Solid-phase synthesis of protected α-amino phosphonic acid oligomers

Article abstract of DOI:10.1039/b912231a

By establishing both a highly efficient phosphonamidate formation and a RuCp-catalyzed cleavage of an allyl linker, the solid-phase synthesis of Fmoc-(GlyP(OBn))₆-OH/DIEA, a protected form of a new type of unnatural peptide α-amino phosphonic a

Full text of DOI:10.1039/b912231a

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Solid-phase synthesis of protected a-amino phosphonic acid oligomerswz
Yoshitaka Ishibashi and Masato Kitamura*
Received (in Cambridge, UK) 22nd June 2009, Accepted 30th September 2009
First published as an Advance Article on the web 14th October 2009
DOI: 10.1039/b912231a
By establishing both
a highly efficient phosphonamidate
formation and a RuCp-catalyzed cleavage of an allyl linker,
the solid-phase synthesis of Fmoc-(GlyP(OBn))6-OH/DIEA, a
protected form of a new type of unnatural peptide a-amino
phosphonic acid oligomer (APO), has been realized.
a-Amino phosphonic acid oligomers (APOs) can be
considered to be the closest analogues of a-amino carboxylic
acid-based peptides. Incorporating both sp3-hybridized
tetrahedral phosphorus(V) atoms and the dibasic acid
properties of phosphonic acid moieties into the peptide
backbone modifies the size and shape of its three-dimensional
structure, as well as the isoelectronic points of the parent
a-amino acids, which possess smaller, flat, monobasic
carboxylic functions.1,2 In addition, the strong hydrogen
bonding ability of the phosphonamidate might be advanta-
geous for constructing higher-order structures.3 These steric
and electronic differences influence the diverse chemical and
biological activities of APOs through inherent competition
with their carboxylic counterparts for the active sites of
enzymes, antibodies or receptors.1a Intense interest in
these so-called ‘‘phosphorus analogues’’ has increasingly
attracted attention with regard to their sequential synthesis
on a solid phase; however, there have been no reports of
their synthesis since 1975, when Yamauchi reported the
solution-phase synthesis of a protected dimer or trimer using
a phosphonochloridate method,4a and the non-sequential
polymerization of a-amino phosphonic acids or esters.4b This
may be partly due to the lack of efficiency in the direct
dehydrative condensation5 between N-protected a-amino
phosphonic acids and a-amino phosphonic esters, and in
the cleavage of the target molecules from supports under
conditions that do not affect the acid-labile P–N bonds.6 In
pursuit of our final goal of creating APOs, we have realized
here, for the first time, the solid-phase synthesis of
Fmoc-(GlyP(OBn))6-OH/DIEA (DIEA = N,N-diisopropyl-
ethylamine) using an N-Fmoc-protected 1-aminomethylphos-
phonic acid monobenzyl ester, Fmoc-GlyP(OBn)-OH (1),y
as the simplest monomer unit.
Fig. 1 The synthetic cycle of a-amino phosphonic acid oligomers.
Fig. 1 illustrates the synthetic cycle, which follows standard
peptide chemistry. The chain is elongated in the N–P direction
on a TentaGel support attached to the monomer via a Jones
a-branched allyl linker.7 One cycle involves Fmoc removal
using piperidine,8 followed by condensation of monomer acid
1 (key point 1). After repetition of this cycle n times, the n-mer
ester is catalytically detached to give Fmoc-(GlyP(OBn))n-OH
(key point 2).
The first key point in Fig. 1 was investigated by the
condensation of
1 with H-GlyP(OBn)-OBn (2), giving
Fmoc-(GlyP(OBn))2-OBn (3) in solution at the outset
(Fig. 2). Successful condensation was attained by the use of
diisopropylcarbodiimide (DIC) and 1-hydroxy-7-azabenzo-
triazole (HOAt), together with DIEA. In 1 h at room
temperature, a modified Carpino’s method quantitatively
converted 1 and 2 in CD3CN to 3 (observed by 31P NMR
analysis). The dimer was isolated in 93% yield. Contrary to
the carboxylic acid case,9 the yield did not exceed 64% in the
absence of DIEA. Removal of Fmoc from 3 was performed
by using piperidine, without any side reactions, to give
H-(GlyP(OBn))2-OBn quantitatively (83% isolated yield).10
31P NMR analysis10 revealed that the reaction proceeds as
shown in Fig. 2. First, the phosphonic acid 1 (d 21.7) was
quantitatively converted into the pyrophosphate anhydride 4
(d 16.7),5,11 10 min after 1 and DIC were mixed in a 1 : 1.5
ratio in CD3CN. The addition of 1.2 equivalents of HOAt to
the solution containing 4 generated the HOAt-activated
Research Center for Materials Science and the Department of
Chemistry, Nagoya University, Chikusa, Nagoya 464-8602, Japan.
E-mail: kitamura@os.rcms.nagoya-u.ac.jp; Fax: +81 52-789-2958
w This article is part of a ChemComm ‘Catalysis in Organic Synthesis’
web-theme issue showcasing high quality research in organic
chemistry. Please see our website (http://www.rsc.org/chemcomm/
organicwebtheme2009) to access the other papers in this issue.
z Electronic Supplementary Information (ESI) available: Experimental
details for the solution-phase and solid-phase synthesis. See DOI:
10.1039/b912231a
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The second issue, detachment of the APO from Jones’ allyl
linker, was investigated by the use of our deallylation catalyst,
[RuCp(P(C6H5)3)(CH3CN)2]PF6,13 and model compound 7a,
prepared from 8a via the condensation of 1, followed by Fmoc
removal and the recondensation of 1.10 The standard
conditions reported ([substrate] = 50 mM, [catalyst] = 0.5 mM,
CH3OH, 25 1C,
(GlyP(OBn))2-OH (7b) (31P NMR: d 24 and 29.5), together
with compounds with resonances at 22.6 and 12.4,
1 h) detached the dipeptide Fmoc-
d
which could be assigned to Fmoc-GlyP(OBn)-OCH3 and
H-GlyP(OBn)-OH, produced by self-acid-catalyzed P–N bond
cleavage. This problem was solved by the addition of DIEA
(50 mM) to the catalytic system to neutralize the acid, giving
7c (d 18.9 and 29.9) in quantitative yield after 3 h, together
with the corresponding branched allyl methyl ether 8b
(branch/normal ratio ca. 3 : 1). Compound 7c was isolated
in >90% yield. As this detachment reaction proceeded
without the need for any additional nucleophiles, such as
potassium carboxylate, alkoxide, morpholine, pyrrolidine,
formate, tributyltin hydride, borohydride or silane, which
are generally required for conventional Pd methods,13 the
isolation of the product will be simplified when applied to its
solid-phase synthesis.
Fig.
2 The supposed reaction pathways of DIC/HOAt/DIEA-
promoted condensation.
phosphonic acid 5 (d 30.7). The signal completely disappeared
30 min after the addition of 1 equivalent of amine 2 (d 29.3)
and 1 equivalent of DIEA, giving a new set of signals at d 26.2
and 28.0, which could be assigned to the phosphorous atoms
of the phosphonic diester and the phosphonamidate in 3,
respectively. The coexistence of DIEA at the stage of forming
5 from 4 gave, however, oxazaphospholine 6 (d 13.9)5 in
ca. 80% yield, which was not further converted to 3 when 2
was added to 6. On the other hand, the reaction of 5 with 2 was
not completed without DIEA, instead giving a mixture of 3, 5
(d 30.7) and another species (d 24), probably the 2/HOAt salt.
The use of N-hydroxybenzotriazole (HOBt) instead of HOAt
stopped the reaction at the stage of anhydride 4 formation.
Next, both the condensation method and the detachment
process established in solution were applied to the solid-
phase synthesis of Fmoc-(GlyP(OBn))6-OH/DIEA. Thus,
initial monomer 1 was connected to a TentaGel-attached
Jones’ branched allyl linker 8c (A (m = 0) in Fig. 1)
(0.26 mmol OH gꢁ1, swelled in CH3CN) by use of the
DIC/HOAt/DIEA method (rt, 10 h). Washing of the
1-connected resin with DMF, CH3OH and CH2Cl2, followed by
drying at 0.1 mmHg, gave B (n = 1), which showed 31P NMR
signals at d 22.6, 23.4 and 24.6 (4 : 85 : 11). A standard
piperidine treatment of B (n = 1) (CH2Cl2, rt, 1 h) released
free amine A (m = 1) (31P NMR signals at d 28.0 and 28.3
(1 : 1)). Repetition of the (1) swelling, (2) condensation,
(3) washing and (4) drying processes afforded B (n = 2)
(31P NMR: d 25 and 29), which was again converted to A
(m = 2) by the (5) piperidine, (6) washing and (7) drying
processes. Three repetitions of the (1)–(7) processes,
followed by one cycle of the (1)–(4) processes afforded
TentaGel-attached B (n = 6). Fig. 3 shows the 31P NMR
spectra obtained in each cycle. The signals labelled J corres-
pond to the phosphorus atom of the diester moiety, the signals
labelled n to those of internal phosphonamidates and the
signals labelled & to those of N-terminal examples. As the
chain is elongated, the intensity of the internal phosphorus
signals increase, although all signals tend to be broadened.
This broadening can be ascribed to the increase in the number
of diastereomers originating from the stereogenic centers of
the phosphorus and carbon atoms. In each step, the 31P NMR
With
benzotriazole-1-yl-oxy-trispyrrolidinophosphonium
hexafluorophosphate (PyBOP), the HOBt-activated ester was
obtained, whereas no reaction with 2 occurred, even with the
assistance of DIEA.
Evidence from a series of experiments clearly indicated
the importance of the N(7) atom in HOAt for facilitating
phosphonamidate P–N bond formation, as has been suggested
in the case of carboxamide.9 The simultaneous addition of 2
and DIEA to 5 would generate a stable DIEA/HOAt
(pKa 3.47)9c complex, moving the equilibrium predominantly
to the side of 3. The generation of the very reactive phosphonyl-
trialkylammonium species, as reported by Smith et al.,5 might
be also involved, although the 31P NMR spectrum of a 1 : 1
mixture of 5 and DIEA showed no signals in region of d 445
assignable to the species. Other methods used for phosphon-
amidate formation between a-amino carboxylic esters and
N-protected a-amino phosphonic monoesters were not
efficient.10 In situ-activation of 1 with diphenylphosphoryl
azide (DPPA)6b or isolation of the phosphonochloridate
(SOCl2, 2 h, rt), followed by the addition of 2 and triethyl-
amine, gave 3, at best, in 80% yield (by 31P NMR analysis),5
which is not high enough for oligomer synthesis.12
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dehydrative-condensation method and achieving a RuCp/
P(C6H5)3-catalyzed detachment from Jones’ allyl linker. Until
now, it has not been possible to regard APO as a synthetic
target because of the instability of the P–N bond. Although
there is no a-substituent and the final product is still protected,
our new method should inspire further research into the design
and preparation of various phosphorus analogues of
higher peptides, which have high potential in peptide and
nucleotide sciences, and future biochemical technologies.
The characteristics of APOs should make our new type of
unnatural peptide an important complement to existing
unnatural biooligomers.14 The synthesis of unprotected
H-(AlaP(OH))6-OH/DIEA is an on-going project in our group.
Notes and references
y a-Amino phosphonic acid abbreviation follows the IUPAC rule
determined for usual a-amino acids by the addition of ‘‘P’’ at the
end of the three character code. e.g. GlyP for a-aminomethyl phosphonic
acid, where H-GlyP(OH)-OH represents H2NCH2P(O)(OH)2.
1 Book and review: (a) Aminophosphonic and Aminophosphinic Acids,
ed. P. V. Kukhar and R. H. Hudson, Wiley-VCH, Weinheim, 1991,
pp. 1–634; (b) P. Kafarski and B. Lejczak, Phosphorus, Sulfur
Silicon Relat. Elem., 1991, 63, 193–215; (c) The first report on
a-amino phosphonic acids: M. Horiguchi and M. Kandatsu,
Nature, 1959, 184, 901–902.
2 M. Kitamura, M. Yoshimura, M. Tsukamoto and R. Noyori,
Enantiomer, 1996, 1, 281–303.
3 B. P. Morgan, J. M. Scholtz, M. D. Ballinger, I. D. Zipkin and
P. A. Bartlett, J. Am. Chem. Soc., 1991, 113, 297–307.
4 (a) K. Yamauchi, Y. Mitsuda and M. Kinoshita, Bull. Chem. Soc.
Jpn., 1975, 48, 3285–3286; (b) K. Yamauchi, J. Synth. Org. Chem.,
Jpn., 1988, 46, 654–666.
5 R. Hirschmann, K. M. Yager, C. M. Tayor, J. Witherington,
P. A. Sprengeler, B. W. Phillips, W. Moore and A. B. Smith III,
J. Am. Chem. Soc., 1997, 119, 8177–8190.
6 (a) N. S. Sampson and P. A. Bartlett, J. Org. Chem., 1988, 53,
4500–4503; (b) K. Yamauchi, S. Ohtsuki and M. Kinoshita, J. Org.
Chem., 1984, 49, 1158–1163; (c) M. Hariharan, R. J. Motekaitis
and A. E. Martell, J. Org. Chem., 1975, 40, 470–473; (d) For a
report on relatively stable phosphonamidates, see: D. A. McLeod,
R. I. Brinkworth, J. A. Ashley, K. D. Janda and P. Wirsching,
Bioorg. Med. Chem. Lett., 1991, 1, 653–658.
7 X. Zhang and R. A. Jones, Tetrahedron Lett., 1996, 37, 3789–3790.
8 L. A. Carpino and G. Y. Han, J. Org. Chem., 1972, 37, 3404–3409.
9 (a) L. A. Carpino, J. Am. Chem. Soc., 1993, 115, 4397–4398;
(b) L. A. Carpino and A. El-Faham, Tetrahedron, 1999, 55,
6813–6830; (c) L. A. Carpino, H. Imazumi, B. M. Foxman,
M. J. Vela, P. Henklein, A. El-Faham, J. Klose and M. Bienert,
Org. Lett., 2000, 2, 2253–2256.
Fig. 3 The 31Pt{1H} NMR spectrum change during APO elongation
on TentaGel (CDCl3, 25 1C). a: Dimer, b: trimer, c: tetramer,
d: pentamer, e: hexamer. The right-hand spectra show the Fmoc-
deprotected oligomers. K: CH(CHQCH2)(CH2)7CONH-TentaGel.
signals of the terminal phosphonamidates appear at around
d 29, and the chemical shifts are lowered to about d 33 after
Fmoc removal. This phenomenon can be understood by
assuming that the Fmoc moiety has a magnetic shielding
effect, an idea that is consistent with our observations in the
conversion of 3 (d 25.8, 27.0 and 28.0 (50 : 4 : 46))
to H-(GlyP(OBn))2-OBn (d 25.6 and 32.3) in CDCl3.10
10 For details, see the ESIz.
11 (a) R. Greenhalgh, R. M. Heggie and M. A. Weinberger, Can. J.
Chem., 1970, 48, 1351–1357; (b) N. S. Corby, G. W. Kenner and
A. R. Todd, J. Chem. Soc., 1952, 1234–1242.
Compound
B (n = 6) was subjected to a cationic
12 Y. Hayakawa, S. Wakabayashi, H. Kato and R. Noyori, J. Am.
Chem. Soc., 1990, 112, 1691–1696.
13 M. Kitamura, S. Tanaka and M. Yoshimura, J. Org. Chem., 2002,
67, 4975–4977.
RuCp/P(C6H5)3-catalyzed detachment13 (B = 80 mg (0.1 mmol),
S/C = 100, [RuCp catalyst] = 1 mM, [DIEA] = 100 mM,
CH3OH, rt, 24 h, >95% conversion) to give Fmoc-
(GlyP(OBn))6-OH/DIEA (31P NMR: d 19, 29.5 and 31.5;
MALDI-TOF MS: 1360.5 (Fmoc-(GlyP(OBn))6-ONa); tR in
HPLC 3.3 min (column Develosil ODS-UG 4.6 mm ꢂ 25 cm,
eluent 8 : 2–7 : 3 CH3OH–H2O, flow rate 1.0 mL minꢁ1)).
In summary, to the best of our knowledge, a protected
a-amino phosphonic acid hexamer, Fmoc-(GlyP(OBn))6-OH/
DIEA, has been synthesized for the first time by solving
two problems: namely, establishing a DIC/HOAt/DIEA
14 (a) Z. Zhang and E. Fan, J. Org. Chem., 2005, 70, 8801–8810;
(b) D. Seebach, A. K. Beck and D. J. Bierbaum, Chem. Biodiversity,
2004, 1, 1111–1239; (c) K. Oh, K.-S. Jeong and J. S. Moore,
Nature, 2001, 414, 889–893; (d) K. Kirshenbaum, R. N.
Zuckermann and K. A. Dill, Curr. Opin. Struct. Biol., 1999, 9,
530–535; (e) S. E. Schneider and E. V. Anslyn, in Advances in
Supramolecular Chemistry, ed. G. W. Gokel, JAI Press,
Greenwich, 1999, vol. 5, pp. 55–120; (f) M. J. Soth and
J. S. Nowick, Curr. Opin. Chem. Biol., 1997, 1, 120–129;
(g) R. P. Cheng, S. H. Gellman and W. F. DeGrado, Chem.
Rev., 2001, 101, 3219–3232.
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This journal is The Royal Society of Chemistry 2009
Chem. Commun., 2009, 6985–6987 | 6987