Synthesis of substituted terpyridine ligands for use in protein purification

Article abstract of DOI:10.1016/j.tet.2014.09.074

A number of 4,4″-disubstituted terpyridines bearing a 4′-thioethanamine linker, and regioisomers thereof, have been synthesised from 4-substituted picolinates. The substituted terpyridines were immobilised onto epoxy-activated Sepharose FF gel, creating functionalised solid supports for the fractionation of proteins - including antibodies - by mixed mode affinity chromatography. The metal chelating properties of the immobilised ligand render the stationary phase also amenable for use in immobilised metal-ion affinity chromatography (IMAC), and have been demonstrated with the purification of suitably tagged green fluorescent protein (GFP).

Full text of DOI:10.1016/j.tet.2014.09.074

Tetrahedron xxx (2014) 1e12
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Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis of substituted terpyridine ligands for use in protein
purification
Chih-Pei Lin, Pas Florio, Eva M. Campi, Chunfang Zhang, Dale P. Fredericks, Kei Saito,
*
W. Roy Jackson, Milton T.W. Hearn
Centre for Green Chemistry, School of Chemistry, Monash University, Clayton, Victoria 3800, Australia
a r t i c l e i n f o
a b s t r a c t
A number of 4,400-disubstituted terpyridines bearing a 40-thioethanamine linker, and regioisomers
thereof, have been synthesised from 4-substituted picolinates. The substituted terpyridines were im-
mobilised onto epoxy-activated SepharoseÔ FF gel, creating functionalised solid supports for the frac-
tionation of proteinsdincluding antibodiesdby mixed mode affinity chromatography. The metal
chelating properties of the immobilised ligand render the stationary phase also amenable for use in
immobilised metal-ion affinity chromatography (IMAC), and have been demonstrated with the purifi-
cation of suitably tagged green fluorescent protein (GFP).
Article history:
Received 20 July 2014
Received in revised form 12 September 2014
Accepted 22 September 2014
Available online xxx
Keywords:
Heterocyclic compound synthesis
40-Terpyridones
40-Halo-terpyridines
Ó 2014 Published by Elsevier Ltd.
4,400-Disubstituted-40-
aminoethanesulphanylterpyridines
Affinity chromatography
Protein purification
1. Introduction
the synthesis of an array of pyridine based ligands for use in mixed
mode affinity chromatography.10 The pyridine nucleus in these li-
Affinity chromatography in its various forms is an important
tool for the analytical and preparative purification of proteins and
other biomolecules.1e6 In recent years there has been a consider-
able interest in the development of low molecular weight affinity
ligands for the functionalisation of support materials for the
chromatographic purification of proteins.7e10 Such efforts typi-
cally seek to address limitations in protein purification such as
specificity, capacity, ligand toxicity and leaching.11,12 The de-
velopment of synthetic ligands for use in affinity chromatography
has utilised traditional ligand discovery approaches ranging from
ligand-based design, structure based design and ligand
screening.13
The use of low molecular weight pyridine-based ligands in
protein purification is well documented.7e10 Such ligands present
opportunities for protein binding through a number of interactions
such as hydrogen bonding, hydrophobic interactions, ionic in-
teractions, thiophilic interactions, etc., and are therefore amenable
to various forms of chromatography. A recent study has reported
gands contained a thioethanamine unit to create both a thiophilic
centre10 and an easy modality for immobilisation onto suitably
activated support materials and solid phase surfaces. Both the na-
ture and arrangement of the substituents on the pyridine nucleus of
these ligands were demonstrated to affect their binding capacities
and specificities for cell culture derived monoclonal antibodies
(mAbs).
As part of an ongoing research program into the synthesis of
hetero-aromatic ligands for use in protein separation,10,14,15
a range of substituted terpyridines bearing a thioethanamine
functionality have now been prepared (Scheme 1). These ligands
incorporate the terpyridine skeleton as a structural motif with
substituents on the peripheral terpyridine rings to influence the
steric and electronic properties of the ligand. Starting from the
appropriately 4-substituted alkyl picolinates 1, the 4,400-di-
substituted terpyridines of interest 2 were prepared via a number
of alternative synthetic strategies building upon modifications to
procedure of Constable et al..16 The advantages of these synthetic
approaches will be discussed, and applications documented of the
target compounds as ligands used to prepare novel chromato-
graphic supports for the separation of antibodies and suitably
tagged proteins.
* Corresponding author. Tel.: þ61 3 9905 4547; fax: þ61 3 9905 8501; e-mail
address: milton.hearn@monash.edu (M.T.W. Hearn).
http://dx.doi.org/10.1016/j.tet.2014.09.074
0040-4020/Ó 2014 Published by Elsevier Ltd.
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C.-P. Lin et al. / Tetrahedron xxx (2014) 1e12
without purification. This was not only desirable from a Green
Chemistry perspective, but the triketones were brightly coloured
and very pervasive and therefore handling was kept to a minimum
to avoid contamination of glassware.
Treatment of the triketones 5aee with ammonium acetate
(Scheme 3) lead to ring closure in a condensation reaction that gave
the terpyridones 3aee as crystalline solids in moderate to good
overall yields (Table 1). The terpyridone 3d was obtained in 58%
yield containing a trace (ca. 5%) of the 4,400-dimethoxyterpyridone
3c (by 1H NMR spectroscopy) resulting from some dimethoxy tri-
ketone 5c formed during the initial Claisen reaction. This impurity
could be readily removed by recrystallisation but for convenience
could be carried through to the next step for subsequent removal.
Scheme 1. Terpyridine targets of interest, where R is a range of substituents having
varying steric and electronic properties, i.e., H, Me, OMe, Cl, Br, NO2.
2. Results and discussion
The target compounds 2 were prepared via the C40-halogenated
terpyridine 4 as the key intermediate (Scheme 2). This intermediate
was accessed from the picolinates (1) via the terpyridone 3.
Scheme 2. Key intermediates in the synthesis of target compounds 2.
2.1. Terpyridone formation
Table 1
Terpyridone formation via Claisen-like condensation of 4-substituted picolinates
with acetone
Based on a two-step procedure established by Constable and
Ward for the preparation of terpyridin-40-ones,16 the 4-substituted
picolinates 1aef were reacted with acetone in a Claisen-like con-
densation to give the triketones 5aee, followed by ring closure with
ammonium acetate to furnish the symmetrical 4,400-disubstituted
terpyridones 3aee, (Scheme 3). Constable and Ward performed the
first step of this procedure using 3 or more equivalents of the
picolinate 1a and 5 equiv of sodium hydride to 1 equiv of acetone.16
These investigators reported an initial exothermic reaction oc-
curred, which, once settled, was refluxed for 6 h. In earlier studies,
we have found that the number of equivalents of the picolinate can
be reduced to 2.25 equiv.17 Furthermore, we have found that good
yields of the triketone 5a can be obtained by performing the re-
action for as little as 2 h at room temperature, as similarly reported
by Wieprecht et al. for the chloro analogue 5d.18
Entry
Picolinate
R
Conditions for step
a Scheme 3
Terpyridone
R
a,b
R0
Yieldc
1
2
3
4
5
1a
1b
1c
1d
1e
H
Et
2 h at 30 ꢀC.
18 h at 30 ꢀC.
18 h at 30 ꢀC.
3 h at 30 ꢀC.
2 h at 30 ꢀC.
3a
3b
3c
3d
3e
H
53%
43%
58%
58%
48%
Me
OMe
Cl
Me
Me
Me
Et
Me
OMe
Cl
Br
Br
a
2.5 equiv of picolinate used in all instances.
Step b) 18 h, reflux.
Represents the overall yield from the corresponding picolinate.
b
c
The unsubstituted terpyridone 3a was, however, consistently iso-
lated as a stable 1:1 adduct of the terpyridone 3a with acetic acid
(Fig.1), as evidenced by NMR spectroscopy and a crystal structure.17
Scheme 3. Preparation of symmetrical 4,400-disubstituted terpyridones from 4-substituted picolinates. (a) NaH, DME, acetone; 2e18 h, 30 ꢀC. (b) NH4OAc, EtOH; 18 h, reflux.
With the exception of the 4-nitropicolinate 1f, the 4-
substituted picolinates 1ae1e reacted readily in the Claisen con-
densation reaction. The 4-nitropicolinate 1f did not react and was
recovered as starting material. Extended reaction times (18 h at
30 ꢀC) or heating under reflux for 2 h did not change this outcome
with 1f. The 4-chloropicolinate 1d was prone to substitution at C-4
by methoxide liberated from the ester group of the picolinate 1d
in the course of the reaction, and therefore it was necessary to
shorten the reaction time considerably. Reaction at 30 ꢀC for 18 h
or refluxing for 6 h lead to the complete displacement of the
chloro substituent at C-4 of the picolinate 1d. However, per-
forming the reaction for 3 h at 30 ꢀC yielded the 4,400-dichloro-
triketone 5d successfully. The 4-bromopicolinate 1e did not
undergo displacement of the halogen by the ethoxide leaving
group, although the Claisen condensation reaction did require
gentle heating to allow the reaction to proceed as mentioned
above. The triketones 5aee were isolated as solids that were used
This acetic acid adduct was employed in subsequent reactions
without causing any side reactions or decrease in product yield. The
other 4,400-disubstituted terpyridones 3bee were isolated as their
free bases, and not as acetic acid adducts.
Fig. 1. Acetic acid adduct of (3a).
2.2. Halogenation at C-40
2.2.1. Iodination via an O-triflate. A number of methods were in-
vestigated for the C-40 halogenation of the terpyridones 3aee.
Whilst Constable and Ward16 have reported the use of POCl3/PCl5 as
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C.-P. Lin et al. / Tetrahedron xxx (2014) 1e12
3
a chlorinating agent for the terpyridone 3a, we sought an alternative
procedure that was chemoselective and more easily scalable. The
halogenation of C-40 via a 40-O-triflate was investigated using the
unsubstituted terpyridone 3a as a model compound (Scheme 4).
yield (Table 3, entry 1). Similarly, 4,400-dichloroterpyridone 3d gave
4,40,400-trichloroterpyridine 8d in 77% yield (Table 3, entry 6). The
majority of the excess POCl3 (ca. 75%) was recovered by distillation
prior to isolation of the product (Scheme 5).
Scheme 4. Iodination at C-40 via a 4-O-triflate. (a) Pyridine, (CF3SO2)2O; 48 h, 30 ꢀC. (b) KI, DMF; 2 h, 160 ꢀC.
The reaction of the terpyridones 3aee with triflic anhydride
gave the 4-O-triflates 6aee in excellent yields (85e93%) (Table 2).
The signal for CF3 groups in the 13C NMR spectrum (ca.
d 120 ppm)
was only observed when the delay time, D1, was set to 15 s and this
was only done with the methoxy compound 6c. Reaction of the
parent triflate 6a and its methyl homologue 6b with potassium
iodide19 in DMF at 160 ꢀC for 2 h gave the corresponding C-40 io-
dides 7a and 7b in excellent yield (Table 2, entries 1and 2).
Scheme 5. Chlorination at C-40 of terpyridones 3a, cee using POCl3/PCl5. (a) POCl3/
PCl5, 4e18 h, reflux (See Table 3 for product numbers).
Table 2
However, attempts to chlorinate the C-40 position of the 4,400-
disubstituted-40-terpyridones 3c and 3e using this procedure lead
to the concomitant substitution (partial or complete) of the aro-
matic substituents at C-4 and C-400 (Table 3, entries 2e5, 7). The
reaction of 4,400-dibromoterpyridone 3e under these conditions
furnished the product of trans-halogenation, namely 4,40,400-tri-
chloroterpyridine 8d in 96% (Table 3, entry 7). Similarly, displace-
ment of aryl methoxy by chlorine occurred in the reaction of 4,400-
dimethoxyterpyridone 3c. The reaction for 2 h using 2.4 equiv of
PCl5 furnished a mixture of the desired 40-chloro-4,400-dimethox-
yterpyridine 8c, (14%), 4,40-dichloro-400-methoxyterpyridine 9,
(29%) and 4,40,400-trichloroterpyridine 8d, (8%) (Table 3, entry 3).
The reaction for 4 h using 1.2 equiv of PCl5 gave 8c, (32%), 9, (39%)
and 8d, (13%) (Table 3, entry 5), while reaction using the literature
conditions (2.4 equiv PCl5, 15 h) (Table 3, entry 2) gave 8c, (21%), 9,
(27%) and 8d, (12%). Performing the reaction for 4 h using 1.2 equiv
of PCl5 at a lower temperature (70 ꢀC) did however result in
a greater proportion of the desired product, (Table 3, entry 4) and
gave 8c, (35%), 9, (30%) and 8d, (6%) Fortunately, the three products
from the reaction could be separated by radial chromatography on
silica. This inadvertently generated additional ligand diversity,
furnishing ligands that would otherwise have been difficult to ac-
cess by rational means.
Iodination of 4,400-disubstituted terpyridones via the triflation/iodination sequence
Entry
Terpyridone
R
Products with yields
Triflate
Iodide
Overall
1
2
3
4
5
3a
3b
3c
3d
3e
H
6a
6b
6c
6d
6e
93%
87%
85%
88%
91%
7a
7b
7c
7d
7e
97%
89%
0%
0%
0%
90%
77%
0%
0%
0%
Me
OMe
Cl
Br
However the other 40-O-triflates 6cee did not react at all with
potassium iodide under these conditions (Table 2). In the case of
the 4,400-dimethoxytriflate 6c, the material recovered was con-
firmed by 1H NMR spectroscopy to be the starting triflate. Attempts
to drive the reaction with increasing temperature (180 ꢀC) led to
decomposition. Other means for the halogenation of C-40 were thus
sought.
2.2.2. Chlorination using POCl3/PCl5. Chlorination at C-40 using
POCl3/PCl5 has been demonstrated to be effective for the parent
terpyridone 3a16 and reaction of terpyridone 3a with POCl3/PCl5
under these conditions furnished 40-chloroterpyridine 8a in 68%
2.2.3. Bromination using PBr5. Since the attempted C-40 chlorina-
tion of 4,400-dibromoterpyridone 3e using POCl3/PCl5 resulted in
trans-halogenation and loss of the bromine substituents, bromi-
nation using phosphorous pentabromide20 was investigated
(Scheme 6). Bromination using PBr5 under the same reaction con-
ditions with the 4,400-dibromoterpyridone 3e (Scheme 6) gave the
tribromide 10e in a moderate 32% yield (Table 4, entry 1).
Table 3
C-40 Chlorination of 4,400-disubstituted terpyridones using POCl3/PCl5
Entry Terpyridone Reaction conditions
Halogenation products Isolated
yield
R
Temp Time Eq. PCl5
R1
R2
1
2
3a
3c
H
OMe
125 ꢀC 18 h 2.4
125 ꢀC 15 h 2.4
8a
8c
9
8d
8c
9
8d
8c
9
8d
8c
9
H
H
OMe
Cl
Cl
OMe
Cl
Cl
OMe
Cl
Cl
OMe
Cl
68%
21%
27%
12%
14%
29%
8%
35%
30%
6%
32%
39%
13%
77%
96%
OMe
OMe
Cl
OMe
OMe
Cl
OMe
OMe
Cl
OMe
OMe
Cl
3
4
5
3c
3c
3c
OMe
OMe
OMe
125 ꢀC 2 h
70 ꢀC 4 h
125 ꢀC 4 h
1.2
1.2
1.2
Scheme 6. Bromination at C-40 of 4,400-disubstituted terpyridones 3d and 3e using
PBr5. (a) PBr5, CHCl3, 0.5 h reflux.
8d
8d
8d
Cl
Cl
Cl
6
7
3d
3e
Cl
Br
125 ꢀC 4 h
125 ꢀC 4 h
1.2
1.2
Cl
Cl
Bromination at C-40 of 4,400-dichloroterpyridone 3d was ex-
pected to generate an intermediate 10d whose subsequent
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C.-P. Lin et al. / Tetrahedron xxx (2014) 1e12
some disubstituted products were apparent in the 1H NMR spec-
trum of the crude material.
Table 4
C-4 Bromination of 4,400-disubstituted terpyridones using PBr5
The tribromoterpyridine 10e also reacted with 2-
aminoethanethiol in DMF under the mild conditions described
above for the parent compound 8a. TLC of the reaction mixture
indicated the presence of the 40- and 400- substituted regioiso-
meric products, in addition to starting material 10e and products
corresponding to di- and tri-substitution. The components of the
reaction mixture were, however, not easily separated by silica
chromatography using DCM/MeOH/NH3 or DCM/EtOH mixtures
as the eluent. This resulted in a lower than expected recovery of
pure materials. The major regioisomer 2e was recovered in 14%
yield (Table 5, entry 7). The 4-substituted isomeric compound
was observed by TLC and in the 1H NMR spectrum of the total
product.
Under the same conditions, the trichloroterpyridine 8d was,
resistant to substitution, even at elevated temperatures and longer
reaction times (18 h at 160 ꢀC). We therefore resorted to a THF/
HMPA solvent mixture used previously by Mountford et al. for
analogous pyridine and quinoline compounds.10 This modification
gave a 1:3 regioisomeric mixture of the 4- and 40- thiosubstituted
terpyridines 13 and 2d, respectively. The isomers were also sepa-
rable by radial chromatography on silica and were isolated in 15%
and 43% yield, respectively (Table 5, entry 6).
In conclusion, the synthesis of a variety of terpyridine-based
compounds bearing a thioethanamine linker has been achieved.
These compounds have a range of substituents in the para position
of the pyridine rings and a thioethanamine moiety in either the 4-
or the 40- position. These ligands were immobilised onto a solid
support (epoxy-activated Sepharose 6 Fast FlowÔ) based on
methods described previously for other classes of affinity li-
gands.10,21 The data in Table 6 give the immobilisation conditions
for the various ligands, the ligand concentration used and the li-
gand density of the resulting adsorbents.
Entry
Terpyridone
R
Time
40-Bromoterpyridines
R
Yield
1
2
3e
3d
Br
Cl
0.5 h
0.5 h
10e
10d
Br
Cl
32%
43%a
a
Mixed with 10e (ca. 8%).
substitution at C-40 with 2-aminoethanethiol would lead to en-
hanced regio- and chemoselectivity for the C-40 position of the
central ring. However, reaction of 3d with PBr5 led to partial trans-
halogenation (w20% by 1H NMR) of the chloro-substituents on the
peripheral rings, giving the tribromide 10e as a byproduct. Fur-
thermore, the yield of the target compound 10d was moderate (ca.
43%) and the products could not be separated by alumina or silica
chromatography using various ratios of EtOAc/hexane, hexane/
DCM or DCM/MeOH as the eluent.
2.2.4. Preparation of (2-aminoethanesulfanyl)terpyridines 2. The
halogenated terpyridines 7a and b, 8a, c and d, 9 and 10e were
reacted with 2-aminoethanethiol under the basic conditions in
DMF at 50 ꢀC as reported by Mountford et al.10 It was expected that
the di-halo- (9) and tri-haloterpyridines 8d, 10e would furnish
regioisomeric mixtures of the substituted-(2-aminoethanesulfanyl)
terpyridines, as well as products of di- and tri-substitution. The
regioisomers resulting from mono-substitution would furnish ad-
ditional targets for testing (Scheme 7).
Scheme 7. Substitution reactions in halo-, dihalo- and trihalo-terpyridines. (a) NaH, 2-
aminoethanethiol, DMF. (b) NaH, 2-aminoethanethiol, THF/HMPA.
Table 6
Results for ligand immobilisation
Reaction of either the 40-chloro- or 40-iodoterpyridine 8a or 7a
as described by Mountford et al.10 gave 2a in very good to excellent
yield (75e86%) (Table 5, entries 1e2). The 4,400-dimethyl analogue
7b required a higher temperature than that required for the
unsubstituted compound 7a in order to give the thioethylamine 2b
in 90% yield (Table 5, entry 3). The 4,400-dimethoxy analogue 8c
required additional heating as well as a much longer reaction time
(Table 5, entry 4) but gave the desired product 2c in comparable
yield. Similarly, the 4,40-dichloro-400-methoxy analogue 9 also re-
quired extended heating for 24 h at 90 ꢀC to yield a 1:2 regioiso-
meric mixture of the 4- and 40-thiolated terpyridines 12 and 11 in
12% and 28%, respectively after radial chromatography on silica.
(Table 5, entry 5). The starting material (26%) was also isolated and
Entry Ligand
Immobilisation at 28 ꢀC,
solvent and time
Ligand Ligand
conc density
mol/g
m
1
2
3
4
5
6
7
40-TerPSEA
2a
75% MeOH, 25% H2O, 21 h 0.2 M
296
205
155
246
228
4,400-diOMe-40-TerPSEA 75% DMF, 25% MeOH, 64 h 0.1 M
2c
4,400-diMe-40-TerPSEA
MeOH, 21 h
0.1 M
0.1 M
0.1 M
2b
4,400-diCl-40-TerPSEA
55% DMF, 20% DCM, 20%
MeOH, 5% H2O, 64 h
55% DMF, 20% DCM, 20%
MeOH, 5% H2O, 64 h
2d
40,400-diCl-4-TerPSEA
13
40,400-diCl-4-TerPSEA
97% DMSO, 3% H2O, 21 h 0.067 M 59
13
4-Cl-400-OMe-40-
TerPSEA
11
95% DMSO, 5% H2O, 21 h 0.1 M
95% DMSO, 5% H2O, 21 h 0.1 M
114
183
Table 5
Substitution of halo-terpyridones with 2-aminoethanethiol
8
40-Cl-400-OMe-4-
TerPSEA
12
Entry
Terpyridine
Reagents and
conditions
Products
Isolated
yield
R1
R2 R3
R1
R2 R3
1
2
3
4
5
8a
7a
7b
8c
9
H
H
Me
Cl
I
I
H
H
Me
50 ꢀC, 3 h
70 ꢀC, 2 h
90 ꢀC, 3 h
2a
2a
2b Me
H
H
SR
SR
SR Me
H
H
75%
86%
90%
OMe Cl OMe 80 ꢀC, 22 h
2c OMe SR OMe 81%
3. Purification of IgG1 by affinity chromatography
Cl
Cl OMe 90 ꢀC, 24 h
11 Cl
12 SR
2d Cl
13 SR
2e Br
SR OMe 28%
Cl OMe 12%
SR Cl
Cl Cl
SR Br
The chromatographic utility of the adsorbents prepared from
the substituted terpyridine ligands to purify monoclonal anti-
bodies, such as humanised immunoglobulin 1 (IgG1), was eval-
uated based on the binding conditions of 25 mM Tris, 600 mM
Na3citrate, pH 8.0 with elution using a buffer of lower pH
6
7
8d
Cl
Cl Cl
Br Br
30 ꢀC, 40 ha
50 ꢀC, 2 h
43%
15%
14%
10e Br
SR¼SCH2CH2NH2.
a
Reaction in THF/HMPA.10
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C.-P. Lin et al. / Tetrahedron xxx (2014) 1e12
5
without salt (e.g., 25 mM MES, pH 5.0). Although high concen-
trations of the kosmotropic salt, Na2SO4, have previously been
favoured for the adsorption of monoclonal antibodies to mixed
mode and hydrophobic interaction chromatographic adsor-
bents,10 we have recently found that replacing this salt with
Na3citrate resulted in improved antibody binding using immo-
bilised 40-TerPSEA.22
Table 6 shows the results for the ligand immobilisation and
conditions employed. Under identical loading conditions with the
same volume of crude IgG1 (ca. 0.6 mg/mL) the adsorbents with
chromatographic elution profiles with these new adsorbents,
namely the dimethoxy gel, (Fig. 2(a), Table 6 entry 2) and the 40,400-
dichloro gel (Fig. 2(b), Table 6 entry 4).
The SDS-PAGE gels of the flow through, wash and elution frac-
tions (Fig. 2(c) and (d)) demonstrate different selectivity profiles for
these two ligands. For example, the elution profiles for the dime-
thoxy gel (Fig. 2(c)), and the 40-Cl-400-OMe gel (Table 6, entries 2 and
8) are similar to that of the adsorbent prepared from the unsub-
stituted 40-TerPSEA ligand, with the major protein band corre-
sponding to the mAb IgG1 (ca. 160 kDa).10 The minor band at ca.
25 kDa has been shown by MALDI-TOF MS in previous in-
vestigations14,15 to be the light chain fragment of the mAb. In
contrast, the SDS-PAGE pattern for the 40,400-dichloro gel (Fig. 2(d)),
(Table 6, entry 4) shows that the elution fractions (both peaks seen
in the chromatogram) contained essentially only the 160 kDa mAb
IgG1.
a ligand loading ca. 200 mmol/g, i.e. the dimethoxy gel, the dimethyl
gel, both dichloro gels and the 40-Cl-400-OMe gel (entries 2, 3, 4, 5
and 8, respectively) exhibited similar binding capacity for the IgG1,
comparable to that found for the adsorbent prepared from the
unsubstituted ligand 40-TerPSEA (Table 6 entry 1). The maximum
binding capacity (Qmax) derived from isothermal measurements for
this adsorbent was 61.4 mg/mL (unpublished results), higher than
that reported for adsorbents prepared from the 2-(pyridin-20-
ylsulfanyl)ethanamine family of mixed mode ligands.10 Not un-
expectedly, adsorbents derived from the same ligand but with
significantly lower ligand densities, e.g. compare entries 5 and 6,
bound significantly less IgG1 and would thus be expected to show
lower Qmax values. Fig. 2 provides illustrative examples of the
These observations raise the possibility that these 40-TerPSEA-
related ligands are capable of selective molecular recognition of
two different classes of binding sites within the intact IgG1, namely
a site within the light chain and a site within the heavy chain as part
of the Fc fragment of the IgG1. On-going investigations have been
designed to resolve this possibility and these results will be re-
ported subsequently.
Fig. 2. Chromatographic profiles for the separation of the same crude IgG1 (6 mg IgG1 in 10 mL) using (a) the Sepharose-4,400-dimethoxy-40-TerPSEA adsorbent and (b) the Se-
pharose-4,400-dichloro-40-TerPSEA adsorbent. In Panels (c) and (d) are shown the corresponding SDS-PAGE gels for the crude sample, and flow through, wash and elution fractions
for the two adsorbents. The chromatographic conditions and procedures as well as the SDS-PAGE protocols are found in the Experimental section.
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6
C.-P. Lin et al. / Tetrahedron xxx (2014) 1e12
4. Purification of tagged proteins by immobilised metal ion
affinity chromatography (IMAC)
400 MHz (1H) or 100 MHz (13C) with a Bruker DRX-400 spec-
trometer or at 600 MHz (1H) or 150 MHz (13C) with a Bruker DRX-
600 spectrometer. The NMR spectra refer to solutions in deuter-
ated DMSO-d6 or base-washed (Na2CO3) CDCl3 where either the
Immobilised metal ion affinity chromatography (IMAC) is
a highly versatile method for protein purification, which involves
the interaction of an immobilised metal complex, via coordination
processes, with a specific amino acid side chain, such as the imi-
dazolyl group of histidine residue, often included in multiple copies
as an affinity tag (e.g. hexahistidine, [His]6) as part of the protein
structure.23 The utility of adsorbents prepared from the unsub-
stituted ligand 40-TerPSEA to purify tagged green fluorescent pro-
tein (GFP) by IMAC was thus evaluated. The immobilised ligand was
charged with Cu(II), Ni(II) and Zn(II) salts as described previously
for the purification of [His]6 and NT1A-tagged glutathione S-
transferase (GST).21,24 Based on previous results with tagged GFP,25
the conditions used for binding of the tagged GFP, expressed in E.
coli and recovered as a cleared crude fermentation, were 50 mM
phosphate buffer, 500 mM NaCl, 10 mM imidazole, pH 7.6 while
elution was achieved using the same buffer and pH conditions with
300 mM imidazole.
The SDS-PAGE analysis of the flow through, wash and elution
fractions from all three adsorbents (i.e., 40-TerPSEA loaded with
Cu(II), Ni(II) and Zn(II)), showed similar selectivity for the target
protein (ca. 37 kDa), which was readily recovered in the elution
fraction. Fig. 3 shows the results for the Ni(II) loaded 40-TerPSEA.
The bands at ca. 20 and 70 kDa have been shown by Western blot
analysis to be GFP related, and thought to correspond to the GFP
dimer and a truncated version, respectively.25
residual solvent peak or tetramethylsilane (TMS) (d 0.00 ppm) was
used as the internal standard for 1H spectra and the solvent peak
used as an internal reference in 13C NMR spectra. Chemical shift
values (d) are given in parts per million (ppm) relative to the re-
sidual solvent peak (or TMS) and coupling constants are given in
Hertz (J Hz). Assignments were determined from J-modulated Spin
Echo experiments for X-nuclei coupled to 1H in order to determine
the number of attached protons. Heteronuclear Multiple Quantum
Correlation (HMQC) was used to determine, which 1H were at-
tached to the corresponding 13C. Heteronuclear Multiple Bond
Correlation (HMBC) was used to determine long range 13Ce1H
connectivity. Absorbance infrared spectra (IR) were recorded neat
on a Bruker Equinox 55 diamond ATR spectrometer. Low resolu-
tion electrospray ionisation mass spectra (ESIþ mode) were
recorded on a Waters ZMD API QMS Electrospray mass spec-
trometer with cone voltage at 25 V or 35 V as solutions in nom-
inated solvents. High resolution electrospray mass spectra (HRMS)
were recorded on an Agilent 6220 Accurate-Mass ESI TOF MS as
acetonitrile solutions (ESIþ mode). With regard to the HRMS (ESIþ)
data for the terpy analogues and their precursors containing
a chloro-group, the observed and calculated m/z values refer to the
35Cl isotope and follow the guidance provided in the ACS Style
Guide, A Coghill et al., American Chemical Society, Washington,
DC, Chapter 13, p 275. Melting points were determined using
a Buchi Melting Point B-545 with a digital thermometer and are
uncorrected. Picolinic acid derivatives were obtained from Sig-
maeAldrich, Australia (4-methyl, methyl ester), Angene In-
ternational, Hong Kong (4-chloro and 4-bromo, methyl ester and
4-nitro, acid), AK Scientific, USA (4-chloro, methyl ester), Combi-
Blocks Inc., USA (4-methoxy, methyl ester) and HetCat, Switzer-
land (4-nitro, acid) and were used as supplied. Solvents and other
reagents were purchased from SigmaeAldrich, Castle Hill, NSW,
Australia. Preparative radial chromatography was carried out using
a Chromatotron model 7924T (Harrison Research) on glass plates
coated with 2 mm silica gel (Merck, 60 PF/UV254). Elemental
analyses were performed by The Campbell Microanalytical Labo-
ratory, Department of Chemistry, University of Otago, Dunedin,
New Zealand.
Fig. 3. SDS-PAGE pattern for the crude protein, flow through, wash and elution frac-
tions using the Ni(II) Sepharose-40-TerPSEA adsorbent. The monomeric form of the
[His]6-GFP is indicated by the full arrow (at ca. 37 kDa), the dimeric form (at ca.
70 kDa) by the dashed arrow and the truncated form at 20 kDa by the hatched arrow.
The procedures used for the SDS-PAGE protocols are found in the Experimental
section.
6.2. General Method A for preparation of terpyridin-40-ones
The terpyridin-40-ones were prepared by following a modifica-
tion of the procedure reported by Constable and Ward16 A solution
of the picolinate ester (1) (2.5 equiv) and acetone (8e30 mmol) in
DME (16e100 mL) was added dropwise to a stirred suspension of
NaH (60% suspension in oil, 5 equiv) in DME (16e100 mL) under an
atmosphere of argon at room temperature. A vigorous evolution of
gas was observed after approximately 15 min and the grey-white
suspension turned into a bright orange suspension. This suspen-
sion was stirred at room temperature overnight. The solvent was
removed in vacuo and the orange paste cautiously treated with
water. The solution was filtered through a pad of Celite and the
filtrate stirred in an ice bath. 5 M HCl was added dropwise with
stirring at 0 ꢀC to adjust the pH to 6.5. The copious amount of
mustard yellow solid so obtained was collected by filtration and
washed with water until the washing appeared colourless. The
solid was oven-dried under vacuum (40 ꢀC, ca. 30 mm) for 2 days to
give the crude triketone 5, which was re-suspended in absolute
EtOH (30 mL). Ammonium acetate (ca. 7 equiv) was added and the
stirred suspension was refluxed overnight to give a dark brown
solution. The solution was cooled and concentrated in vacuo to half
the original volume and cooled to room temperature. The solid that
5. Conclusion
In this study,
a
range of 4,400-disubstituted-40-(2-
aminoethanesulphanyl)terpyridines and their regioisomers have
been prepared from 4-substituted picolinates. These compounds
were immobilised onto epoxy-activated SepharoseÔ 6 FF to gen-
erate a versatile family of chromatographic supports for protein
fractionation. These studies indicate that these terpyridine ligands
have significant potential in the purification of antibodies (mAbs
such as humanised IgG1) by mixed mode chromatography and
tagged proteins by metal ion affinity chromatography.
6. Experimental
6.1. General
Nuclear magnetic resonance spectra were recorded at 300 MHz
(1H) or 75 MHz (13C) with a Bruker DPX-300 spectrometer, at
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C.-P. Lin et al. / Tetrahedron xxx (2014) 1e12
7
precipitated was collected by filtration, washed well with Et2O and
air-dried to give the terpyridone 3.
1264m, 1224w, 1203m, 1184s, 1101s, 1073w, 1058w, 1020s, 992m,
941m, 865s, 824s, 785s, 772s cmꢂ1 1H NMR (400 MHz, CDCl3):
8.58 (2H, d, J 5.7 Hz, H6,600), 7.395 (2H, d, J 2.4 Hz, H3,300), 7.02 (2H,
s, H30,50), 6.925 (2H, dd, J 5.7, 2.4 Hz, H5,500), 3.94 (6H, s, OCH3). 1H
NMR (400 MHz, DMSO-d6):
8.50 (2H, d, J 5.6 Hz, H6,600), 8.08 (2H,
d, J 2.5 Hz, H3,300), 7.85 (2H, s, H30,50), 7.04 (2H, dd, J 5.6, 2.5 Hz,
.
The acetic acid adduct of 2,20:60,200-terpyridin-40(10H)-one (3a)
d
was obtained as grey-white fibrous crystals (53%). Mp 132e136 ꢀ
C
(lit.26 mp 128e131 ꢀC). The 1H NMR spectral data were consistent
d
with literature data for the free base.27
H5,500), 3.94 (6H, s, OCH3). 13C NMR (100 MHz, DMSO-d6):
d 166.21
6.2.1. Synthesis of 4,400-dimethyl-2,20:60,200-terpyridin-40(10H)-one
(3b). A solution of methyl 4-methyl-2-picolinate (1b) (3.0 g,
19.8 mmol) and acetone (0.59 mL, 7.94 mmol) in DME (16 mL) was
added dropwise to a stirred suspension of NaH (60% suspension in
oil, 1.60 g, 39.7 mmol) in DME (16 mL) under an atmosphere of
argon at room temperature. Reaction as described above in General
Method A. gave a bright orange suspension, which was stirred at
room temperature overnight. The solvent was removed in vacuo
from the resultant dark orange suspension and the orange paste
slowly treated with water (50 mL). By aid of mild heating and
stirring, the paste dissolved to give an intense orange-coloured
solution. This solution was filtered through a pad of Celite and
the filtrate stirred in an ice bath. 5 M HCl was added dropwise with
stirring at 0 ꢀC to adjust the pH to 6.5. The copious amount of
mustard yellow solid so obtained was collected by filtration and
washed with water until the washing appeared colourless. The
solid was oven-dried under vacuum (40 ꢀC, ca. 30 mm) for 2 days to
give the crude triketone 5b as a yellow solid (1.68 g). Most of this
solid (1.56 g) was re-suspended in absolute EtOH (30 mL). Am-
monium acetate (2.81 g, 36.4 mmol) was added and the stirred
suspension was refluxed overnight to give a dark brown solution.
The solution was allowed to cool to room temperature and the
solvent removed in vacuo. The brown residue was dissolved in DCM
(30 mL) and washed with saturated NaHCO3(aq) (3ꢁ20 mL). The
organic layer was evaporated in vacuo and the residue triturated
with Et2O. The residual solid was collected by filtration and air-
dried, then purified by flash column chromatography on silica gel
using MeOH:DCM (gradient from 2:98 to 5:95) as the eluent to give
the title compound 3bþas a light orange solid (0.97 þg, 43%). Mp
164e167 ꢀC. HRMS (ESI ): Found m/z 278.1289 (MþH) , C17H16N3O
requires 278.1293. nmax (neat): 1630m, 1606m, 1580s, 1561s, 1501s,
1470m, 1435s, 1379s, 1353s, 1301m, 1271m, 1233m, 1178w, 1157m,
1115w, 1087w, 1036w, 1017w, 990m, 832s, 819s, 778s, 750m, 704w,
(C40), 165.82 (C4,400), 157.00 (C2,200), 156.25 (C20,60), 150.59 (C6,600),
110.34 (C5,500), 108.51 (C30,50), 106.43 (C3,300), 55.31 (OCH3).
6.2.3. Synthesis of 4,400-dichloro-2,20:60,200-terpyridin-40(10H)-one
(3d). Reaction of methyl 4-chloro-2-picolinate (1d) (10.0 g,
58.3 mmol) and acetone (1.72 mL, 23.3 mmol) in DME (48 mL) with
a stirred suspension of NaH (60% suspension in oil, 4.68 g,
117 mmol) in DME (47 mL) as described above in General Method A
gave a bright orange suspension, which was stirred at room tem-
perature for 3 h. The solvent was removed in vacuo and the red-
orange paste slowly treated with water (200 mL). The resultant
insoluble mixture was acidified as described previously to pH 6.5
and the mustard yellow solid so obtained collected by filtration,
washed with water until the washing appeared colourless and
oven-dried under vacuum (40 ꢀC, ca. 30 mm) for 2 days to yield the
crude triketone 5d as a yellow solid (8.83 g). Most of this solid
(6.40 g) was re-suspended in absolute EtOH (130 mL). Reaction
with ammonium acetate (10.2 g, 133 mmol) as described in General
Method A gave a dark brown solution. A cream coloured precipitate
formed after 1 h of reflux, and copious amounts were present after
overnight reflux. The mixture was allowed to cool to room tem-
perature and the solid was collected by filtration and washed with
Et2O to yield the title compound 3d as a pink fibrous crystalline solid
(3.0 g, 58%). Mp 222e224 ꢀC. HRMS (ESIþ): Found m/z 318.0203
(MþH)þ, C15H10Cl2N3O requires 318.0201. nmax (neat): 1609w,
1577m,1567m,1548s,1477m,1458m,1433m,1378s,1326m,1284m,
1253m, 1200s, 1129w, 1104w, 1065w, 1044w, 1004w, 989m, 962w,
900w, 878m, 854w, 829s, 756s, 719m, 688m, 661m cmꢂ1. 1H NMR
(400 MHz, DMSO-d6):
d
8.65 (2H, d, J 5.3 Hz, H6,600), 8.56 (2H, d, J
2.1 Hz, H3,300), 7.86 (2H, s, H30,50), 7.60 (2H, dd, J 5.3, 2.1 Hz, H5,500).
13C NMR (100 MHz, DMSO-d6):
d
166.10 (C40), 156.73 (C2,200), 155.30
(C20,60), 150.62 (C6,600), 144.11 (C4,400), 124.20 (C5,500), 120.60
(C3,300),109.15 (C30,50). The 1H and 13C NMR data were consistent
with literature data.18,28
669w cmꢂ1
.
1H NMR (400 MHz, CDCl3):
d
8.60 (2H, d, J 5.1 Hz,
H6,600), 7.69 (2H, s, H3,300), 7.21 (2H, d, J 5.1 Hz, H5,500), 7.02 (2H, s,
H30,50), 2.45 (6H, s, CH3). 13C NMR (100 MHz, CDCl3):
d
181.37 (C40),
6.2.4. Synthesis of 4,400-dibromo-2,20:60,200-terpyridin-40(10H)-one
(3e). A solution of ethyl 4-bromo-2-picolinate (1e) (92%, 20.6 g,
82.3 mmol) and acetone (2.43 mL, 32.9 mmol) in DME (100 mL) was
added dropwise to a stirred suspension of NaH (60% suspension in
oil, 6.6 g, 165 mmol) in DME (100 mL) under an atmosphere of
argon at room temperature. The temperature was slowly increased
until reflux was achieved, and a vigorous evolution of gas was ob-
served after 10 min and the brown suspension turned dark red. The
reaction mixture was allowed to cool to room temperature and was
stirred for 2 h. The solvent was removed in vacuo and the red-
orange paste slowly treated with water (200 mL). The resultant
insoluble mixture was acidified as described in previously to pH 6.5
and the copious amount of yellow solid so obtained was collected
by filtration and washed with water until the washing appeared
colourless. The solid was oven-dried under vacuum (40 ꢀC, ca.
30 mm) for 2 days to yield the crude triketone 5e as a yellow solid
(15.3 g). This solid (5.1 g) was re-suspended in absolute EtOH
(400 mL). Reaction with ammonium acetate (19.1 g, 248 mmol) as
described in General Method A gave a cream yellow solid, which
was collected by filtration and washed with Et2O. The filtrate from
the wash was concentrated to give a second crop of precipitate. The
solids were combined and dried under high vacuum to yield a light
grey crystalline solid (8.29 g), which was purified by flash column
149.09 (C6,600), 148.87 (C4,400), 148.68 (C2,200), 144.22 (C20,60), 125.94
(C5,500), 121.05 (C3,300), 113.50 (C30,50), 21.26 (CH3).
6.2.2. Synthesis of 4,400-dimethoxy-2,20:60,200-terpyridin-40(10H)-one
(3c). Reaction of methyl 4-methoxy-2-picolinate (1c) (5.0 g,
29.9 mmol) and acetone (0.89 mL, 12.0 mmol) in DME (25 mL) with
a stirred suspension of NaH (60% suspension in oil, 2.39 g,
59.8 mmol) in DME (25 mL) as described above in General Method
A gave a bright orange suspension after stirring overnight. The
solvent was removed in vacuo and the red-orange paste slowly
treated with water (150 mL). The resultant insoluble mixture was
treated with 5 M HCl dropwise with stirring at 0 ꢀC to adjust the pH
to 6.5. The copious amount of mustard green solid so obtained was
collected by filtration, washed with water and oven-dried under
vacuum (40 ꢀC, ca. 30 mm) for 2 days to give the crude triketone 5c
as a yellow-green solid (4.28 g). Most of this solid (4.14 g) was re-
suspended in absolute EtOH (100 mL). Reaction with ammonium
acetate (6.81 g, 88.3 mmol) as described in General Method A gave
a cream solid. The solid was collected by filtration and washed with
Et2O to yield the title compound 3c as yellow-brown crystalline
solid (2.07 g, 58%). Mp 253e255 ꢀC. HRMS (ESIþ): Found m/z
310.1186 (MþH)þ, C17H16N3O3 requires 310.1192. nmax (neat):
1633w, 1597s, 1581m, 1558s, 1504s, 1470m, 1450s, 1317m, 1286m,
chromatography on
a short column of alumina gel using
Please cite this article in press as: Lin, C.-P.; et al., Tetrahedron (2014), http://dx.doi.org/10.1016/j.tet.2014.09.074

8
C.-P. Lin et al. / Tetrahedron xxx (2014) 1e12
MeOH:DCM (gradient from 5:95 to 15:85) as the eluent to give the
title compound 3e as a white solid (6.50 g, 48%). Mp 204e207 ꢀC.
HRMS (ESIþ): Found m/z 405.9184 (MþH)þ, C15H10Br2N3O requires
405.9191. nmax (neat): 1604w, 1572m, 1543s, 1476w, 1455m, 1432m,
1378m, 1328m, 1282w, 1252w, 1221w, 1198s, 1110w, 1065w, 991m,
866m, 851m, 808s, 756s, 699w, 661w, 606w cmꢂ1
(400 MHz, CDCl3):
8.53 (2H, d, J 5.6 Hz, H6,600), 8.41 (2H, s, H30,50),
8.12 (2H, d, J 2.6 Hz, H3,300), 6.91 (2H, dd, J 5.6, 2.6 Hz, H5,500), 3.98
(6H, s, OCH3). 13C NMR (100 MHz, CDCl3): 166.72 (C4,400), 158.60
.
1H NMR
d
d
(C20,60), 158.35 (C40), 156.17 (C2,200), 150.73 (C6,600), 118.69 (CF3),
113.50 (C30,50), 110.44 (C5,500), 107.66 (C3,300), 55.30 (OCH3). (Note,
13C NMR was re-run with D1 set to 15 s for acquisition of the CF3
signal).
903w, 881m, 827s, 733s, 679w, 661w cmꢂ1
DMSO-d6):
7.86 (2H, s, H30,50), 7.77 (2H, dd, J 5.2, 1.9 Hz, H5,500). 13C NMR
.
1H NMR (400 MHz,
d
8.72 (2H, d, J 1.9 Hz, H3,300), 8.59 (2H, d, J 5.2 Hz, H6,600),
(100 MHz, DMSO-d6): d
166.06 (C40), 156.48 (C2,200), 155.28 (C20,60),
150.50 (C6,600), 133.30 (C4,400), 127.23 (C5,500), 123.60 (C3,300), 109.17
(C30,50).
6.3.3. Synthesis of 4,400-dichloro-40-trifluoromethanesulfonyloxy-
2,20:60,200-terpyridine (6d). Triflic anhydride (0.15 mL, 0.88 mmol)
was reacted with the 4,400-dichloroterpyridin-40-one 3d (0.253 g,
0.80 mmol) in pyridine (5 mL) as described in General Method B.
Precipitation of a yellow solid was observed. The mixture was
stirred as described previously and work-up gave a solid, which was
collected by filtration, washed with water and dried under high
vacuum to yield the title compound 6d as a pale brown solid
(0.314 g, 88%). Mp 160e163 ꢀC. HRMS (ESIþ): Found m/z 449.9687
(MþH)þ, C16H9Cl2F3N3O3S requires 449.9694. nmax (neat): 3090w,
1590w, 1568m, 1551s, 1464w, 1427s, 1361m, 1289w, 1222s, 1207s,
1133s, 1062m, 991m, 941m, 909m, 886m, 875m, 828m, 814s, 767w,
6.2.5. Attempted synthesis of 4,400-dinitro-2,20:60,200-terpyridin-
40(10H)-one (3f). Methyl 4-nitropicolinate (1f) was prepared in 65%
yield by esterification of 4-nitropicolinic acid using catalytic conc.
H2SO4 in dry MeOH at 25 ꢀC for 18 h. A solution of methyl 4-nitro-2-
picolinate (1f) (1.0 g, 6.62 mmol) and acetone (0.195 mL,
2.65 mmol) in DME (5 mL) was added dropwise to a stirred sus-
pension of NaH (60% suspension in oil, 0.5 g, 13.23 mmol) in DME
(5 mL) under an atmosphere of argon at room temperature. Stirring
at 30 ꢀC for 2 h or 18 h followed by refluxing the mixture for 2 h did
not result in the evolution of gas and work up resulted in recovery
of starting material.
746m, 715w, 686m, 661m cmꢂ1. 1H NMR (400 MHz, CDCl3):
d 8.61
(2H, d, J 5.2 Hz, H6,600), 8.57 (2H, d, J 2.0 Hz, H3,300), 8.43 (2H, s,
H30,50), 7.41 (2H, dd, J 5.2, 2.0 Hz, H5,500). 13C NMR (100 MHz,
6.3. General Method B for preparation of terpyridine 40-
triflates
CDCl3)): d
158.45 (C40), 157.76 (C20,60), 155.48 (C2,200), 150.29
(C6,600), 145.49 (C4,400), 125.00 (C5,500), 121.74 (C3,300),114.13 (C30,50).
The triflates were prepared by following a modified procedure
6.3.4. Synthesis of 4,400-dibromo-40-trifluoromethanesulfonyloxy-
2,20:60,200-terpyridine (6e). Triflic anhydride (0.57 mL, 3.40 mmol)
was reacted with the 4,400-dibromoterpyridin-40-one 3e (0.923 g,
2.27 mmol) in pyridine (10 mL) as described in General Method B.
Precipitation of a yellow solid was observed. The mixture was
stirred at 0 ꢀC for 30 min before being allowed to warm to room
temperature. The solids redissolved to give a dark brown solution,
which was stirred for a further 2 days. Work-up gave a solid, which
was collected by filtration, washed with water and dried under high
vacuum to yield the title compounþd 6e as a light brown solid (1.11 þg,
91%). Mp 177e180 ꢀC. HRMS (ESI ): Found m/z 537.8678 (MþH) ,
reported by Holbrey et al.27
40-(Trifluoromethanesulfonyloxy)-2,20:60,200-terpyridine
(6a)
was prepared in 88% yield. The 1H NMR spectral data were con-
sistent with literature data.27
6.3.1. Synthesis of 40-trifluoromethanesulfonyloxy-4,400-dimethyl-
2,20:60,200-terpyridine (6b). Triflic anhydride (0.51 mL, 3.02 mmol)
was added to a stirred solution of the 4,400-dimethylterpyridin-40-
one 3b (0.701 g, 2.74 mmol) in pyridine (10.0 mL) under an atmo-
sphere of argon at 0 ꢀC. The solution was stirred at 0 ꢀC for 30 min
before being allowed to warm to room temperature and stirred for
a further 2 days. The dark brown solution was poured into an ice-
water slurry (60 mL) and the mixture stirred for 30 min. A solid
precipitated from the solution and was collected by filtration,
washed with water and dried under high vacuum to yield the title
compound 6b as a light-orange solid (0.971 g, 87%). Mp 113e115 ꢀC.
HRMS (ESIþ): Found m/z 410.0788 (MþH)þ, C18H15F3N3O3S requires
410.0786. nmax (neat): 2928w, 1606m, 1590m, 1562s, 1476w, 1416s,
1368s, 1245m, 1204s, 1137s, 1121s, 1102m, 1039w, 994m, 947s,
C15H9Br2F3N3O3S requires 537.8683. nmax (neat): 3087w, 1589w,
1565m, 1551m, 1462w, 1426m, 1358m, 1288w, 1222m, 1207s, 1133s,
1062m, 990w, 939s, 877m, 827m, 811s, 764w, 728m, 677m, 660w
cmꢂ1. 1H NMR (400 MHz, CDCl3): 8.73 (2H, d, J 1.9 Hz, H3,300), 8.52
d
(2H, d, J 5.2 Hz, H6,600), 8.42 (2H, s, H30,50), 7.57 (2H, dd, J 5.2, 1.9 Hz,
H5,500). 13C NMR (100 MHz, CDCl3):
d
158.38 (C40), 157.68 (C20,60),
155.20 (C2,200), 150.11 (C6,600), 134.12 (C4,400), 128.03 (C5,500), 124.74
(C3,300), 114.15 (C30,50).
877m, 856s, 829s, 800m, 757w, 702w, 666m cmꢂ1
(400 MHz, CDCl3):
8.57 (2H, d, J 5.1 Hz, H6,600), 8.41 (2H, s, H3,300),
8.40 (2H, s, H30,50), 7.21 (2H, d, J 5.1 Hz, H5,500), 2.52 (6H, s, CH3). 13C
.
1H NMR
6.4. General Method C for preparation of 40-iodoterpyridines
d
The 40-iodoterpyridines were prepared by following a procedure
reported by Di Fabio et al.19
NMR (100 MHz, CDCl3):
d
159.06 (C40), 158.34 (C20,60), 154.17
(C2,200),149.23 (C6,600),148.22 (C4,400),125.63 (C5,500),122.13 (C3,300),
113.22 (C30,50), 21.31 (CH3).
The 40-iodo-2,20:60,200-terpyridine (8a) was isolated as a purple
solid (93%). Mp 145e147 ꢀC (lit.29 mp 147 ꢀC). The 1H and 13C NMR
spectral data were consistent with literature data.29
6.3.2. Synthesis of 40-trifluoromethanesulfonyloxy-4,400-dimethoxy-
2,20:60,200-terpyridine (6c). Triflic anhydride (0.15 mL, 0.90 mmol)
was reacted with the 4,400-dimethoxyterpyridin-40-one 3c (0.253 g,
0.82 mmol) in pyridine (5 mL) as described in General Method B.
Precipitation of a yellow-orange solid was observed. The mixture
was stirred as described previously and work-up gave a solid, which
was collected by filtration, washed with water and dried under high
vacuum to yield the title compound 6c as a pale pink solid (0.307 þg,
85%). Mp 164e167 ꢀC. HRMS (ESIþ): Found m/z 442.0683 (MþH) ,
6.4.1. Synthesis
of
40-iodo-4,400-dimethyl-2,20:60,200-terpyridine
(7b). Potassium iodide (0.75 g, 4.52 mmol) was added to a solution
of the 4,400-dimethylterpyridine 40-triflate 6b (0.924 g, 2.26 mmol)
in DMF (6.0 mL) in one portion under an atmosphere of argon. The
solution was refluxed for 3 h then allowed to cool to room tem-
perature. The dark brown solution was slowly added to a stirred
ice-water slurry (50 mL), which a dark purple solid quickly pre-
cipitated. The solid was collected by filtration and dried under high
vacuum to give the title compound 7b as a purple solid (0.779 g,
89%). Mp 195e196 ꢀC HRMS (ESIþ): Found m/z 388.0308 (MþH)þ,
C
18H15F3N3O5S requires 442.0685. nmax (neat): 2839w, 1599m,
1561s, 1478m, 1435s, 1404m, 1377m, 1324w, 1293m, 1244m, 1226s,
1208s, 1141s, 1055w, 1033s, 991m, 951s, 922m, 905m, 894w, 878w,
C17H15IN3 requires 388.0311. nmax (neat): 3048w, 1671w, 1601m,
Please cite this article in press as: Lin, C.-P.; et al., Tetrahedron (2014), http://dx.doi.org/10.1016/j.tet.2014.09.074

C.-P. Lin et al. / Tetrahedron xxx (2014) 1e12
9
1546s, 1467m, 1414m, 1357s, 1263m, 1109s, 1071w, 990m, 927w,
889m, 830s, 759w, 745m, 672m cmꢂ1. 1H NMR (400 MHz, CDCl3):
2.6 Hz, H300), 7.35 (1H, dd, J 5.2, 2.0 Hz, H5), 6.88 (1H, dd, J 5.6,
2.6 Hz, H500), 3.91 (3H, s, OCH3). 13C NMR (100 MHz, CDCl3):
d
8.84 (2H, s, H30,50), 8.56 (2H, d, J 5.0 Hz, H6,600), 8.38 (2H, s, H3,300),
d
166.94 (C400), 156.98, 156.74, 156.69 and 155.62 (C2, C20, C60, C200),
7.18 (2H, d, J 5.0 Hz, H5,500), 2.51 (6H, s, CH3). 13C NMR (100 MHz,
150.82 (C600), 150.29 (C6), 146.66 (C4), 145.45 (C40), 124.63 (C5),
122.22, 121.91 and 121.84 (C3, C30, C50), 110.44 (C500), 107.84 (C300),
55.59 (OCH3). The 1H NMR spectral data of were consistent with
literature data.16
CDCl3):
d
155.96 (C20,60), 154.70 (C2,200), 149.03 (C6,600), 148.10
(C4,400), 130.25 (C30,50), 125.15 (C5,500), 122.10 (C3,300), 159.06 (C40),
21.32 (CH3).
Reaction of the 4,400-dimethoxyterpyridine 40-triflate 6c, the
4,400-dichloroterpyridine 40-triflate 6d and the 4,400-dibromo-
terpyridine 40-triflate 6e with potassium iodide as described in
General Method C resulted only in recovered starting material (i.e.
the triflates).
8c: mp 165e167 ꢀC. HRMS (ESIþ): Found m/z 328.0849 (MþH)þ,
C
17H15ClN3O2 requires 328.0853. nmax (neat): 3089w, 2939w,
2840w, 1597s, 1552s, 1470s, 1403m, 1372s, 1315w, 1290s, 1264w,
1223s, 1173m, 1109w, 1091m, 1030s, 991m, 913w, 898w, 877s,
829w, 816m, 787m, 685w, 661 m cmꢂ1. 1H NMR (400 MHz, CDCl3):
d
8.49 (2H, d, J 5.6 Hz, H6,600), 8.43 (2H, s, H30,50), 8.07 (2H, d, J
6.5. General Method D for preparation of 40-
chloroterpyridines
2.6 Hz, H3,300), 6.85 (2H, dd, J 5.6, 2.6 Hz, H5,500), 3.94 (6H, s, OCH3).
13C NMR (100 MHz, CDCl3): 166.83 (C4,400), 156.90 (C2,200), 156.68
d
(C20,60), 150.71 (C6,600), 146.42 (C40), 121.66 (C30,50), 110.40 (C5,500),
107.63 (C3,300), 55.44 (OCH3).
The 40-chloroterpyridines were prepared by following the pro-
cedure reported by Constable and Ward.16
A similar reaction (also using 1.2 equiv of PCl5) after reflux for
4 h gave the title compounds 8d (13%), 9 (39%) and 8c (32%), re-
spectively after purification (total yield of the three analogues 84%)
while reaction at 70 ꢀC for 4 h gave 8d (6%), 9 (30%) and 8c (35%),
respectively after purification (total yield of the three analogues
71%). A reaction using 2.4 equiv of PCl5 after reflux for 15 h gave the
title compounds 8d (12%), 9 (27%) and 8c (21%), respectively after
purification (total yield of the three analogues 60%).
40-Chloro-2,20:60,200-terpyridine (5a) was prepared in 77% yield.
The 1H and 13C NMR spectral data were consistent with literature
data.30,31
6.5.1. Synthesis of 4,40,400-trichloro-2,20:60,200-terpyridine (8d), 4,40-
dichloro-400-methoxy-2,20:60,200-terpyridine (9) and 40-chloro-4,400-
dimethoxy-2,20:60,200-terpyridine (8c). Phosphorus oxychloride
(35 mL) was added to a mixture of the 4,400-dimethoxyterpyridin-
40-one 3c (1.746 g, 5.64 mmol) and phosphorus pentachloride
(1.41 g, 6.77 mmol, 1.2 equiv) to give a yellow suspension, which
was stirred and heated at reflux under an atmosphere of argon. The
solids gradually dissolved as temperature increased to finally give
a brown solution at refluxing temperature. After reflux for 2 h, the
mixture was allowed to cool slightly and transferred to a distillation
apparatus. Under a light vacuum, the excess phosphorus oxy-
chloride was removed by distillation at ca. 30 mm, aided with
careful heating. The beige solid residue so obtained was cooled in
an ice bath, water was cautiously added followed dropwise addi-
tion of a KOH solution (5 M) with stirring until the pH was ca. 9e10
to give a cream solid precipite. The solid was collected by filtration
and washed with water until the washings were neutral. The solid
was air-dried, pulverised, then dried under high vacuum to give
a solid, which was dissolved in DCM and passed through a short
pad of alumina to yield the crude product as a light brown solid
(1.10 g). Purification by radial chromatography (2 mm silica plate,
DCM followed by EtOH/DCM graded from 1% to 10% then EtOH)
gave the title compounds 8d (0.153 g, 8%), 9 (0.544 g, 29%) then 8c
(0.261 g, 14%) as a white fibrous crystalline solid, a white solid and
a pale-orange solid, respectively (total yield of the three analogues
51%).
6.5.2. Synthesis of 4,40,400-trichloro-2,20:60,200-terpyridine (8d). Phos-
phorus oxychloride (40 mL) was added to a mixture of 4,400-
dichloroterpyridin-40-one 3d (2.144 g, 6.74 mmol) and phosphorus
pentachloride (1.684 g, 8.09 mmol) to give a stirred light brown
suspension, which was stirred and heated at reflux under an at-
mosphere of argon. A fine yellow solid was observed after 90 min.
After reflux for 4 h, the mixture was allowed to cool slightly and the
excess phosphorus oxychloride was removed by distillation at ca.
30 mm, aided with careful heating as described in the General
Method D. The yellow solid residue so obtained was cooled in an ice
bath and water (20 mL) cautiously added to quench any remaining
phosphorus compounds, which produced a brown solution, which
was basified as described previously with an aqueous KOH solution
(10 M) to pH ca. 9e10. The white precipitate (1.10 g) was collected
by filtration and washed with water until the washings were
neutral. The solid was air-dried, pulverised and dissolved in
refluxing 1:1 THF:CHCl3 (150 mL) to give a brown solution. The
solution was allowed to cool to room temperature while stirring
overnight to precipitate the title compound 8d (1.201 g) as a white
solid that was collected by filtration and washed with minimal
amount of ice-cold CHCl3. The filtrate was concentrated in vacuo to
give a second crop of product (0.550 g), also collected by filtration
and washed with chloroform. (Combined weight 1.75 g, 77%). The
spectral data were consistent with those described above for 8d.
8d: mp 214e215 ꢀC (lit.32 mp 212e213 ꢀC). The 1H NMR spectral
data were consistent with literature data.16 HRMS (ESIþ): Found m/
z 335.9857 (MþH)þ, C15H9Cl3N3 requires 335.9862. nmax (neat):
3080w, 1925w, 1795w, 1666w, 1567s, 1546s, 1461m, 1412m, 1355m,
1256m, 1118m, 1095m, 1066m, 990m, 901m, 889m, 845s, 824s,
A
similar reaction of phosphorus pentachloride (0.153 g,
0.737 mmol) and 4,400-dibromoterpyridin-40-one 3e (0.25 g,
0.614 mmol) in phosphorus oxychloride (5 mL) after reflux for 4 h
gave, after workup, the 4,40,400-trichloro-2,20:60,200-terpyridine (8d)
in 96% yield. The 1H NMR and mass spectral data were consistent
with those described above for 8d and showed no evidence of Br
content.
753w, 736s, 662m cmꢂ1. 1H NMR (400 MHz, CDCl3):
d
8.59 (2H, d, J
5.2 Hz, H6,600), 8.57 (2H, d, J 2.0 Hz, H3,300), 8.50 (2H, s, H30,50), 7.38
(2H, dd, J 5.2, 2.0 Hz, H5,500). 13C NMR (100 MHz, CDCl3):
d156.51
(C2,200), 155.94 (C20,60), 150.36 (C6,600), 146.89 (C40), 145.61 (C4,400),
124.83 (C5,500), 122.37 (C30,50), 121.98 (C3,300).
9: mp 143e144 ꢀC. HRMS (ESIþ): Found m/z 332.0357 (MþH)þ,
6.6. General Method E for preparation of 40-
bromoterpyridines
C
16H12Cl2N3O requires 332.0357. nmax (neat): 3090w, 3052w,
2974w, 2941w, 2835w, 1600m, 1559s, 1546s, 1479m, 1464m,
1405m, 1361m, 1313w, 1289m, 1243w, 1198m, 1182w, 1122w,
1058w, 1036m, 989w, 903w, 869m, 835m, 816m, 782m, 723m,
The 40-bromoterpyridines were prepared following a literature
procedure.20
662m, 616w cmꢂ1 1H NMR (400 MHz, CDCl3):
. d 8.57 (1H, d, J
5.2 Hz, H6), 8.53 (1H, d, J 2.0 Hz, H3), 8.51 (1H, d, J 5.6 Hz, H600),
8.48 (1H, d, J 2.0 Hz) and 8.44 (1H, d, J 2.0 Hz, H30,50), 8.08 (1H, d, J
6.6.1. Synthesis of 4,40,400-tribromo-2,20:60,200-terpyridine (10e). Phos-
phorus pentabromide (0.528 g, 1.23 mmol) was added in one
Please cite this article in press as: Lin, C.-P.; et al., Tetrahedron (2014), http://dx.doi.org/10.1016/j.tet.2014.09.074

10
C.-P. Lin et al. / Tetrahedron xxx (2014) 1e12
portion into a stirred suspension of the 4,400-dibromoterpyridin-40-
one 3e (0.333 g, 0.82 mmol) in CHCl3 (10 mL) to give a bright or-
ange suspension. The mixture was refluxed for 30 min then
allowed to cool to room temperature. The solvent was removed in
vacuo and DMF (10 mL) added, resulting in the evolution of a white
fume and a red solution, which was then added slowly, with stir-
ring, into an aqueous mixture of 7:3 NaOH (2 M):Na2S2O3 (1 M)
(100 mL) cooled in an ice bath. An orange suspension resulted,
which was filtered through a glass sinter to remove red-coloured
particulates. The tea-green aqueous phase so obtained was
extracted with CHCl3 (3ꢁ50 mL). The organic phase dried with
Na2SO4, filtered and solvent removed in vacuo to give a dark
green residue. This residue was dissolved in a small amount DCM
and passed down a short column of alumina using 100 mL DCM to
yield the pure title compound 10e as a white fibrous crystalline
solid (0.12 g, 32%). Mp 216e217 ꢀC. HRMS (ESIþ): Found m/z
467.8332 (MþH)þ, C15H9Br3N3 requires 467.8347. nmax (neat):
3082m, 3047w, 1922w, 1796w, 1660w, 1564m, 1535s, 1457s, 1405m,
1349s, 1288w, 1252m, 1120m, 1085m, 1064m, 988m, 960w, 900m,
124.86 (C5,500), 122.13 (C3,300), 118.27 (C30,50), 40.87 (CH2N), 35.07
(SCH2), 21.30 (CH3).
6.7.2. Synthesis of 40-(2-aminoethanesulfanyl)-4,400-dimethoxy-
2,20:60,200-terpyridine
(2c). 2-Aminoethanethiol hydrochloride
(0.088 g, 0.773 mmol) was added to a stirred suspension of sodium
hydride (95%, 0.049 g, 1.93 mmol) in DMF (8.0 mL) at room tem-
perature under an atmosphere of argon. The resultant mixture was
stirred for 15 min to give a white-yellow suspension. 40-Chloro-
4,400-dimethoxyterpyridine 8c (0.211 g, 0.644 mmol) was then
added, the temperature increased to 80 ꢀC and the mixture stirred
overnight to give an orange suspension. Additional cysteamine
hydrochloride (0.053 g, 0.464 mmol) and sodium hydride (0.025 g,
0.965 mmol) was added after TLC analysis revealed incomplete
reaction. The mixture was heated at 80 ꢀC for a further 2 h, to give
a complete reaction. The mixture was allowed to cool to room
temperature, then slowly added into a stirred ice-water slurry
(20 mL) to form a white precipitate, which was collected by filtra-
tion and washed with water. A second crop of precipitate was ob-
served in the aqueous filtrate and was also collected. The white
solid was dried under high vacuum to give the title compound 2c
(0.193 g, 81%). Mp 125e127 ꢀC. HRMS (ESIþ): Found m/z 369.1374
891m, 873m, 823m, 752w, 736w, 701s, 661w cmꢂ1
(400 MHz, CDCl3):
8.72 (2H, d, J 1.9 Hz, H3,300), 8.66 (2H, s, H30,50),
8.52 (2H, d, J 5.2 Hz, H6,600), 7.55 (2H, dd, J 5.2, 1.9 Hz, H5,500). 13C
.
1H NMR
d
NMR (100 MHz, CDCl3):
d
155.93 (C2,200), 155.35 (C20,60), 149.95
(MþH)þ,
C19H21N4O2S
requires 369.1385.
nmax (neat):
(C6,600), 135.25 (C40), 134.03 (C4,400), 127.59 (C5,500), 125.18 (C30,50),
124.77 (C3,300).
3500e3100bw, 3347w, 3079w, 2993w, 2940w, 2834w, 1604m,
1576m, 1555s, 1484m, 1473s, 1406m, 1371m, 1319m, 1292m, 1266w,
1225s, 1180w, 1097w, 1037m, 988w, 913w, 862w, 828w, 814w,
784m, 664w cmꢂ1. 1H NMR (400 MHz, CDCl3):
d 8.51 (2H, d, J 5.6 Hz,
6.7. General Method F for preparation of (2-
aminoethanesulfanyl)terpyridines
H6,600), 8.34 (2H, s, H30,50), 8.13 (2H, d, J 2.6 Hz, H3,300), 6.87 (2H, d, J
5.6, 2.6 Hz, H5,500), 3.97 (6H, s, CH3), 3.28 (2H, t, J 6.4 Hz, SCH2), 3.08
(2H, t, J 6.4 Hz, CH2N), 1.68 (2H, br s, NH2). 13C NMR (100 MHz,
The (2-aminoethanesulfanyl) terpyridines were prepared by
following a modified procedure reported by Mountford et al.10
40-(2-Aminoethanesulfanyl)-2,20:60,200-terpyridine (2a) was
prepared from reaction of the 40-chloroterpyridine 8a (at 50 ꢀC for
3 h) or the 40-iodoterpyridine 7a (at 70 ꢀC for 2 h) with 2-
aminoethanethiol hydrochloride and sodium hydride in DMF. The
reactions gave the target compound 2a in 75% and 86% yields, re-
spectively. The 1H and 13C NMR spectral data of 2a were consistent
with literature data.10,33
CDCl3): d
166.87 (C4,400), 157.91 (C20,60), 155.04 (C2,200), 150.89 (C40),
150.60 (C6,600), 118.63 (C30,50), 110.30 (C5,500), 107.57 (C3,300), 55.47
(CH3), 41.14 (CH2N), 34.95 (SCH2).
6.7.3. Synthesis
of
40-(2-aminoethanesulfanyl)-4,400-dibromo-
(2e). 2-Aminoethanethiol hydrochloride
2,20:60,200-terpyridine
(0.138 g, 1.21 mmol) was added to a stirred suspension of sodium
hydride (95%, 0.064 g, 2.54 mmol) in DMF (10 mL) at room tem-
perature under an atmosphere of argon. The resultant mixture was
stirred for 15 min to give a white-yellow suspension. 4,40,400-Tri-
bromoterpyridine 10e (0.542 g, 1.15 mmol) was then added and
reaction mixture heated to 50 ꢀC for 2 h to give an orange sus-
pension, which was allowed to cool to room temperature. The
mixture was cautiously added into an ice-water slurry (50 mL) and
stirred for 30 min. The aqueous suspension was extracted with
CHCl3 (3ꢁ25 mL). The organic layer was dried with Na2SO4,
filtered and solvent removed in vacuo to give the crude product as
an orange oil (0.312 g). Purification by radial chromatography
(2 mm silica plate, DCM followed by EtOH/DCM graded from 10% to
50% then EtOH) gave recovered starting material 10e (0.029 g, 5%)
6.7.1. Synthesis
of
40-(2-aminoethanesulfanyl)-4,400-dimethyl-
2,20:60,200-terpyridine (2b). 2-Aminoethanethiol hydrochloride
(0.218 g, 1.92 mmol) was added to a stirred suspension of sodium
hydride (95%, 0.10 g, 3.97 mmol) in DMF (4.0 mL) at room tem-
perature under an atmosphere of argon. The resultant mixture was
stirred for 15 min to give a white-yellow suspension. 40-Iodo-4,400-
dimethyl-2,20:60,200-terpyridine (7b) (0.496 g, 1.28 mmol) was then
added and reaction mixture heated to 70 ꢀC for 3 h to give an or-
ange suspension. TLC analysis showed a slow consumption of
starting material, hence, an extra 1.0 and 1.5 equiv of cysteamine
hydrochloride and sodium hydride, respectively, was added to the
reaction mixture. This addition was repeated once more after 4.5 h.
TLC analysis after 5 h showed consumption of starting material, and
mixture was allowed to cool to room temperature. The mixture was
cautiously added into a stirred ice-water slurry (40 mL) to form
a light orange precipitate, which was collected by filtration. The
solid was dried under high vacuum to give the title compound 2b as
a white solid (0.529 g, 90%). Mp 109e110 ꢀC. HRMS (ESIþ): Found m/
z 337.1512 (MþH)þ, C19H21N4S requires 337.1487. nmax (neat):
2918m, 1657bw, 1605m, 1571s, 1547s, 1467s, 1414m, 1360s, 1317m,
1180m, 1114m, 1070m, 1035m, 989m, 909w, 888w, 868m, 826s,
and title compound 2e as
a yellow solid (0.074 g, 14%).
Mp131e133 ꢀC. HRMS (ESIþ): Found m/z 464.9376 (MþH)þ,
C17H15Br2N4S requires 464.9384. nmax (neat): 1563s, 1535s, 1458s,
1431m, 1412m, 1349s, 1287w, 1226w, 1100w, 1066m, 1026w, 987w,
900w, 860m, 822m, 767w, 727w, 709s, 671m, 660m, 618w cmꢂ1. 1H
NMR (400 MHz, CDCl3): d
8.72 (2H, d, J 1.9 Hz, H3,300), 8.48 (2H, d, J
5.2 Hz, H6,600), 8.34 (2H, s, H30,50), 7.51 (2H, dd, J 5.2, 1.9 Hz, H5,500),
3.27 (2H, t, J 6.4 Hz, SCH2), 3.10 (2H, t, J 6.4 Hz, CH2N) 1.63 (2H, br s,
NH2). 13C NMR (100 MHz, CDCl3):
d
156.81 (C2,200), 153.90 (C20,60),
151.26 (C40), 149.77 (C6,600), 133.96 (C4,400), 127.19 (C5,500), 124.71
(C3,300), 118.81 (C30,50), 40.73 (CH2N), 34.95 (SCH2).
785m, 689m, 668 m cmꢂ1. 1H NMR (400 MHz, CDCl3):
d 8.54 (2H, d,
J 4.7 Hz, H6,600), 8.39 (2H, s, H3,300), 8.33 (2H, s, H30,50), 7.15 (2H, d, J
6.7.4. Synthesis of 40-(2-aminoethanesulfanyl)-4-chloro-400-me-
thoxy-2,20:60,200-terpyridine (11) and 4-(2-aminoethanesulfanyl)-40-
chloro-400-methoxy-2,20:60,200-terpyridine (12). 2-Aminoethanethiol
hydrochloride (0.235 g, 2.07 mmol) was added to a suspension of
4.7 Hz, H5,500), 3.27 (2H, t, J 6.4 Hz, SCH2), 3.08 (2H, t, J 6.4 Hz,
CH2N), 2.49 (6H, s, CH3). 13C NMR (100 MHz, CDCl3):
d155.67
(C2,200), 155.33 (C20,60), 150.53 (C40), 148.92 (C6,600), 147.98 (C4,400),
Please cite this article in press as: Lin, C.-P.; et al., Tetrahedron (2014), http://dx.doi.org/10.1016/j.tet.2014.09.074

C.-P. Lin et al. / Tetrahedron xxx (2014) 1e12
11
NaH (95%, 0.099 g, 4.13 mmol) in DMF (24 mL) at room temperature
under argon. The mixture was stirred for 10 min before the slow
addition of HMPA (1 mL). 4,40-Dichloro-400-methoxyterpyridine 9
(0.572 g, 1.72 mmol) was then added and the mixture stirred at
90 ꢀC for 24 h. The reaction mixture was allowed to cool to room
temperature then cautiously added into an ice-water slurry
(100 mL) and the mixture stirred for 30 min. The aqueous sus-
pension was extracted with DCM (3ꢁ50 mL). The organic layer was
dried with Na2SO4, filtered and solvent removed in vacuo to obtain
the crude product as a yellow solid (0.558 g). Purification by radial
chromatography (2 mm silica plate, DCM followed by EtOH/DCM
graded from 2.5% to 50% then EtOH gave recovered starting mate-
rial 9 (0.150 g, 26%) followed by title compounds 12 (0.078 g, 12%)
and finally 11 (0.179 g, 28%).
s, H30,50), 7.35 (2H, dd, J 5.4, 1.9 Hz, H5,500), 3.27 (2H, t, J 6.4 Hz,
SCH2), 3.10 (2H, t, J 6.4 Hz, CH2N), 1.56 (2H, br s, NH2). 13C NMR
(150 MHz, CDCl3):
d
157.08 (C20,60), 153.98 (C2,200), 151.30 (C40),
149.93 (C6,600), 145.21 (C4,400), 124.18 (C5,500), 121.68 (C3,300) 118.81
(C30,50), 40.77 (CH2N), 35.02 (SCH2).
13: mp 58e60 ꢀC. HRMS (ESIþ): Found m/z 377.0400 (MþH)þ,
C
17H15Cl2N4S requires 377.0394. nmax (neat): 3356bw, 3082w,
3051w, 2918w, 2850w, 1667bw, 1565s, 1546s, 1461m,1408m, 1353s,
1288w, 1120w, 1064w, 988w, 870m, 822m, 754w, 733m, 662m,
618w cmꢂ1. 1H NMR (400 MHz, CDCl3): 8.57 (1H, d, J 5.2 Hz, H600),
d
8.53 (1H, d, J 2.0 Hz, H300), 8.46 (1H, d, J 5.3 Hz, H6), 8.45 (1H, d, J
2.0 Hz, H50), 8.44 (1H, d, J 2.0 Hz, H30), 8.43 (1H, d, J 2.0 Hz, H3), 7.35
(1H, dd, J 5.2, 2.0 Hz, H500), 7.19 (1H, dd, J 5.3, 2.0 Hz, H5), 3.21 (2H, t,
J 6.0 Hz, SCH2), 3.12 (2H, t, J 6.0 Hz, CH2N), 1.51 (2H, br s, NH2). 13
C
11: mp 104e105 ꢀC. HRMS (ESIþ): Found m/z 373.0894 (MþH)þ,
NMR (150 MHz, CDCl3): d
156.66 (C20,200), 155.61 (C60), 154.63 (C2),
C
18H18ClN4OS requires 373.0890. nmax (neat): 3352w, 3271w,
150.32 (C600), 150.16 (C4), 149.02 (C6), 146.73 (C40), 145.43 (C400),
124.64 (C500), 122.20 (C50), 121.90 and 121.88 (C30,300), 121.56 (C5),
118.71 (C3), 41.03 (CH2N), 35.25 (SCH2).
3069w, 3005w, 2920w, 1597w, 1562s, 1544s, 1480m, 1461m,
1407m, 1355m, 1313m, 1287m, 1245w, 1203m, 1177w, 1126w,
1093w, 1038m, 987w, 891w, 862m, 827m, 780m, 720m, 662m,
619w cmꢂ1. 1H NMR (400 MHz, CDCl3):
d
8.44 (1H, d, J 5.2 Hz, H6),
6.8. Ligand immobilisation
8.43 (1H, d, J 2.0 Hz, H3), 8.39 (1H, d, J 5.6 Hz, H600), 8.23 (1H, d, J
1.8 Hz, H30), 8.20 (1H, d, J 1.8 Hz, H50), 7.98 (1H, d, J 2.6 Hz, H300), 7.22
(1H, dd, J 5.2, 2.0 Hz, H5), 6.76 (1H, dd, J 5.6, 2.6 Hz, H500), 3.87 (3H, s,
OCH3), 3.17 (2H, t, J 6.4 Hz, SCH2), 2.99 (2H, t, J 6.4 Hz, CH2N), 2.11
Epichlorohydrin activated Sepharose 6 Fast FlowÒ was prepared
by the method described by Jiang et al.21 Sepharose 6 Fast FlowÒ
(500 g) (Amersham Pharmacia Biotechnology, Uppsala, Sweden;
now GE Healthcare) was filtered and washed extensively with
distilled water (5ꢁ bed volumes of wet gel, 2.5 L). The gel was
suction dried and transferred to a Schott bottle. A 2 M NaOH so-
lution (500 mL) containing NaBH4 (0.937 g) (1.875 mg/mL) was
added and the resulting suspension mixed vigorously at 28 ꢀC for
2 h at 175 rpm on an Axyos orbital shaker. Epichlorohydrin
(300 mL) was added and the gel slurry was gently agitated for
a further 21 h. The epoxy-activated resin was collected by vacuum
filtration and washed with distilled water (2.5 L). The activated
resin was stored in 20% (v/v) aqueous EtOH at 4 ꢀC until required for
ligand immobilisation.
The ligands were immobilised onto the activated Sepharose
based on the methods described by Jiang et al.21 A 0.2 M solution of
the ligand was prepared by dissolving the heterocyclic compound
in an appropriate solvent (e.g. 80% (v/v) aqueous MeOH or 75%
DMF/25% MeOH). The suction dried epichlorohydrin-activated
resin was added to the solution and the suspension mixed thor-
oughly on a rotating wheel at 28 ꢀC for 21 h at ambient tempera-
ture. The resulting adsorbent was collected by vacuum filtration,
washed with the appropriate solvent (ca. 10 bed volumes) followed
by distilled water (ca. 5 bed volumes). The adsorbent was stored in
20% (v/v) aqueous EtOH solution at 4 ꢀC.
(2H, br s, NH2). 13C NMR (100 MHz, CDCl3):
d
166.76 (C400), 157.34
and 157.32 (C20, C60), 155.03 (C200), 153.75 (C2), 151.00 (C40), 150.47
(C600), 149.98 (C6), 145.20 (C4), 124.15 (C5), 121.75 (C3), 118.74 (C30),
118.50 (C50), 110.10 (C500), 107.60 (C300), 55.42 (OCH3), 40.84 (CH2N),
34.82 (SCH2).
12:HRMS (ESIþ): Found m/z 373.0885 (MþH)þ, C18H18ClN4OS
requires 373.0890. nmax (neat): 3358bm, 2966m, 2935w, 1658w,
1597m, 1557s, 1482m, 1465m, 1433w, 1409m, 1361m, 1314w,
1294m, 1255w, 1203m, 1179w, 1160w, 1127w, 1035m, 989w, 951w,
865m, 821m, 782m, 724m, 663w, 617w cmꢂ1. 1H NMR (600 MHz,
CDCl3): d
8.49 (1H, dd, J 5.6, 0.3 Hz, H600), 8.45 (1H, dd, J 5.2, 0.6 Hz,
H6), 8.43 (1H, d, J 1.9 Hz, H50), 8.41 (1H, d, J 1.9 Hz, H30), 8.41 (1H, dd,
J 2.0, 0.6 Hz, H3), 8.05 (1H, d, J 2.6 Hz, H300), 7.16 (1H, dd, J 5.2, 2.0 Hz,
H5), 6.85 (1H, dd, J 5.6, 2.6 Hz, H500), 3.95 (3H, s, OCH3), 3.18 (2H, t, J
6.2 Hz, SCH2), 3.08 (2H, t, J 6.2 Hz, CH2N), 1.52 (2H, br s, NH2). 13
C
NMR (150 MHz, CDCl3):
d
166.85 (C400), 157.83 (C200), 156.73 (C60),
156.40 (C20), 154.86 (C2), 150.76 (C600), 149.90 (C4), 148.98 (C6),
146.51 (C40), 121.76 and 121.70 (C30, C50), 121.39 (C5), 118.71 (C3),
110.39 (C500),107.69 (C300), 55.49 (OCH3), 40.95 (CH2N), 35.11 (SCH2).
6.7.5. Synthesis
of
40-(2-aminoethanesulfanyl)-4,400-dichloro-
2,20:60,200-terpyridine (2d) and 4-(2-aminoethanesulfanyl)-40,400-di-
chloro-2,20:60,200-terpyridine (13). 2-Aminoethanethiol hydrochlo-
ride (0.126 g, 1.10 mmol) was added to a suspension of NaH (95%,
0.062 g, 2.59 mmol) in THF (5 mL). The mixture was stirred for
10 min before the slow addition of HMPA (1 mL). 4,40,400-Tri-
chloroterpyridine (8d) (0.249 g, 0.74 mmol) was then added and
the mixture stirred at 30 ꢀC for 40 h. The reaction mixture was
quenched with water (10 mL) and the THF removed under reduced
pressure. Ice (50 mL) was added to the residue and the mixture
allowed to stand until the ice melted. The resulting suspension was
filtered and the collected solid washed with water to give a beige
solid (0.249 g). The crude product was purified by radial chroma-
tography (2 mm silica plate, DCM followed by EtOH/DCM graded
from 2.5% to 50% then EtOH) to yield recovered starting material 8d
(0.013 g, 5%) followed by the titled compounds 13 (0.041 g, 15%) and
finally 2d (0.119 g, 43%).
The extent of ligand immobilisation onto the epichlorohydrin
activated Sepharose 6 Fast FlowÒ was determined by the nitrogen
elemental analysis. In brief, the adsorbent (approximately 1 mL wet
gel) was collected by filtration, washed with 25% (v/v) aqueous
acetone followed by 50% (v/v), then 75% (v/v) and finally 100% ac-
etone (ca. 2 bed volumes) and dried in vacuo to a constant weight.
The dried resin was analysed for total nitrogen content to give the
amount of immobilised ligand per gram of dry resin.
6.9. Purification of IgG1 by chromatography
Each resin was packed into a 1 mL Tricon 5/50 column (GE
Healthcare) and chromatography was run on an AKTA purifier (GE
Healthcare) at a flow rate of 1 mL/min. Buffer A consisted of 25 mM
Tris, 600 mM Na3citrate, pH 8.0 while Buffer B was 25 mM MES, pH
5.0. The crude IgG sample was diluted 5-fold with a higher strength
buffer to achieve a final condition the same as Buffer A. The sample
(10 mL) was loaded onto the column and the column washed with
15 mL Buffer A. Protein elution was achieved by a gradient from 0%
to 100% Buffer B in 10 mL, followed by washing with 10 mL Buffer B.
2d: mp 136e137 ꢀC. HRMS (ESIþ): Found m/z 377.0409 (MþH)þ,
C
17H15Cl2N4S requires 377.0394. nmax (neat): 3354w, 3052w, 2918w,
1565s, 1540s, 1471m, 1458m, 1412m, 1349s, 1117m, 1092w, 1065m,
988m, 899w, 887w, 867m, 829m, 754w, 734s, 662m, 619w cmꢂ1. 1H
NMR (400 MHz, CDCl3): d
8.57e8.56 (4H, m, H6,600, H3,300), 8.36 (2H,
Please cite this article in press as: Lin, C.-P.; et al., Tetrahedron (2014), http://dx.doi.org/10.1016/j.tet.2014.09.074

12
C.-P. Lin et al. / Tetrahedron xxx (2014) 1e12
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NuPAGEÒ loading buffer (Life Technologies) and electrophoresed
using the Hoefer miniVE vertical electrophoresis system (Amer-
sham Biosciences) in 4e12% Bis-Tris NuPAGEÒ gradient gels (Life
Technologies). Proteins were visualised by Coomassie blue staining.
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was 50 mM phosphate buffer, 500 mM NaCl, 300 mM imidazole, pH
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Cu(NO3)2, washed with water (5 mL) then equilibrated with Buffer
A. E. Coli cell lysate containing expressed tagged GFP (1 mL) was
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Acknowledgements
The support of the Australian Research Council, Linkage Grant
0561064, and NovoNordisk A/S, Bargsvaed, Denmark, for funding
this research is gratefully acknowledged. C-HP was a recipients of
a Faculty Of Science (Monash) Dean’s Postgraduate Research
Scholarship and
Scholarship.
a Centre for Green Chemistry Postgraduate
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M. T. W. Biotechnol. Prog. 2011, 27, 1048e1053.
Supplementary data
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Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.tet.2014.09.074.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
References and notes
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Please cite this article in press as: Lin, C.-P.; et al., Tetrahedron (2014), http://dx.doi.org/10.1016/j.tet.2014.09.074