Synthesis of a biotin-derived alkyne for Pd-catalyzed coupling reactions

Article abstract of DOI:10.1021/ol060458r

An efficient synthesis of a terminal alkyne derived from d-biotin was developed to provide an alternative for carboxyi-based biotinylation. This approach was illustrated by the preparation of alkyne- and alkene-linked phenylalanine derivatives using palladium-catalyzed Sonogashira and Oh methodology. (Strept)avidin binding was observed using soluble and immobilized receptors. These results demonstrate the applicability of carbon-linked biotin derivatives for use in affinity-based purification and bioanalytical applications.

Full text of DOI:10.1021/ol060458r

ORGANIC
LETTERS
2006
Vol. 8, No. 9
1883-1886
Synthesis of a Biotin-Derived Alkyne for
Pd-Catalyzed Coupling Reactions
Cesear Corona, Bj K. Bryant, and Jeffrey B. Arterburn*
New Mexico State UniVersity, P.O. Box 30001MSC 3C,
Las Cruces, New Mexico 88003
jarterbu@nmsu.edu
Received February 22, 2006
ABSTRACT
An efficient synthesis of a terminal alkyne derived from d-biotin was developed to provide an alternative for carboxyl-based biotinylation. This
approach was illustrated by the preparation of alkyne- and alkene-linked phenylalanine derivatives using palladium-catalyzed Sonogashira and
Oh methodology. (Strept)avidin binding was observed using soluble and immobilized receptors. These results demonstrate the applicability
of carbon-linked biotin derivatives for use in affinity-based purification and bioanalytical applications.
The interaction between biotin (Vitamin H) 1 and the
glycoprotein avidin is one of the strongest noncovalent
associations known (Ka ∼ 2.5 × 1015 M-1). Avidin is
tetrameric, and each subunit is capable of binding a biotin
ligand. The high binding affinity and exceptionally slow
dissociation rates of biotin result from a network of hydrogen
bonds between the receptor and the heterocyclic 7-oxo-3-
thia-6,8-diazabicyclo[3.3.0]oct-4-yl ligand. The ureido ni-
trogens of biotin form hydrogen bonds with Thr35 and
Asn118, the oxygen contacts of Ser16 and Tyr33. Additional
hydrogen bonding occurs between the biotin carboxylic acid
and avidin residues Ala39, Thr40, and Ser75. The hydro-
phobic tetrahydrothiophene interacts with Phe79, Trp97, and
Trp110.1
and Tyr43. Interactions also exist between Asn49 and Ser88
and the biotin carboxylic acid group. This network of
hydrogen bonding in conjunction with hydrophobic interac-
tions with four Trp residues (Trp 79, 92, 108, 120) and the
tetrahydrothiophene result in high binding affinity.3
Biotin-(strept)avidin systems have been used for a variety
of applications such as affinity isolation and purification,
immunoassay, diagnostics, and localization.4-6 Nearly any
biological entity can be labeled with biotin including
peptides, proteins, oligonucleotides, and antibodies. Antibody-
(strept)avidin conjugates are used in the pretargeting ap-
proach to deliver radiolabeled biotin therapeutics and imaging
agents.7 Other applications include drug delivery8 and
material science.9
Avidin and the related tetrameric protein streptavidin share
∼33% of the conserved amino acids and a strong biotin
binding affinity (Ka ∼ 1.0 × 1012 M-1).2 Specific interactions
include hydrogen bonding between biotin urea nitrogens with
Ser45 and Asp128 and oxygen contacts with Asn23, Ser27,
(3) (a) Chilkoti, A.; Stayton, P. S. J. Am. Chem. Soc. 1995, 117, 10622.
(b) Klumb, L. A.; Chu, V.; Stayton, P. S. Biochemistry 1998, 37, 7657.
(4) Wilchek, M.; Bayer, E. A. Anal. Biochem. 1988, 171, 1.
(5) Diammandis, E. P.; Christopoulos, T. K. Clin. Chem. 1991, 35, 625.
(6) Immunoassay; Diamandis, E. P., Christopoulos, T. K., Eds.; Acedemic
Press: San Diego, CA, 1996.
(1) Puglise, L.; Coda, A.; Malcovati, M.; Bolognesi, M. J. Mol. Biol.
1993, 231, 698.
(2) Pahler, A.; Hendrickson, W. A.; Kolks, M. A. G.; Argarana, C. E.;
Cantor, C. R. J. Biol. Chem. 1987, 29, 13933.
(7) Boerman, O. C.; van Schaijk, F. G.; Oyen, W. J. G.; Corstens, F. H.
M. J. Nucl. Med. 2003, 44, 400.
(8) Bhunilya, S.; Park, S. M.; Kim, B. H. Org. Lett. 2005, 7, 1741.
(9) Niemeyer, C. M. Angew. Chem., Int. Ed. 2001, 40, 4128.
10.1021/ol060458r CCC: $33.50
© 2006 American Chemical Society
Published on Web 03/31/2006

A variety of biotin-labeling agents are commercially
available.10 Activated esters (N-hydroxysuccinimdyl ester and
p-nitrophenyl ester), maleimide, and iodoacetyl derivatives
are frequently used for coupling to substrates possessing
amine or thiol groups.11 In vivo applications of amide-linked
biotin derivatives can be problematic because of cleavage
by the endogenous enzyme biotinidase.12,13
Scheme 1. Synthesis of Biotin-Alkyne
Several structural modifications have been described to
prevent biotinidase cleavage; however, these derivatives
exhibit increased dissociation rates from (strept)avidin. Biotin
amide derivatives containing a small R-substituent have
provided the most effective balance of biotinidase stability
and (strept)avidin affinity.14 Both N-methylation of the
biotinamide linkage15 and homologation of the valeric acid
chain provided increased resistance to cleavage16 as did
replacement of the biotinamide connection with thiourea.
Unfortunately, increased dissociation rates were observed
with all of these linkage-modified derivatives.17
We have been interested in developing biotin conjugates
of hydrophobic receptor ligands for affinity purification.
Available biotin-coupling reagents are not suitable for
derivatizing compounds that lack reactive functional groups
such as amine, carboxylate, or thiol. The installation of polar
substituents on a hydrophobic receptor substrate is possible
but may decrease receptor-binding affinity because of
unfavorable electrostatic interactions, modified solvation
characteristics, different lipophilicities (as measured by log
P octanol/water), and increased steric interactions.18 As an
alternative, we considered using nonpolar linkages synthe-
sized via catalytic C-C bond-coupling methodology. Re-
placement of the biotin carboxamide with an alkyne or alkene
connection would eliminate chemical or enzymatic hydroly-
sis. The success of this approach depends on the resulting
C-C linked biotin substrates maintaining (strept)avidin
affinity.
1).20 The iodide 5a and bromide 5b were prepared by halide
substitution of 4.
Low yields of the desired alkyne 6 were obtained from
direct lithium acetylide substitution reactions of tosylate 4
or iodide 5a. Fortunately, the alkyl bromide 5b underwent
efficient displacement with lithium acetylide-ethylenedi-
amine in DMSO with careful temperature control e15 °C
and produced the desired alkyne 6 in high yield. This
synthesis provided alkyne-biotin derivative 6 with a com-
bined yield of 66% over five steps from biotin.
We evaluated avidin binding of 6 in solution. Competitive
displacement of 2-(4′-hydroxyphenylazo)benzoic acid (HABA)
with biotin derivatives provides a convenient spectroscopic
method for assessing avidin binding. In solution, HABA
forms a complex with the biotin binding site of avidin that
is characterized by an absorbance band at 500 nm. Displace-
ment of the HABA substrate by biotin results in decreased
absorbance at 500 nm. This method has been widely used
as a qualitative assay for evaluation of biotinylated sub-
strates.21
We constructed a standardized biotin response curve for
the HABA-avidin complex in 0.1 M phosphate buffer
solution. The decrease in absorbance accompanying additions
of 5 µL aliquots of d-biotin reference solution (5.0 × 10-4
M in 0.10 M NaH2PO4) was measured in triplicate and
plotted against the concentration of added biotin. A response
curve was generated analogously for alkyne 6 by addition
to the standardized HABA-avidin solution. A decrease in
absorbance was observed with the addition of 6 demonstrat-
ing HABA displacement. From the results of this assay, it
can be concluded that the association constant of 6 remained
high because HABA (Ka ) 6 × 106 M-1) is displaced.21
Although the HABA displacement was attenuated relative
to biotin, effective binding of 6 was retained despite
replacement of the carboxyl group with alkyne.
We elected to replace the carboxylic acid group with an
alkyne that would allow entry into a wide variety of metal-
catalyzed coupling procedures.19 The synthesis was initiated
by acid-catalyzed esterification of biotin. Selective reduction
of the ester 2 was accomplished using diisobutylaluminum
hydride (DIBAL) at -78 °C, affording alcohol 3 in 96%
yield. Reaction of 3 with toluenesulfonyl chloride in pyridine
at 0 °C provided the sulfonate ester 4 in 94% yield (Scheme
(10) Haugland, R. P. In The Handbook: A Guide to Fluorescent Probes
and Labeling Technologies, 10th ed.; Spence, M. T. Z., Ed.; Invitrogen:
Eugene, OR, 2005; Chapter 4, p 141.
(11) Foulon, C. F.; Alston, K. L.; Zalutsky, M. R. Bioconjugate Chem.
1997, 8, 179.
(12) Singh, R.; Maloney, E. K. Anal. Biochem. 2002, 304, 147.
(13) Bogusiewicz, A.; Mock, N. I.; Mock, D. M. Anal. Biochem. 2004,
331, 260.
(14) Wilbur, D. S.; Hamlin, D. K.; Chyan, M.; Kegley, B. B.; Pathre, P.
M. Bioconjugate Chem. 2001, 12, 616.
(15) Pazy, Y.; Kulik, T.; Bayer, E. A.; Wilchek, M.; Livnah, O. J. Biol.
Chem. 2002, 277, 30892.
(16) Wilbur, D. S.; Hamlin, D. K.; Pathare, P. M. Bioconjugate Chem.
1997, 8, 572.
(17) Wilbur, D. S.; Chyan, M.; Pathare, P. M.; Hamlin, D. K. Biocon-
jugate Chem. 2000, 11, 569.
(18) Schneider, C.; Scholer, H. F.; Schneider, R. J. Steroids 2004, 69,
245.
(20) DeLaLuz, P. J.; Golinski, M.; Watt, D. S.; Vanaman, T. C.
Bioconjugate Chem. 1995, 6, 558.
(21) Hermanson, G. T. Bioconjugate Techniques; Academic Press: San
Diego, CA, 1996; p 591.
(19) Negishi, E.; Anastasia, L. Chem. ReV. 2003, 103, 1979.
1884
Org. Lett., Vol. 8, No. 9, 2006

Encouraged by the affinity exhibited by 6, we proceeded
to synthesize a phenylalanine biotin derivative. Amino acids
labeled with biotin are important in peptide synthesis.8,22,23
Sonogashira cross-coupling reactions have been used to
produce alkynyl phenylalanine derivatives.24,25 To increase
the solubility of 6 in organic solvents, we protected the urea
as the di-tert-butyl carbamate 7. The 4-iodo-phenylalanine
derivative 8 was coupled with alkyne 7 under standard
conditions using catalytic Pd(OAc)2, PPh3, and CuI in
diethylamine to give conjugate 9 in 91% yield (Scheme 2).
Scheme 3. Synthesis of Alkene Derivatives
Scheme 2. Synthesis of Alkyne Derivatives
The sp2-hybridized carbon in alkene 13 corresponds to the
position of the carboxyl in biotinamide derivatives and can
be considered to be isosteric although not capable of
hydrogen bonding. The linear alkyne connection in 10 is
comparable to the overall length of homologated biotinamide
conjugates. These minor differences in length and geometry
did not significantly alter the observed binding affinity of
10 and 13. These derivatives exhibited decreased affinity for
avidin relative to biotin as expected from the deletion of the
carboxyl group.
Hydrolysis of the ester with LiOH(aq) in MeOH/THF at
0 °C, followed by deprotection of the Boc groups with
Solid-supported (strept)avidin sorbents have been widely
used for batch affinity isolation and purification of biotiny-
lated substrates. Nonbinding components are easily removed
by elution, and isolation of biotinylated substrates is ac-
complished by dissociation from the supported (strept)-
avidin.27 The strong biotin-(strept)avidin interaction presents
a problem in these applications because harsh conditions are
required to dissociate the biotin from the support matrix.
Biotin derivatives with reduced binding affinity such as
desthiobiotin and 2-imunobiotin are advantageous because
elution occurs with milder conditions. We prepared the
fluorescent alkyne-linked phenylalanine 15 to evaluate the
affinity for immobilized (strept)avidin (Scheme 4).
t
trifluoroacetic acid (TFA) in CH2Cl2 gave the alkyne-linked
phenylalanine conjugate 10. Spectroscopic characterization
by NMR and HPLC-MS confirmed the product identity.
We wanted to investigate the effect of shortening the
linkage between the biotin heterocycle and the phenylalanine
on avidin affinity. Recently, Oh et al. reported a palladium-
catalyzed insertion of arylboronic acids into terminal alkynes.26
Following this procedure, alkyne 7 was coupled with
protected 4-boronic acid derivative 11 to afford alkene 12
in 73% yield (Scheme 3). The alkene protons appeared as
1
doublets at δ 5.21 and 5.01 (J ) 1.3 Hz) in the H NMR
spectrum, consistent with the expected alkene. No other
1
alkene isomers were observed by H NMR.
The ester 12 was hydrolyzed with LiOH, followed by tBoc
deprotection with TFA/CH2Cl2. The product was character-
ized spectroscopically and by HPLC-MS. The exocyclic
alkene isomerized to the more substituted (E)-alkene 13
during deprotection. The vinyl proton signal was observed
as a triplet at δ 5.78 (J ) 7.1 Hz), and the allylic methyl
group appeared as a singlet at δ 1.96.
Scheme 4. Synthesis of a Biotin Alexafluor Conjugate
The avidin affinities of 10 and 13 were evaluated using
the HABA assay as described for 6. Both derivatives
displaced HABA from avidin with comparable efficiency.
(22) Gallivan, J. P.; Lester, H. A.; Dougherty, D. A. Chem. Biol. 1997,
4, 739.
(23) Kumar, V.; Aldrich, J. V. Org. Lett. 2003, 5, 613.
(24) Sonogashira, K.; Tohda, Y.; Hagihara, N. Tetrahedron Lett. 1975,
The R-amino group of 10 was coupled with Alexafluor
546 succinimidyl ester 14 to give 15. Compound 15 was
dissolved in 0.1 M phosphate buffer, pH 7.2, at a concentra-
50, 4467.
(25) Hoffmanns, U.; Metzler-Nolte, N. Bioconjugate Chem. 2006, 17,
204.
(26) Oh, C. H.; Jung, H. H.; Kim, K. S.; Kim, N. Angew. Chem., Int.
Ed. 2003, 42, 805.
(27) Techincal Document #25-0524, StreptaVidin Agarose: For Affinity
Chromatography and Immunopecipitation of Biotinylated Proteins; Invit-
rogen: Eugene, OR, 2003.
Org. Lett., Vol. 8, No. 9, 2006
1885

tion of 3.27 × 10-6 M. A 3 mL aliquot (50% of the
manufacturer’s recommended loading) was added to a
centrifuge tube containing (strept)avidin (5 mL) on agarose
support. The tube was agitated for 15 min and centrifuged
for 5 min, and the supernate containing unbound 15 was
isolated. The tube was washed with 10 bed volumes of water
to collect unbound substrate 15. The concentration of free
15 was determined spectroscopically (Figure 1). The im-
(Kd) of ca. 3.8 × 10-9 M was determined for avidin-bound
15 using the method described by Srivastava et al.29 This
dissociation constant lies between the values of desthiobiotin
(Kd ) 5.0 × 10-13 M)30 and 2-imunobiotin (Kd ) 8.0 ×
10-6 M)31 and is also comparable to other recently reported
biotin-fluorophore conjugates.28,32
This study evaluated the potential for carbon-linked biotin
conjugates in affinity-based methods. Alkyne 6 was ef-
ficiently synthesized from d-biotin and exhibited strong
binding in the HABA assay despite the deletion of the
carboxylic acid. The tBoc-protected alkyne 7 was effectively
coupled using palladium-catalyzed reactions. Synthetic phe-
nylalanine derivatives 10 and 13 and fluorescent probe 15
exhibited affinity for soluble and immobilized (strept)avidin,
as expected for binding to the preserved 7-oxo-3-thia-6,8-
diazabicyclo[3.3.0]oct-4-yl core. This approach enables
conjugation to substrates that cannot be labeled with activated
carboxylate derivatives of biotin. The covalent carbon linkage
that replaces the carboxamide group of traditional biotin
conjugates decreases relative (strept)avidin binding but
retains sufficient affinity for bioanalytical applications and
provides complete protection from chemical or enzymatic
hydrolysis. This approach may be extended to other alkyne-
coupling and cycloaddition procedures.
Figure 1. Emission spectrum of 15.
Acknowledgment. This research was supported by NIH/
SCORE GM08136. C.C. was supported by NIH/RISE
GM61222. The instrumentation facility was funded by NIH
RR16480 from the NCRR-INBRE Program.
mobilized (strept)avidin retained 84% of 15, confirming the
potential for C-linked derivatives of 6 in batch isolation
methods.
Supporting Information Available: General methods,
1
experimental details, copies of H NMR and 13C NMR
The avidin binding of 15 was studied by fluorescence
titration. It was observed that 15 displayed higher emission
intensity in the presence of avidin. Lo et al. have attributed
the observed fluorescence enhancement to greater hydro-
phobicity within the binding pocket. A standardized solution
of solubilized avidin was prepared and titrated with 15.
Fluorescence emission enhancement was monitored to
determine the concentration required to saturate the recep-
tor.28 From this titration experiment, a dissociation constant
spectra of compounds 1-3, 5-7, 9, 10, 12, 13, and HABA
titration curves. This material is available free of charge via
the Internet at http://pubs.acs.org.
OL060458R
(29) Wang, Z.; Kumar, N. R.; Srivastava, D. K. Anal. Biochem. 1992,
206, 376.
(30) Muller, W.; Ringsdorf, H.; Rump, E.; Wildberg, G.; Zhang, X.;
Angermaier, L.; Knoll, W.; Liley, M.; Spinke, J. Science 1993, 262, 1706.
(31) Reznik, G. O.; Vajda, S.; Sano, T.; Cantor, C. R. Proc. Natl. Acad.
Sci. U.S.A. 1998, 95, 13525.
(28) (a) Lo, K. K.; Hui, W.; Ng, D. C. J. Am. Chem. Soc. 2002, 124,
9344. (b) Lo, K. K.; Hui, W. Inorg. Chem. 2005, 44, 1992.
(32) Marek, M.; Kaiser, K.; Gruber, H. J. Bioconjugate Chem. 1997, 8,
560. (b) Lo, K. K.; Lee, T. K. Inorg. Chem. 2004, 43, 5275.
1886
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