Design and development of the first peptide-chelating bisphosphane bioconjugate from a novel functionalized phosphorus(III) hydride synthon

Article abstract of DOI:10.1002/(sici)1521-3773(19990712)38:13/14<2020::aid-anie2020>3.0.co;2-r

Remarkable oxidative stability is shown by the carboxylate- functionalized primary bisphosphane 1. Compound 1 can be used in conjugation reactions with biomolecules for functionalization through the COOH group without prior protection of PH₂ groups. A novel water-soluble bisphosphane was obtained by reaction of the PH₂ groups of 1 with formaldehyde under mild conditions.

Full text of DOI:10.1002/(sici)1521-3773(19990712)38:13/14<2020::aid-anie2020>3.0.co;2-r

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In the case of the FP-LMTO calculations, we made use of valence band s, p,
and d basis functions for both Al and In. For In pseudocore 4d states were
included in the basis as well; that is, the resulting basis always formed a
single, fully hybridizing basis set. The exchange ± correlation potential was
parametrized according to Hedin and Lundqvist.[5d] For sampling the
irreducible wedge of the BZ we used the special k points with a Gaussian
smearing of width 20 mRy.
Design and Development of the First Peptide-
Chelating Bisphosphane Bioconjugate from a
Novel Functionalized Phosphorus(iii) Hydride
Synthon**
Hariprasad Gali, Srinivasa R. Karra,
To ensure convergency several standard tests were performed for both
methods, such as increasing the number of k points used in the summation
over the BZ. The calculated energy differences (Ec/a Efcc) and band
structures were found to be virtually identical for the two full-potential
techniques. The presented energy differences and density of states were
obtained from FP-LMTO calculations, the presented band structures from
FLAPW calculations.
V. Sreenivasa Reddy, and Kattesh V. Katti*
Dedicated to Professor S. S. Krishnamurthy
on the occasion of his 60th birthday
The incorporation of metal-chelating units into peptide
backbones (and related biomolecules) has become an area of
intense interest because of the potential applications to
catalysis[1] and biomedicine.[2] Among various ligands avail-
able, phosphanes are unique because they display versatile
coordination chemistry with transition metals.[3] The secon-
dary and tertiary structures of peptides to which they are
attached may subsequently help in controlling the reactivity
of phosphane-coordinated transition metals. Specifically, the
chirality and related important stereospecific characteristics
associated with biomolecules (e.g. peptides or proteins) may
be transferred to the transition metals if peptides are
immobilized with chelating units that are capable of coordi-
nating with transition metals.[4] This approach of conjugating
catalytically active transition metals to chiral biomolecules
provides a straightforward route to chiral compounds with
potential applications in enantioselective catalysis.[4] The
incorporation of phosphanes into peptides (and proteins) will
also help to engineer metal binding sites, which may
eventually provide conformational integrity, biospecificity,
and enhanced enzymatic activities.[5]
Received: January 18, 1999 [Z12929IE]
German version: Angew. Chem. 1999, 111, 2155 ± 2159
Keywords: ab initio calculations ´ aluminum ´ bond theory ´
elemental structures ´ indium
[1] J. Donohue, The Structure of the Elements, Wiley, New York, 1974.
[2] V. Heine, D. Weaire, Solid State Phys. 1970, 24, 249.
[3] J. Hafner, V. Heine, J. Phys. F 1983, 13, 2479.
[4] P. Söderlind, O. Eriksson, B. Johansson, J. M. Wills, A. M. Boring,
Nature (London) 1995, 374, 524; R. Ahuja, O. Eriksson, J. M. Wills, B.
Johansson, Phys. Rev. Lett. 1995, 75, 280; S. I. Simak, U. Häussermann,
I. A. Abrikosov, O. Eriksson, J. M. Wills, S. Lidin, B. Johansson, Phys.
Rev. Lett. 1997, 79, 1333.
[5] a) Program pacakge WIEN97: P. Blaha, K. Schwarz, J. Luitz, Program
WIEN97: A Full Potential Linearized Augmented Plane Wave Package
for Calculating Crystal Properties (K. Schwarz, Vienna University of
Technology, Austria), 1999 (ISBN 3-9501031-0-4); b) J. M. Wills,
unpublished results; J. M. Wills, B. R. Cooper, Phys. Rev. B 1987, 36,
3809; c) J. P. Perdew, Y. Wang, Phys. Rev. B 1992, 45, 13244; d) L.
Hedin, B. I. Lundqvist, J. Phys. C 1971, 4, 2064.
Bioconjugation of cytotoxic transition metals to receptor-
active peptides may eventually provide effective vehicles for
delivering cytotoxic moieties to specific tumors through
receptor-mediated agonist or antagonist interactions.[6] In this
context, peptides (or receptor-binding biomolecules) contain-
ing phosphane substituents are important in the design and
development of tumor-specific radiopharmaceuticals.[7, 8] De-
spite significant catalytic and biomedical applications offered
by such peptides (and proteins), synthetic strategies for
producing such bioconjugates are still in their infancy. The
elegant work by Gilbertson and co-workers on the incorpo-
ration of aryl- and cyclohexylphosphanes into specific pep-
tides has provided impetus to this potentially burgeoning field
of chemical and biomedical sciences.[9]
[6] D. G. Pettifor, Bonding and Structure of Molecules and Solids,
Clarendon Press, Oxford, 1995.
[7] U. Häussermann, S. I. Simak, I. A. Abrikosov, S. Lidin, Chem. Eur. J.
1997, 3, 904.
[8] For the calculation of the band structures we used a tetragonal body
p
centered (bct) unit cell with the appropriate c/a ratio of 2. Thus, the
two different nearest neighbor distances d1 and d2 (Figure 1) are both
equal to the lattice constant a, and in reciprocal space the two
directions G-Z and G-X become degenerate (Figure 5).
[9] J. K. Burdett, Chemical Bonding in Solids, Oxford University Press,
Oxford, 1995.
[10] C. E. Moore, Atomic Energy Levels, National Bureau of Standards,
Washington, DC, 1949. ep was taken as the negative experimental
ionization energy from the corresponding p orbital; es was calculated
as ep minus the lowest s !p excitation energy yielding the same spin
multiplicity.
[11] J. P. Desclaux, At. Data Nucl. Data Tables 1973, 12, 311.
[12] R. Ahuja, S. I. Simak, unpublished results.
As part of our ongoing research on the development of
functionalized phosphanes for biomedical and catalytic ap-
[*] Prof. Dr. K. V. Katti, H. Gali, Dr. S. R. Karra, Dr. V. S. Reddy
Department of Radiology
Center for Radiological Research
Allton Building Laboratories, Room 106
University of Missouri-Columbia
Columbia, MO 65211 (USA)
Fax : (‡ 1)573-884-5679
E-mail: kattik@missouri.edu
[**] We thank Professor Wynn A. Volkert for valuable scientific input and
support. This work was supported by DuPont Pharmaceuticals, USA,
and the US Department of Energy.
Supporting information for this article is available on the WWW
under http://www.wiley-vch.de/home/angewandte/ or from the author.
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plications,[10] we have recently developed a novel strategy
which allows bisphosphanes with heteroatoms and chelating
functionalities to be directly incorporated into peptide back-
bones, under mild conditions, to produce the first example of a
biomolecule functionalized with hydrophilic phosphane che-
lating units. We discuss here the synthetic utility of 3-bromo-
propylphosphane (1) in the development of a functionalized,
water-soluble phosphane based on the dithio-bisphosphane
(P2S2) framework of 6,8-bis[(3-phosphanylpropyl)thio]octa-
noic acid (2). We also demonstrate that the COOH group in 2
can be efficiently conjugated to peptides while retaining the
phosphane moieties.
groups in these compounds toward molecules containing a
wide spectrum of functional groups (including carboxylic
acids, amines, thiols, and proteins) was remarkably low or
nonmeasurable. To our knowledge, this is the first report of
compounds containing phosphanyl groups which exhibit such
a degree of oxidative stability and lack of reactivity toward
solvents and chemical functionalities. Preliminary AM1
calculations carried out with the MOPAC program[12a] on 2
and related molecules suggest there are atomic orbital
contributions from the heteroatom (e.g. S in 2) to the frontier
molecular orbitals. It is conceivable, therefore, that there is
negative hyperconjugation involving specific orbitals of S and
the PIII centers in 2. This electronic effect may explain the
unusual stability towards oxidation of 2 and related molecules
with heteroatoms on their backbone.[12b] More detailed
calculations are underway. This electronic effect appears to
operate even in the hydroxymethylated phosphanes 3. For
example, 3 is stable towards oxidation in aqueous media over
several days (Figure 1). Such stability of alkyl-substituted
phosphanes is remarkable.
Our approach to the synthesis of ligand framework 2 is
shown in Scheme 1. It involves the use of 1 as a key synthon
which was recently obtained in our laboratory by the
COOH
COOH
a
HS
SH
98%
S
S
D,L-thioctic acid
b
c
OEt
Br
P
Br
PH2
d
Br
Br
OEt
O
77%
1
85%
O
COOH
COOH
OH
S
S
S
S
S
P
S
P
base
e
+
+
acid
P
PH2 H2P
80%
P
OH
OH
HO
HO
HO
HO
OH
OH
>95%
3
2
HO
OH
Scheme 1. a) NaBH4 (3.0 equiv), H2O, EtOH, 258C, 5 h; b) P(OEt)3
~
(0.2 equiv), , 1 h; c) LiAlH4 (1.5 equiv), AlCl3 (4.6 equiv), tetraglyme,
Figure 1. 31P NMR spectra of 3 (aqueous sodium bicarbonate solution,
pH 8.5) at various timepoints. Peaks at d ˆ 23.5 and 55.4 refer to 3 and the
corresponding phosphane oxide, respectively.
~
0 !258C, 12 h; d) NaH (4.1 equiv), THF, , 12 h; e) HCHO (4.0 equiv),
EtOH, 258C, 15 min.
reduction of diethyl 3-bromopropylphosphonate with dichlor-
oaluminum hydride.[11] Reaction of 1 with the dianion of 6,8-
dithiooctanoic acid produced the COOH-functionalized P2S2
framework 2. The proton-coupled 31P NMR of 2 showed two
merged triplets (1JP,H ˆ 193.2 Hz), which confirmed the pres-
ence of two different phosphanyl groups. The thermally stable
PIII hydride 2 was then formylated with 37% formaldehyde in
ethanol to produce the corresponding water-soluble phos-
phane framework 3 in greater than 95% yields (Scheme 1).
To determine the feasibility of linking compounds contain-
ing PH2 groups to biomolecules, a P2S2-d-Lys conjugate was
prepared and then used to synthesize a P2S2 conjugate of a
luteinizing hormone releasing hormone peptide, the d-
Lys6 LHRH conjugate 4, by automated solid-phase peptide
synthesis (SPPS; Scheme 2). This method involved repeated
use of a variety of chemicals in high concentrations (including
trifluoroacetate (TFA) for cleavage of the peptide from the
resin. Peptide 4 was purified by HPLC and analyzed by 31P
NMR spectroscopy and mass spectrometry (Figure 2). These
data demonstrate that the peptide conjugate 4 was formed in
high yields with no modification of the PH2 groups. These
results also confirm that the PH2 groups of 2 are resistant to
oxidation and are unreactive towards other functional groups
in the LHRH peptide and the reagents used in SPPS. The
synthesis of such biomolecules which contain PH2 groups
allows their conversion into hydrophilic alkylphosphanes.
Thus, formaldehyde reacted rapidly with the PH2 groups of 4
1
Both 2 and 3 were characterized by H, 13C, and 31P NMR
spectroscopy, and HR-FAB mass spectrometry.
The co-existence of phosphanyl and carboxyl groups within
the same molecule is difficult to achieve because the reaction
conditions that are used to reduce a P(O)(OEt)2 group to a PH2
group can also reduce COOH groups. Thus, 1 can be used as a
versatile synthon to produce compounds in which a chemical
functionality that is susceptible to reduction (e.g. a carboxyl or
amide group) and a highly reduced phosphanyl group coexist.
In general, primary phosphanes tend to be unstable towards
oxidation. For example, PH3 and RPH2 (R ˆ Me, Et) are
oxidized instantaneously upon contact with atmospheric
oxygen and water or during reactions in various organic
solvents. In contrast, the new primary phosphane 2 exhibited a
much improved stability profile under an even wider range of
chemical conditions. Furthermore, the reactivity of the PH2
to produce the peptide-functionalized phosphane
5
(Scheme 2) now containing additional P C bonds. Either
PH2 groups or their disubstituted analogues PR2 may be used
as a part of the chelator framework of biomolecules to form
well-defined metalated conjugates by complexation with
transition metals.
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transition metal compounds for catalytic applications. More-
over, the metalation of functionalized peptides with cytotoxic
metals, for example PtII, will also enable the design and
development of site-specific drugs for delivery of cytotoxic
agents to specific cancer sites through receptor-mediated
agonist ± antagonist interactions. Therefore, the new biocon-
jugate chemistry reported herein has implications in both
catalysis and biomedicine fields.
PH2 H2P
S
S
O
NH
NH
H2N
O
NH
HN
N
OH
O
O
O
O
H
N
H
H
N
H
N
H
N
N
N
O
N
N
N
N
H
NH2
H
H
H
O
O
O
O
O
Experimental Section
NH
Diethyl 3-bromopropylphosphonate was synthesized by the Arbuzov
OH
reaction.[13] Typically, triethyl phosphite in
a tenfold excess of 1,3-
4
dibromopropane was heated at reflux for 1 h to yield diethyl 3-bromopro-
pylphosphonate in 95% yield after distillation under reduced pressure
(858C, 2 mmHg). d,l-6,8-dithiooctanoic acid was synthesized according to
the literature procedure.[14]
a, b
HO
HO
OHOH
1: To
a well-stirred cold (08C) suspension of LiAlH4 powder (5 g,
135 mmol) in tetraglyme (100 mL) was added anhydrous AlCl3 powder
(55 g, 412 mmol) over 15 min. After the reaction mixture was stirred for
30 min at 08C, diethyl 3-bromopropylphosphonate (23 g, 89 mmol) was
added dropwise over 20 min. Stirring was continued at 258C for 12 h, and
then the reaction mixture was distilled under reduced pressure (508C,
2.5 mm of Hg) to afford 1 (12 g, 77%), which was collected in a cooled
receiver at 778C. 1H NMR (300 MHz, CDCl3, 258C): d ˆ 1.99 (brm, 2H),
2.32 (brm, 2H), 2.98 (brm, 2H), 3.32 (m, 2H); 13C NMR (75 MHz, CDCl3):
d ˆ 12.59 (d, JP,C ˆ 9.2 Hz), 33.72 (s), 35.81 (s); 31P NMR (121 MHz,
CDCl3): d ˆ 138.37 (s).
P
S
P
S
O
NH
NH
H2N
O
NH
HN
N
OH
O
O
O
O
2: d,l-6,8-dithiooctanoic acid (7.8 g, 37.8 mmol) in dry tetrahydrofuran
(25 mL) was added dropwise to a well-stirred suspension of 60% NaH
(6.2 g, 155 mmol) in dry tetrahydrofuran (200 mL) at 08C. After the
reaction mixture was stirred for 20 min at 08C, a solution of 1 (11.8 g,
76.1 mmol) in dry tetrahydrofuran (25 mL) was added. The reaction
mixture was then heated at reflux under nitrogen for 12 h. Excess NaH was
quenched with a minimum of 10% HCl, the mixture was filtered through
silica gel, and the solvent was evaporated under reduced pressure. The
crude product was purified by column chromatography on silica gel with
hexane/ethyl acetate (3:2) to give pure 2 (10.8 g, 80%) as a colorless
viscous oil. 1H NMR (300 MHz, CDCl3): d ˆ 1.44 ± 1.61 (m, 6H), 1.69 ± 1.78
(m, 6H), 2.29 ± 2.34 (m, 2H), 2.46 ± 2.66 (m, 12H), 2.93 ± 2.99 (m, 4H), 11.45
(brs, 1H); 13C NMR (75 MHz, CDCl3): d ˆ 13.46 (t, JP,C ˆ 13.8 Hz), 24.9 (s),
26.62 (s), 29.72 (s), 31.23 (d, 2JP,C ˆ 8.8 Hz), 33.05 (m), 33.38 (d, 3JP,C ˆ 3 Hz),
34.34 (s), 35.02 (s), 35.08 (s), 44.1 (s), 180.34 (s); 31P NMR (121 MHz,
CDCl3): d ˆ 136.38 (s), 136.34 (s); 31P NMR (121 MHz, proton coupled,
CDCl3): d ˆ 136.29 (t, JP,H ˆ 193.2 Hz), 136.24 (t, JP,H ˆ 193.2 Hz); IR
H
N
H
H
N
H
N
H
N
N
N
O
N
N
N
N
H
NH2
H
H
H
O
O
O
O
O
NH
OH
5
Scheme 2. a) 37% aq. HCHO, 0.1n HCl, 258C, 5 min; b) 1m aq. NaHCO3,
258C, 5 min.
(NaCl): nÄ ˆ 2294 (P H), 1723 (C O) cm 1; HR-MS (FAB): m/z calcd for
ˆ
‡
[M ‡H]: 357.1240; found: 357.1237.
3: To a solution of 2 (1 g, 2.8 mmol) in degassed ethanol (5 mL) at room
temperature (258C) was added 37% aqueous formaldehyde solution
(0.92 mL, 11.3 mmol), and the reaction mixture was stirred for 1 h under
nitrogen. Removal of the solvent under reduced pressure afforded 3 (1.3 g,
97%) as a colorless viscous oil. 31P NMR (121 MHz, D2O): d ˆ 25.16 (s),
25.03 (s). For characterization and all other studies 3 was converted into
its bisphosphonium chloride salt by the addition of 5n hydrochloric acid
(0.5 mL) and 37% aqueous formaldehyde (0.5 mL, 5.6 mmol) to a solution
of 3 (1.3 g, 2.7 mmol) in degassed ethanol (5 mL) at room temperature
(258C). After removal of solvent under reduced pressure the crude product
was purified by chromatography on a C-18 Sep-Pak reverse phase column
with water/methanol (3:2) to afford a pure bisphosphonium chloride (1.6 g,
94%) as a colorless viscous oil. 1H NMR (300 MHz, D2O): d ˆ 1.18 ± 1.23
(m, 2H) 1.31 ± 1.36 (m, 4H), 1.50 ± 1.63 (m, 2H), 1.65 ± 1.80 (m, 4H), 2.10 ±
2.31 (m, 6H), 2.42 ± 2.46 (m, 6H), 2.55 ± 2.60 (m, 1H), 4.32 (brs, 12H);
Figure 2. Proton-coupled 31P NMR spectrum of peptide ± phosphane
conjugate 4.
The chemistry described herein demonstrates for the first
time that primary phosphanes containing thioether groups
possess high oxidative and thermal stabilities. Furthermore,
the COOH group of such primary phosphanes provides an
elegant opportunity for their incorporation into peptide
backbones. Metalated analogues of 5 and related biomole-
cules may open up new avenues in the design of chiral
13C NMR (75 MHz, D2O): d ˆ 12.0 (d, JP,C ˆ 10.56 Hz), 12.54 (d, JP,C
ˆ
ˆ
ˆ
1
10.5 Hz), 20.52 (s), 20.98 (s), 23.62 (s), 25.2 (s), 27.91 (s), 29.76 (d, JP,C
3
1
15.7 Hz), 31.32 (d, JP,C ˆ 15.8 Hz), 33.36 (s), 43.66 (s), 49.64 (d, JP,C
54.4 Hz), 178.27 (s); 31P NMR (121 MHz, D2O): d ˆ 28.98 (s), 28.99(s);
‡
HR-MS (FAB): m/z calcd for [M
Cl]: 573.1641; found: 573.1649.
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4: Conjugate 4 was synthesized on a solid-phase peptide synthesizer.
Standard fmoc-protected amino acids (fmoc ˆ 9-fluorenylmethoxycarbon-
yl) were used for the synthesis. Conjugate 4 was purified by HPLC and
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analyzed with 31P NMR spectroscopy and mass spectrometry (Figure 1). 31
P
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coupled, D2O) d ˆ 137.90 (t, JP,H ˆ 190.2 Hz). LR-MS (FAB) m/z calcd for
‡
[M ‡H]: 1591.9; found: 1592.0.
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5: To a solution of 4 (0.5 mg) in ethanol (400 mL) and DMF (100 mL) were
added 0.1n HCl (25 mL) and 37% aqueous formaldehyde (25 mL), and the
reaction mixture was stirred at room temperature (258C) for 5 min. The
formation of P2S2-d-Lys6-LHRH hydroxymethylphosphonium chloride was
confirmed by the 31P NMR signal at d ˆ 31.39(s). The P2S2-d-Lys6-LHRH
hydroxymethylphosphonium chloride was converted into 5 by the addition
of 1m aqueous sodium bicarbonate (30 mL) in near quantitative yields as
demonstrated by the 31P NMR chemical shift at d ˆ 24.23.
Received: December 9, 1998
Revised version: February 9, 1999 [Z12764IE]
German version: Angew. Chem. 1999, 111, 2152 ± 2155
Keywords: bioorganic chemistry
´ peptide conjugates ´
phosphanes
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Modeling a Nitrogenase Key Reaction:
‡
The N2-Dependent HD Formation by D2/H
Exchange**
Dieter Sellmann* and Anja Fürsattel
Dedicated to Professor Helmut Werner
on the occasion of his 65th birthday
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Biological N2 fixation is one of the fundamental natural
synthetic processes and is catalyzed by FeMo, FeV, or FeFe
nitrogenases.[1] X-ray structure analyses have revealed the
molecular structure of FeMo nitrogenase and its active
centers, in particular the structure of the FeMo cofactors
(FeMoco).[2] However, the intimate molecular mechanism of
biological N2 reduction and the concomitant ªobligatory
dihydrogen evolutionº (OHE) has remained a mystery. The
OHE is an integral part of enzymatic N2 reduction and cannot
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[6] a) T. Janaky, A. Juhasz, Z. Rekasi, P. Serfözö, J. Pinski, L. Bokser, G.
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D. L. Nosco, K. F. Deutsch, E. Deutsch, J. Nucl. Med. 1992, 33, 850;
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[8] For recent reviews and papers in radiolabeled biomolecules see:
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Higginbotham, G. L. Sieckman, W. A. Volkert, Bioconjugate Chem.
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[*] Prof. Dr. D. Sellmann, Dipl.-Chem. A. Fürsattel
Institut für Anorganische Chemie der Universität Erlangen-Nürnberg
Egerlandstrasse 1, D-91058 Erlangen (Germany)
Fax: (‡49)9131-852-7367
E-mail: sellmann@anorganik.chemie.uni-erlangen.de
[**] Transition Metal Complexes with Sulfur Ligands. Part 137. This work
was supported by the Deutsche Forschungsgemeinschaft and the
Fonds der Chemischen Industrie. Part 136: D. Sellmann, J. Utz, F. W.
Heinemann, Inorg. Chem. submitted.
Angew. Chem. Int. Ed. 1999, 38, No. 13/14
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