Tetrabiphenylporphyrin-based receptors for protein surfaces show sub-nanomolar affinity and enhance unfolding

Article abstract of DOI:10.1016/S0960-894X(03)00551-1

A family of tetrabiphenylporphyrin-based receptors has been synthesized. Receptor 7 showed sub-nanomolar affinity (K_d=0.67 nM) in binding to the surface of cytochrome c. In addition, a stoichiometric amount of the receptor 7 caused a lowering in the T_m of cytochrome c from 85 to 35°C.

Full text of DOI:10.1016/S0960-894X(03)00551-1

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2651–2654
Tetrabiphenylporphyrin-Based Receptors for Protein Surfaces
Show Sub-Nanomolar Affinity and Enhance Unfolding
Toshihiro Aya and Andrew D. Hamilton*
Department of Chemistry, Yale University, New Haven, CT 06520-8107, USA
Received 25 March 2003; revised 13 May 2003; accepted 1 June 2003
Abstract—A family of tetrabiphenylporphyrin-based receptors has been synthesized. Receptor 7 showed sub-nanomolar affinity
=0.67 nM) in binding to the surface of cytochrome c. In addition, a stoichiometric amount of the receptor 7 caused a lowering
(
K
d
ꢀ
in the Tm of cytochrome c from 85 to 35 C.
#
2003 Elsevier Ltd. All rights reserved.
The design of synthetic agents that modulate protein
function remains an important area of study in modern
bioorganic chemistry. While the development of inhibi-
tors that bind small, well-defined cavities in enzymes is
electron transfer and apoptosis. The region on cyto-
chrome c that predominantly interacts with its natural
redox partner proteins contains a hydrophobic patch
near the edge of the heme surrounded by several invar-
1
7
common, synthetic receptors that bind to the exterior
2
iant Lys and Arg residues. The complementary
surface of proteins have been largely unexplored. We
domains on the partner proteins, such as cytochrome c
peroxidase or cytochrome c oxidase, have a hydro-
phobic area and invariant Asp and Glu residues. This
simple matching of hydrophobic and charged domains
encouraged us to design artificial receptors for cyto-
chrome c in which a hydrophobic core was surrounded
by negatively charged substituents (Fig. 1). Our early
results with receptor 1 suggested that improved recog-
nition might be achieved by increasing the hydrophobic
surface area and the negative charge density that sur-
rounds it. This can be accomplished by expanding the
hydrophobic core scaffold from tetraphenylporphyrin,
have previously reported that synthetic receptors con-
taining multiple peptide loops on the periphery of a
calix[4]arene scaffold can bind to the exterior of certain
3
proteins. More recently, we have shown that the tetra-
phenylporphyrin scaffold provides an excellent platform
for protein surface recognition and have described
receptor 1 which binds to cytochrome c with a K of 20
d
4
nM. The best of these early protein surface receptor
ꢁ
7
designs bind to their targets with affinities in the 10
ꢁ
–
M range. However, many protein–protein inter-
8
1
actions involve large interfacial surface area contacts
0
˚
>1000 A ) and very high affinities (K values of 10
2
ꢁ9
˚
(
1
–
M). In order to disrupt these strong protein–
with a diameter of 15.5 A, to tetrabiphenylporphyrin
d
ꢁ
12
5
˚
which measures 24.0 A across (Fig. 2).
0
protein complexes it is essential to develop synthetic
receptors that bind their target with sub-nanomolar affi-
nity. In this paper, we describe a new family of protein
binding agents that contain a large hydrophobic core sur-
rounded by a periphery of negatively charged substituents
and the best of which bind to the positively charged pro-
The meso-tetrakis-(4-carboxybiphenyl)porphyrin 2 and
meso-tetrakis-(3,5-dicarboxy-4,4 -biphenyl)porphyrin 5
0
were prepared as hydrochloride salts by a modification of
8
Adler’s method [acetic acid and Zn(OAc) ] from the cor-
2
responding aldehydes 10a and 10b, respectively, followed
by hydrolysis (LiOH). The aldehydes 10a and 10b were
synthesized by Suzuki coupling between the arylbromides
and arylboronic acids using Pd(PPh3)4 as catalyst
(Scheme 1). The protected forms of the receptors were
prepared from the corresponding acids 2 or 5 by initial
ꢁ
9
ꢁ10
tein target, cytochrome c, with 10 –10 M affinity.
Cytochrome c is a well characterized 12.5 kDa protein
6
with a pI of 10.4 that plays key roles in mitochondrial
conversion to acid chlorides [(COCl) /DMF] followed
2
by coupling reactions with hydrochloride salts of
appropriately protected aspartic acid derivatives in the
*Corresponding author. Tel.:+1-203-432-5570; fax:+1-203-432-3221;
e-mail: andrew.hamilton@yale.edu
0
960-894X/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00551-1

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T. Aya, A. D. Hamilton / Bioorg. Med. Chem. Lett. 13 (2003) 2651–2654
Figure 1. Schematic representation of cytochrome c binding by synthetic receptors.
Figure 2. Comparison of tetraphenylporphyrin and tetra-biphenyl-
phorphyrin scaffolds.
The binding affinities of receptors 1–7 for horse heart
cytochrome c were investigated by fluorescence quench-
ing measurements. To a solution of each receptor (10
nM), aliquots of cytochrome c solution were added and
the fluorescence intensity at 650 nm was recorded (exci-
tation at 420 nm) after each titration. An amphiphilic
nonionic detergent, Tween20, was added (0.05 v/v%) to
the aqueous 5 mM sodium phosphate buffer (pH 7.4) to
9
minimize aggregation effects from the receptor. In the
best cases, a dramatic quenching of the fluorescence
emission of the porphyrin was seen on addition of the
protein (Fig. 3). The plots of fluorescence emission
intensity vs. receptor concentration for 2–7 (as well as 1
in comparison) fitted well to a 1-to-1 binding isotherm
Scheme 1. Reagents and conditions: (a) Pd(PPh
(
HCl (11a: 11%, 11b: 16%); (e) LiOH, dioxane, MeOH; (f) HCl (2:
quant, 5: 86%).
3 4 2 3
) , K CO , DMF
2
10a: 93%, 10b: 62%); (b) AcOH, Zn(OAc) , Á, air; (c) DDQ; (d) 18%
(within 15% standard error except for the case of 7) to
give dissociation constants (Fig. 4, Table 1). The dis-
sociation constants indicated a clear relationship
between binding affinity and the number of anionic
groups on the periphery. Receptors 2 and 3, which bear
four carboxyl groups, were found to bind to cytochrome
presence of base (diisopropylethylamine). Final depro-
tection by trifluoroacetic acid gave trifluoroacete salts of
receptors 3, 4, 6 and 7 (Scheme 2). All the compounds
were identified by H NMR and mass spectroscopy, and
the purity was confirmed by HPLC.
1
c with a K of 12–17 mM. In contrast, receptors 4, 5 and
d

T. Aya, A. D. Hamilton / Bioorg. Med. Chem. Lett. 13 (2003) 2651–2654
2653
Figure 4. Fluorescence quenching profiles of 1 (*) and 7 (*) upon
addition of cytochrome c ([1]=[7]=10 nM). The titrations were carried
out in 5 mM sodium phosphate buffer, pH 7.4, 0.05% Tween 20, rt.
a
Table 1. Dissociation constants and charge of synthetic receptors 1–
7
Receptors
K
d
(nM)
Charge
b
1
2
3
4
5
6
7
220 (ꢂ17), 20 (ꢂ5)
ꢁ8
ꢁ4
ꢁ4
ꢁ8
ꢁ8
ꢁ8
ꢁ16
17,000 (ꢂ840)
12,000 (ꢂ950)
1300 (ꢂ130)
1500 (ꢂ170)
1700 (ꢂ97)
0.67 (ꢂ0.34)
Scheme 2. Reagents and conditions: (a) (COCl)
Asp(Ot-Bu)-OMe or H-Asp(Ot-Bu)-Ot-Bu, DIEA (12a: 54%, 12b:
3%, 13a: 68%, 13b: quant.); (c) TFA (3: 59%, 4: quant., 6: 49%, 7:
quant.).
2
, DMF; (b) H-
aDetermined in 5 mM sodium phosphate buffer, pH 7.4, 0.05% Tween
20, rt. Standard error is given in parentheses.
Determined in 5 mM sodium phosphate buffer, pH 7.4, rt see ref. 4.
6
b
6
, which bear eight carboxyl groups, showed stronger
knowledge, this is the strongest binding affinity of all
synthetic protein surface receptors reported.
affinity to cytochrome c (K =1.3–1.7 mM). The binding
d
of receptor 1 (K =220 nM), with essentially the same
d
composition of hydrophobic and anionic groups, to
cytochrome c was shown to be about 6-fold stronger,
indicating that flexible binding groups might better fit the
recognition features on the protein surface. Most inter-
estingly, the fluorescence quenching profile for receptor 7
carrying 16 carboxyl groups revealed significantly stron-
The selectivity of receptor 7 for cytochrome c relative to
other proteins was investigated by fluorescence quench-
ing at the same receptor concentration of 10 nM. Titra-
tions were carried out (Fig. 5) with closely related
cytochrome c
kDa, pI=4.7 ) and ferredoxin (from Clostridium pas-
1
(from Pseudomonas aeruginosa, 9.3
51
5
6
0
ger affinity to cytochrome c (K =0.67 nM). To our
d
teurianum, 6.2 kDa, pI=2.75 ) and the affinity of 7 was
found to be, respectively, 270- and 25,000-fold weaker
(
K =180 and 17,000 nM) than cytochrome c, suggest-
d
ing that charge and size matching are required for
strong binding.
To further investigate the influence of the receptors on
cytochrome c, circular dichroism (CD) spectroscopy
was performed to trace the thermal denaturation of the
protein in the presence and absence of the receptors
(5 mM sodium phosphate buffer, pH 7.4). The dena-
turation profile of horse heart cytochrome c at 222 nm
ꢀ
showed a clear melting temperature (T ) of 85 C (10
m
mM cytochrome c). Earlier work had shown that a slight
excess of the receptor 1 (12 mM) lowers the T of the
m
ꢀ
receptor 7 the T decreased to 35 C (Fig. 6). Titration of
receptor 7 into cytochrome c solution (2 mM) at 65 C
showed a saturation for the effect at one equiv, suggesting
11
protein to about 64 C. However, in the presence of
ꢀ
m
Figure 3. Fluorescence quenching of 7 upon addition of cytochrome c
([7]=10 nM, [cyt. c]=0–36 nM). The titrations were carried out in 5
mM sodium phosphate buffer, pH 7.4, 0.05% Tween 20, rt.
ꢀ

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T. Aya, A. D. Hamilton / Bioorg. Med. Chem. Lett. 13 (2003) 2651–2654
Figure 7. CD-monitored thermal denaturation profile (y at 222 nm) of
cytochrome c as a function of [7]/[cyt. c] at 65 C. Titrations were
carried out in 5 mM sodium phosphate buffer, pH 7.4.
Figure 5. Fluorescence quenching profiles of 7 ([7]=10 nM) upon
addition of cytochrome c (*), cytochrome c551 (*) and ferredoxin
(~). The titrations were carried out in 5 mM sodium phosphate buf-
fer, pH 7.4, 0.05% Tween 20, rt.
ꢀ
Acknowledgements
This work was supported by the National Institutes of
Health (GM35208).
References and Notes
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*: cyt. c + 7 (10+12 mM); ~: cyt. c + 7 + NaCl (10+12 mM + 50
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4
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higher T , indicating that electrostatic interactions
m
between the receptor and cytochrome c have a significant
1
2
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In conclusion, we have designed and synthesized a
family of protein surface receptors based on a tetra-
biphenylporphyrin scaffold. Receptor 7 showed a
remarkably strong affinity to cytochrome c. Stoichio-
metric amounts of receptor 7 lower the melting tem-
9
. A plot of [7] from 5 to 90 nM against fluorescence emission
intensity (at 650 nm, excited at 420 nm) was linear, suggesting
no aggregation effects at these concentrations.
ꢀ
perature of cytochrome c to 35 C. These results may be
1
5
1
4
0. Dutton, J. E.; Rogers, L. J. Biochim. Biophys. Acta 1978,
37, 501.
1. Jain, R. K.; Hamilton, A. D. Angew. Chem. Int. Ed. 2002,
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extended to the development of other subnanomolar
protein surface receptors. The findings may also provide
an insight to the design of protein denaturants that
works at stoichiometric concentration and physiological
temperature.
12. Wilson, A. J.; Groves, K.; Jain, R. K.; Park, H. S.;
Hamilton, A. D. J. Am. Chem. Soc. 2003, 125, 4420.