Modular synthesis of bifunctional linkers for materials science

Article abstract of DOI:10.1002/ejoc.201001666

A practical synthesis of α,ω-bifunctional linkers is described that is based on three building blocks, namely, thioctic acid, a spacer-arm of seven ethylene glycol units, and a functional motif dedicated to the selective immobilization of purposely tagged proteins. Such representative motifs are biotin, haloacetamide, maleimide, and nitrilotriacetic acid derivatives. The building blocks are connected through alkyl, amide, and/or carbamate linkages. Copyright

Full text of DOI:10.1002/ejoc.201001666

SHORT COMMUNICATION
DOI: 10.1002/ejoc.201001666
Modular Synthesis of Bifunctional Linkers for Materials Science
Julie Moreau[a] and Jacqueline Marchand-Brynaert*[a]
Keywords: Oligoethylene glycol / Sulfur / Synthesis design / Amino alcohols / Immobilization
A practical synthesis of α,ω-bifunctional linkers is described
that is based on three building blocks, namely, thioctic acid,
a spacer-arm of seven ethylene glycol units, and a functional
motif dedicated to the selective immobilization of purposely
tagged proteins. Such representative motifs are biotin,
haloacetamide, maleimide, and nitrilotriacetic acid deriva-
tives. The building blocks are connected through alkyl,
amide, and/or carbamate linkages.
als, metal oxides, semiconductors, or glasses have been al-
ready described for forming dense self-assembled mono-
layers (SAMs).[4] However, the most commonly encoun-
tered class of SAMs is derived from the chemisorption of
alkanethiols or disulfides onto gold due to strong S–Au in-
teractions.[5]
Introduction
The stable immobilization of biomacromolecules onto
materials or nanoparticles has received growing interest
over the last few decades.[1a] A challenging goal in biotech-
nology, molecular electronics, and biosensors is the grafting
of proteins that retain their full activity on the surfaces of
the devices.[1b] The synthesis of appropriate linkers capable
of connecting surfaces to specific proteins is still a difficult
task; most often, detailed protocols are not available or the
reactions suffer from poor reproducibility, poor yields, and
unspecified purity of the prepared bifunctional molecules.
Several protocols were recently proposed by using either
high molecular weight molecules as spacer arms, usually
polyethylene glycols (PEGs),[2a–2c] or on the contrary very
short ones.[2d] Heterotelechelic oligoethylene glycols
(OEGs), in particular thiol end-functionalized OEGs, are
also commercially available and used for gold surface modi-
fication.[2e,2f] Nevertheless, α,ω-bifunctional linkers based
precisely on seven ethylene glycol units [i.e., OEG of 14
CH2 groups named (EG)7] are not marketed, and their syn-
thesis is rarely mentioned in the literature.[2g]
We describe here a modular and practical route to syn-
thesize a series of (EG)7-containing bifunctional linkers de-
signed to immobilize different tagged proteins and mutants
onto metal surfaces in a regularly oriented manner (Fig-
ure 1). In the course of a program dedicated to the label-
free detection of analytes through the use of biosensors
based on sum-frequency generation (SFG) spectroscopy,[3a]
we needed to graft proteins as dedicated receptors onto the
surface of silver- or gold-coated optical elements.[3b] Vari-
ous chemical functionalities able to bind selectively to met-
Figure 1. Bifunctional linkers.
In our study, the disulfide function (F1 in Figure 1) of
thioctic acid (TA) was chosen to interact with a gold or
silver surface, as it was already shown that this “masked”
dithiol motif allows the corresponding linkers to be handled
without risk of any side reactions, leading to stable double
anchorage on the device.[1a]
The functions selected for protein immobilization (F2 in
Figure 1) were (i) a biotin molecule giving stable complexes
with avidin-tagged proteins;[6] (ii) a halogenoacetyl residue
or (iii) a maleimide moiety, both susceptible to fix cysteine-
tagged proteins by nucleophilic substitution or Michael ad-
dition respectively;[7] and (iv) a nitrilotriacetic acid molecule
(NTA) able to complex histidine-tagged proteins with the
aid of a NiII cation.[8]
The design of the spacer arm linking the F1 and F2 mo-
tifs was inspired from our previous work dedicated to ger-
manium-ATR devices.[9] The alkyl chain of TA connected
to a few ethylene glycol units through a peptide bond is
expected to favor the formation of SAMs. In order to get
accurate spectroscopic detection at the interface, we consid-
ered an organic layer as thin as possible. Hence, a short
[a] Institute of Condensed Matter and Nanosciences (IMCN),
Molecules, Solids and Reactivity (MOST), Université catholique
de Louvain, Bâtiment Lavoisier,
Place Louis Pasteur 1, 1348 Louvain-la-Neuve, Belgium
Fax: +32-10474168
E-mail: jacqueline.marchand@uclouvain.be
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201001666.
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J. Moreau, J. Marchand-Brynaert
SHORT COMMUNICATION
OEG chain of seven units was chosen, as this length is m = 3 in Scheme 1). Monotosylation of triethylene glycol
known to be the minimum length for inducing nonfouling on a 10-gram scale was first realized in the presence of silver
behavior.[10] Thus, the bifunctional linkers (Figure 1) were oxide[11] with an optimized yield of 95% of 1 after
constructed by combining building blocks A, B, and C, as chromatography. Formation of corresponding azide 2 fol-
shown in Figure 2. It is worth noting that ester linkages lowed by catalytic hydrogenation afforded amine 3, which
between the blocks are excluded from our synthetic plan after tert-butyloxycarbonyl protection provided intermedi-
for stability reasons in biological fluids (X in Figure 1).
ate 4 in 82% yield (overall yield for four steps). Activation
of the hydroxy function by mesylation to afford 5 allowed
the (EG)3 chain to be lengthened by SN2 reaction with
tetraethylene glycol in the presence of concentrated aqueous
NaOH in hot toluene (100 °C); spacer arm 6 was recovered
by liquid–liquid extraction in 86% yield (overall yield for
six steps: 62.5%). Amino alcohol 6 was then converted in
three conventional steps into corresponding monoprotected
diamine block B2. Tosylation to afford 7, substitution with
azide to afford 8, and reduction by hydrogen in the presence
of KCl led to spacer arm 9 in 64.4% yield over three steps
(overall yield for nine steps: 40.2%). The other building
blocks are commercially available (A, C1, and C2; Figure 2)
or easily prepared. N-(Ethoxycarbonyl)maleimide (C3) was
obtained according to Keller.[12] The trimethyl ester of NTA
(C4) was synthesized from ε-N-(benzyloxycarbonyl)-(l)-
lysine in three steps following the protocol of McGeary.[13]
In principle, two modes for the assembly of the building
blocks could be considered, that is, the formation of A and
B followed by the coupling to C, or the formation of C and
B followed by the coupling to A. Practically, we found that
the latter mode (although it is the less convergent one) is
the most efficient, because it requires only one chromato-
graphic purification step on the disulfide-containing mole-
Figure 2. Selected building bocks. TA = thioctic acid; NTA = ni-
trilotriacetic acid.
Results and Discussion
Seven-unit monoprotected amino-OEG (block B1) was cules. Biotinylated derivative 10 was obtained by treating 9
first synthesized in six steps from commercially available with biotin (C1, Scheme 2). Different activation methods
triethylene and tetraethylene glycols (Scheme 1). The (EG)7 of C1 were evaluated, that is, the in situ activation with
motif could be constructed by nucleophilic substitution dicyclohexylcarbodiimide and an additive (DMAP, NHS,
(ether bond-forming reaction), either from activated (EG)4 HOBT, etc.), preformation of the N-hydroxysuccinimidyl
(i.e., 5 with n = 3) coupled to (EG)3 or from activated ester or preformation of the pentafluorophenyl (PFP) es-
(EG)3 (i.e., 5 with n = 2) coupled to (EG)4. Both synthetic ter.[14a] The highest yields of 10 were recorded by using the
schemes have been tested, but taking into account the yields PFP ester method, whose performance was already men-
of individual steps and the easiness of intermediates purifi- tioned in the field of polymer chemistry[14b] and bioorganic
cation, we recommend the latter strategy, (i.e., n = 2 and chemistry.[14c] Iodoacetamide derivative 11a could not be
Scheme 1. Synthesis of α,ω-functionalized EG heptamers. TsCl = tosyl chloride; Boc = tert-butyloxycarbonyl; MsCl = mesyl chloride;
DMAP = dimethylaminopyridine.
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Modular Synthesis of Bifunctional Linkers for Materials Science
Scheme 2. Synthesis of linkers assembled with 9. PFP = pentafluorophenyl; DMF = N,NЈ-dimethylformamide; TFA = trifluoroacetic
acid; NHS = N-hydroxysuccinimidyl.
isolated (highly unstable compound). Bromoacetamide 11b
The NTA-containing linker was constructed by using a
was prepared by adding bromoacetyl bromide to 9 in different strategy because two nucleophilic partners,
dichloromethane. A low temperature and a short reaction namely, blocks B1/B2 (OEG spacer) and C4 (derived from
time were required to avoid N-Boc deprotection. Rapid l-lysine with free ε-NH2), have to be connected. To shorten
neutralization with aqueous NaHCO3 allowed the product the total synthesis, spacer 6 (instead of 9) was considered
to be recovered in 40% yield after purification. Compound for activation by a bis-electrophilic reagent containing one
11b is light and air sensitive. At last, chloroacetamide deriv- carbon (phosgene-type reagent). In this case, the created
ative 11c was readily obtained from 9 and chloroacetyl link between the building blocks is a carbamate function
chloride; it could be purified and stored without significant (Scheme 3). Thus, imidazole carboxylate derivative 16 was
degradation (Scheme 2). Maleimide derivative 12 was syn- synthesized from alcohol 6 and carbonyldiimidiazole (CDI)
thesized from 9 and C3 in equimolar amounts, in ice-cold in dichloromethane and was directly engaged with amine
aqueous sodium hydrogen carbonate,[15] in 71% yield after C4 in the presence of DBU (10 equiv.). Carbamate 17 was
chromatography (Scheme 2). Boc cleavage from precursors obtained after purification in 78% yield over two steps. TA
10, 11b, 11c, and 12 was realized in the presence of tri- coupling was then realized as described previously to obtain
fluoroacetic acid (TFA), and the TFA salts of the corre- triester 18 in 95% yield. Saponification with lithium hy-
sponding primary amines were neutralized by addition of a droxide[17] in water furnished quantitatively final target 19.
tertiary amine before coupling with TA (block A). The
Bifunctional linkers 13, 14b, 14c, 15, and 19 were fully
NHS-ester of A previously prepared and isolated[16] was characterized by IR, 1H NMR, and 13C NMR spectroscopy
used as a pure reagent to obtain better yields of final targets and HRMS (13C spectra are given in the Supporting Infor-
13, 14b, 14c, and 15, respectively, than by applying in situ mation). Their stability in solution (≈10–2 m, CD3OD,
activation with carbodiimide. The PFP-ester of A gave sim- 25 °C) was monitored by NMR spectroscopy over several
ilar results.
months: 13 and 19 are stable and 15 and 14c very slowly
Scheme 3. Synthesis of linker assembled with 6. CDI = carbonyldiimidazole; DBU = diazabicycloundecane.
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J. Moreau, J. Marchand-Brynaert
SHORT COMMUNICATION
degrade (half-life ≈ 4–6 months). In the pure state, the link-
ers can be preserved in the freezer without damage.
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We have disclosed optimized protocols for the synthesis
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Acknowledgments
The authors thank the Région Wallone (Belgium) for financial sup-
port (contract 816890). We are grateful to S. Devouge, F. Billard,
and J. Amalric for their contribution to the organic syntheses. We
thank M. Henry and S. Demoustier-Champagne from the Unité de
chimie et de Physique des hauts polymères (POLY), UCL Louvain-
la-Neuve, for the QCM-D experiments. J.M.-B. is a senior research
associate at the Fonds de la Recherche Scientifique (FNRS, Bel-
gium).
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Received: December 13, 2010
Published Online: February 15, 2011
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Eur. J. Org. Chem. 2011, 1641–1644