Synthesis of phosphonamidate peptides by Staudinger reactions of silylated phosphinic acids and esters

Article abstract of DOI:10.1039/c0cc02472d

The Staudinger reaction of unprotected azido-peptides with silylated phosphinic acids and esters on the solid support offers a straightforward acid-free entry to different phosphonamidate peptide esters or acids under mild conditions in high purity and yield.

Full text of DOI:10.1039/c0cc02472d

View Online / Journal Homepage / Table of Contents for this issue
COMMUNICATION
www.rsc.org/chemcomm | ChemComm
Synthesis of phosphonamidate peptides by Staudinger reactions of
silylated phosphinic acids and esterswzy
Ina Wilkening, Giuseppe del Signore and Christian P. R. Hackenberger*
Received 9th July 2010, Accepted 6th August 2010
DOI: 10.1039/c0cc02472d
The Staudinger reaction of unprotected azido-peptides with silylated
phosphinic acids and esters on the solid support offers a straight-
forward acid-free entry to different phosphonamidate peptide esters
or acids under mild conditions in high purity and yield.
the final protecting group removal of interfering nucleophilic side
chains.24 Consequently, only few reports of peptides 3 with a free
OH at phosphorus have been reported, which utilize an
additional basic hydrolysis8,10,16,18 or hydrogenation step to
convert ester 2 into acid 3 (Scheme 1).
Phosphon- and phosphoramidates are of great interest in
various areas of modern organic chemistry, including
applications in asymmetric catalysis1–4 as well as bioorganic
and pharmaceutical research.5–12 Phosphoramidates have
recently received attention for bioconjugation in chemoselective
protein modification strategies, which yield phosphoramidate
peptides 1.13,14 In contrast to the rather stable phosphoramidates,
phosphonamidates are known to be more prone towards
P(QO)–NH bond cleavage under acidic conditions, in particular
when a free OH group is present at the phosphorous
atom.11,15,16 Nevertheless, phosphonamidate peptides 2 and
3, in which an amide bond is replaced by P(QO)–OR,NH (2)
or P(QO)–OH,NH (3), have found considerable attention as
promising protease inhibitors. Upon incorporation into a
peptide backbone phosphonamidates can be regarded as
analogues of the transition state during peptide bond cleavage
by proteases and thereby mimic the natural substrate
(Scheme 1).5,8,11,17–20 This potency makes phosphonamidate
peptides 2 and 3 interesting synthetic targets for the development
of protocols to probe different proteases5–12 in the elucidation
of a variety of biological processes and as targets for drug
discovery for the treatment of diseases like HIV6,10 and others.
Although several synthetic pathways to phosphonamidate
peptides 2 and 3 have already been developed, they still face
limitations due to the acid lability of the P(QO)–NH bond. Most
of the synthetic protocols are based on nucleophilic substitution
at P(v), in which phosphonochloridates 4 are reacted with side-
chain protected amino acids or peptides 6 under rather harsh
reaction conditions (Scheme 1).21–23 Extensive purification steps
caused by side product formation and subsequent removal of the
commonly acid-labile peptide side chain protecting groups after
the P(QO)–NH bond formation are therefore particularly
challenging. Alternatively, nucleophilic substitution of P(III)
phosphonochloridites 5 with peptides 6 followed by oxidation
can be performed, however with respect to the same limitation of
In order to stress the importance of a direct and acid-free
synthetic route to phosphonamidate peptides 2 and 3, we first
conducted stability tests of phosphonamidate esters with a phenyl
(2a) and a benzyl (2b) substituent at nitrogen under standard
TFA-deprotection conditions. These studies showed that both
compounds are cleaved in TFA within a short period of time.
Compounds containing an alkyl residue at nitrogen showed even
higher instability due to the electron-donating influence.
Based on these results, we decided to develop a convenient
synthetic route under acid-free conditions to both phospho-
namidate peptides 2 and 3 (Scheme 2). We reasoned that the
Staudinger reaction25 between azides26 and trivalent phosphorus
species in the presence of water would be suitable even for
peptides 3, since the reaction is mild and does not require the
protection of nucleophilic side chains, as demonstrated recently
in the bioorthogonal functionalization of whole proteins.13,14
In order to access phosphonamidates, a phosphonite has to be
used in the Staudinger reaction with azides. Since the synthesis of
phosphonites is difficult due to their high tendency to oxidize, we
propose to use phosphinic esters (H-phosphinates) 7 as starting
materials. These compounds are air stable and can be converted
by silylation into the trivalent silylated phosphonite species 8
in situ, which can be directly used in the subsequent Staudinger
reaction with azides 9 to deliver phosphonamidates 2. Previously,
the silylation of 7 was already applied for the synthesis of
phosphinic peptides12 and for modified nucleotides27–29 but to
the best of our knowledge not demonstrated for unprotected
peptides 2 or 3.
The first goal of our strategy was to find efficient silylating
conditions.27–30 Both, TMSCl and BSA (bistrimethylsilyl
acetamide), showed a quantitative conversion of the methyl
phenylphosphinate (7a) in CH3CN. After the addition of phenyl
azide (9a), the reaction was stirred for 16 h at room temperature
before desilylation was performed with TBAF to form phos-
phonamidate 2a in excellent 91% yield (Table 1, entry 1).
Alternatively, deprotection with sodium hydroxide solution
(1 N) led to similar yields (entry 2). Afterwards, different
substituted azides 9 were employed and in all cases the desired
products 2 could be obtained in good to high yields after column
chromatography (entries 3–10). In case of para-azido benzoic
acid 9f HFꢀpyridine was favoured over TBAF for the desilylation,
because the phosphonamidate 2h precipitated from the
reaction mixture under these conditions (entry 9). To further
probe another silylated alkyl phosphonite 8, which is
expected to be more sensitive towards oxidation, methyl
Freie Universitat Berlin, Institut fur Chemie und Biochemie,
¨
Takustr. 3, 14195 Berlin, Germany.
¨
E-mail: hackenbe@chemie.fu-berlin.de; Fax: +49 30 838 52551;
Tel: +49 30 838 52451
w This article is part of the ‘Emerging Investigators’ themed issue for
ChemComm.
z Electronic supplementary information (ESI) available: General
synthetic procedures, synthesis and characterisation of compounds.
See DOI: 10.1039/c0cc02472d
y This paper is dedicated to Prof. Helmut Vorbruggen on the occasion
¨
of his 80th birthday.
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 349–351 349

View Online
Scheme 1 Phosphonamidate synthesis.
Table 1 Synthesis of phosphonamidates 2 (Scheme 2)a
Phosphinic Silylating Desilylation Yield
Entry Azide 9 esters 7 reagent reagent (%) Product 2
1
2
BSA
BSA
TBAF
91
95
NaOH
(1 N)
9a
7a
7a
2a
3
4
5
BSA
TBAF
TBAF
TBAF
54
82
80
Scheme 2 Silylation and Staudinger reaction of phosphinic esters 7.
Et3N/
TMSCl
octylphosphinate 7d (for preparation see ESIz) was silylated
and reacted with 9a. The corresponding phosphonamidate 2j
was formed in a moderate yield (30%), in which hydrolysis
byproducts complicated the isolation by column chromatography.
After the synthesis of small phosphonamidates 2a–j in
solution, we next attempted to transfer the synthetic route to
solid supported aryl azido peptides (Scheme 3), in which we used
the salt-free BSA silylation condition. The advantages of such a
solid supported process include the easy removal of reagents and
additives and the possibility to combine this route with standard
Fmoc-based solid-phase peptide synthesis (SPPS).
9a
9a
BSA
BSA
6
7
7a
7a
TBAF
TBAF
61
72
For this purpose, a base-labile commercially available
HMBA resin was chosen as the solid support, which enables
after SPPS a side chain deprotection by TFA to deliver an
immobilized unprotected azido peptide 10. This allows a
Staudinger reaction with silyl-phosphonites 8 on the solid
support before the peptide is desilylated and cleaved from
the resin by basic treatment, thereby evading acidic conditions
after the P(QO)–NH bond formation (Scheme 3).
BSA
BSA
8
9
7a
7a
TBAF
76
86
Three model peptides 10a–c with para-azido benzoic acid at
the N-terminus and different functional groups in the side
chains were synthesized, to which an excess of BSA and three
different aryl and alkyl phosphinic esters 7 were added
(Scheme 3 and ESIz). In addition to the formation of silyl-
phosphonites 8 the peptide side chains are assumed to be also
silylated during the reaction. The reaction was then allowed to
proceed at room temperature under slight agitation. Finally,
the phosphonamidate peptides 2k–o were cleaved with 1 N
NaOH from the solid support. For all peptides, LC-MS
analysis showed full consumption of the azide and good to
high conversions into the corresponding phosphonamidate
peptides as determined by UV (see Scheme 3, Fig. 1 and ESIz).
After this successful synthesis of peptide esters 2 we additionally
probed the synthesis of the very labile phosphonamidate acid
peptides 3. Three phosphinic acids 11a–c were synthesized
according to literature protocols (see ESIz) and applied in
HFꢀ
BSA
pyridine
10
7a
BSA
BSA
TBAF
TBAF
90
30
11
9a
a
Conditions: (1) 1 equiv. 9, 1 equiv. 7, 4–6 equiv. BSA or TMSCl/
Et3N, CH3CN, r.t., 16–20 h; (2) TBAF or HFꢀpyr, r.t., 30 min.–1 h, r.t.
See also ESI.
c
350 Chem. Commun., 2011, 47, 349–351
This journal is The Royal Society of Chemistry 2011

View Online
acid peptides 3. The target compounds were obtained in very
good to excellent yields in solution and in high purities on the
solid support without the need of acidic protecting group
manipulations. The synthesis of phosphonamidate peptides from
alkyl azido peptides for protease inhibition studies as well as other
phosphinic acid derivatives for analogous Staudinger reactions is
currently under investigation.
The authors acknowledge financial support from the
German Science Foundation (Emmy-Noether program,
HA 4468/2-1), the SFB 765, the Max-Buchner Stiftung and
the Fonds der chemischen Industrie (FCI).
Notes and references
Scheme 3 Synthesis of phosphonamidate peptides 2 and 3. Conversions
1 P. Jiao, D. Nakashima and H. Yamamoto, Angew. Chem., Int. Ed.,
2008, 47, 2411.
2 D. Nakashima and H. Yamamoto, J. Am. Chem. Soc., 2006, 128, 9626.
3 M. Rueping, B. J. Nachtsheim, S. A. Moreth and M. Bolte, Angew.
Chem., Int. Ed., 2008, 47, 593.
were determined by UV measurement at 280 nm. For conditions and LC-
a
MS analysis see Fig. 1 and ESI.z Isolated yield after HPLC purification.
4 S. E. Denmark and G. L. Beutner, Angew. Chem., Int. Ed., 2008,
47, 1560.
5 P. A. Bartlett and C. K. Marlowe, Biochemistry, 1983, 22, 4618.
6 N. P. Camp, P. C. D. Hawkins, P. B. Hitchcock and D. Gani,
Bioorg. Med. Chem. Lett., 1992, 2, 1047.
7 R. E. Galardy, Biochemistry, 1982, 21, 5777.
8 N. E. Jacobsen and P. A. Bartlett, J. Am. Chem. Soc., 1981, 103, 654.
9 A. P. Kaplan and P. A. Bartlett, Biochemistry, 1991, 30, 8165.
10 D. A. Mc Leod, R. I. Brinkworth, J. A. Ashley, K. D. Janda and
P. Wirsching, Bioorg. Med. Chem. Lett., 1991, 1, 653.
11 K. A. Mookhtiar, C. K. Marlowe, P. A. Bartlett and H. E.
Van Wart, Biochemistry, 1987, 26, 1962.
12 A. Yiotakis, D. Georgiadis, M. Matziari, A. Makaritis and
V. Dive, Curr. Org. Chem., 2004, 8, 1135.
13 (a) R. Serwa, I. Wilkening, G. Del Signore, M. Muehlberg,
I. Claussnitzer, C. Weise, M. Gerrits and C. P. R. Hackenberger,
Angew. Chem., Int. Ed., 2009, 48, 8234; (b) R. Serwa, P. Majkut,
B. Horstman, J.-M. Swiecicki, M. Gerrits, E. Krause and C. P.
R. Hackenberger, Chem. Sci., 2010, DOI: 10.1039/c0sc00324g;
(c) D. M. M. Jaradat, H. Hamouda and C. P. R. Hackenberger,
Eur. J. Org. Chem., 2010, DOI: 10.1002/ejoc.201000627.
14 V. Boehrsch, R. Serwa, P. Majkut, E. Krause and C. P. R.
Hackenberger, Chem. Commun., 2010, 46, 3176.
15 P. de Medina, L. S. Ingrassia and M. E. Mulliez, J. Org. Chem.,
2003, 68, 8424.
16 A. Mucha, J. Grembecka, T. Cierpicki and P. Kafarski, Eur. J.
Org. Chem., 2003, 4797.
17 J. L. Radkiewicz, M. A. McAllister, E. Goldstein and K. N. Houk,
J. Org. Chem., 1998, 63, 1419.
18 J. Grembecka, A. Mucha, T. Cierpicki and P. Kafarski, J. Med.
Chem., 2003, 46, 2641.
19 H. M. Holden, D. E. Tronrud, A. F. Monzingo, L. H. Weaver and
B. W. Matthews, Biochemistry, 1987, 26, 8542.
20 M. Izquierdomartin and R. L. Stein, J. Am. Chem. Soc., 1992, 114, 1527.
21 R. Hirschmann, K. M. Yager, C. M. Taylor, J. Witherington,
P. A. Sprengeler, B. W. Phillips, W. Moore and A. B. Smith,
J. Am. Chem. Soc., 1997, 119, 8177.
22 W. P. Malachowski and J. K. Coward, J. Org. Chem., 1994, 59, 7616.
23 A. Mucha, P. Kafarski, F. Plenat and H. J. Cristau, Tetrahedron,
1994, 50, 12743.
24 M. D. Fernandez, C. P. Vlaar, H. Fan, Y. H. Liu, F. R. Fronczek
and R. P. Hammer, J. Org. Chem., 1995, 60, 7390.
25 Y. G. Gololobov and L. F. Kasukhin, Tetrahedron, 1992, 48, 1353.
26 S. Braese, C. Gil, K. Knepper and V. Zimmermann, Angew. Chem.,
Int. Ed., 2005, 44, 5188.
27 T. Kline, M. S. Trent, C. M. Stead, M. S. Lee, M. C. Sousa,
H. B. Felise, H. V. Nguyen and S. I. Miller, Bioorg. Med. Chem.
Lett., 2008, 18, 1507.
28 R. A. Fairhurst, S. P. Collingwood and D. Lambert, Synlett, 2001, 473.
29 R. A. Fairhurst, S. P. Collingwood, D. Lambert and E. Wissler,
Synlett, 2002, 763.
Fig. 1 (A) HRMS analysis and HPLC profile of crude 2k; (B) HRMS
analysis, 31P-NMR and HPLC profile of crude 3a. For conditions see ESI.z
an analogous silylation and Staudinger reaction sequence with
immobilized peptide 10a (Scheme 3). We were delighted to find
that peptides 3a–c were formed as major products. However,
small amounts (7–25%) of the corresponding amino peptides
appeared in the LC-MS traces, which are presumably formed
by P(QO)–NH bond cleavage during the HPLC measurements.
The high conversion to 3a was further validated by 31P-NMR
measurement of the crude peptide 3a in which only one phospho-
namidate species was present. Addition of phenylphosphonic acid
to the NMR sample indicated that the previous NMR signal did
not result from the expected phosphorous cleavage product of 3a.
Finally, we attempted to access a phosphonamidate acid
peptide 3 by light cleavage of the nitro benzyl ester 2n. This
procedure would allow the synthesis of a relatively stable phospho-
namidate peptide, which could be converted to the more labile
peptide 3 during biological applications. After ten minutes of UV
irradiation concomitant P(QO)–NH bond cleavage occurred in
addition to the formation of 3a (see ESIz). Currently, we are further
optimizing this procedure by the synthesis of electron rich nitro-
benzyl phosphinates which allows milder irradiation conditions.
In conclusion, we have demonstrated that the Staudinger
reaction of silylated phosphinic acids and esters with different
aryl azides in solution as well as on the solid support represents an
efficient and straightforward acid-free protocol for the synthesis of
various phosphonamidates including sensitive phosphonamidate
30 H. Vorbruggen, Silcon-mediated Transformations of Functional
¨
groups, Wiley-VCH, Weinheim, 2004.
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 349–351 351