Synthesis of a Non-Heme Template for Attaching Four Peptides: An Approach to Artificial Iron (II)-Containing Peroxidases

Article abstract of DOI:10.1021/jo035157z

We are developing all-synthetic model cofactor-protein complexes in order to define the parameters controlling non-natural cofactor activity. The long-term objective is to establish the theoretical and practical basis for designing novel enzymes. A non-heme pentadentate ligand (N4Py) is being developed as a template for the site-specific attachment of a designed four-helix bundle. Previously, we attached two unprotected peptides via CH ₂Cl handles to N4Py. In the presence of hydrogen peroxide, the iron(II) complex of this ligand (2a) generates an Fe^IIIOOH intermediate (3a) that can oxidize a wide variety of organic compounds. Here, we describe the synthesis of 27, a N4Py derivative in which four three-carbon spacers have been introduced, and show that four copies of an unprotected, single-cysteine peptide can be coupled via a thioether linkage to the ligand. In addition, a divergent synthesis route to tetrabromide ligand lb has also been developed, providing the opportunity to prepare alternative pentadentate ligands efficiently by four cross-coupling reactions on a single molecule. Also, two of the four bromides of lb can be selectively addressed by magnesium-bromide exchange.

Full text of DOI:10.1021/jo035157z

Syn th esis of a Non -Hem e Tem p la te for Atta ch in g F ou r P ep tid es:
An Ap p r oa ch to Ar tificia l Ir on (II)-Con ta in in g P er oxid a ses
Marco van den Heuvel, Tieme A. van den Berg, Richard M. Kellogg,
Christin T. Choma,*,† and Ben L. Feringa*
Department of Organic and Molecular Inorganic Chemistry, University of Groningen,
Nijenborgh 4, 9747 AG Groningen, The Netherlands
b.l.feringa@chem.rug.nl; chomac@rpi.edu
Received August 6, 2003
We are developing all-synthetic model cofactor-protein complexes in order to define the parameters
controlling non-natural cofactor activity. The long-term objective is to establish the theoretical and
practical basis for designing novel enzymes. A non-heme pentadentate ligand (N4Py) is being
developed as a template for the site-specific attachment of a designed four-helix bundle. Previously,
we attached two unprotected peptides via CH2Cl handles to N4Py. In the presence of hydrogen
peroxide, the iron(II) complex of this ligand (2a ) generates an FeIIIOOH intermediate (3a ) that can
oxidize a wide variety of organic compounds. Here, we describe the synthesis of 27, a N4Py derivative
in which four three-carbon spacers have been introduced, and show that four copies of an
unprotected, single-cysteine peptide can be coupled via a thioether linkage to the ligand. In addition,
a divergent synthesis route to tetrabromide ligand 1b has also been developed, providing the
opportunity to prepare alternative pentadentate ligands efficiently by four cross-coupling reactions
on a single molecule. Also, two of the four bromides of 1b can be selectively addressed by
magnesium-bromide exchange.
In tr od u ction
dendrimers,6 and a cyclotribenzylene macrocycle7 are
some examples of applied non-metal-binding templates.
Metal-ion-assisted self-assembly processes of peptides8
and chelating molecules, such as porphyrins,4e,9 have
been employed to prepare de novo designed metallopro-
teins. For instance, the incorporation of a heme group,
With an increasing understanding of the principles
governing protein structure and function, together with
initiatives over the past decade in the de novo design of
proteins, simple, stable proteins that fold as predicted
and exhibiting rudimentary catalytic activity can now be
designed with some success.1 One approach to aid the
folding of designed proteins is template-assembled syn-
thetic proteins (TASP),2 where the peptide chains are
attached to a template that helps direct the folding of
the protein. Cavitands,3 (cyclic) peptides,4 carbohydrates,5
(3) (a) Gibb, B. C.; Mezo, A. R.; Causton, A. S.; Fraser, J . R.; Tsai,
F. C. S.; Sherman, J . C. Tetrahedron 1995, 51, 8719-8732. (b) Gibb,
B. C.; Mezo, A. R.; Sherman, J . C. Tetrahedron Lett. 1995, 36, 7587-
7590. (c) Mezo, A. R.; Sherman, J . C. J . Am. Chem. Soc. 1999, 121,
8983-8994.
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Eggleston, I. M.; Mutter, M. Macromol. Symp. 1996, 101, 397-404.
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Org. Chem. 1999, 64, 7114-7120. (e) Rau, H. K.; DeJ onge, N.; Haehnel,
W. Angew. Chem., Int. Ed. Engl. 2000, 39, 250-253.
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Brask, J .; J ensen, K. J . J . Pept. Sci. 2000, 6, 290-299. (c) Brask, J .;
Dideriksen, J . M.; Nielsen, J .; J ensen, K. J . Org. Biomol. Chem. 2003,
1, 2247-2252.
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1375. (b) Higashi, N.; Koga, T.; Niwa, N.; Niwa, M. Chem. Commun.
2000, 361-362. (c) Sakamoto, M.; Ueno, A.; Mihara, H. Chem.
Commun. 2000, 1741-1742.
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27.
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1992, 114, 4000-4002. (c) Mutz, M. W.; Case, M. A.; Wishart, J . F.;
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A° kerfeldt, K. S.; Kim, R. M.; Camac, D.; Groves, J . T.; Lear, J . D.;
DeGrado, W. F. J . Am. Chem. Soc. 1992, 114, 9656-9657. (d) Arai, T.;
Kobata, K.; Mihara, H.; Fujimoto, T.; Nishino, N. Bull. Chem. Soc. J pn.
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* To whom correspondence should be addressed. (B.L.F.) Tel: +31-
50-3634235. Fax: +31-50-3634296. (C.T.C.) Tel: +1-518-276-2804.
Fax: +1-518-276-4887.
† Current address: Department of Chemistry, Rensselaer Polytech-
nic Institute, Troy, NY 12180-3590.
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S. F.; Raleigh, D. P.; DeGrado, W. F. Curr. Opin. Struct. Biol. 1993, 3,
601-610. (c) Bryson, J . W.; Betz, S. F.; Lu, H. S.; Suich, D. J .; Zhou,
H. X.; O’Neil, K. T.; DeGrado, W. F. Science 1995, 270, 935-941. (d)
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10.1021/jo035157z CCC: $27.50 © 2004 American Chemical Society
Published on Web 12/20/2003
250
J . Org. Chem. 2004, 69, 250-262

Non-Heme Template for Attaching Four Peptides
SCHEME 1
either by covalent attachment or by coordination of the
metallo-heme unit to peptide-bound histidine, can in-
troduce functionality into a protein structure.10
Here, we address the design and preparation of a first-
generation non-heme iron complex for incorporation into
a four-helix bundle, a simple protein consisting of four
R-helices. We ultimately aim to construct a functional
non-heme peroxidase mimic in which the catalytic cofac-
tor is integrated into a designed four-helix bundle. The
simplicity of such catalyst-protein complexes will permit
the factors controlling protein-mediated modulation of
cofactor activity to be defined. Eventually, the size, shape,
and hydrophobicity of the active site and the substrate
binding interactions in the four-helix bundle will be
altered in order to tune selective substrate oxidation.
The template chosen for peptide attachment is the
neutral pentadentate ligand N4Py (1a ) (Scheme 1),11
which consists of four pyridine rings anchored via one-
carbon bridges to a central nitrogen atom.12 In the
presence of hydrogen peroxide, its iron(II) complex
N4PyFe 2a generates a short-lived FeIIIOOH intermedi-
ate (3a )13 that can oxidize a wide variety of organic
compounds.14 This non-heme iron system has successfully
been transformed into an effective DNA cleaving agent
that mimics iron bleomycin.15
F IGURE 1. Crystal structure of N4PyFe 2a superimposed
on the crystal structure of GCN4-p1-LI, homo-tetra-
meric coiled-coil (side chains are omitted for clarity). Left: a
7-residue, 10 Å slice of GCN4-p1-LI,17 showing N4PyFe inter-
digitating between the helices of the bundle; right: a 47 Å slice,
showing the full-length coiled-coil.
a
hydrogen peroxide in water.16 These results indicated
that the incorporation of the N4PyFe catalyst into a
designed four-helix bundle environment is feasible.
The challenge addressed in this paper is to modify the
N4Py ligand to host four peptide chains. This essentially
entails two key issues. The first is the development of a
new synthesis route to tetrafunctionalized N4Py ligands.
The second is a stability issue: the implementation of
the previously developed methodology (the use of -CH2-
Cl groups for peptide attachment) resulted in the intrin-
sically unstable ligand 26 (Scheme 5). A stable ligand
was obtained by extending the linker to a three-carbon
spacer to provide template 27. As both 26 and 27 were
prepared via the same synthesis route, their syntheses
will be discussed simultaneously.
The geometry of the N4PyFe complex appears to
complement the spacial arrangement of the four peptide
chains that constitute the core of the four-helix bundle
(Figure 1). In a preliminary study, two peptide chains
were attached to a disubstituted N4Py derivative by
means of cysteine thiol-mediated nucleophilic substitu-
tion on -CH2Cl groups.16 The corresponding iron(II)
complex retained its catalytic oxidation activity with
Resu lts a n d Discu ssion
(10) Lombardi, A.; Nastri, F.; Pavone, V. Chem. Rev. 2001, 101,
3165-3189.
Con ver gen t Syn th esis Str a tegy. From visual in-
spection of molecular models of 2a , it was deduced that
the linkers for peptide binding should be one methylene
group long (C1-spacer) and attached at the 5-positions
of the pyridine rings. Consequently, the 2-positions are
to be utilized to construct the N4Py core (Scheme 1). In
the final iron(II) complex, the linkers at the 5-position
will thus direct the peptide chains away from the iron
center. According to the models, this architecture pro-
vides an unstrained linkage between the folded four-helix
bundle and the cofactor. A convergent synthesis strategy
was chosen wherein the required spacer and functionality
for the target template are incorporated into the building
blocks before the core of the ligand itself is constructed.
Several procedures are known for the preparation of
2,5-disubstituted pyridine synthons for the construction
of functionalized N4Py derivatives.16,18-20 In a departure
from these methods, tetrasubstituted N4Py derivatives
(11) Abbreviations used: N4Py, N-[di(2-pyridinyl)methyl]-N,N-bis-
(2-pyridinylmethyl)amine; N4PyFe, the iron(II) complex of N4Py.
(12) Lubben, M.; Meetsma, A.; Wilkinson, E. C.; Que, L., J r.;
Feringa, B. L. Angew. Chem., Int. Ed. Engl. 1995, 34, 1512-1514.
(13) (a) Ho, R. Y. N.; Roelfes, G.; Feringa, B. L.; Que, L., J r. J . Am.
Chem. Soc. 1999, 121, 264-265. (b) Roelfes, G.; Lubben, M.; Chen, K.;
Ho, R. Y. N.; Meetsma, A.; Genseberger, S.; Hermant, R.; Hage, R.;
Mandal, S. K.; Young, V. G., J r.; Zang, Y.; Kooijman, H.; Spek, A. L.;
Que, L., J r.; Feringa, B. L. Inorg. Chem. 1999, 38, 1929-1936. (c) Ho,
R. Y. N.; Roelfes, G.; Hermant, R.; Hage, R.; Feringa, B. L.; Que, L.,
J r. Chem. Commun. 1999, 2161-2162. (d) Roelfes, G.; Vrajmasu, V.;
Chen, K.; Ho, R. Y. N.; Rohde, J .-U.; Zondervan, C.; la Crois, R. M.;
Schudde, E. P.; Lutz, M.; Spek, A. L.; Hage, R.; Feringa, B. L.; Munck,
E.; Que, L., J r. Inorg. Chem. 2003, 42, 2639-2653.
(14) (a) Roelfes, G.; Lubben, M.; Leppard, S. W.; Schudde, E. P.;
Hermant, R. M.; Hage, R.; Wilkinson, E. C.; Que, L., J r.; Feringa, B.
L. J . Mol. Catal. A: Chem. 1997, 117, 223-227. (b) Roelfes, G.; Lubben,
M.; Hage, R.; Que, L., J r.; Feringa, B. L. Chem. Eur. J . 2000, 6, 2152-
2159.
(15) Roelfes, G.; Branum, M. E.; Wang, L.; Que, L., J r.; Feringa, B.
L. J . Am. Chem. Soc. 2000, 122, 11517-11518.
(16) Choma, C. T.; Schudde, E. P.; Kellogg, R. M.; Robillard, G. T.;
Feringa, B. L. J . Chem. Soc., Perkin Trans. 1 1998, 769-773.
J . Org. Chem, Vol. 69, No. 2, 2004 251

van den Heuvel et al.
SCHEME 2a
SCHEME 3a
a
Conditions: (i) (1) n-BuLi, THF, -80 °C, 40 min, (2) DMF,
-80 °C, 1 h; (3) H2O; (ii) NaBH4, MeOH, 0 °C to rt, 15 min; (iii)
SOCl2, TEA, DCM, rt, 18 h.
As noted above, molecular modeling indicates that a
single methylene group as spacers would provide optimal
unstrained linkage between the folded bundle and the
ligand. However, due to stability problems with the
ligand containing C1-spacers (vide infra), it was neces-
sary to also investigate the synthesis of the ligand with
C3-spacers. To obtain key building block 10 for the
construction of the N4Py ligand 24, with a C3-spacer,
aldehyde 5 was converted to the R,â-unsaturated ester 8
by a Horner-Emmons reaction using triethyl phospho-
noacetate (Scheme 2). Subsequent reduction by LiAlH4
to the saturated alcohol 9 was followed by a silylation to
afford the TBDMS ether 10 in 81% yield after purifica-
tion.
Depending on the spacer length in the final ligand, the
required 2-picolyl chlorides 15 and 16 can be obtained
from building blocks 7 and 10, respectively (Scheme 3).
To this end, these building blocks were lithiated with
n-BuLi in THF at -80 °C and quenched with DMF to
provide the aldehydes 11 and 12 (80% and 72% yield,
respectively). Alternatively, the bromo-metal exchange
can be accomplished by the use of i-PrMgBr in THF at
room temperature,22 which furnishes 11 and 12 in similar
yields. These aldehydes can in principle be reduced in
situ to the picolyl alcohols 13 and 14. However, as both
aldehydes are also required for the synthesis of bis(2-
pyridinyl)methylamine 21 and 22 (Scheme 4), these
reductions were carried out in a separate reaction step.
Finally, by reaction with thionyl chloride in the presence
of TEA alcohols 13 and 14 were quantitatively converted
to the corresponding 2-picolyl chlorides 15 and 16, and
isolated in 66% and 75% yield, respectively, after puri-
fication. Flash column chromatography of these com-
pounds proved to be crucial to minimize decomposition
of the product during purification.23
a
Conditions: (i) (1) n-BuLi, Et2O, -80 °C, 1 h, (2) DMF, -80
°C, 1 h, (3) H3O+; (ii) NaBH4, MeOH, 0 °C to rt, 15 min; (iii)
TBDMSCl, TEA, DMF, 15 min; (iv) (EtO)2P(O)CH2CO2Et, NaH,
THF/Et2O, 0 °C to rt, 1 h; (v) LiAlH4, THF, 0 °C to rt, 75 min.
23 and 24 were constructed starting from commercially
available 2,5-dibromopyridine (4). This enables a versa-
tile and controlled introduction of various functionalities
at the desired positions of the pyridine ring (vide infra)
and consequently facilitates the preparation of a wide
range of tetrafunctionalized N4Py derivatives.
Liga n d Syn th esis. For the synthesis of the N4Py
ligand 23, with a C1-spacer, pyridine derivative 7 was
required. The C1-spacer in building block 7 was intro-
duced by preparing aldehyde 5 from 2,5-dibromopyridine
(4) by a regioselective lithiation21 with n-BuLi in diethyl
ether at -80 °C, followed by quenching with N,N-
dimethylformamide (DMF) (Scheme 2).
Although the 2-position in 4 is more electron-deficient
than the 5-position, mono-metalation under these condi-
tions occurs selectively at the latter position,21 providing
a powerful tool for regioselective functionalization. Al-
dehyde 5 was subsequently reduced with NaBH4 in
MeOH to furnish alcohol 6, which was protected with tert-
butyldimethylsilyl chloride (TBDMSCl) in DMF in the
presence of triethylamine (TEA) to provide the essential
building block 7 in 78% yield from 4.
(17) Harbury, P. B.; Zhang, T.; Kim, P. S.; Alber, T. Science 1993,
262, 1401-1406.
(18) (a) Abblard, J .; Decoret, C.; Cronenberger, L.; Pacheco, H. Bull.
Soc. Chim. Fr. 1972, 2466-2481. (b) Guthikonda, R. N.; Cama, L. D.;
Quesada, M.; Woods, M. F.; Salzmann, T. N.; Christensen, B. G. J .
Med. Chem. 1987, 30, 871-880.
(19) (a) Boekelheide, V.; Linn, W. J . J . Am. Chem. Soc. 1954, 76,
1286-1291. (b) Hardegger, E.; Nikles, E. Helv. Chim. Acta 1957, 249,
2428-2433. (c) Langlois, Y.; Potier, P. Tetrahedron 1975, 31, 419-
422. (d) Patent, Lonza AG, Gampel/Wallis (CH), EP 0084118 B1, 1982.
(20) Roelfes, J . G. In Models for Non-Heme Iron Containing Oxida-
tion Enzymes. Ph.D. Thesis, Groningen, The Netherlands, 2000 (http://
www.ub.rug.nl/eldoc/dis/science/j.g.roelfes/).
(21) (a) Bolm, C.; Ewald, M.; Felder, M.; Schlingloff, G. Chem. Ber.
1992, 125, 1169-1190. (b) Wang, X.; Rabbat, P.; O’Shea, P.; Tillyer,
R.; Grabowski, E. J . J .; Reider, P. J . Tetrahedron Lett. 2000, 41, 4335-
4338.
The key step in the synthesis of tetrasubstituted N4Py
ligands is the coupling of two functionalized pyridine
rings via a methylene bridge, preceding the formation of
the required bis(2-pyridinyl)methylamines 21 and 22.
This coupling reaction was carried out by allowing
lithiated 7 or 10 to react with the 2-formylpyridine
(22) Tre´court, F.; Breton, G.; Bonnet, V.; Mongin, F.; Marsais, F.;
Que´guiner, G. Tetrahedron 2000, 56, 1349-1360.
(23) The pure picolyl chlorides 15 and 16 are reasonably stable at
room temperature and can be stored under nitrogen at -20 °C for many
months.
252 J . Org. Chem., Vol. 69, No. 2, 2004

Non-Heme Template for Attaching Four Peptides
SCHEME 4a
in the presence of cesium carbonate furnished the tetra-
functionalized N4Py derivatives 23 and 24 (Scheme 5).
Although similar results were obtained for diisopropyl-
ethylamine (DIEA) as the base in acetonitrile, it was
found that the use of cesium carbonate resulted in a
cleaner reaction. Compounds 23 and 24 were obtained
only in moderate yields, partially due to incomplete
dialkylation of amines 21 and 22 (even in the presence
of an excess of the picolyl chloride derivative) and difficult
separation of the mono- and dialkylated products by
column chromatography.
To obtain the tetrachloride N4Py derivative 26, the
silyl protecting groups of 23 were first removed by dilute
hydrochloric acid in ethanol. Various strategies were
investigated to convert 25 to the tetrachloride N4Py
derivative 26, either to be isolated as such or to be
converted in situ to a sulfide by reaction with benzyl
mercaptan. Tetra-alcohol 25 was treated with (a) thionyl
chloride in the presence of TEA in CH2Cl2 and pyridine
as cosolvent; (b) methanesulfonyl chloride, lithium chlo-
ride, and collidine in DMF;25 or (c) triphenylphosphine
and carbon tetrachloride in acetonitrile with or without
pyridine as base or cosolvent. These conditions were
attempted at various temperatures and with different
orders of addition of the reagents, but in all cases the
N4Py derivative decomposed and no characterizable
products were obtained. Tetrachloride 26 was detected
by electrospray ionization mass analysis as its HCl salt
from the reaction of 25 with thionyl chloride in DMF at
0 °C.26 However, this compound decomposed immediately
when water or a base was added.
a
Conditions: (i) (1) n-BuLi, THF, -80 °C, 40 min, (2) 11 or 12,
-80 °C, 1 h, (3) H2O; (ii) MnO2, CHCl3, reflux, 18 h; (iii)
NH2OH‚HCl, TEA, EtOH, reflux, 1 h; (iv) Zn, NH4OAc, NH4OH,
EtOH, reflux, 3 h.
derivatives 11 or 12, respectively (Scheme 4). The crude
intermediate bis(2-pyridinyl)methanols were oxidized by
manganese dioxide to furnish bis(2-pyridinyl) ketones 17
and 18 in 78% and 80% yield, respectively, after purifica-
tion by column chromatography. Next, 17 and 18 were
converted to the bis(2-pyridinyl) ketoximes 19 and 20 in
83% and 93% yield, respectively, by a reaction with
hydroxylammonium chloride in the presence of TEA.
These oximes were then converted to bis(2-pyridinyl)-
methylamines 21 and 22 in near-quantitative yields by
a mild reduction using zinc powder under basic condi-
tions.24
Replacement of the four hydroxyl groups in 25 by
(chloride) leaving groups apparently leads to the forma-
tion of an intrinsically unstable compound. The observed
instability is probably inherent to the structure of 26.
Although at this moment the exact mechanism of de-
composition can only be speculated upon, it might be
triggered by hydrogen chloride elimination, which is
The dialkylation of bis(2-pyridyl)methylamines 21 and
22 with the 2-picolyl chlorides 15 and 16, respectively,
SCHEME 5a
a
Conditions: (i) Cs2CO3, MeCN, reflux, 48 h; (ii) H3O+, EtOH, 15 min; (iii) (1) NaH, DMF, 0 °C, 30 min, (2) MeI, 0 °C to rt, 2 h; (iv)
Ph3PBr2, CH2Cl2, rt, 18 h.
J . Org. Chem, Vol. 69, No. 2, 2004 253

van den Heuvel et al.
SCHEME 6. Con ver gen t vs Diver gen t Syn th esis Rou tes of Tetr a su bstitu ted N4P y Der iva tives
attributed to the double benzylic, tertiary hydrogen in
26 and the reactive ‘benzylic’ leaving groups.27,28 In
evidence that it is not the structure of 25 that is unstable,
this N4Py derivative was successfully converted into the
tetramethoxy derivative 1c by first deprotonating the
four hydroxyl groups of 25 and subsequently quenching
with methyl iodide (Scheme 5). The rather low 40% yield
in this case is due to loss of product during purification
by column chromatography.
In view of the seemingly unsolvable problems associ-
ated with the instability of the tetrachloride 26, we
decided to increase the N4Py ligand spacer length. A
dimethylene (C2) linker was rejected, because HCl
elimination to form vinyl-pyridine derivatives can obvi-
ously occur. The synthesis of the C3-spacer ligand 27 is
outlined in Schemes 2-5 for n ) 3. To enhance the
leaving group ability at the nonbenzylic position, the silyl
ether groups in 24 were converted directly into bromides
by a reaction with triphenylphosphine dibromide (Scheme
5).29,30 As a result, the tetrafunctionalized N4Py deriva-
tive 27 was obtained in 12 steps from 2,5-dibromopyri-
dine (4) in an overall yield of 5%.
initially using the bromide at the 2-position of the
pyridine ring to construct the core of the N4Py scaffold.
The remaining bromides in 1b are then available for
further functionalization by, for instance, metal-catalyzed
cross-coupling methodologies or via bromide-metal ex-
change reactions.
As indicated above, mono-metalation of 4 by n-BuLi
occurs preferentially at the 5-position in diethyl ether at
-80 °C.31 However, at low concentrations in noncoordi-
nating solvents such as toluene or dichloromethane,
lithiation occurs preferentially at the 2-position rather
than the 5-position.21b This reversed regioselectivity
enabled the synthesis of bispyridinyl ketone 30, which
was achieved by quenching the in situ prepared 5-bromo-
2-lithiopyridine in toluene at -80 °C with 0.5 equiv of
diethyl carbonate (Scheme 7). The low yield is partially
due to incomplete regioselectivity under these conditions.
Subsequent conversion of the ketone 30 into the bispyr-
idinyl ketoxime 31 by reaction with hydroxylamine, and
a highly efficient reduction of the ketoxime 31 using zinc
in acetic acid at room temperature provided bispyridinyl
methylamine 32 in 94% yield.
The required picolyl chloride derivative 36 can be
obtained from 5-bromo-2-picoline18 (33) by oxidation with
m-CPBA to the N-oxide 34 in 98% yield (Scheme 8).
Subsequent reaction of 34 with trifluoroacetic acid an-
hydride (TFAA), and hydrolysis of the resulting acetate
furnished 5-bromo-2-(hydroxymethyl)pyridine (35) in
81% yield. Finally, this picolyl alcohol was converted with
thionyl chloride to 36 in 97% yield.
Analogously to the previously described preparation of
N4Py derivatives, the dialkylation of amine 32 with 2
equiv of picolyl chloride 36 in the presence of base
enabled the construction of tetrabromo N4Py 1b (Scheme
Diver gen t Syn th esis Str a tegy. The problems of
instability encountered with 26 and the consequent need
to prepare the more stable 27 suggested that a divergent
synthetic route, permitting the modification of the N4Py
ligand after its construction, would facilitate a versatile
preparation of alternative ligand systems. A potential
N4Py framework, tetrabromide N4Py derivative 1b, that
could enable a variety of tetrafunctionalized N4Py de-
rivatives 29 to be generated via divergent synthesis is
presented in Scheme 6. Again starting from 2,5-dibro-
mopyridine (4), tetrabromo N4Py 1b can be prepared by
(24) Niemers, E.; Hiltmann, R. Synthesis 1976, 593-595.
(25) Collington, E. W.; Meyers, A. I. J . Org. Chem. 1971, 36, 3044-
3045.
(28) Attempts to replace the double benzylic proton by a methyl
group have been unsuccessful. Lithiation of 23 at -80 °C in THF in
the presence of N,N,N′,N′-tetramethylethylenediamine (TMEDA) fol-
lowed by quenching with methyl iodide resulted repeatedly only in the
recovery of 23 and not to the desired product. These conditions were
successful when applied to N4Py (ref 20).
(26) The molecular ion was observed at m/z 560.1 [M + H]+ for the
main isotope peak, which corresponded to the expected mass of 559.1
[M]+, and the detected isotope distribution pattern matched the
calculated arrangement.
(27) A dichloride N4Py derivative was previously prepared as
described in ref 16. The two benzylic chlorides were located in the upper
half of the N4Py scaffold (see Scheme 5 for compound 26) and did not
prevent its formation. However, in the tetrachloride N4Py derivative
26 the double benzylic proton in the bottom half of the N4Py ligand is
now flanked by two pyridine groups that each contain a benzylic
chloride moiety.
(29) Despite the more reactive bromine atoms, 27 can be stored
under nitrogen at -20 °C for at least
temperature for at least 2 days.
2 months and at room
(30) The electrospray mass analysis of 27 ([M + H]+ at m/z 848.0)
corresponded to the calculated isotope distribution pattern.
(31) Parham, W. E.; Piccirilli, R. M. J . Org. Chem. 1977, 42, 257-
260.
254 J . Org. Chem., Vol. 69, No. 2, 2004

Non-Heme Template for Attaching Four Peptides
SCHEME 7a
SCHEME 9a
a
Conditions: (i) PhB(OH)2, Pd(PPh3)4, Na2CO3 (aq), toluene,
reflux, 18 h; (ii) (1) i-PrMgBr, THF, rt to reflux, 1 h, (2) H2O, D2O,
or DMF.
carbonate afforded the cross-coupled product 1f in 80%
yield (Scheme 9). 1H NMR and mass analysis of the crude
product indicated that the tetrasubstituted cross-coupled
product was obtained with no detectable amounts of
mono-, di-, or trisubstituted products.34
a
Conditions: (i) (1) n-BuLi, toluene, -80 °C, 2 h, (2) (EtO)2CO,
-80 °C to rt; (ii) NH2OH‚HCl, py, EtOH, reflux, 1.5 h; (iii) Zn,
AcOH, rt, 30 min; (iv) 36, DIEA, MeCN, reflux, 48 h.
To test the susceptibility of tetrabromo N4Py 1b
toward halogen-metal exchange and subsequent quench-
ing with electrophiles, 1b was treated at room temper-
ature with 4.5 equiv of i-PrMgBr as a solution in THF
(Scheme 9). Mass analysis of a product sample, obtained
after quenching with water, revealed the presence of only
the dibromide N4Py derivative 37a . The addition of more
i-PrMgBr did not result in a complete magnesium-
bromide exchange, even after heating at reflux tempera-
ture. When a sample was quenched with D2O, mass
analysis revealed the incorporation of three deuterium
atoms with two bromine atoms still present, which
suggested the formation of 37b. Not only does this
indicate that the double benzylic position in 1b is
deprotonated, but also that magnesium-bromide ex-
change occurs only at two of the four sites. The use of
DMF as an electrophile afforded the dialdehyde deriva-
tive 38, which has been characterized by COSY NMR
experiments and mass analysis.35 The anion at the double
benzylic position apparently does not react with DMF,
and is therefore reprotonated during workup. These
findings indicate that the bottom half of 1b is not
susceptible toward a metal-halogen exchange, probably
due to the delocalization of the negative charge at the
SCHEME 8a
a
Conditions: (i) m-CPBA, CH2Cl2, rt, 18 h; (ii) (1) TFAA, (2)
NaHCO3 (aq); (iii) SOCl2, CH2Cl2, 0 °C to rt, 18 h.
7). The best results were obtained by heating at reflux
for 2 days in the presence of a large excess of DIEA and
a minimal amount of acetonitrile to dissolve the reac-
tants. After purification of the crude product by short
path flash column chromatography, traces of mono-
alkylated product could be removed by precipitation of
the product in diethyl ether to afford 1b in 58% yield
(16% yield from 4).
F u n ction a liza tion of Tetr a br om o N4P y 1b. For the
functionalization of tetrabromo N4Py 1b via cross-
coupling reactions we have focused on the Suzuki-
Miyaura reaction.32,33 Heating 1b for 16 h with 10
equivalents of phenylboronic acid and 12 mol % Pd(PPh3)4
as the catalyst (i.e., 3 mol % of palladium catalyst per
bromide) in a mixture of toluene and aqueous sodium
(33) For bromopyridines in Suzuki reactions, see: (a) Thompson,
W. J .; Gaudino, J . J . Org. Chem. 1984, 49, 5237-5243. (b) Stavenuiter,
J .; Hamzink, M.; Van der Hulst, R.; Zomer, G.; Westra, G.; Kriek, E.
Heterocycles 1987, 26, 2711-2716. (c) Thompson, W. J .; J ones, J . H.;
Lyke, P. A.; Thies, J . E. J . Org. Chem. 1988, 53, 2052-2055. (d)
Aliprantis, A. O.; Canary, J . W. J . Am. Chem. Soc. 1994, 116, 6985-
6986. (e) Gong, Y.; Pauls, H. W. Synlett 2000, 829-831.
(34) In test reactions carried out on several bromopyridines, it was
established that THF as a solvent generally resulted in superior
efficiencies over toluene. However, when toluene was replaced by THF
as the solvent for the Suzuki reaction with 1b, only
a single,
structurally related, but unidentified compound was obtained within
a few minutes as observed by 1H NMR, without any trace of the desired
product 1f. Presumably, a very reactive species is formed under these
conditions, which results in the formation of this undesired product.
(35) The observed fragmentation with m/z 329, 254, and 122 can
only be ascribed to the proposed structure of 38 and not its regioisomer.
(32) (a) Miyaura, N.; Suzuki, A. Chem. Rev. 1995, 95, 2457-2483.
(b) Suzuki, A. In Metal-catalyzed Cross-coupling Reactions; Diederich,
F., Stang, P. J ., Eds.; Wiley-VCH Verlag GmbH: Germany, 1998; pp
49-97. (c) Suzuki, A. J . Organomet. Chem. 1999, 576, 147-168.
J . Org. Chem, Vol. 69, No. 2, 2004 255

van den Heuvel et al.
TABLE 1. Selected Bon d Dista n ces for 2b a n d 2ca ,b
SCHEME 10a
bond (Å)
2a 12
2b
2c
Fe-Namine
Fe-Npy
N1
N2
N3
N4
N5
N6
1.961(3)
1.976(3)
1.967(3)
1.968(3)
1.975(3)
1.915(3)
0.207(5)
1.965(3)
1.965(3)
1.956(3)
1.962(3)
1.970(3)
1.927(4)
0.207(1)
1.958(3)
1.957(3)
1.960(3)
1.960(3)
1.967(3)
1.928(3)
0.197(1)
Fe-NMeCN
Fe-Npy-mean
plane
a
b
Standard deviations are given in parentheses. X-ray diffrac-
tion of 2a was performed at 130 K, 2b at 90 K, and 2c at 100 K.
TABLE 2. Sp ectr oscop ic Da ta of th e F eII- a n d F eIIIOOH
Com p lexes of N4P y (1a ) a n d Its Der iva tives 1b-d in
Aceton e a t Room Tem p er a tu r ea
a
Conditions: N-Ac-Cys-OMe or peptide N-Ac-CGLHELLKG,
Cs2CO3 (aq), pH 9, DMF, rt, 18 h.
[(L)FeII(MeCN)](ClO4)2
[(L)FeIIIOOH](ClO4)2
double benzylic anion over the two adjacent pyridine
rings. Incidentally, the very selective dimetalation of 1b
with i-PrMgBr provides the opportunity to introduce two
different sets of spacers at the periphery of the N4Py
ligand: after quenching the dimetalated species of 1b
with an appropriate electrophile, the two remaining
bromides in the resulting product can be submitted to
further functionalization by cross-coupling reactions.
P ep tid e Cou p lin g. Using our previously developed
methodology for coupling unprotected single-cysteine
peptides to the ligand via halogen displacement by the
cysteine thiolate,16 tetracysteine derivative 1d was pre-
pared by reaction of 27 with methyl N-acetylcysteine and
purified by reversed-phase HPLC. Similarly, the model
nonapeptide Ac-Cys-Gly-Leu-His-Glu-Leu-Leu-Lys-Gly-
NH2 was coupled quantitatively to the tetrabromide
template 27 to provide 1e (Scheme 10).36 It should be
noted that the sequence of the unprotected test peptide
was chosen only for its representative content of nucleo-
philic groups in the amino acids side chains that might
interfere with the halogen displacement, and was not
designed to fold into a four-helix bundle.
ligand
(L)
λmax
(nm)
ꢀ (× 102
λmax
(nm)
ꢀ (× 102
M-1 cm-1
)
M-1 cm-1
)
1a
457 (455)
464 (460)
461 (457)
456 (n.d.)
53 (48)
70 (88)
41 (59)
37 (n.d.)
539 (520)
553 (544)
538 (522)
525 (n.d.)
11 (7.2)
11 (8.9)
10 (7.8)
3.4 (n.d.)
1b
1c
1d b
a
The results in parentheses refer to acetonitrile as solvent. n.d.
b
) not determined. At 0 °C, λmax,2d 457 nm, ꢀ 26 × 102 M-1 cm-1
;
λmax,3d 531 nm, ꢀ 3.7 × 102 M-1 cm-1
.
2).12,20 For 2d , the pH appeared to have only a minor
effect on its extinction coefficient (λmax 452 nm, ꢀpH 35
× 102 M-1 cm-1, ꢀpH 38 × 102 M-1 cm-1, ꢀpH 33 ×4102
7
10
M-1 cm-1) at room temperature.
Crystals of the iron(II) complexes 2b and 2c suitable
for X-ray analysis were obtained by the slow diffusion of
ethyl acetate into their solutions in acetonitrile. The
molecular structures are shown in Figure 2. Five coor-
dination sites to the iron center are occupied by the
chelating ligand and a solvent-derived acetonitrile mol-
ecule functions as an axial ligand. This mode of coordina-
tion to the metal ion is identical to that previously
observed for N4PyFe 2a .12,13b As can be seen in Figure 2,
the bromine atoms in 2b and the methoxymethyl groups
in 2c are directed away from the iron center in the
complex. The same spatial arrangement can, therefore,
also be predicted for the iron-complexes of other tetra-
substituted N4Py ligands with functional groups at the
same positions, such as in 2d and 2e. Although the
tetraphenyl N4Py derivative 1f could be complexed
successfully with iron perchlorate in acetonitrile,37 no
suitable crystals of the iron complex could be obtained
for X-ray diffraction.
The iron-nitrogen bond lengths of 2b and 2c are in
the range of 1.91-1.98 Å, which is typical in bond lengths
for low-spin iron(II) centers.38 Complexes 2b and 2c show
only a small variation when compared to those found for
N4PyFe 2a (Table 1).12,13b Electronic effects alone are,
in our opinion, insufficient to explain these features.
Therefore, these minor differences in bond lengths in
2a -c are most likely due to the differences in tempera-
ture during the X-ray analysis (see footnote in Table 1).
Syn th esis a n d Ch a r a cter iza tion of Ir on Com -
p lexes of N4P y Der iva tives. By addition of iron(II)
perchlorate to the pentadentate ligands 1a -f in aceto-
nitrile the corresponding iron complexes 2a -f were
1
obtained (Scheme 1). The H NMR spectra of 2b and 2c
in CD3CN revealed signals in the diamagnetic range of
0-10 ppm, which is distinctive for low-spin iron(II)
complexes of N4Py ligands.12,20 The electrospray ioniza-
tion mass analyses (ESI-MS) were also in accordance
with the postulated composition of the complexes 2b and
2c.
Upon complexation of the tetrapeptide-bound N4Py
ligand 1e with iron(II) perchlorate, the metalated com-
plex 2e was obtained in 18% yield after preparative
reversed-phase HPLC. Its chemical integrity was verified
by MALDI mass analysis. The calculated mass for 2e
corresponded to the detected mass (calcd for [M -
2(ClO4)- - (MeCN)]2+ m/z 6121.3 amu (monoisotopic),
6125.1 amu (average isotope composition), found 6124.0
amu). The UV-vis absorption bands of 2b-d correlate
well with those of the previously prepared low-spin iron-
(II) complexes of N4Py derivatives, with maximum
absorptions typically around 380 and 460 nm (Table
(37) Formation of the iron complex [(1f)FeII(MeCN)](ClO4)2 (2f) was
confirmed by ESI-MS analysis of the dark purple solid that was
obtained after evaporation of the solvent.
(38) Hawker, P. N.; Twigg, M. V. In Comprehensive Coordination
Chemistry; Wilkinson, G., Ed.; Pergamon: Oxford, 1987; Vol. IV, p
1179.
(36) MALDI analysis of the crude iron(II) complex of the peptide
coupled N4Py ligand revealed only the tetrasubstituted product. The
masses for mono-, di-, or trisubstituted peptide complexes were not
observed.
256 J . Org. Chem., Vol. 69, No. 2, 2004

Non-Heme Template for Attaching Four Peptides
UV-vis absorption data are compiled in Table 2. The
nature of the FeIIIOOH species was previously elucidated
for N4PyFe 2a .13b Based on the UV-vis data, we propose
similar FeIIIOOH structures for complexes 3b-e. The
mechanism of its formation and further reactions are also
considered to be applicable to the new N4Py complexes
described here. The complex 2d was prepared as a model
for peptide-bound N4PyFe complexes such as 2e in order
to study the stability of the complex and in particular
its sulfide linkage toward the applied oxidative condi-
tions. However, the FeIIIOOH species 3d was found to
be considerably less stable than 3a , and could not be
detected by a stepwise titration of the iron(II) complex
with hydrogen peroxide in acetone at room temperature.
Conversely, the addition of a single aliquot of excess
hydrogen peroxide to 2d resulted in the formation and
observation of the hydroperoxo species 3d (∼2 min
lifetime). Its lifetime was significantly extended to ap-
proximately 15 min by reducing the temperature of the
solution to 0 °C.
Oxid a tion Activity of Ir on (II) Com p lexes of N4P y
a n d Der iva tives. The peroxidase activity and the pH
dependency of N4PyFe 2a and complex 2e were deter-
mined by monitoring spectrophotometrically the forma-
tion of the ABTS•+ radical cation40 over time at pH 3.5
(ꢀ660 1.47 × 104 M-1 cm-1) (Figure 3).
This probe had previously been used to establish the
peroxidase-like activity of the dipeptide-bound N4PyFe
catalyst.16 Under these conditions complex 2a is deacti-
vated within 200 s, due to an oxidative degradation of
the complex, which is attributed to hydroxyl radicals
formed during the reaction.14b,20 Indeed, the addition of
a radical scavenger (2,4,6-tri-tert-butylphenol) increased
the lifetime of the catalyst, but reduced the reaction rate
of the ABTS oxidation. When compared to N4PyFe 2a ,
the N4PyFe-peptide complex 2e displayed a substantially
lower activity for the peroxidation of ABTS under identi-
cal conditions (4.4 turnovers for 2e vs 27 for 2a at t ) 50
s, Figure 3). This result may be attributed to steric
interactions between the unfolded peptide chains on the
catalyst and the ABTS substrate. Note that this is in
contrast to our earlier findings16 when two unfolded
peptides, covalently attached to N4PyFe, had no effect
on the catalyst’s activity.
F IGURE 2. Pluto representation of 2b (top) and 2c (bottom).
Hydrogen atoms and counterions are omitted for clarity.
The cyclic voltammetry measurements for 2b in
acetonitrile displayed a reversible oxidation wave with
a redox potential for the FeII/FeIII couple of 1198 mV vs
SCE and an observed peak-to-peak separation of 105 mV.
Forward and reverse differential pulse voltammetry at
a scan rate of 10 mV s-1 confirmed this process to be
reversible with a redox potential for the FeII/FeIII couple
of 1196 mV vs SCE. The redox potential of the tetrabromo
N4PyFe complex 2b was found to be substantially higher
than that of N4PyFe 2a (E1/2 ) 1010 mV vs SCE)12 and
a dibromo N4PyFe derivative20,39 (E1/2 ) 1100 mV vs
SCE). The general trend infers that the introduction of
a greater number of electron-withdrawing groups into the
N4Py ligand results in an increase in the redox potential
of the corresponding iron(II) complex. This in turn gives
Con clu sion s
The synthesis of the tetrabromide N4Py derivative 1b
could be accomplished by taking advantage of a change
in regioselectivity in the lithiation of 2,5-dibromopyridine
by substituting a coordinating solvent by a noncoordi-
nating solvent. This enabled the construction of the N4Py
core while preserving the remaining bromide on the
pyridine ring for further functionalization of the final
ligand 1b. The potential of this tetrabromo N4Py deriva-
tive to be functionalized further by cross-coupling
methodologies was demonstrated by the successful prepa-
ration of tetraphenyl N4Py 1f. This reaction entails four
palladium-mediated Suzuki reactions on a single mol-
ecule with no detectable products from incomplete cross-
coupling reactions on 1b. In view of the positive results
in the Suzuki reaction with 1b it is very likely that other
rise to the observed relatively large red shift for the FeIII
-
OOH species 3b (vide infra).
For m ation of Ir on (III) Hydr oper oxo Species. Upon
the addition of excess hydrogen peroxide to 2a -e, the
iron complexes are converted to the corresponding purple
iron(III) hydroperoxo species 3a -e (Scheme 1). The
(39) The iron(II) comlex of N,N-bis[(5-bromo-2-pyridinyl)methyl]-
N-[di(2-pyridinyl)methyl]amine.
(40) Adams, P. A. J . Chem. Soc., Perkin Trans. 2 1990, 1407-1414.
J . Org. Chem, Vol. 69, No. 2, 2004 257

van den Heuvel et al.
F IGURE 3. Formation of the ABTS•+ radical cation over time at 20 °C, expressed in turnover numbers (TON): background
corrected oxidation of ABTS by 2a , 2e, and iron(II) perchlorate at pH 3.5 and by 2a in the presence of the radical scavenger
2,4,6-tri-tert-butylphenol (TBP).
a solution of 5 (9.97 g, 53.6 mmol) in dry THF/diethyl ether
(250 mL, 1:1) was added over 10 min. After 15 min, water was
added and the phases were separated. The aqueous layer was
extracted with diethyl ether. The combined organic layer was
washed with brine and dried over Na2SO4. Evaporation of the
solvent produced 8 as a brown solid. Recrystallization from
hexane yielded analytically pure product as white needles (9.96
g pure E-isomer, 75%). From the concentrated filtrate a further
1.24 g of 8 (85% combined yield) was obtained after flash
column chromatography over silica using hexane/diethyl ether
(9:1) as the eluent. Rf ) 0.53 (SiO2, hexane/ethyl acetate )
3:1). Mp 83.6-84.1 °C. 1H NMR (CDCl3, 300 MHz) δ 1.33 (t, J
) 7.3 Hz, 3H), 4.26 (q, J ) 7.3 Hz, 2H), 6.48 (d, J ) 16.1 Hz,
1H), 7.50 (d, J ) 8.4 Hz, 1H), 7.59 (d, J ) 16.1 Hz, 1H), 7.67
(dd, J ) 8.4, 2.2 Hz, 1H), 8.47 (d, J ) 2.2 Hz, 1H). 13C NMR
(CDCl3, 50.3 MHz) δ 14.2, 60.9, 121.2, 128.3, 129.5, 136.2,
139.3, 143.4, 149.8, 166.0. HRMS: calcd for C10H10NO2Br
254.989, found 254.990. Anal. Calcd for C10H10BrNO2: C,
46.90; H, 3.90; N, 5.50; Br, 31.20. Found: C, 46.96; H, 3.97;
N, 5.43; Br, 31.18.
cross-coupling reactions can also be applied to provide a
range of tetrasubstituted N4Py derivatives.
All described iron complexes of tetrafunctionalized
N4Py derivatives show spectroscopic data that are char-
acteristic for low-spin iron(II) complexes. X-ray crystal-
lographic data of 2b and 2c have revealed that the four
functionalities at the periphery of the N4Py ligand are
directed away from the iron metal center. A similar
orientation can therefore be expected for 2e and other
peptide-bound N4PyFe complexes. Complexes 2b and 2c
also show a catalytic activity comparable to 2a in the
oxidation of organic compounds with hydrogen peroxide.41
After the successful preparation of several tetrasub-
stituted N4Py derivatives one, 27, has been used as a
functional template for the covalent attachment of four
unprotected peptides. Although the C3-spacer on 27 is
too long to be compatible with a folded four-helix bundle,
the work described here provides a foundation for syn-
thesizing alternative derivatives of the ligand.
3-(6-Br om o-3-p yr id in yl)-1-p r op a n ol (9). To a stirred
suspension of LiAlH4 (1.90 g, 50.1 mmol) in dry diethyl ether
(75 mL) at 0 °C was added a solution of 8 (6.40 g, 25.0 mmol)
in dry diethyl ether (75 mL) over 20 min. After 10 min at 0
°C, the reaction mixture was allowed to warm to room
temperature and stirred for 45 min. The reaction was quenched
by carefully adding Na2SO4‚10H2O and stirring the mixture
for 1 h. The solids were removed by filtration and washed with
diethyl ether. Evaporation of the solvent yielded 9 as a light
brown oil (3.27 g, 61%), which was used without any further
Exp er im en ta l Section
Rep r esen ta tive P r oced u r e for th e P r ep a r a tion of
TBDMS Eth er s. To a stirred solution of 6 (21.8 g, 116 mmol)
and TEA (25.0 mL, 179 mmol, 1.5 equiv) in dry DMF (50 mL)
was added tert-butyldimethylsilyl chloride (18.8 g, 125 mmol,
1.05 equiv). After being stirred for 15 min, protected from
moisture, the mixture was diluted with diethyl ether and
washed with water and brine. The organic phase was dried
over Na2SO4 and concentrated by rotary evaporation to yield
a yellow/brown oil (35.0 g, quant). Purification by flash column
chromatography over silica using a gradient of hexane/diethyl
ether (19:1 to 9:1) yielded 29.8 g of 7 as colorless oil.
1
purification. Rf ) 0.29 (SiO2, hexane/ethyl acetate ) 1:1). H
NMR (CDCl3, 300 MHz): δ 1.84 (m, 2H), 2.68 (t, J ) 7.9 Hz,
2H), 3.66 (t, J ) 6.0 Hz, 2H), 7.38 (s, 1H), 7.39 (s, 1H), 8.19 (s,
1H). 13C NMR (CDCl3, 75.5 MHz): δ 28.2, 33.3, 60.9, 127.6,
136.7, 138.8, 139.0, 149.8. HRMS: calcd for C8H10NOBr
214.995, found 214.996.
2-Br om o-5-[[[ter t-bu tyl(d im eth yl)silyl]oxy]m eth yl]p yr -
id in e (7). Yield: 85%. Rf ) 0.43 (SiO2, hexane/diethyl ether
) 9:1). 1H NMR (CDCl3, 300 MHz): δ 0.09 (s, 6H), 0.92 (s,
9H), 4.69 (s, 2H), 7.43 (d, J ) 8.1 Hz, 1H), 7.52 (dd, J ) 8.1,
2.2 Hz, 1H), 8.30 (d, J ) 2.2 Hz, 1H). 13C NMR (CDCl3, 75.5
MHz): δ -5.4, 18.2, 25.7, 61.9, 127.5, 136.0, 136.5, 140.4,
148.0. HRMS: calcd for C12H20BrNOSi 303.048, found 303.048.
Eth yl (E)-3-(6-Br om o-3-p yr id in yl)-2-p r op en oa te (8). A
solution of triethyl phosphonatoacetate (12.0 g, 53.6 mmol) in
dry diethyl ether (50 mL) was added over 10 min to a
suspension of NaH (2.35 g, 58.8 mmol) in dry diethyl ether
(130 mL) at 5 °C. The icebath was removed, and after 10 min
Rep r esen ta tive P r oced u r es for th e P r ep a r a tion of
2-P yr id in eca r ba ld eh yd es. Meth od A. To a solution of 7
(10.4 g, 34.3 mmol) in dry THF (450 mL) at -80 °C under a
nitrogen atmosphere was slowly added n-BuLi (24.0 mL, 38.4
mmol, 1.1 equiv). After 40 min at -80 °C, dry DMF (3.0 mL,
39 mmol, 1.1 equiv) was added to the red solution. The reaction
mixture was allowed to warm to room temperature after 1 h
at -80 °C. Brine and diethyl ether were added, and the layers
were separated. The aqueous layer was extracted with diethyl
ether. The combined organic layers were washed with brine
and dried over Na2SO4. Concentration by rotary evaporation
afforded a light brown oil (8.98 g). Purification by flash column
chromatography over silica using a gradient of hexane/diethyl
ether (9:1 to 3:1) yielded 6.88 g of 11 as a light brown oil.
Meth od B. To a solution of isopropylmagnesium bromide
(11 mL, 22 mmol, 2 M in THF) in dry THF (10 mL) was added
(41) van den Heuvel, M. In Non-Haem Ligands as Functional
Templates for Peptide Attachment. Ph.D. Thesis, Groningen, The
Netherlands, 2002 (http://www.ub.rug.nl/eldoc/dis/science/m.van.den.
heuvel/).
258 J . Org. Chem., Vol. 69, No. 2, 2004

Non-Heme Template for Attaching Four Peptides
7 (6.02 g, 19.9 mmol). Heat was evolved during the reaction,
and after 1 h dry DMF (1.7 mL, 22 mmol) was added. The
reaction was quenched after an additional 1 h by the addition
of a saturated aqueous NH4Cl solution (20 mL). Diethyl ether
was added, and the layers were separated. The aqueous layer
was extracted with diethyl ether. The combined organic layers
were washed with water and brine and dried over Na2SO4.
Evaporation of the solvent furnished an orange/brown oil (5.12
g).
5-[[[ter t-Bu tyl(d im eth yl)silyl]oxy]m eth yl]-2-p yr id in e-
ca r ba ld eh yd e (11). Yield: 80%. Rf ) 0.54 (SiO2, hexane/ethyl
acetate ) 3:1). 1H NMR (CDCl3, 300 MHz): δ 0.12 (s, 6H),
0.94 (s, 9H), 4.84 (s, 2H), 7.82 (d, J ) 8.1 Hz, 1H), 7.94 (d, J
) 8.1 Hz, 1H), 8.72 (s, 1H), 10.06 (s, 1H). 13C NMR (CDCl3,
75.5 MHz): δ -5.4, 18.2, 25.8, 62.4, 121.9, 134.4, 141.7, 148.0,
151.8, 193.1. HRMS: calcd for C13H21NO2Si 251.134, found
251.133.
Rep r esen ta tive P r oced u r e for Na BH4 Red u ction s. To
a solution of 11 (5.87 g, 23.3 mmol) in methanol (90 mL) at 0
°C was slowly added NaBH4 (0.44 g, 12 mmol, 0.5 equiv). The
reaction mixture was allowed to warm to room temperature,
and after 30 min dilute HCl (2 M) was added until pH 7,
followed by a saturated aqueous Na2CO3 solution. The solvent
was evaporated, and the residue was extracted with diethyl
ether. The organic layer was washed with brine, dried over
Na2SO4, and concentrated by rotary evaporation to yield 13
as a light yellow oil (5.73 g, 97%). The crude product was used
without any further purification.
portions of 4.4 g. The reaction mixture was refluxed overnight.
After evaporation of the solvent the residue was filtered
through Celite and washed with diethyl ether. The filtrate was
dried over Na2SO4 and concentrated by rotary evaporation to
yield a brown solid (8.97 g, 90%), which was purified over silica
using hexane/ethyl acetate (5:1) as the eluent.
Bis[5-[[[ter t-bu tyl(d im eth yl)silyl]oxy]m eth yl]-2-p yr id i-
n yl]m eth a n on e (17). Yield: 78%. Rf ) 0.40 (SiO2, hexane/
ethyl acetate ) 3:1). 1H NMR (CDCl3, 300 MHz): δ 0.12 (s,
12H), 0.94 (s, 18H), 4.84 (s, 4H), 7.84 (d, J ) 8.1 Hz, 2H), 8.08
(d, J ) 8.1 Hz, 2H), 8.69 (s, 2H). 13C NMR (CDCl3, 75.5 MHz):
δ -5.4, 18.2, 25.8, 62.4, 124.9, 134.0, 139.8, 147.0, 153.2, 192.5.
HRMS: calcd for C25H40N2O3Si2 472.258, found 472.258.
R ep r esen t a t ive P r oced u r e for t h e P r ep a r a t ion of
Dip yr id ylk etoxim es. A mixture of hydroxylammonium chlo-
ride (2.60 g, 37.4 mmol, 2 equiv) and TEA (7.8 mL, 56 mmol,
3 equiv) in ethanol (50 mL) was stirred at room temperature
for 0.5 h. The salts were removed by filtration, and the filtrate
was added to 17 (8.84 g, 18.7 mmol). After heating under reflux
for 1 h, the solvent was removed by rotary evaporation, and
the residue was extracted with ethyl acetate. The organic layer
was washed with brine, dried over Na2SO4, and concentrated
to give a brown oil (7.51 g) that solidified on standing.
Analytically pure product was obtained by recrystallization
from ethanol/water to yield 19 as white plates.
Bis[5-[[[ter t-bu tyl(d im eth yl)silyl]oxy]m eth yl]-2-p yr id i-
n yl]m eth a n on e Oxim e (19). Yield: 83%. Mp: 106.3-107.4
1
°C. H NMR (CDCl3, 300 MHz): δ 0.11 (s, 6H), 0.12 (s, 6H),
0.93 (s, 9H), 0.94 (s, 9H), 4.81 (s, 4H), 7.64 (d, J ) 8.4 Hz,
1H), 7.75 (dd, J ) 8.1, 2.0 Hz, 1H), 7.80 (dd, J ) 8.4, 2.0 Hz,
2H), 8.55 (s, 1H), 8.59 (s, 1H). 13C NMR (CDCl3, 75.5 MHz): δ
-5.4, 5.3, 18.3, 25.8, 25.9, 62.3, 62.6, 123.8, 124.7, 134.9, 135.2,
136.6, 138.4, 143.6, 146.5, 150.0, 150.1, 153.5. HRMS: calcd
for C25H41N3O3Si2 487.269, found 487.268. Anal. Calcd for
[5-[[[ter t-Bu tyl(dim eth yl)silyl]oxy]m eth yl]-2-pyr idin yl]-
m eth a n ol (13). 1H NMR (CDCl3, 300 MHz): δ 0.09 (s, 6H),
0.92 (s, 9H), 3.93 (br‚s, 1H), 4.74 (s, 4H), 7.21 (d, J ) 8.1 Hz,
1H), 7.64 (d, J ) 8.1 Hz, 1 H), 8.48 (s, 1H). 13C NMR (CDCl3,
75.5 MHz): δ -5.3, 18.3, 25.8, 62.5, 64.1, 120.2, 134.9, 135.3,
146.5, 158.1. HRMS: calcd for C13H23NO2Si 253.150, found
253.149.
Rep r esen ta tive P r oced u r e for th e P r ep a r a tion of
2-P icolyl Ch lor id es. To a solution of 13 (5.98 g, 23.6 mmol)
and TEA (6.6 mL, 47 mmol, 2 equiv) in dry CH2Cl2 (100 mL)
at 0 °C under a nitrogen atmosphere was slowly added SOCl2
(2.6 mL, 35 mmol, 1.5 equiv). The reaction mixture was
allowed to warm to room temperature and stirred overnight.
Saturated aqueous NaHCO3 solution was added. After separa-
tion, the aqueous layer was extracted with CH2Cl2. The
combined organic layers were washed with brine and dried
over Na2SO4, and the solvent was evaporated by rotary
evaporation. The product was obtained as a dark red/brown
oil (6.08 g, 95%). The crude product was purified over alumina
using hexane/ethyl acetate/TEA (200:9:1) as the eluent to
provide 4.24 g of pure 15.
C
25H41N3O3Si2: C, 61.56; H, 8.47; N, 8.61. Found: C, 61.48;
H, 8.58; N, 8.50.
R ep r esen t a t ive P r oced u r e for t h e P r ep a r a t ion of
Dip yr id ylm eth yla m in es. A mixture of 19 (2.00 g, 4.10 mmol)
and NH4OAc (0.42 g, 5.5 mmol, 1.3 equiv) in ethanol (25 mL)
and NH4OH (15 mL, 25% v/v) was heated under reflux. Zinc
powder (1.34 g, 20.5 mmol, 5 equiv) was added in portions
through the condenser at 15 min intervals over 2 h. After
refluxing for an additional 1 h, the reaction mixture was cooled
to room temperature and concentrated NaOH was added until
pH∼12 was reached. After filtration through Celite and
washing with diethyl ether the layers were separated. The
aqueous layer was extracted with diethyl ether. The organic
phase was washed with brine, dried over Na2SO4, and con-
centrated in vacuo to yield 1.91 g of pure 21 as a green/brown
viscous oil.
Bis[5-[[[ter t-bu tyl(d im eth yl)silyl]oxy]m eth yl]-2-p yr id i-
n yl]m eth yla m in e (21). Yield: 99%. 1H NMR (CDCl3, 300
MHz): δ 0.06 (s, 12H), 0.90 (s, 18H), 2.34 (br‚s, 2H), 4.70 (s,
4H), 5.31 (s, 1H), 7.33 (d, J ) 8.1 Hz, 2H), 7.58 (dd, J ) 8.1,
2.2 Hz, 2H), 8.48 (d, J ) 2.2 Hz, 2H). 13C NMR (CDCl3, 75.5
MHz): δ -5.4, 18.3, 25.8, 61.8, 62.5, 121.3, 134.7, 135.0, 147.2,
161.5. MS CI: [M + H]+ calcd for C25H44N3O2Si2 m/z 474.3,
found 474.0.
Rep r esen ta tive P r oced u r e for th e P r ep a r a tion of
N4P y Der iva tives. A mixture of 21 (0.21 g, 0.44 mmol), 15
(0.36 g, 1.3 mmol, 3 equiv), and Cs2CO3 (0.44 g, 1.4 mmol, 3
equiv) in dry acetonitrile (30 mL) was heated under reflux
under a nitrogen atmosphere for 60 h. The reaction mixture
was filtered through Celite, washed with diethyl ether, and
concentrated in vacuo. Purification by flash chromatography
over alumina using a gradient of hexane/ethyl acetate/TEA
(20:9:1 to 0:9:1) afforded 0.23 g of 23 as a brown viscous oil.
N-[Bis[5-[[[ter t-bu tyl(dim eth yl)silyl]oxy]m eth yl]-2-pyr -
idin yl]m eth yl]-N,N-bis[[5-[[[ter t-bu tyl(dim eth yl)silyl]oxy]-
m eth yl]-2-p yr id in yl]m eth yl]a m in e (23). Yield: 56%. 1H
NMR (CDCl3, 300 MHz): δ 0.08 (s, 24H), 0.91 (s, 36H), 3.92
(s, 4H), 4.69 (s, 4H), 4.70 (s, 4H), 5.30 (s, 1H), 7.61 (m, 8H),
5-[[[ter t-Bu t yl(d im et h yl)silyl]oxy]m et h yl]-2-(ch lor o-
m eth yl)p yr id in e (15). Yield: 66%. Rf ) 0.32 (Al2O3, hexane/
diethyl ether ) 9:1). 1H NMR (CDCl3, 300 MHz): δ 0.10 (s,
6H), 0.93 (s, 9H), 4.66 (s, 2H), 4.75 (s, 2H), 7.43 (d, J ) 8.1
Hz, 1H), 7.69 (dd, J ) 8.1, 2.2 Hz, 1H), 8.51 (d, J ) 2.2 Hz,
1H). 13C NMR (CDCl3, 75.5 MHz): δ -5.4, 18.2, 25.8, 47.5,
62.5, 120.7, 134.5, 134.6, 147.3, 160.5. HRMS calcd for C13H22
ClNOSi 271.116, found 271.118.
-
Rep r esen ta tive P r oced u r e for th e P r ep a r a tion of
Dip yr id yl Keton es. To a solution of 7 (6.36 g, 21.0 mmol) in
dry THF (250 mL) at -80 °C was added n-BuLi (14.0 mL, 22.5
mmol, 1.07 equiv) under a nitrogen atmosphere. After 40 min
at -80 °C, a solution of 11 (5.29 g, 21.0 mmol) in dry THF (50
mL) was added to the red reaction mixture. The blue/purple
solution was stirred at -80 °C for 1 h and then allowed to
warm to room temperature. Brine and diethyl ether were
added, and the layers were separated. The aqueous layer was
extracted with diethyl ether. The combined organic layers were
washed with brine, dried over Na2SO4, and concentrated by
rotary evaporation to yield an orange/brown oil. This residue
was then dissolved in chloroform (130 mL). Activated MnO2
(4.4 g, 43 mmol) was added, and the suspension was heated
under reflux. At 1 h intervals, 13.2 g MnO2 was added in
J . Org. Chem, Vol. 69, No. 2, 2004 259

van den Heuvel et al.
8.43 (s, 2H), 8.49 (s, 2H). 13C NMR (CDCl3, 75.5 MHz): δ -5.4,
18.2, 25.8, 56.6, 62.6, 62.6, 71.0, 122.3, 123.4, 134.1, 134.4,
134.5, 134.7, 147.0, 147.2, 158.6, 158.7. ESI-MS: [M + H]+
calcd for C51H86N5O4Si4 m/z 944.8, found 944.6.
(s, 2H), 8.34 (s, 2H). 13C NMR (CDCl3, 75.5 MHz): δ 30.5, 30.6,
32.5, 32.6, 33.3, 33.4, 56.6, 71.1, 122.3, 123.4, 133.6, 133.8,
136.1, 136.2, 148.8, 149.1, 157.5, 157.7. ESI-MS: [M + H]+
calcd for C35H42N5Br4 m/z 848.0, found 848.0.42
Tetr a cystein e N4P y Der iva tive 1d . Methyl N-acetylcys-
teine (114 mg, 0.64 mmol) was dissolved in nitrogen-purged
DMF (1 mL) and titrated with a Cs2CO3 solution (∼100 mg/
mL) to pH 9 under a nitrogen atmosphere. A solution of 27
(53 mg, 0.062 mmol) in DMF (1 mL) was added, and the
mixture was stirred at room temperature overnight. The
solution was diluted with water and lyophilized. The brown
residue was purified by preparative reverse-phase HPLC with
a gradient of 0.05 M ammonium acetate/MeCN (50:50) to 100%
MeCN over 10 min. The product eluted at tR ) 7.2 min, was
collected and lyophilized to yield a brown oil (9.5 mg, 12%).
1H NMR (CDCl3, 500 MHz): δ 1.88 (m, 8H), 2.04 (s, 12H), 2.55
(m, 8H), 2.68 (m, 8H), 3.00 (m, 8H), 3.78 (m, 12H), 3.90 (s,
4H), 4.83 (m, 4H), 5.28 (s, 1H), 6.39 (br‚d, 4H), 7.47 (m, 8H),
8.35 (s, 2H), 8.41 (s, 2H). ESI-MS: [M + H]+ calcd for
[6-[[[Bis[5-(h ydr oxym eth yl)-2-pyr idin yl]m eth yl][[5-(h y-
d r oxym eth yl)-2-p yr id in yl]m eth yl]a m in o]m eth yl]-3-p yr -
id in yl]m eth a n ol (25). Dilute HCl (2 mL, 1 M) was added to
a solution of 23 (0.22 g, 0.23 mmol) in ethanol (10 mL). After
0.5 h, the reaction mixture was extracted with diethyl ether.
The aqueous layer was neutralized with solid Na2CO3 and
lyophilized or azeotropically removed with ethanol. The residue
was taken up in dry ethanol, and after filtration and evapora-
tion of the solvent 25 was obtained as a viscous brown,
hygroscopic oil (0.12 g, 100%). 1H NMR (CD3OD, 300 MHz):
δ 3.86 (s, 4H), 4.58 (s, 4H), 4.91 (s, 4H), 5.32 (s, 1H), 7.39 (d,
J ) 8.1 Hz, 2H), 7.63 (d, J ) 8.1 Hz, 4H), 7.73 (d, J ) 8.1 Hz,
1H), 7.74 (d, J ) 8.1 Hz, 1H), 8.38 (s, 2H), 8.49 (s, 2H). 13C
NMR (CD3OD, 75.5 MHz): δ 58.3, 62.3, 62.4, 74.3, 124.6,
125.3, 137.0, 137.0, 137.1, 137.7, 148.7, 149.3, 158.8, 159.4.
ESI-MS: [M + H]+ calcd for C27H30N5O4 m/z 488.2, found
488.0.
C
59H82N9O12S4 m/z 1236.5, found 1236.1.
Tetr a p ep tid e N4P y d er iva tive 2e was first prepared as
described for 1d , using the peptide sequence Ac-Cys-Gly-Leu-
His-Glu-Leu-Leu-Lys-Gly-NH2.43 After stirring overnight, the
solution was acidified with 0.1% TFA in water and Fe(ClO4)2
(1.1 equiv) was added as a solution in MeCN. The orange/red
solution was purified by preparative reverse-phase HPLC
using a linear gradient of 20-40% solvent B over 10 min. The
product eluted at tR ) 8.6 min and was collected and lyophi-
lized to yield a yellow/orange solid (2.1 mg, 18%). MALDI:
calcd for [M - 2(ClO4)- - (MeCN)]2+ m/z 4620.38 (monoiso-
topic), 4623.43 (average isotope composition), found 4619.2.
Bis(5-br om o-2-p yr id in yl)m eth a n on e (30). To a suspen-
sion of 2,5-dibromopyridine (10.7 g, 44.3 mmol) in dry toluene
(1 L) at -80 °C was slowly added n-BuLi (29 mL, 46 mmol,
1.05 equiv) under a nitrogen atmosphere. After 2 h at -80 °C
the reaction was quenched with freshly distilled diethyl
carbonate (2.7 mL, 22 mmol, 0.5 equiv). After further workup
and extractions using ethyl acetate, removal of the solvent
furnished a dark brown oil, which solidified on standing. By
triturating the crude product with diethyl ether pure 30 was
obtained as a light brown solid (2.77 g, 37%). Recrystallization
from diethyl ether afforded the analytically pure product.
Mp: 191.8-192.7 °C. 1H NMR (CDCl3, 300 MHz): δ 7.98 (d, J
) 8.2 Hz, 2H), 8.03 (dd, J ) 8.2, 2.0 Hz, 2H), 8.77 (d, J ) 2.0
Hz, 2H). 13C NMR (CDCl3, 50.3 MHz): δ 124.7, 126.3, 139.4,
150.3, 152.1, 191.0. HRMS: calcd for C11H6N2OBr2 339.885,
found 339.885. Anal. Calcd for C11H6N2OBr2: C, 38.60; H, 1.80;
N, 8.20; Br, 46.70. Found: C, 38.33; H, 1.80; N, 8.01; Br, 47.04.
Bis(5-br om o-2-pyr idin yl)m eth an on e Oxim e (31). A mix-
ture of 30 (2.77 g, 8.10 mmol), hydroxylammonium chloride
(3.0 g, 42 mmol, 5 equiv), and pyridine (3.4 mL, 42 mmol, 5
equiv) in ethanol (60 mL) was refluxed for 1.5 h and subse-
quently cooled to room temperature. The mixture was poured
into ice-water and stirred in an ice bath for 1 h. The solids
were collected by filtration and washed with cold dilute ethanol
(50%). After drying in air the product was obtained as a light
brown solid (2.63 g, 91%). Recrystallization from ethanol/water
afforded analytically pure 31 as fine, lilac/grey needles. Mp:
N-[Bis[5-(m et h oxym et h yl)-2-p yr id in yl]m et h yl]-N,N-
bis[[5-(m eth oxym eth yl)-2-p yr id in yl]m eth yl]a m in e (1c).
To a stirred suspension of NaH (55 mg, 1.4 mmol, 8 equiv) in
dry DMF (1 mL) at 0 °C was added a solution of 25 (83 mg,
0.17 mmol) in dry DMF (1 mL). After 30 min, MeI (85 µL, 1.4
mmol, 8 equiv) was added at 0 °C. The reaction mixture was
allowed to warm to room temperature and stirred for 2 h.
Water was added and the mixture extracted with CH2Cl2. The
organic layer was washed with water and brine. After drying
over Na2SO4 and evaporation of the solvent, the product was
obtained as a red/brown oil (83 mg, 89%). Purification by flash
column chromatography over alumina using hexane/ethyl
acetate/TEA (10:18:2) yielded 1c as a light brown oil (37 mg,
40%). 1H NMR (CDCl3, 300 MHz): δ 3.33 (s, 12 H), 3.91 (s,
4H), 4.83 (s, 8H), 5.30 (s, 1H), 7.60 (s, 4H), 7.62 (s, 4H), 8.40
(s, 2H), 8.46 (s, 2H). 13C NMR (CDCl3, 75.5 MHz): δ 56.8, 58.1,
58.2, 71.4, 71.9, 122.5, 123.5, 131.4, 131.7, 135.7, 135.8, 148.4,
148.6, 159.3, 159.4. ESI-MS: [M + H]+ calcd for C31H38N5O4
m/z 544.3, found 544.4.
[(1c)F e(MeCN)](ClO4)2 (2c). To a solution of 1c (32 mg,
0.059 mmol) in acetonitrile (1.5 mL) was added Fe(ClO4)2‚H2O
(17 mg, 0.066 mmol, 1.1 equiv). The deep red solution was
placed in a sealed container, and ethyl acetate was allowed to
diffuse slowly into the solution. A dark red oil was formed after
1 week. The solution was removed and the oil dissolved in
acetonitrile. A fine red powder was formed which was isolated
and redissolved in acetonitrile. Slow diffusion of ethyl acetate
provided 2c as red crystals after 2 weeks (9 mg, 18%). 1H NMR
(CD3CN, 300 MHz): δ 3.29 (s, 6H), 3.34 (s, 6H), 4.32 (dd, J AB
) 27.9, 8.1 Hz, 4H), 4.38 (s, 4H), 4.47 (s, 4H), 6.31 (s, 1H),
7.04 (d, J ) 8.1 Hz, 2H), 7.63 (d, J ) 7.7 Hz, 2H), 7.85 (m,
4H), 8.82 (s, 2H), 8.92 (s, 2H). ESI-MS: [M - (ClO4)-
-
(MeCN)]+ calcd for C31H38ClFeN5O8 m/z 698.2, found 698.0;
[M - 2(ClO4)-]2+ calcd for C33H40FeN6O4 m/z 320.1, found
320.3; [M - 2(ClO4)- - (MeCN)]2+ calcd for C31H37FeN5O4 m/z
299.6, found 300.0.
N-[Bis[5-(3-br om op r op yl)-2-p yr id in yl]m eth yl]-N,N-bis-
[[5-(3-br om op r op yl)-2-p yr id in yl]m eth yl]a m in e (27). PPh3-
Br2 was freshly prepared by the addition of Br2 (75 µL, 1.4
mmol, 4.7 equiv) to a stirred solution of Ph3P (395 mg, 1.51
mmol, 5. equiv) in dry CH2Cl2 (1 mL) under a nitrogen
atmosphere. To the resulting yellow suspension was added a
solution of 24 (321 mg, 0.304 mmol) in dry CH2Cl2 (1 mL).
The reaction mixture was stirred at room temperature until
all the solids had disappeared (2 days) and extracted with 1
M HCl. The aqueous layer was neutralized with solid NaHCO3
and extracted with CH2Cl2. The organic layer was washed with
brine and dried over Na2SO4. Evaporation of the solvent
1
191.1-191.7 °C. H NMR (CD3OD, 300 MHz): δ 7.63 (d, J )
8.4 Hz, 1H), 7.76 (d, J ) 8.4 Hz, 1H), 7.92 (dd, J ) 8.4, 1.8 Hz,
1H), 7.99 (dd, J ) 8.4, 1.8 Hz, 1H), 8.65 (d, J ) 1.8 Hz, 1H),
1
8.70 (d, J ) 1.8 Hz, 1H). H NMR (CD3OD, 300 MHz): δ 7.56
(d, J ) 8.4 Hz, 1H), 7.83 (d, J ) 8.4 Hz, 1H), 8.01 (dd, J ) 8.4,
2.2 Hz, 1H), 8.10 (dd, J ) 8.4, 2.2 Hz, 1H), 8.50 (d, J ) 2.2 Hz,
1H), 8.68 (d, J ) 2.2 Hz, 1H). 13C NMR (CDCl3, 50.3 MHz): δ
119.8, 120.3, 122.8, 127.0, 138.7, 139.4, 149.5, 150.0, 150.6,
153.0, 153.9. HRMS: calcd for C11H7N3OBr2 354.895, found
(42) Observed isotope pattern corresponds to the calculated isotope
pattern.
(43) The peptide was synthesized by standard automated methods
on a peptide synthesizer using Fmoc-protected amino acids and purified
by preparative reversed-phase HPLC.
1
afforded 27 as a red oil (190 mg, 74%). H NMR (CDCl3, 300
MHz): δ 2.07 (t, J ) 6.6 Hz, 8H), 2.67 (t, J ) 6.6 Hz, 8H),
3.32 (m, 8H), 3.84 (s, 4H), 5.22 (s, 1H), 7.34-7.58 (m, 8H), 8.28
260 J . Org. Chem., Vol. 69, No. 2, 2004

Non-Heme Template for Attaching Four Peptides
354.896. Anal. Calcd for C11H7N3OBr2: C, 37.00; H, 2.00; N,
11.80; Br, 44.80. Found: C, 36.58; H, 2.06; N, 11.41; Br, 44.58.
Bis(5-br om o-2-p yr id in yl)m eth yla m in e (32). Zinc dust
(1.1 g, 17 mmol, 3.4 equiv) was carefully added in small
portions over 5 min to a suspension of 31 (1.79 g, 5.01 mmol)
in acetic acid (30 mL) and water (15 mL). The mixture was
stirred at room temperature for 30 min, filtered over Celite,
and washed with water. After evaporation of the solvent the
residue was neutralized with concentrated ammonia (25% v/v)
and extracted with CH2Cl2. The organic layer was washed with
brine, dried over Na2SO4, and concentrated in vacuo to furnish
pure 32 as a brown viscous oil that solidified on standing (1.62
g, 94%). 1H NMR (CDCl3, 300 MHz): δ 2.38 (br. s, 2H), 5.24 (s,
1H), 7.29 (d, J ) 8.4 Hz, 2H), 7.72 (dd, J ) 8.4, 1.8 Hz, 2H),
8.55 (d, J ) 1.8 Hz, 2H). 13C NMR (CDCl3, 75.5 MHz): δ 61.1,
119.1, 123.0, 139.2, 150.1, 160.6. HRMS: calcd for C11H9N3-
Br2 340.916, found 340.916.
5-Br om o-2-p icolin e N-Oxid e (34). A mixture of 5-bromo-
2-picoline18 (11.7 g, 68.0 mmol) and m-CPBA (20.9 g, 97.0
mmol, 1.4 equiv) in CH2Cl2 (300 mL) was stirred at room
temperature overnight. Saturated Na2CO3 (300 mL) was
added, and after stirring for 30 min the layers were separated.
The water layer was extracted with CH2Cl2, and the combined
organic layers were washed with brine and dried over Na2-
SO4. Evaporation of the solvent yielded 34 as a light yellow
solid that was recrystallized from diethyl ether (12.5 g, 98%).
Mp: 119.3-120.4 °C (lit.44 mp 117-118 °C). 1H NMR (CDCl3,
300 MHz): δ 2.43 (s, 3H), 7.11 (d, J ) 8.4 Hz, 1H), 7.29 (dd, J
) 8.4, 1.1 Hz, 1H), 8.38 (d, J ) 1.1 Hz, 1H). 13C NMR (CDCl3,
75.5 MHz): δ 17.2, 117.1, 126.3, 128.2, 140.5, 147.9. HRMS:
calcd for C6H6BrNO 186.963, found 186.963.
(5-Br om o-2-pyr idin yl)m eth an ol (35). Protected from mois-
ture, trifluoroacetic acid anhydride (10 mL, 71 mmol, 5 equiv)
was carefully and slowly added to 34 (2.63 g, 14.0 mmol). After
the vigorous thermal reaction had ceased, the orange mixture
was stirred at room temperature for 30 min and then refluxed
for 30 min. Saturated NaHCO3 was carefully added after
cooling to room temperature until pH 8 was reached. The
resulting red solution was stirred at room temperature under
nitrogen overnight. The mixture was extracted with CH2Cl2,
and the combined organic layers were washed with brine and
dried over Na2SO4. Evaporation of the solvent yielded 35 as a
dark brown oil (2.15 g, 81%), which was used without any
further purification. 1H NMR (CDCl3, 300 MHz): δ 3.41 (br. s,
1H), 4.72 (s, 2H), 7.20 (d, J ) 8.3 Hz, 1H), 7.81 (dd, J ) 8.3,
2.2 Hz, 1H), 8.60 (d, J ) 2.2 Hz, 1H). 13C NMR (CDCl3, 75.5
MHz): δ 63.9, 118.8, 121.9, 139.3, 149.3, 158.5. HRMS: calcd
for C6H6NOBr 186.963, found 186.965.
5-Br om o-2-(ch lor om eth yl)p yr id in e (36). Thionyl chlo-
ride (0.89 mL, 12 mmol, 1.5 equiv) was slowly added to a
solution of crude 35 (1.51 g, 8.03 mmol) in CH2Cl2 (30 mL) at
0 °C, protected from moisture. After being allowed to warm to
room temperature the reaction mixture was stirred overnight.
Saturated NaHCO3 was added, and the layers were separated.
The water layer was extracted with CH2Cl2. The combined
organic layers were washed with brine and dried over Na2-
SO4. Concentration by rotary evaporation furnished 36 as a
dark brown oil (1.63 g, 97%) that was used without further
purification.45 1H NMR (CDCl3, 300 MHz): δ 4.61 (s, 2H), 7.37
(d, J ) 8.4 Hz, 1H), 7.84 (dd, J ) 8.4, 2.2 Hz, 1H), 8.62 (d, J
) 2.2 Hz, 1H). 13C NMR (CDCl3, 75.5 MHz): δ 45.8, 120.0,
124.0, 139.5, 150.3, 155.0. HRMS: calcd for C6H6NClBr
204.929, found 204.929.
the solvent, the residue was taken up in CH2Cl2, washed with
water and brine, and dried over Na2SO4. Concentration by
rotary evaporation afforded a dark brown, viscous oil that
solidified on standing. After purification by flash chromato-
graphy over aluminum oxide using a gradient of hexane/ethyl
acetate/TEA (40:9:1-30:9:1) and evaporation of the solvents,
the residue was precipitated from diethyl ether to furnish 1b
as a white solid (0.83 g, 58%). Recrystallization from CH2Cl2/
diethyl ether afforded pure 1b as white needles. Mp: 151.6-
1
152.2 °C. H NMR (CDCl3, 300 MHz): δ 3.84 (s, 4H), 5.21 (s,
1H), 7.40 (d, J ) 8.4 Hz, 2H), 7.55 (d, J ) 8.4 Hz, 2H), 7.72
(dd, J ) 8.4, 2.2 Hz, 2H), 7.78 (dd, J ) 8.4, 2.2 Hz, 2H), 8.56
(d, J ) 2.2 Hz, 2H), 8.59 (d, J ) 2.2 Hz, 2H). 13C NMR (CDCl3,
75.5 MHz): δ 56.3, 70.7, 119.0, 119.5, 124.4, 125.2, 138.9, 139.0,
150.1, 150.3, 157.7. MS CI: [M + H]+ calcd for C23H18Br4N5
m/z 679.8, found 679.9. Anal. Calcd for C23H17Br4N5‚H2O: C,
39.41; H, 2.73; N, 9.99; Br, 45.59. Found: C, 39.54; H, 2.39;
N, 9.96; Br, 45.60.
[(1b)F e(MeCN)](ClO4)2 (2b). To a suspension of 1b (93 mg,
0.14 mmol) in methanol (3 mL) was added Fe(ClO4)2‚6H2O (56
mg, 0.15 mmol, 1.1 equiv) in acetonitrile (1.5 mL). The
resulting clear dark red/brown solution was stirred at room
temperature for 15 min. Slow diffusion of ethyl acetate into
1
this solution furnished 2b as red crystals (115 mg, 87%). H
NMR (CD3CN, 300 MHz): δ 4.31 (dd, J AB ) 51.8, 18.5 Hz, 4H),
6.32 (s, 1H), 7.00 (d, J ) 8.4 Hz, 2H), 7.75 (d, J ) 8.4 Hz, 2H),
7.88 (dd, J ) 8.4, 1.8 Hz, 2H), 8.10 (dd, J ) 8.4, 1.8 Hz, 2H),
9.01 (d, J ) 1.8 Hz, 2H), 9.10 (d, J ) 1.8 Hz, 2H). ESI-MS:
[M - (ClO4)- - (MeCN)]+ calcd for C23H17Br4ClFeN5O4 m/z
833.7, found 833.6; [M - 2(ClO4)- - (MeCN)]2+ calcd for C23H17
-
Br4FeN5 m/z 367.4, found 367.8. Anal. Calcd for C25H20Br4Cl2-
FeN6O8: C, 30.68; H, 2.07; N, 8.59. Found: C, 30.48; H, 2.27;
N, 8.36.
N-[Bis(5-p h en yl-2-p yr id in yl)m eth yl]-N,N-bis[(5-p h en -
yl-2-p yr id in yl)m eth yl]a m in e (1f). A mixture of 1b (0.20 g,
0.30 mmol), phenylboronic acid (0.36 g, 3.0 mmol, 10 equiv),
Pd(PPh3)4 (40 mg, 34 µmol, 12 mol %), aqueous Na2CO3 (2 M,
2 mL), and 4 droplets of ethylene glycol was heated at reflux
in toluene (4 mL) under a nitrogen atmosphere overnight. The
layers were separated, and the aqueous phase was extracted
with CH2Cl2. The combined organic layers were washed with
aqueous Na2CO3 (2 M) and brine and dried over Na2SO4.
Evaporation of the solvent afforded the pure product as a dark
1
yellow oil that solidified on standing (0.16 g, 80%). H NMR
(300 MHz, CDCl3): δ 4.10 (s, 4H), 5.50 (s, 1H), 7.30-7.90 (m,
28H), 8.72 (d, J ) 2.2 Hz, 2H), 8.82 (s, 2H). 13C NMR (50.3
MHz, CDCl3): δ 57.3, 72.4, 115.6, 119.0, 123.0, 123.8, 126.8,
126.9, 127.8, 128.2, 128.4, 128.8, 131.8, 132.0,134.6, 134.7,
134.9, 137.5, 147.2, 147.6, 158.3, 158.6. MS CI: [M + H]+ calcd
for C47H38N5 m/z 672.3, found 672.0 (fragmentations m/z 352,
323, 170).
[(1f)F e(MeCN)](ClO4)2 (2f) was prepared as described for
2c. After failed crystallization attempts the solution was
concentrated and analyzed by mass analysis. ESI-MS: [M-
(MeCN)] calcd for C47H37Cl2FeN5O8 m/z 925.1, found 925.1; [M
- (ClO4)- - (MeCN)]+ calcd for C47H37ClFeN5O4 m/z 826.3,
found 826.3; [M - 2(ClO4)-]2+ calcd for C49H40FeN6 m/z 384.1,
found 384.3; [M - 2(ClO4)- - (MeCN)]2+ calcd for C47H37FeN5
m/z 363.6, found 363.7.
6-[[[Bis(5-br om o-2-p yr id in yl)m eth yl][(5-for m yl-2-p yr -
id in yl)m eth yl]a m in o]m eth yl]n icotin a ld eh yd e (38). To 1b
was added i-PrMgBr (4.5 equiv, 2 M in THF), and the mixture
was stirred at room temperature for 1 h. ESI-MS analysis of
a sample quenched with water revealed the molecular ion of
37a ([M + H]+ 526). When quenched with D2O, the molecular
ion of 37b ([M + H]+ 529) was observed. More i-PrMgBr (4
equiv) was added, and the mixture was heated to reflux for 1
h. Dry DMF (15 equiv) was added. The mixture was washed
with water and brine and dried over Na2SO4. Evaporation of
N-[Bis(5-br om o-2-p yr id in yl)m eth yl]-N,N-bis[(5-br om o-
2-p yr id in yl)m eth yl]a m in e (1b). A mixture of 32 (0.72 g, 2.1
mmol), 36 (1.8 g, 8.7 mmol, 4 equiv), DIEA (7.2 mL, 41 mmol,
20 equiv), and dry acetonitrile (1.5 mL) was heated at 80 °C
under a nitrogen atmosphere for 2 days. After evaporation of
(44) Blanz, E. J ., J r.; French, F. A.; DoAmaral, J . R.; French, D. A.
J . Med. Chem. 1970, 13, 1124-1130.
(45) The product can be stored at -20 °C under nitrogen for many
months without any detectable signs of degradation.
1
the solvent afforded a brown oil. H NMR (300 MHz, CDCl3):
δ 4.03 (s, 4H), 5.27 (s, 1H), 7.52 (d, J ) 8.2 Hz, 2H), 7.71 (d, J
) 8.2 Hz, 2H), 7.76 (dd, J ) 8.2, 1.8 Hz, 2H), 8.07 (dd, J )
J . Org. Chem, Vol. 69, No. 2, 2004 261

van den Heuvel et al.
8.2, 1.8 Hz, 2H), 8.58 (d, J ) 1.8 Hz, 2H), 8.93 (d, J ) 1.8 Hz,
2H), 10.04 (s, 2H). 13C NMR (50.3 MHz, CDCl3): δ 58.0 (t),
71.8 (d), 120.5 (s), 124.1 (d), 126.0 (d), 130.9 (s), 136.9 (d), 139.8
(d), 150.2 (d), 152.4 (d), 158.2 (s), 166.2 (s), 191.1 (d). MS CI:
[M + H]+ calcd for C25H19Br2N5O2 m/z 582.3, found 582.3
(fragmentations m/z 329, 254, 122).
ABTS Oxid a tion . The ABTS oxidations were performed
in 0.1 M acetate buffers at pH 3.5 using a magnetically stirred,
thermostatically controlled quartz cuvette at 20 °C. The
increase in absorption due to the ABTS+• radical cation
Netherlands, is gratefully acknowledged for performing
the crystallographic analyses of complexes 2b and 2c.
Special recognition goes to Dr. R. Hage and Dr. S.
Killeen from Unilever Research Laboratory, The
Netherlands, for performing cyclic voltammetry mea-
surements on 2b. Dr. J . A. W. Kruijtzer from the
University of Utrecht, The Netherlands, is kindly
acknowledged for the preparation of the peptide used.
formation was monitored by a UV-vis spectrophotometer at
Su p p or tin g In for m a tion Ava ila ble: Experimental pro-
cedures and analytical data of compounds 5, 6, 10, 12, 14, 16,
18, 20, 22, and 24, and a summary of the crystallographic data
and the cyclic voltammetry measurements. This material is
available free of charge via the Internet at http://pubs.acs.org.
Further details of the crystal structure may be obtained from
the Cambridge Crystallographic Data Centre by quoting the
references CCDC 210556 for 2b and CCDC 210557 for 2c.
40
660 nm (ꢀ 1.47 × 104 M-1cm-1
)
at intervals of 1 s for 400 s
with an incremental cycle time of 10% after an initial time of
60 s. The experimental conditions were identical to those
described previously.16
Ack n ow led gm en t . We thank the Council for
Chemical Sciences of The Netherlands Organization for
Scientific Research (NWO-CW) for supporting grants.
Auke Meetsma from the University of Groningen, The
J O035157Z
262 J . Org. Chem., Vol. 69, No. 2, 2004