Double selective synthetic approach to the n-functionalized 1,4,7-triazacyclononane derivatives: chelating compounds for controllable protein orientation

Article abstract of DOI:10.1021/jo902504d

"Chemical Equation Presented" The double selective synthetic approach is proposed for easy preparation of asymmetrically N-substituted derivatives of 1,4,7-triazacyclononane (tacn, [9]aneN₃) with the same or different pendant arms. The syntheti

Full text of DOI:10.1021/jo902504d

pubs.acs.org/joc
A selective, stable, and nondestructive immobilization of
Double Selective Synthetic Approach to the
N-Functionalized 1,4,7-Triazacyclononane
Derivatives: Chelating Compounds for Controllable
Protein Orientation
proteins on surfaces in a sterically defined form8 is a pre-
requisite for the investigation of molecular interactions.
Currently, the predominant strategy for spatially oriented
immobilization of proteins is based on specific interaction
between a metal chelator, usually nitrilotriacetic acid (NTA)
functionalized SAMs, and histidine-tagged proteins.3 NTA
are very frequently employed for His-tag protein purifica-
tion.9a More than 60% of the proteins produced for struc-
tural studies include His6-tag9b and can be directly used for
selective and oriented binding to chelator-functionalized
surfaces. However, complexes between His-tag and NTA-
Ni2þ are of very low affinity (Kd values in the 1-10 μM
range).10 SAMs presenting the Multivalent Chelators based
on NTA were prepared with increased stability of the
chelator-His-tag complexes.11 The chips based on SAMs
that present “tacn” ligand offer a major advantage over
current NTA-based chelator systems, i.e., the triazamacro-
cyclic ligand forms stable (nondissociable) complexes with a
Ni ion and forms stable complexes with His-tagged pro-
teins.12 The protein binding selectivity also has been exam-
ined with the “tacn”-modified sorbent.13
“Tacn” chelators also have been used for an affinity-
capturing system of His-tagged proteins in SAMDI experi-
ment14 on SAMs prepared from maleimide-terminated
OEG-alkyl disulfides. These SAMs were incubated with
thiol-terminated “tacn” after formation of a monolayer.
However, SAMs have more often been formed from OEG-
alkanethiols than from unsymmetrical disulfides. The latter
are known for reduced kinetics of chemisorption on gold.15
SAMs with reactive carboxyls, often in “active ester” form,
are the most widely used for coupling with desired ligands.16
Formation of an amide bond mediated by NHS/EDC is
simple but suffers from sensitivity of the transiently formed
active NHS esters to hydrolysis,17 which complicates appli-
cation of this method, especially in microspotting. Hence,
ꢀ
Tomasz D. Sobiesciak* and Piotr Zielenkiewicz
Institute of Biochemistry and Biophysics, Polish Academy of
Sciences, Pawinꢀskiego 5a, Warsaw 02-106, Poland
tomasz_s@ibb.waw.pl
Received November 24, 2009
The double selective synthetic approach is proposed for
easy preparation of asymmetrically N-substituted deri-
vatives of 1,4,7-triazacyclononane (tacn, [9]aneN3) with
the same or different pendant arms. The synthetic routes
simplify the synthesis of “tacn” ligands for functionaliza-
tion of other carboxy terminated molecules/supports and
also facilitate access to azamacrocycle functionalized self-
assembled monolayer (SAMs) or nanopatterns for spe-
cific and stable Histidine-tagged protein immobilization.
The analysis of protein complexes and protein-protein
interactions is currently a subject of great interest.1 Studies of
these interactions often require ligands which are linked to
biocompatible surfaces and specifically and selectively inter-
act with biomolecules.2 Protein interactions can be detected
by SPR measurement on biochips3 that are prepared from
SAM on gold,4 and by the method that combines SAMs with
MALDI-TOF-MS (also called as SAMDI).5 Oligo(ethylene
glycol)-terminated6 SAMs are inert in nonspecific interac-
tions with proteins.7
(8) (a) Gamsjaeger, R.; Wimmer, B.; Kahr, H.; Tinazli, A.; Picuric, S.;
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Lata, S.; Tampe, R.; Maulet, Y.; Gruber, H. J.; Hinterdorfer, P.; Romanin,
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M.; Schweitzer, B.; Predki, P. F. Curr. Opin. Biotechnol. 2005, 16, 447–452.
(c) Cha, T.; Guo, A.; Zhu, X.-Y. Proteomics 2005, 5, 416–419.
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Flagiello, A.; Pucci, P. Expert Rev. Proteomics 2009, 6, 159–169. (b) Fuentes,
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DOI: 10.1021/jo902504d
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J. Org. Chem. 2010, 75, 2069–2072 2069
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Sobiesciak and Zielenkiewicz
JOCNote
separate synthesis of fully ligand-functionalized OEG-
alkane-thiols and formation of SAMs from compounds
which are already ready for interaction with their affinity
counterparts is the best way.18
SCHEME 1. Application of the Methodology to the Synthesis of
1,4,7-Triazonane Derivatives with Aminoalkyl Linker and Two
Pendant Arms
“Tacn” and its derivatives coordinate facially to metal ions
and form a very stable19a and diverse range of metal com-
plexes.19b The preparation of special “tacn” derivatives for
functionalization of other molecules/supports is not simple.
This results from synthetic difficulties involved in the differen-
tiation of three equivalent secondary amino groups on aza-
crown. Even the synthesis of pure “tacn” is low-yielding and
troublesome. Syntheses of monosubstituted or asymmetrically
functionalized “tacn” derivatives with more than one type of
pendant donor generally yielded a mixture of products.20
Thus, we looked for a convenient, inexpensive, facile, and
efficient synthesis of “tacn” derivatives from commercially
available precursors which does not require special reaction
conditions, exclude the occurrence of the statistically substituted
derivatives, and offer the possibility of simple modulation of
coordination properties of azamacrocycle. The introduction of
specific, various functionalities at the periphery of the coordinat-
ing moiety may allow for fine-tuning of the properties of the
complex.21 The choice of “tacn orthoamide” as a starting
compound provides efficient routes to “tacn” derivatives incor-
porating different functional groups. The “tacn-orthoamide”
was initially used effectively for the synthesis of monofunctio-
nalized “tacn” derivatives.22 Then, many applications of this
general reaction scheme apeared with a simple change in the type
of azacrown substituent.23 Subsequent simple modifications of
this procedure involve introduction of the monoamidinium salt
of phthalimide-tacn as precursors to “tacn” with two different
pendant arms with a potential for intermolecular cross-linking.24
These derivatives we found to be basic model compounds for the
search for an expanded generally applicable synthetic approach.
Here, we have demonstrated a new and practical synthetic
method utilizing the “double selective asymmetric functio-
nalization” of “tacn” (Scheme 1). This was done in the first
selective stage (I) of synthesis, by adapting “tacn orthoa-
mide” and taking advantage of self-protection of azacrown
after revealing a masked formyl group resulting from the
At each stage of the synthesis: 100% conversion. I and II denote the
two major selective stages of synthesis.
(18) Luk, Y.-Y.; Tingey, M. L.; Hall, D. J.; Israel, B. A.; Murphy, C. J.;
Bertics, P. J.; Abbott, N. L. Langmuir 2003, 19, 1671–1680.
(19) (a) Yang, R.; Zompa, L. J. Inorg. Chem. 1976, 15, 1499–1502.
(b) Wainwright, K. P. Coord. Chem. Rev. 1997, 166, 35–90.
(20) (a) Kovacs, Z.; Sherry, A. D. Tetrahedron Lett. 1995, 36, 9269–9272.
(b) Pulacchini, S.; Watkinson, M. Tetrahedron Lett. 1999, 40, 9363–9365.
(c) Ellis, D.; Farrugia, L. J.; Peacock, R. D. Polyhedron 1999, 18, 1229–1234.
(d) Biot, C.; Dessolin, J.; Ricard, I.; Dive, D. J. Organomet. Chem. 2004, 689,
4678–4682.
(21) Nonat, A.; Gateau, C.; Fries, P. H.; Mazzanti, M. Chem.;Eur. J.
2006, 12, 7133–7150.
(22) (a) Weisman, G. R.; Vachon, D. J.; Johnson, V. B.; Gronbeck, D. A.
J. Chem. Soc., Chem. Commun. 1987, 12, 886–887. (b) Alder, R. W.;
Mowlam, R. W.; Vachon, D. J.; Weisman, G. R. J. Chem. Soc., Chem.
alkylation reaction. In the second selective stage (II) of
synthesis, this was done by differentiation of amine groups
by trifluoroacetylation, i.e., by selective monotrifluoroace-
tylation of primary over secondary amine and subsequent
attachment of the third pendant arm, or by protection of the
secondary amine. Our synthetic methodology allows the
synthesis of a new type of triazamacrocyclic chelate that
can possess an amino-terminated linker of variable length,
introduced into the structure in order to increase the mobility
of the final molecule and retain protein functionality, and
one or two pendant arms. In our approach the “tacn”
derivative is the key reaction product at every step of
€
Commun. 1992, 6, 507–508. (c) Schulz, D.; Weyhermuller, T.; Wieghardt, K.;
Nuber, B. Inorg. Chim. Acta 1995, 240, 217–229. (d) Stockheim, C.; Hoster,
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Ross, S. A.; Schroder, M. J. Chem. Soc., Dalton Trans. 1996, 4379–4387.
(23) For example: (a) Ellis, D.; Farrugia, L. J.; Hickman, D. T.; Lovatt,
P. A.; Peacock, R. D. Chem. Commun. 1996, 15, 1817–1818. (b) Blake, A. J.;
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Fallis, I. A.; Heppeler, A.; Parsons, S.; Ross, S. A.; Schroder, M. J. Chem.
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M. J. Chem. Soc., Perkin Trans. 1 1999, 9, 1211–1213. (d) McGowan, P. C.;
Podesta, T. J.; Thornton-Pett, M. Inorg. Chem. 2001, 40, 1445–1453.
(24) (a) Warden, A.; Graham, B.; Hearn, M. T. W.; Spiccia, L. Org. Lett.
2001, 3, 2855–2858. (b) Belousoff, M. J.; Battle, A. R.; Graham, B.; Spiccia,
L. Polyhedron 2007, 26, 344–355.
(25) (a) For example: Pale-Grosdemange, C.; Simon, E. S.; Prime, K. L.;
Whitesides, G. M. J. Am. Chem. Soc. 1991, 113, 12–20. (b) For example:
Hoffmann, Ch.; Tovar, G. E. M. J. Colloid Interface Sci. 2006, 295, 427–435.
(c) Yam, C. M.; Lopez-Romero, J. M.; Gu, J.; Cai, C. Chem. Commun. 2004,
2510–2511. (d) Lasseter, T. L.; Clare, B. H.; Abbott, N. L.; Hamers, R. J.
J. Am. Chem. Soc. 2004, 126, 10220–10221.
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Sobiesciak and Zielenkiewicz
JOCNote
synthesis, without byproduct with another distribution pat-
tern of substituents. The usefulness of these ligands has been
further demonstrated by the synthesis of a “tacn” analogue
linked to OEG-alkene, which is commonly used as a pre-
cursor in the synthesis of OEG terminated alkanethiols25a or
silanes25b for SAMs formation and can be directly and
readily used for functionalization of hydrogen-terminated
silicon25c,d and diamond surfaces25d for SAMs formation.
We assume that all synthetic steps in our approach should
meet some of the demands of “positional synthesis”
(mechanosynthesis), i.e., the synthetic route should minima-
lize unwanted reactions and strongly favor desired reactions
and should yield exclusively the desired product in a highly
controlled fashion. The synthesis of “tacn”26 and “tacn-
orthoamide” (1)27 is readily accomplished by standard pro-
cedures. Compund 8a was prepared according to the syn-
thetic strategy outlined in Scheme 1, starting with “tacn
orthoamide” (1). The compounds 2-5a were synthesized
following known methods, i.e., “tacn” monofunctionaliza-
tion method,22 azacrown amine alkylation after revealing the
formyl group.22e,23,35 Compounds 2-5a have been pre-
viously described.24
Selective protection of primary amine in the presence of
secondary amines without requirement for a chromatographic
separation of mono- and diprotected mixtures of “tacn” com-
pounds was best accomplished by the reaction of 5a with ethyl
trifluoroacetate28 in methanol solution in the presence of an
amine base. No trace of a ditrifluoroacetylated byproduct was
detected after reaction at -10 to 0 °C. The Boc protected
“tacn” derivative 7a was prepared by reaction of 6a with Boc
anhydride in THF. The resulting compound was treated with
NaOH in water/MeOH at rt in order to remove the trifluor-
oacetyl protecting group, leading to free aminopropyl linker
substituted “tacn” derivative 8a.29
SCHEME 2. Alternative Functionalization Route
the presence of acid/base sensitive methyl ester group.
The sodium borohydride/ethanol system,31 similar to the
K2CO3/water/methanol system,32 is inappropriate in the
case of the methyl ester group. A similar procedure starting
with amidinium salt 2, but differing in the use of ethyl
bromoacetate instead of benzyl chloride, was adopted for
preparation of 8b (Scheme 1). Accordingly, the secondary
amino group in azacrown 3 was alkylated with ethyl bro-
moacetate yielding compound 4b. Compound 7b was ob-
tained in three consecutive steps: hydrolysis, monotrifluo-
roacetylation, and Boc protection. We found that sodium
borohydride in THF/absolute ethanol removed the trifluo-
roacetyl group effectively in compound 7b, but also reduced
the methyl ester pendant arm group to alcohol.33 Therefore,
deprotection was performed by using NaBH4 in THF at reflux,
which afforded compound 8b. These conditions did not affect
the methyl ester functionality in the molecule.34
To check the possibility of derivatization of 6a via an
alkylation reaction with ethyl bromoacetate,35 the route pre-
sented in Scheme 2 was performed. The “tacn” derivative 6a
was functionalized with the methylenecarboxylate group, in-
stead of the Boc group introduced on the “tacn” ring pre-
viously, giving compound 9.36 This approach illustrated the
suitability of monotrifluoroacetylated “tacn” derivatives as
precursors to asymmetrically N-functionalized azamacro-
cycles with different pendant arms.
In exploring the alternative synthetic strategy to com-
pounds with better solubility in aqueous buffers usually used
for protein maintenance, we propose a reaction sequence in
which we take advantage of self-protection of “tacn” by a
formyl group. The synthesis of 11 followed the shortened
route presented in Scheme 1. Thus, monoformyl “tacn” with
phthalimide-protected aminopropyl linker and methylene-
carboxylic acid t-Bu ester pendant arm 10 was obtained after
four steps. Removal of the phthalimide protecting group by
hydrazine afforded the “tacn” derivative with free unpro-
tected aminopropyl linker 11.37 These conditions did not
affect the more stable t-Bu ester function in the molecule, but
suffer from the possibility of deprotection of the formyl
group by hydrazine derivatives.38
Removal of the trifluoroacetyl group requires treatment
with a base30a-c or reductive deprotection with NaBH4 in
ethanol.30a,d The “tacn” derivative bearing the methyl ester
pendant arm 7b was synthesized in search of appropriate
deprotection conditions for the trifluoroacetyl moiety in
(26) Richman, J. E.; Atkins, T. J. J. Am. Chem. Soc. 1974, 96, 2268–2270.
(27) Atkins, T. J. J. Am. Chem. Soc. 1980, 102, 6364–6365.
(28) Xu, D.; Prasad, K.; Repic, O.; Blacklock, T. J. Tetrahedron Lett.
1995, 36, 7357–7360.
(29) In NMR spectra characteristic peaks are observed (see the Support-
ing Information). The 1H NMR spectrum indicates that 7a is still present
(5.3 (br s, CONH)). The deprotection of the N-trifluoroacetyl group with 1 M
NaOH was sluggish; see: Rosso, V. W.; Pazdan, J. L.; Venit, J. J. Org. Proc.
Res. Dev. 2001, 5, 294–298.
ꢁ
(30) For example: (a) Calmes, M.; Escale, F.; Rolland, M.; Martinez, J.
Tetrahedron: Asymmetry 2003, 14, 1685–1689. (b) Boger, D. L.; Yohannes,
D. J. Org. Chem. 1989, 54, 2498–2502. (c) Jackson, R. F. W.; Rilatt, I.;
Murray, P. J. Chem. Commun. 2003, 1242–1243. (d) Weygand, F.; Frauen-
dorfer, E. Chem. Ber. 1970, 103, 2437–2449.
(31) (a) Seki, H.; Koga, K.; Matsuo, H.; Ohki, S.; Matsuo, I.; Yamada,
S-i. Chem. Pharm. Bull. 1965, 13, 995–1000. (b) Brown, M. S.; Rapoport, H.
J. Org. Chem. 1963, 28, 3261–3263.
“Bis-formyl-tacn” derivative 13 was obtained39 in a simi-
lar way after convenient formylation with ammonium for-
mate40 and subsequent hydrazinolysis (Scheme 3).
The aminoalkyl functionalized “tacn” ligands are important
intermediates for the synthesis of azamacrocycle terminated
(32) Kaestle, K. L.; Anwer, M. K.; Audhya, T. K.; Goldstein, G.
Tetrahedron Lett. 1991, 32, 327–330.
(33) For example, CF3CONH- and -COOMe groups are stable in the
presence of NaBH4/THF/H2O; see ref 30c.
(34) Selective deprotection of the trifluoroacetyl group in the presence of
intact methyl ester group was also achieved with NaOMe/MeOH (see ref 30c)
and with K2CO3/H2O/MeOH (see ref 30b). Nonetheless, Kudzin et al.
reported that NaOMe-mediated deprotection is not effective due to the
(36) In NMR spectra characteristic peaks are observed (see the Support-
ing Information).
(37) The structure of this compound was analyzed by 2D NMR, IR, and
ESI-MS as well (see the Supporting Information).
(38) Geiger, R.; Siedel, W. Chem. Ber. 1968, 101, 3386–3391.
(39) The structure of this compound was analyzed by 2D NMR, IR, and
ESI-MS as well (see the Supporting Information).
(40) Reddy, P. G.; Kumar, G. D. K.; Baskaran, S. Tetrahedron Lett. 2000,
41, 9149–9151.
_
formation of stable amidate salts; see: Kudzin, Z. H.; yyzwa, P.; yuczak, J.;
Andrijewski, G. Synthesis 1997, 44–46.
(35) Schultz, R. A.; White, B. D.; Dishong, D. M.; Arnold, K. A.; Gokel,
G. W. J. Am. Chem. Soc. 1985, 107, 6659–6668.
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Sobiesciak and Zielenkiewicz
JOCNote
SCHEME 3. Synthetic Route to the Bis-Formyl “tacn” Deri-
vatives
bonds,47 splitting/broadening of signals caused by long-range
fluorine coupling (JFC and JFH) in trifluoroacetamide deriva-
tives,48 the NMR shielding effect of a benzene ring, occurrence
of CH-π interaction between the benzene ring and -CH
protons,49 and appearance of t-Bu in a Boc group as two
singlets (or three, two of which may coincide) in the 1H NMR
spectrum.50 Thus, the structure of synthesized compounds was
determined in a straightforward manner by using one-bond
(1H-13C) correlation NMR spectra and confirmed by the
results of ESI mass spectrometric analysis. Nevertheless, for
unambiguous assignment of all resonances to proper confor-
mers, additional NMR correlation spectra, especially NOESY,
and 1D-NOE difference NMR spectra are desirable.
SCHEME 4. Representative Synthesis of “Tacn”-Terminated
OEG-Alkene
Therefore, in order to clearly show the selectivity of the
trifluoroacetylation stage, resulting in a lack of ditrifloro-
acetylated product, electrospray ionization mass spectro-
metry was applied (see the Supporting Information). ESI-
MS is the technique of choice for solution mechanistic
studies in chemistry and biochemistry.51
In summary, we developed a general method for the synth-
esis of selectively N-functionalized “tacn” with aminoalkyl
linker. Our approach is not only effective, but also offers
attractive features such as operational simplicity and conve-
nience, startingmaterialavailability, mildreactionconditions,
air-stability of all synthetic steps, and reduced number of
chromatographic separations. The double selective syn-
thetic method allows for the ordered multistep synthesis of
“tacn” ligands. These ligands are ideal candidates for
attachment, via the standard carbodiimide coupling, to
carboxy-terminated oligo(oxyethylene)alkyl derivatives
and facilitate access to “tacn”-functionalized SAMs.
oligo(oxyethylene)alkyl derivatives. The synthesized amino-
propyl linker functionalized azamacrocyclic ligand 13 was
coupled to tri(ethylene glycol)-alkene with carboxyl function-
ality 14 via amide linkage to give the corresponding structure
15.41 Couplings of the “tacn” ligand 13 to carboxylic acid (14)
was carried out with standard reagents DCC (or EDC) and
HOBt in acetonitrile (Scheme 4).
ESI-MS and 1H-13C gHSQC NMR spectroscopy were
applied to monitor the synthesis process. In the HSQC spectra,
characteristic signals are clearly distinguishable from reso-
nances of impurities. The room temperature 1H NMR spectra
of formyl protected “tacn” derivatives have double and/or
broad signals due to the slow NMR time scale of hindered
rotation around the exocyclic N-CO bond.22e,24a We observed
that trifluoroacetyl- and Boc-protected “tacn” derivatives ex-
hibit similar multiplication and broadening of signals. The
restricted rotation about the N-CO/NdCO bond42 and occur-
rence of conformational isomerism was described for trifluor-
oacetamides43 and Boc-protected amines.44 These conformers
of “tacn” derivatives exist in solution and therefore assignment
by NMR was not easy. Broadening of NMR signals can also be
attributed at least in part to the heterogenity of the sample.
Moreover, the structural analysis by NMR is complicated by
inversion of the azamacrocycle ring,21,45 signals broadening
and affecting chemical shifts upon complexation of the ca-
tions,46 formation of inter- and/or intramolecular hydrogen
Experimental Section
General Procedure for the Selective Trifluoroacetylation. Eth-
yl trifluoroacetate (1.2 equiv) was added dropwise to a solution
of the aminoalkyl-“tacn” (1 equiv) and triethylamine (1 equiv) in
anhydrous methanol at -10 to 0 °C. The resulting solution was
stirred for 2 h. Subsequently, the reaction mixture was stirred
overnight at ambient temperature. The solvent was then re-
moved under reduced pressure to afford monotrifluoroacety-
lated compound. ESI-MS clearly shows the selectivity of the
trifluoroacetylation reaction step.
Data for 6a: HRMS (ESI) calcd for C18H28F3N4O [(M þ
H)þ]:373.22152, found 373.22148.
Acknowledgment. ESI-MS and NMR experiments were
performed by a local facility.
(41) (a) The structure of this compound was analyzed by 2D NMR, IR, and
ESI-MS as well (see the Supporting Information. (b) The “tacn”-OEG-alkene was
further thioacetylated (AcSH, AIBN). Subsequent smooth deprotection of formyl
and acetyl groups was performed under acidic conditions (see the Supporting
Information).
Supporting Information Available: Experimental proce-
dures and characterization data. This material is available free
of charge via the Internet at http://pubs.acs.org.
(42) Stewart, W. E.; Siddall, T. H. Chem. Rev. 1970, 70, 517–551.
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