Soluble peptidyl phosphoranes for metal-free, stereoselective ligations in organic and aqueous solution

Article abstract of DOI:10.1021/ol202627h

Protocols for solid-phase syntheses of soluble peptidyl phosphoranes are presented. Various supported phosphoranylidene acetates were prepared on Rink amide or via alkylation of trialkyl- and triarylphosphines with bromoacetyl Wang ester. C-Acylation was

Full text of DOI:10.1021/ol202627h

ORGANIC
LETTERS
2012
Vol. 14, No. 1
14–17
Soluble Peptidyl Phosphoranes for
Metal-Free, Stereoselective Ligations
in Organic and Aqueous Solution
,†,‡
Ahsanullah,‡ Samer I. Al-Gharabli,§,‡ and Jorg Rademann*
€
Institute of Pharmacy, University of Leipzig, Bru€derstrasse 34, 04103 Leipzig, Germany,
€
Leibniz Institute of Molecular Pharmacology (FMP), Robert-Rossle-Strasse 10, 13125
Berlin, Germany, and Chemical-Pharmaceutical Engineering, German-Jordanian
University, 35247 Amman, Jordan
rademann@uni-leipzig.de
Received September 29, 2011
ABSTRACT
Protocols for solid-phase syntheses of soluble peptidyl phosphoranes are presented. Various supported phosphoranylidene acetates were
prepared on Rink amide or via alkylation of trialkyl- and triarylphosphines with bromoacetyl Wang ester. C-Acylation was conducted racemization-
free with activated Fmoc-amino acids, followed by SPPS (solid-phase peptide synthesis). Acidic conditions released decarboxylated peptidyl
phosphoranes into solution. The protocol allowed for the electronic variation of peptidyl phosphoranes which were investigated in ligation
reactions with azides in organic and aqueous solvents.
Chemoselective organic reactions have had a tremen-
dous impact on chemical biology during recent years.1
Ideally, such reactions tolerate the presence of water
and other biologically relevant nucleophiles and occur
at ambient temperature or up to 37 °C and over a
broad range of concentrations. The involved func-
tionalities should be stable under physiological redox
conditions and toward reactive groups occurring fre-
quently in biological systems. In the past decade, the
copper(I)-catalyzed dipolar cycloaddition of an azide
with an alkyne moiety has become an especially pro-
minent chemoselective reaction, mostly denominated
simply as a “click reaction”.2 Despite the advantages
associated with this reaction in terms of kinetics and
regioselectivity, it is not ideal for use in cellular
systems or even living organisms due to the toxicity
of copper. The scope of click materials is limited by
residual traces of copper.3 In addition, the reactions
can be severely impeded in copper-coordinating en-
vironments. Therefore, metal-free, biocompatible re-
actions are highly desirable.
One early example for the development of a metal-
free chemoselective ligation reaction has been made by
introduction of the Staudinger ligation though this
approach is impeded by comparably slow reaction
rates and background phosphine oxidation.4 In addi-
tion, strained alkynes have been used in the click
reaction though this variant of a metal-free reaction
† University of Leipzig.
‡ Leibniz Institute of Molecular Pharmacology.
§ German-Jordanian University.
(3) (a) Tirrell, D. A.; Link, A. J. J. Am. Chem. Soc. 2003, 125, 11164.
(b) Tirrell, D. A.; Link, A. J.; Vink, M. K. S. J. Am. Chem. Soc. 2004,
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(b) Hackenberger, C. P.; Schwarzer, D. Angew. Chem., Int. Ed. 2008, 47,
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Sharpless, K. B. Angew. Chem., Int. Ed. 2002, 41, 2596.
r
10.1021/ol202627h
Published on Web 12/02/2011
2011 American Chemical Society

reactions, the biocompatibility of phosphoranes needs
more consideration.7 Differing from phosphines, their
direct precursors, peptidyl phosphoranes, are stable
toward most oxidants. For cleavage, strong oxidants
such as ozone and dimethyldioxirane are required. In
principle, acylphosphoranes can react with carbonyl
compounds; however, most aldehydes including aro-
matic and peptidyl aldehydes as well as ketones re-
quire heating and prolonged reaction times. Only
aliphatic, nonhydrated aldehydes may react at room
temperature in a water-free environment. Hydroly-
sis of peptidyl phosphoranes was found only under
strongly basic conditions and under acidic conditions
with heating.7a Therefore, it appeared to be attractive
to investigate the potential of this reaction for chemo-
selective ligations. As the first step toward this goal, a
robust synthesis of soluble peptidyl phosphoranes had
to be established. Initially, Rink amide resin was
employed for this purpose (Scheme 1). The poly-
mer was acylated with 4-carboxyphenyldiphenylpho-
sphine toward 1 and subsequently alkylated with tert-
butyl 2-bromoacetate yielding 2. Acylation with an
Fmoc-amino acid using BTFFH for activation deliv-
ered 3, and peptide elongation furnished the peptidyl
phosphorane 4, which released by decarboxylating
cleavage under acidic conditions the phosphonium
salt 5a which was isolated in 78% overall yield. Treat-
ment of 5a with sodium hydroxide precipitated 5b with
95% yield. In the first ligation reaction, 5b was reacted
with 4-azidobenzoic acid. At room temperature, the
reaction proceeded smoothly and delivered the triazole
product (Table 1). The ligation proceeded, however, sig-
nificantly slower when phosphorane 5b was reacted with
azido peptide 14. Only 50% conversion was obtained in 18 h
and did not increase over the extended reaction time of
3 days (see the Supporting Information (Figure S1)).
Scheme 1. Synthesis of Soluble Peptidyl Phosphoranes Based on
Rink Amide Phosphine Resin 1
Variation of the phosphorus substituents was expected
to alter the electronic nature and thus the reactivity of the
obtained phosphoranes. For this reason, a protocol allow-
ing for the flexible variation of peptide sequence and
phosphorus substituents was devised (Scheme 2). The
central idea was to incorporate phosphorus by alkylation
of trisubstituted phosphines with polymer-bound electro-
philes. Thus, 4-hydroxymethyl phenoxide resin (“Wang
resin”) was O-acylated with bromoacetyl bromide yielding
polymer 6. Triphenylphosphine 7a was alkylated with
the bromoacetate residue, and treatment with Et3N pro-
vided the attached phosphorus ylide 8a. The latter was
C-acylated employing a BTFFH-activated Fmoc-amino
acid and yielded the Fmoc-amino acylphosphorane 9a.
Following Fmoc-deprotection, the sequence of a simple
peptide was constructed using diisopropylcarbodiimide and
1-hydroxybenzotriazole for activation. The N-terminal
amino group was deblocked and acetylated furnishing
N-acetyl-peptidyltriphenylphosphoranylidene acetate
ester 10a. Linker cleavage with trifluoroacetic acid
(TFA) led to the soluble peptidyl phosphonium salt 11a,
and the phosphorane ylide 13a was precipitated upon
addition of an aqueous solution of NaOH in pure form.
requires considerable synthetic effort for the prepara-
tion of cyclooctyne derivatives and is not stereo-
selective.5 Recently, the dipolar cycloaddition reac-
tions between azides and polymer-attached peptidyl
phosphoranes have been reported.6 The reaction was
found to proceed smoothly, stereoselectively, and
without metal catalysis. Both involved functionalities
that possess remarkable stability in biological sys-
tems. While azides are broadly established in ligation
€
(6) (a) Ahsanullah; Schmieder, P.; Kuhne, R.; Rademann, J. Angew.
Chem., Int. Ed. 2009, 48, 5042. (b) Ahsanullah; Rademann, J. Angew.
Chem., Int. Ed. 2010, 49, 5378. (c) El-Dahshan, A.; Weik, S.; Rademann,
J. Org. Lett. 2007, 9, 949–952.
(7) (a) El-Dahshan, A.; Ahsanullah; Rademann, J. Biopolymers
Peptide Sci. 2010, 94, 220. (b) El-Dahshan, A.; Nazir, S.; Ahsanullah;
Ansari, F. L.; Rademann, J. Eur. J. Org. Chem. 2011, 730.
Org. Lett., Vol. 14, No. 1, 2012
15

were obtained and investigated for C-acylation. Unexpect-
edly, acylation of the tri-n-butylphosphoranylidene resin 8c,d
failed in the first set of experiments despite using a list of
activating agents including BTFFH, TFFH, PyBOP, HATU,
and MSNT. Fmoc-amino acyl fluorides, however, which were
prepared in advance,8 succeeded in the C-acylation of the
ylides 8c,d affording the N-Fmoc-aminoacyl-tributylpho-
sphoranylidene acetate 9c and N-Fmoc-aminoacyl-tricyclo-
hexylphosphoranylidene acetate 9d. Cleavage of the linker
with TFA afforded pure N-Fmoc-aminoacyl-tributylpho-
sphorane 12c indicating the complete acylation of the pre-
cursor judging from peak integration in the NMR spectrum.
While the aminoacyl-tributylphosphorane 12c was stable
toward strong acidic conditions, the supported N-Fmoc-
aminoacyl-tributylphosphoranylidene acetate 9c was found
to degrade on treatment with 20% piperidine with breakage
of the carbonÀphosphorus bond. The same was observed for
the tricyclohexyl derivative 9d obtained from tricyclohexyl-
phosphine as the precursor.
Scheme 2. Synthesis of Soluble Peptidyl Phosphoranes Based on
2-Bromoacetyl Wang Resin 6
Scheme 3. Ligation Reaction of Peptidyl Phosphoranes 13a,b
and Azido Peptide 14 in Aqueous Medium
In the first ligation reaction, 13a was reacted with
only 1.2 equiv of 4-azidobenzoic acid. At room tem-
perature, the reaction proceeded smoothly and deliv-
ered the triazole product. When, however, the same pep-
tidyl phosphorane was ligated at rt with azido peptide
14 carrying the phenyl azide attached to the amino
terminus, the reaction to the ligation product 15 pro-
ceeded significantly slower with only 48% conversion.
Considering its mechanism, the rate of this reaction
should be increased either by lowering the energy level
of the lowest unoccupied molecular orbital (LUMO) of
the azide dipole or by raising the energy level of the
highest occupied molecular orbital (HOMO) of the
phosphorane. While the electronic effects on the side
of the azide have already been investigated, here we focused on
variations on the side of the phosphorane. In order to accel-
erate the reaction, aromatic phosphines with increased elec-
tron density (7b with R = 4-methoxyphenyl) and 7e with R =
2,4,6-trimethoxy) were employed first. In addition, the alipha-
tic trialkylated phospines 7c (R = n-butyl) and 7d (R =
cyclohexyl) were used. All five alkylation products 8aÀe
In contrast, the acylation of the tris-(2,4,6-trimeth-
oxyphenyl)-phosphoranylidene acetate 8e failed either
by using the condensation agents or by employing Fmoc-
amino acyl fluorides. C-Acylations with the triaryl phosphor-
anes 8a and 8b proceeded with ease using amino acyl fluoride
(8) Kaduk, C.; Wenschuh, H.; Beyermann, M.; Forner, K.; Carpino,
L. A.; Bienert, M. Lett. Pept. Sci. 1995, 2, 285.
16
Org. Lett., Vol. 14, No. 1, 2012

peptide 14 in organicÀaqueous solvent mixtures (DMF/
H2O) (Scheme 3). The reactivity was considerably enhanced
in the case of tris(4-methoxyphenyl) 13b compared to
the analogous triphenylphosphorane 13a. More than
85% conversion of the phosphorane was observed with
a 1.2 equiv excess of the azide at a 0.06 M concentration
over 18 h based on LCMS. Ligation product 15 was
purified by preparative HPLC in 58% yield and ana-
lyzed by NMR spectroscopy.
In summary, we have presented the first synthesis of
soluble peptidyl phosphoranes via solid phase peptide
synthesis. The synthetic route is flexible allowing for
reactivity control of the peptidyl phosphoranes with
altering the trivalent phosphine precursor. The pepti-
dyl phosphoranes have been demonstrated to ligate the
peptide component to aryl azide functionalities in the
presence of water. Next, we will investigate the poten-
tial of soluble peptidyl phosphoranes in target guided
synthesis9 and in template-assisted ligation screening10
through either azideÀphosphorane or Wittig reactions.
Table 1. Effect of Differentially Substituted Phosphoranes and
Azide on the Conversion in the Ligation Reactionsa
conv
no.
phosphorane
azide
(%)
1
2
3
4
5
6
5b (PPh2Ph-CONH2) 4-azidobenzoic acid
98
45
98
50
48
85
5b (PPh2Ph-CONH2) N3Bz-Ser-Ser-Val-NH2 (14)
13a (PPh3)
4-azidobenzoic acid
5b (PPh2Ph-CONH2) N3Bz-Asp-Ser-Gly-NH2
13a (PPh3)
N3Bz-Ser-Ser-Val-NH2 (14)
N3Bz-Ser-Ser-Val-NH2 (14)
13b (P(4-MeOPh)3)
a Conversions determined by LCMS. Reaction conditions: DMF/
H2O (60:40), 0.06 M, 18 h, rt, 1.2 equiv of excess of azide.
or BTFFH for activation. Fmoc-removal succeeded for 9a,b,
and the liberated amine could be used for peptide synthesis.
Following the described protocol (coupling of amino acids,
Fmoc-deprotection, N-acetylation of the N-terminus, and
acidic cleavage) the peptidyl triphenylphosphorane 11a (R=
Ph) and peptidyl tris-(4-methoxyphenyl)-phosphorane 11b
(R = 4-MeO-Ph) were isolated and fully characterized. The
corresponding ylide 13a,b was obtained by treatment with
base, and the ligations were investigated with the azido
Acknowledgment. We gratefully acknowledge the Higher
Education Commission (HEC) Pakistan and the Deutscher
Akademische Austauschdienst (DAAD) for a stipend granted
to A. and the DFG (RA895/11,12 FOR 806, and SFB 765). J.
R. thanks the Fonds der Chemischen Industrie for continuous
support.
(9) (a) Marchot, P.; Bourne, Y.; Kolb, H. C.; Radic, Z.; Sharpless,
K. B.; Taylor, P. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 1449.
(b) Senapati, S.; Cheng, Y. H.; McCammon, J. A. J. Med. Chem.
2006, 49, 6222.
Supporting Information Available. Experimental pro-
cedure and characterization data for all new compounds.
This material is available free of charge via the Internet
http://pubs.acs.org.
(10) (a) Schmidt, M. F.; El-Dahshan, A.; Keller, S.; Rademann, J.
Angew. Chem., Int. Ed. 2008, 47, 3275. (b) Schmidt, M.; Isidro-Llobet,
A.; El-Dahshan, A.; Lisurek, M.; Tan, J.; Hilgenfeld, R.; Rademann, J.
Angew. Chem., Int. Ed. 2009, 48, 6346. (c) Schmidt, M.; Rademann, J.
Trends Biotechnol. 2009, 27, 512.
Org. Lett., Vol. 14, No. 1, 2012
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