleavage (e.g. by bio-
attribute this to the rigorous purification of both the complex tinidase, holocarboxylase synthetase etc.) might make this
(by HPLC) and protein (by size exclusion) prior to otherwise difficult to address.
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4.66–4.59 (3H, m, NHCHCH, CHNCH2), 4.42 (1H, dd, J = 7.9,
4.5 Hz, NHCHCH2), 3.99 (2H, t, J = 4.9 Hz, CH2O), 3.79–3.73
(2H, m, CH2O), 3.70–3.60 (8H, m, 4 × OCH2), 3.34 (1H, dt, J =
Experimental
Preparation of biotin alkyne 4
(3aS,4S,6aR)-4-(Hex-5-yn-1-yl)tetrahydro-thieno[3,4-d]imid- 9.7, 5.1 Hz, SCH), 3.27–3.17 (2H, m, CH2NH2), 2.99 (1H, dd, J =
azole-2-one 4. Biotin methyl ester34 (0.320 g, 1.24 mmol) was 13.0, 5.0 Hz, CHAHBS), 2.79 (1H, d, J = 13.0 Hz, CHAHBS), 2.77
dissolved in anhydrous DCM (8 mL), and the solution was (2H, t, J = 7.3 Hz, CH2CCH), 1.82–1.52 (4H, m, 2 × CH2),
cooled to −78 °C. DIBAL-H (2.09 mL, 2.30 mmol, 1.1 M solu- 1.49–1.36 (2H, m, CH2); 13C NMR δ (126 MHz, D2O) 165.33
tion in cyclohexane) was added over 10 min, and the mixture (CO), 148.13 (C), 123.57 (CH), 69.64 (CH2), 69.58 (CH2), 69.50
stirred at −78 °C for 4 h. The excess DIBAL-H was quenched (CH2), 69.45 (CH2), 68.83 (CH2), 66.35 (CH2), 62.08 (CH), 60.23
with anhydrous methanol (5 mL), and the reaction mixture (CH), 55.44 (CH), 49.90 (CH2), 39.64 (CH2), 39.09 (CH2), 28.23
was allowed to warm to 0 °C. Potassium carbonate (0.340 g, (CH2), 27.65 (CH2), 27.63 (CH2), 24.13 (CH2); m/z (ESI+, MeOH)
2.46 mmol), Ohira–Bestmann reagent (0.290 g, 1.51 mmol), 465 ([M + Na]+, 5%), 443 ([M + H]+, 100); HRMS (ESI+, MeOH)
and anhydrous methanol (5 mL) were added and the reaction [M + H]+ C19H35O4N6S requires 443.2435, found 443.2438.
stirred for 24 h. Further Ohira–Bestmann reagent (0.290 g,
Triazole-linked biotin PEG3 Gd-DOTA complex 12. Triazole-
1.51 mmol) was added and the reaction was stirred for a linked biotin amine 13 (0.020 g, 0.045 mmol) was dissolved in
further 18 h. Rochelle’s salt (10 mL) and ethyl acetate (20 mL) a minimum volume borax solution (∼0.5 mL; 0.08 M aq.) and
were added and the mixture stirred vigorously for 1 h. The cooled to 0 °C. To this was added a solution of the NHS-DOTA-
organic layer was separated, washed with brine (3 × 20 mL), ester (0.052 mg, 0.068 mmol) in borax solution (0.5 mL; 0.08 M
dried over magnesium sulfate, and the solvent was removed aq.). The solution was adjusted to pH 7 with NaOH (1.0 M aq.)
under reduced pressure. The crude mixture was purified by then warmed to room temperature and stirred for 24 h.
column chromatography (DCM–MeOH, 90 : 10) to give termi- The crude product was lyophilised to give a colourless solid
nal biotin alkyne 4 as a colourless solid (0.13 g, 0.58 mmol, that was purified by preparative RP-HPLC using a C18 column
47%). Rf (DCM–MeOH, 90 : 10) = 0.5; mp 158–162 °C; IR 3242 and H2O–acetonitrile + 0.1% TFA as mobile phase to give the
(NH), 2108 (CuCH), 1703 (CvO); 1H NMR δ (500 MHz, CDCl3) triazole-linked DOTA ligand as a colourless oil (0.010 g, 27%);
4.97 (1H, s, NHCHCH), 4.89 (1H, s, NHCHCH2), 4.55 (1H, dd, Rt = 37 min (SI Method 2). The triazole-linked DOTA ligand
J = 7.7, 4.6 Hz, NHCHCH), 4.37 (1H, dd, J = 7.7, 4.6 Hz, (0.010 g, 0.012 mmol) was then dissolved in H2O (1.0 mL) and
NHCHCH2), 3.21 (1H, ddd, J = 8.0, 6.6, 4.6 Hz, SCH), 2.97 (1H, the solution was adjusted to pH 7 using KOH (0.1 M aq.).
dd, J = 12.8, 5.0 Hz, CHAHBS), 2.77 (1H, d, J = 12.8 Hz, GdCl3·6H2O (0.0045 g, 0.012 mmol) was dissolved in H2O
CHAHBS), 2.26 (2H, td, J = 6.5, 2.5 Hz, CH2CuCH), 2.00 (1H, t, (1.0 mL) and then added to the ligand solution. After stirring
J = 2.5 Hz, CuCH), 1.75–1.65 (2H, m, CH2), 1.62–1.55 (4H, m, for 15 minutes the solution was readjusted to pH 6 using KOH
2 × CH2); 13C NMR δ (126 MHz, CDCl3) 162.9 (CO), 84.1 (C), (0.1 M aq.) and then the reaction mixture was stirred at 60 °C
68.8 (CH), 61.9 (CH), 60.1 (CH), 55.3 (CH), 40.6 (CH2), 28.1 for 4 h. The solution was cooled to room temperature and
(CH2), 28.0 (CH2), 27.9 (CH2), 18.1 (CH2); m/z (ESI+, MeOH) again adjusted to pH 6 using KOH (0.1 M aq.). Lyophilisation
471 ([2M + Na]+, 100%), 247 ([M + Na]+, 21), 225 ([M + H]+, 11); gave a colourless solid that was purified by preparative
HRMS (ESI+, MeOH) [2M + Na]+ found 471.1864, C11H16N2OS RP-HPLC using a C18 column and H2O–acetonitrile + 0.1%
requires 471.1859.
TFA as mobile phase, to give triazole-linked biotin PEG3
Gd-DOTA complex 12 as a colourless solid (0.0077 g, 65%).
HPLC Rt = 38 min (SI Method 2); m/z (ESI+, MeOH–NH4OAc)
Preparation of biotinidase-resistant complex 12
Triazole-linked biotin PEG3 amine 13. Biotin alkyne 4 1006 ([158GdM + Na]+, 46%), 984 ([158GdM + H]+, 57), 595 (100).
(0.059 g, 0.263 mmol) and TBTA (0.1 eq.) were dissolved in The appropriate isotope pattern was observed.
tBuOH–H2O (2 : 1, 30 mL) and allowed to stir for 15 min.
Sodium ascorbate (0.009 g, 0.044 mmol) was then added and
allowed to stir for 15 min followed by copper(II) sulfate
Acknowledgements
(0.006 g, 0.022 mmol). After a further 15 min 1-amino-
11-azido-3,6,9-trioxaundecane (2.19 mL, 0.219 mmol; 0.1 M in
H2O) was added and the solution stirred under nitrogen at
room temperature for 16 h. QuadraPure-IDA metal scavenger
resin was added and the reaction mixture was gently shaken
overnight. The resin was removed by filtration, and the
aqueous solution extracted with EtOAc (2 × 20 mL). The
aqueous solution was then lyophilised to afford crude triazole
as a colourless solid. The crude product was purified by pre-
parative RP-HPLC using a C18 column and H2O–acetonitrile +
0.1% TFA as mobile phase to give triazole-linked biotin amine
13 as a colourless solid (76 mg, 65%). HPLC Rt = 38 min (SI
Method 2); 1H NMR δ (500 MHz, D2O) 7.92 (1H, NCCHN),
We thank the Wellcome Trust, University of Edinburgh (IKTF)
and EC FP6 (Marie Curie Fellowship to JRH; contract
MEST-CT-2005-020744) for financial support of this work. JL
and DJC thank the BBSRC for funding (BB/I013687/1).
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