Phosphoramidate-peptide synthesis by solution- and solid-phase Staudinger-phosphite reactions

Article abstract of DOI:10.1002/psc.1236

The chemoselective incorporation of phosphoramidate moieties into peptides by a Staudinger-phosphite reaction of azides can be performed in many solvents, including water. In this report, we present two strategies for an efficient synthesis of phosphorami

Full text of DOI:10.1002/psc.1236

Rapid Communication
Received: 11 December 2009
Revised: 12 February 2010
Accepted: 9 March 2010
Published online in Wiley Online Library: 28 June 2010
(wileyonlinelibrary.com) DOI 10.1002/psc.1236
Phosphoramidate-peptide synthesis by
solution- and solid-phase Staudinger-
phosphite reactions‡
Remigiusz A. Serwa, Jean-Marie Swiecicki, Denise Homann and Christian
P. R. Hackenberger∗
The chemoselective incorporation of phosphoramidate moieties into peptides by a Staudinger-phosphite reaction of azides
can be performed in many solvents, including water. In this report, we present two strategies for an efficient synthesis of
phosphoramidate-containing peptides, in which the Staudinger-phosphite reaction is performed either on the solid support or
in solution with aryl azido-containing peptides. The corresponding Staudinger reactions proceed in high conversion rates and
deliver phosphoramidate peptides, in which the modification site is located in the middle of the peptide sequence. Copyright
c
ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd.
Supporting information may be found in the online version of this article
Keywords: Staudinger-phosphite reaction; solid supported reaction; phosphoramidate; aryl azides; chemoselective reaction
Organic azides are very popular functionalities [1], especially
among biological chemists since the first reports addressing them
in chemoselective transformations, for instance, in the Staudinger
ligation and 1,3-dipolar cycloadditions, became available [2–9].
This reporter moiety has been incorporated in a number of
biologically relevant macromolecules [10–12], including peptides
and proteins [13,14], and addressed in chemoselective reactions
that allowed a site-specific incorporation of many biophysical
probes or functional modules, including fluorophores [15],
biotin [16], PEG [17], or lipids [18]. Additionally, aryl azides
can undergo a light-induced transformation to nitrenes, which
are first converted into benzazirines or azacycloheptatetraenes
beforenucleophilesaddspontaneouslytoproducestablecovalent
linkages [19].
Recently,ourgrouphasemployedachemoselectiveStaudinger-
phosphite reaction for the transformation of azides into phos-
phoramidates, in which the phosphoramidate moiety mimics a
naturally phosphorylated tyrosine in a protein [20]. In those stud-
ies, a Staudinger reaction of P(III)-compounds was successfully
performed on p-azido-L-phenylalanine (Pap), containing pep-
tides in organic solvents, as well as in buffers at physiological
pH. All peptide substrates that participated in these transforma-
tions were prepared utilizing a standard Fmoc-based solid-phase
peptide synthesis (SPPS) and always contained Pap at the N-
terminus.
performed (Scheme 1A). In this regard, a Boc-based SPPS that
minimizes the exposure to nucleophiles appears to be beneficial
[23]. However, we additionally observed instability of Pap under
acidic conditions during the cleavage of the peptide from the solid
support upon prolonged treatment with 95% trifluoroacetic acid
(TFA). Although in this case the degradation of Pap proceeds to
a limited extent, a repeated N-Boc-deprotection step performed
under acidic conditions could result in a major damage to the
azide, thus questioning the suitability of Boc-SPPS for the prepa-
ration of Pap containing peptides. In an alternative strategy, Pap
can be conveniently installed into peptides by diazotization of
p-aminophenylalanine (Apa) in solution after cleavage from the
solid support, which has been demonstrated to proceed in the
presence of various functional groups. The efficiency of the trans-
formation of Apa into Pap seems to be sequence dependent,
yet the specific amino acids responsible for decreasing the con-
version could not be identified based on the available literature
reports [24–33]. Furthermore, when Met is present in the peptide
sequence, the thioether is expected to undergo oxidation during
the diazotization reaction in water [24].
In order to provide general synthetic protocols for the prepa-
ration of phosphoramidate peptides, we have now developed
two synthetic routes, which involve a Staudinger-phosphite re-
action of aryl azidopeptides either in solution or on the solid
Insubsequentinvestigations, weintendedtosynthesizearylazi-
dopeptides to allow for a later incorporation of phosphoramidates
by a Staudinger-phosphite reaction regardless of the peptide se-
quence. In contrast to the synthesis of alkyl azidopeptides, which
can be prepared by standard SPPS protocols in high yields [21,22],
we encountered problems during the SPPS of aryl azidopeptides
1. Specifically, we observed that additional peptide coupling and
deprotection cycles after installation of Pap led to the degradation
ofthearylazideandthatthelevelofitsdecompositionwasapprox-
imately proportional to the number of deprotections/couplings
∗
Correspondence to: Christian P. R. Hackenberger, Institut fu¨r Chemie und
¨
Biochemie, Freie Universitat Berlin, Takustr. 3, 14195 Berlin, Germany.
E-mail: hackenbe@chemie.fu-berlin.de
¨
Institut fu¨r Chemie und Biochemie, Freie Universitat Berlin, Takustr. 3, 14195
Berlin, Germany
‡
Special issue devoted to contributions presented at the Symposium ‘‘Probing
Protein Function through Chemistry’’, 20–23 September 2009, Ringberg Castle,
Germany.
c
J. Pept. Sci. 2010; 16: 563–567
Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd.

SERWA ET AL.
support (Scheme 1B). Whereas the Staudinger-phosphite reaction
is performed in the latter strategy during SPPS, the solution-
phase protocol employs an azide on the full length peptide as a
substrate. It is important to note that each method would have
certain advantages. A solution-phase strategy can be considered
as convergent with respect to the formation of the phospho-
ramidate moiety. This represents an ideal strategy when sensitive
modules (to a base or to an acid) are introduced by the Staudinger-
phosphite reaction, since these functionalities could suffer from
conventional Fmoc-deprotection or cleavage conditions. A solid-
phase strategy applied for more robust phosphoramidates would
allow the removal of the remaining phosphite reaction part-
ners or their hydrolytic byproducts before the peptide is cleaved
from the resin. Furthermore, phosphoramidate peptides contain-
ing non-oxidized Met could be potentially prepared utilizing the
solid-phase approach.
We started our investigations of the Staudinger-phosphite
reaction in solution by accessing a model aryl azidopeptide
3a (Asp-Ala-Asp-Glu-Apa-Leu-Ile-Pro-Gln-Gln-Gly), which is an
epidermal growth factor receptor fragment (988–998). Standard
conditions for the diazotization of anilines, which can be
incorporated into peptides with the commercially available
Fmoc-Apa(Boc)-OH building block, utilizing NaNO2 followed by
the addition of NaN3 under acidic conditions were applied
to 3a (Scheme 2). The corresponding aryl azidopeptide 1a
(Asp-Ala-Asp-Glu-Pap-Leu-Ile-Pro-Gln-Gln-Gly) was isolated by
semi-preparative HPLC in 29% overall yield including SPPS. In
comparison, the Fmoc-based SPPS route in analogy to Scheme 1A
with commercially available Fmoc-Pap-OH delivered the same
peptide 1a in only 0.5% isolated yield. Aryl azidopeptide
1a was then subjected to the Staudinger-phosphite reaction
in solution in tris buffer at pH 8.2 with a water-soluble o-
nitrobenzyl-phosphite [20], which delivered peptide 2a bearing
a caged phosphorylated tyrosine analog in 69% isolated yield.
Furthermore, the application of the solid-phase strategy for the
preparation of 2a was unsuccessful due to an intrinsic light-
sensitivity of this phosphoramidate.
After these solution-phase strategies for the acquisition and
transformation of aryl azidopeptides 1, we turned our attention
to a solid-phase Staudinger-phosphite reaction (Scheme 1B).
Since the Fmoc-based SPPS with an Fmoc-Pap-OH building
block delivers only small quantities of aryl azidopeptides 1
(Scheme 1A), we aimed for a step-wise protocol, in which the
peptide is reacted with phosphites directly after coupling of
the Fmoc-Pap-OH building block. Afterwards, SPPS coupling
cycles are continued and the final phosphoramidate peptide 2
is cleaved from the solid support by a standard TFA treatment.
We first investigated the stability of phosphoramidate peptides
2 in the presence of piperidine or TFA. Phosphoramidate peptide
2b (Phe[p-NHP(O)(OBu)2]-Ala-Asp-Glu-Phe-Leu) was prepared
by Fmoc-based SPPS of the corresponding azide precursor on a
Wang resin, TFA cleavage and Staudinger-phosphite reaction in
solution with tributylphosphite according to our previous report
(Scheme 3A, Route II) [20]. Subsequent treatment either with 20%
piperidine in DMF for 24 h or with 95% aqueous TFA at room
temperature for 2 h caused no degradation of 2b under these
conditions. Since aryl phosphoramidates showed the necessary
stability, we focused on performing the Staudinger-phosphite
reaction on the solid support (Scheme 3A, Route I). A previously
employed aryl azidopeptide 1b (Pap-Ala-Asp-Glu-Phe-Leu) was
incubated on the solid support with trimethylphosphite or trib-
utylphosphite in DMSO to form immobilized phosphoramidate
100
90
80
70
60
50
40
30
20
10
0
(A)
2d
8
12
16
20
24
28
32
Time (min)
100
90
80
70
60
50
40
30
20
10
0
(B)
2e
10
14
18
22
26
30
34
Time (min)
Figure 1. LC-UV230 nm trace for crude peptides 2d (A) and 2e (B) released
from the solid support.
peptides. Resins were washed with DMF and CH2Cl2, and peptides
2b and 2c (Phe[p-NHP(O)(OMe)2]-Ala-Asp-Glu-Phe-Leu) were
released from the solid support under the same acid-promoted
cleavage conditions used during the stability measurement.
LC-MS analysis of both crude mixtures indicated the presence
of phosphoramidate peptides 2b and 2c as the only reaction
products (Supporting Information). Encouraged by these results,
we targeted phosphoramidate analogs of a phosphorylated
adhesion and degranulation promoting adapter protein
fragment (586–600), peptides 2d (Cys-Arg-Pro-Ile-Glu-Asp-Asp-
Gln-Glu-Val-Phe[p-NHP(O)(OMe)2]-Asp-Asp-Val-Ala-Glu) and 2e
(Cys-Arg-Pro-Ile-Glu-Asp-Asp-Gln-Glu-Val-Phe{p-NHP(O)[OBn(2-
NO2)]2}-Asp-Asp-Val-Ala-Glu), as a more challenging synthetic
aim, in which the phosphoramidate is located more than ten
amino acids away from the N-terminus (Scheme 3B). A rink amide
resin bound aryl azidopeptide 1c (Pap-Asp-Asp-Val-Ala-Glu)
was incubated with trimethylphosphite or tris(2-nitrobenzyl)
phosphite in DMF, followed by further amino acid couplings.
Final TFA cleavage delivered full length peptides, 2d and 2e.
HPLC and HRMS analysis for the crude peptide material indicated
peptides, 2d and 2e as single major peaks (Figure 1), which were
isolated by semi-preparative HPLC in 12% and 10% overall yield,
respectively.
In summary, we have succeeded in the preparation of
phosphoramidate-containing peptides from aryl azidopeptides,
in which the phosphoramidate moiety can be placed distant
from the peptide N-terminus. In order to synthesize these
molecules,theStaudinger-phosphitereactionwasperformedboth
insolutionandonthesolidsupport,whichdeliveredlight-sensitive
c
wileyonlinelibrary.com/journal/psc Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2010; 16: 563–567

PHOSPHORAMIDATE-PEPTIDE SYNTHESIS BY STAUDINGER-PHOSPHITE REACTIONS
(A)
1. FmocAaaOH
HBTU/HOBT
2. Piperidine
1. FmocAaaOH
HBTU/HOBT
2. Piperidine
O
PG
H
O
N
Peptide
Peptide
N
COOH
Peptide
H2N
H2N
N
H
OH
O
H
then TFA
1. FmocPapOH
HBTU/HOBT
2. Piperidine
N3
N3
aryl azido peptides 1
very low yield
(B)
PG
O
O
Solid-phase
peptide
synthesis
(SPPS)
H
N
Peptide
Peptide
Peptide
FmocHN
N
H
COOH
N
H
H2N
O
NH2
N3
Solution phase
diazotization
Solid phase
Staudinger-Phosphite
reaction
O
PG
H
O
N
Peptide
Peptide
N
COOH
Peptide
H2N
FmocHN
N
H
O
H
O
P
OR
N3
N
H
OR
SPPS
then TFA
Solution phase
Staudinger-Phosphite
reaction
O
H
N
Peptide
Peptide
N
H
COOH
H2N
O
O
P
OR
OR
N
H
phosphoramidate-peptides 2
Scheme 1. (A) Fmoc-based SPPS for the incorporation of Pap. (B) Solution- and solid-phase strategies for the synthesis of phosphoramidate peptides.
1. NaNO2, H3O+
P(OR)3
Peptide
Peptide
Peptide
H2N
COOH
NH2
H2N
COOH
N3
H2N
COOH
O
2. NaN3, H2O
Tris pH 8.2
P
N
OR
H
OR
3a
1a
29 % (incl.SPPS)
2a
69 %
Peptide = AspAlaAspGluApaLeuIleProGlnGlnGly
O
O
R =
O
5
O2N
Scheme 2. Diazotization of aniline peptide 3a in solution to aryl azidopeptides 1a, and subsequent Staudinger-phosphite transformation into
photo-sensitive peptide phosphoramidate 2a. Phe indicates the azide position.
analogs of phosphorylated peptides in high yields. Current
investigation in our laboratory focuses on the development of
diazotization protocols, which can be performed on the solid
support, which allow a convergent synthesis of phosphoramidate
peptides.
were purchased from Novabiochem or Bachem. LC-UV and LC-MS
spectra were recorded on an Agilent 6210 TOF LC/MS system,
Agilent Technologies, Santa Clara, CA, USA. Spray voltage was set
to 4 kV. Drying gas flow rate was set to 25 psi. Separation of the
sample was performed on a Luna C18(2) 100 A column (5 µm,
4.6 mm × 150 mm) at a flow rate of 0.6 ml/min. The following
solvent (A = 1% AcOH in H2O, B = 1% AcOH in MeCN) gradient
was applied: 0% B 0–5 min; 0–60% B 5–25 min; 60% B 25–32 min.
Preparative HPLC purification for peptides was performed on a
JASCO LC-2000 Plus system using a Kromasil RP18 column (25 mm
× 250 mm) at a flow rate of 16 ml/min. The following solvent (A
= 0.1% TFA in H2O, B = 0.1% TFA in MeCN) gradient was applied:
0% B 0–5 min; 0–60% B 5–55 min; 60–100% B 35–40 min; 100%
Materials and Methods
Peptide syntheses were carried out with an automated peptide
synthesizer ABI 433A (Applied Biosystems) utilizing standard
Fmoc SPPS conditions (amide coupling: HBTU/HOBt/DIPEA;
deprotection: 25% piperidine in DMF). All canonical and unnatural
[Fmoc-Pap-OH, Fmoc-Apa(Boc)-OH] L-amino acid building blocks
c
J. Pept. Sci. 2010; 16: 563–567 Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/psc

SERWA ET AL.
(A)
PG
O
95% TFA
P(OR)3, DMSO
H2N
AlaAspGluPheLeu
quantitative
conversion
N
H
Route I
O
O
P
OR
PG
N
OR
O
H
H2N
H2N
AlaAspGluPheLeu
AlaAspGluPheLeu
COOH
N
H
N
H
O
P
= Wang
N3
N
OR
OR
H
1b
2b: R = n-Bu
2c: R = Me
Route II
O
P(On-Bu)3, DMSO
95% TFA
H2N
AlaAspGluPheLeu
N3
COOH
N
quantitative
conversion
H
(B)
PG
PG
AspAspValAlaGlu
O
O
P(OR)3, DMF
H2N
H2N
AspAspValAlaGlu
N
N
H
H
O
P
= Rink Amide
N
H
OR
N3
OR
1c
1. Fmoc SPPS
2. 95% TFA
O
H
N
CysArgProIleGluAspAspGlnGluVal
AspAspValAlaGlu
H2N
CONH2
N
H
O
O
P
2d: R = Me (12 %)
N
OR
2e: R = Bn(2-NO2) (10 %)
H OR
Scheme 3. (A) Preparationofpeptidephosphoramidatesinsolution(RouteII)andonthesolidsupport(RouteI).(B)Synthesisofpeptidephosphoramidates
2d and 2e on the solid support.
B 40–50 min. Phosphoramidate peptide 2b, tris(2-nitrobenzyl)
phosphite and tris(4(2,5,8,11,14-pentaoxahexadecan-16-yloxy)-5-
methoxy-2-nitrobenzyl) phosphite were prepared according to
the literature protocols [20,34].
and lyophilized to give white solids (12 mg, 12% and 11 mg, 10%,
respectively); HRMS for 2d: m/z = 1001.4174 [M + 2H]2+, calcd.:
1001.4170; HRMS for 2e: m/z = 1122.4383 [M + 2H]2+, calcd.:
1122.4337.
Reactions of Resin Bound Peptides
Reactions of Peptides in Solution
Resin bound peptides 2b and 2c were prepared from 1b (25 µmol)
upon overnight incubation at RT in DMSO (1.5 ml) contained
tributyl-andtrimethylphosphite(125 µmol),respectively.Peptides
2b and 2c were cleaved from the resin to give white solids (24 mg,
90% crude and 21 mg, 86% crude, respectively), and they were
analyzed by LC-UV, to show that desired phosphoramidates were
the only detectable products (Supporting Information). Identities
of products were confirmed by HRMS, 2b: m/z = 948.4369 [M
+ H]+, calcd.: 948.4532; 2c: m/z = 864.3625 [M + H]+, calcd.:
864.3593. Resin bound peptides 2d and 2e were prepared
upon overnight incubation at RT of 1c (50 µmol) in DMF
with trimethyl- and tris(2-nitrobenzyl) phosphite (250 µmol),
respectively, followed by standard Fmoc SPPS. Peptides 2d and
2e were cleaved from the resin, purified by semi-preparative HPLC
To a solution of crude peptide 3a released from the resin (6.8 mg,
5.4 µmol) in 8 M HCl (300 µl) at 0 ◦C, NaNO2 (0.7 mg, 10 µmol)
in water (50 µl) was added. The solution was stirred for 15 min,
followed by the addition of NaN3 (1.1 mg, 15 µmol) in water
(50 µl). After 15 min at 0 ◦C, the mixture was neutralized with
aqueous NaOH (1 M), and peptide 1a was isolated by preparative
HPLC, followed by lyophilization to give a white solid (2.8 mg,
41% yield of the transformation in solution; 29% overall yield
including SPPS). HRMS for 1a: m/z = 1273.5801 [M + H]+, calcd.:
1273.5808. Peptide 2a (400 mg) was prepared from 1a upon
overnight incubation with tris(4-(2,5,8,11,14-pentaoxahexadecan-
16-yloxy)-5-methoxy-2-nitrobenzyl) phosphite (8 mg, 5.5 µmol) in
tris buffer (400 µl, 1 M, pH 8.2) at RT. Peptide 2a was isolated from
the mixture via preparative HPLC and lyophilized to give a white
c
wileyonlinelibrary.com/journal/psc Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2010; 16: 563–567

PHOSPHORAMIDATE-PEPTIDE SYNTHESIS BY STAUDINGER-PHOSPHITE REACTIONS
solid (0.45 mg, 69%). HRMS for 2a: m/z = 1079.4680 [M + 2H]2+
,
17 Deiters A, Cropp TA, Summerer D, Mukherji M, Schultz PG. Site-
specific PEGylation of proteins containing unnatural amino acids.
Bioorg. Med. Chem. Lett. 2004; 14: 5743–5745.
calcd.: 1079.4662.
18 Musiol HJ, Dong S, Kaiser M, Bausinger R, Zumbusch A, Bertsch U,
Moroder L. Toward semisynthetic lipoproteins by convergent
strategies based on click and ligation chemistry. ChemBioChem.
2005; 6: 625–628.
19 Iddon B, Meth-Cohn O, Scriven EFV, Suschitzky H, Gallagher PT.
Developments in arylnitrene chemistry: syntheses and mechanisms.
Angew. Chem. Int. Ed. 1979; 18: 900–917.
20 Serwa R, Wilkening I, Del Signore G, Mu¨hlberg M, Claussnitzer I,
Weise C, Gerrits M, Hackenberger CPR. Chemoselective Staudinger-
phosphite reaction of azides for the phosphorylation of proteins.
Angew. Chem. Int. Ed. 2009; 48: 8234–8239.
21 Isaad ALC, Papini AM, Chorev M, Rovero P. Side chain-to-side chain
cyclization by click reaction. J. Pept. Sci. 2009; 15: 451–454.
22 Poot AJ, van Ameijde J, Slijper M, van den Berg A, Hilhorst R,
Ruijtenbeek R, Rijkers DTS, Liskamp RMJ. Development of selective
bisubstrate-based inhibitors against protein kinase C (PKC) isozymes
by using dynamic peptide microarrays. ChemBioChem. 2009; 10:
2042–2051.
23 Wieland T, Dungen AV, Birr C. Peptide syntheses .51. Antamanide
.9. Synthesis of an antitoxic variant of antamanide with P-
azidophenylalanine in position 6. Liebigs Ann. Chem. 1971; 752:
109–114.
24 Escher E,Couture R,Champagne G,Mizrahi J,Regoli D.Synthesisand
biological activities of photoaffinity labeling analogs of substance P.
J. Med. Chem. 1982; 25: 470–475.
25 Escher EHF, Nguyen TMD, Robert H, St-pierre SA, Regoli DC.
Photoaffinity labeling of angiotensin-II receptor. 1. Synthesis and
biological activities of labeling peptides. J. Med. Chem. 1978; 21:
860–864.
26 Fahrenholz F, Toth G, Crause P, Eggena P, Schwartz IL. [1,6-
Alpha-aminosuberic acid, 3-(para-azidophenylalanine), 8-arginine]
vasopressin: a new photoaffinity label for hydroosmotic hormone. J.
Biol. Chem. 1983; 258: 14861–14867.
Acknowledgements
The authors acknowledge financial support from the German
Science Foundation (DFG) within the Emmy-Noether program (HA
4468/2-1), the SFB 765 and the Fonds der Chemischen Industrie
(FCI). We thank Silvia Muth for experimental contributions and
helpful discussions.
Supporting information
Supporting information may be found in the online version of this
article.
References
1 Bra¨se S, Gil C, Knepper K, Zimmermann V. Organic azides: an
exploding diversity of a unique class of compounds. Angew. Chem.
Int. Ed. 2005; 44: 5188–5240.
2 Agard NJ, Prescher JA, Bertozzi CR. A strain-promoted [3 + 2] azide-
alkyne cycloaddition for covalent modification of biomolecules in
living systems. J. Am. Chem. Soc. 2004; 126: 15046–15047.
3 Rostovtsev VV,Green LG,Fokin VV,Sharpless KB.AstepwiseHuisgen
cycloaddition process: copper(I)-catalyzed regioselective ‘‘ligation’’
of azides and terminal alkynes. Angew. Chem. Int. Ed. 2002; 41:
2596–2599.
4 Saxon E, Bertozzi CR. Cell surface engineering by a modified
Staudinger reaction. Science. 2000; 287: 2007–2010.
5 Hackenberger CPR, Schwarzer D. Chemoselective ligation and
modification strategies for peptides and proteins. Angew. Chem.
Int. Ed. 2008; 47: 10030–10074.
6 Jessani N, Cravatt BF. The development and application of methods
for activity-based protein profiling. Curr. Opin. Chem. Biol. 2004; 8:
54–59.
27 Hasegawa M, Hidaka Y, Matsumoto Y, Sanni T, Shimonishi Y.
Determination of the binding site on the extracellular domain of
guanylyl cyclase C to heat-stable enterotoxin. J. Biol. Chem. 1999;
274: 31713–31718.
28 Landis G, Lui G, Shook JE, Yamamura HI, Burks TF, Hruby VJ.
Synthesis of highly mu-opioid and delta-opioid receptor selective
peptides containing a photoaffinity group. J. Med. Chem. 1989; 32:
638–643.
29 Mackiewicz Z, Belisle S, Bellabarba D, Gallopayet N, Lehoux JG,
Lagace G, Escher E. Synthesis and placental binding potencies
of photosensitive analogues of luteinizing-hormone-releasing
hormone (Lhrh) with agonistic and antagonistic structures. Helv.
Chim. Acta 1987; 70: 423–429.
7 Prescher JA, Bertozzi CR. Chemistry in living systems. Nat. Chem. Biol.
2005; 1: 13–21.
8 van Swieten PF, Leeuwenburgh MA, Kessler BM, Overkleeft HS.
Bioorthogonal organic chemistry in living cells: novel strategies
for labeling biomolecules. Org. Biomol. Chem. 2005; 3: 20–27.
9 Tornoe CW, Christensen C, Meldal M. Peptidotriazoles on solid
phase: [1,2,3]-triazoles by regiospecific copper(I)-catalyzed 1,3-
dipolar cycloadditions of terminal alkynes to azides. J. Org. Chem.
2002; 67: 3057–3064.
10 Agard NJ, Prescher JA, Bertozzi CR. A new bioorthogonal ligation for
labelling of azide-bearing glycoconjugates. Glycobiology. 2004; 14:
1197–1197.
30 Yonezawa H, Wada T. Photoaffinity-labeling of pepsin. Bull. Chem.
Soc. Jpn. 1989; 62: 3730–3732.
31 Kurose T, Pashmforoush M, Yoshimasa Y, Carroll R, Schwartz GP,
Burke GT, Katsoyannis PG, Steiner DF. Cross-linking of
a B25
11 Comstock LR, Rajski SR. Methyltransferase-directed DNA strand
azidophenylalanine insulin derivative to the carboxyl-terminal
region of the α-subunit of the insulin receptor. J. Biol. Chem. 1994;
269: 29190–29197.
scission. J. Am. Chem. Soc. 2005; 127: 14136–14137.
12 Dube DH,Prescher JA,Quang CN,Bertozzi CR.Profilingglycosylation
using azidosugars in vivo. Glycobiology. 2004; 14: 1073–1074.
13 Chin JW, Santoro SW, Martin AB, King DS, Wang L, Schultz PG.
Addition of P-azido-L-phenylalanine to the genetic code of
Escherichia coli. J. Am. Chem. Soc. 2002; 124: 9026–9027.
14 Kiick KL, Saxon E, Tirrell DA, Bertozzi CR. Incorporation of azides
into recombinant proteins for chemoselective modification by the
Staudinger ligation. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 19–24.
15 Wang Q, Chan TR, Hilgraf R, Fokin VV, Sharpless KB, Finn MG.
Bioconjugation by copper(I)-catalyzed azide-alkyne [3 + 2]
cycloaddition. J. Am. Chem. Soc. 2003; 125: 3192–3193.
16 Dieterich DC, Link AJ, Graumann J, Tirrell DA, Schuman EM. Selective
identification of newly synthesized proteins in mammalian cells
using bioorthogonal noncanonical amino acid tagging (BONCAT).
Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 9482–9487.
32 Garbay-Jaureguiberry C, Robichon A, Dauge V, Rossignol P,
Roques BP. Highly selective photoaffinity labeling of µ and δ opioid
receptors. Proc. Natl. Acad. Sci. U.S.A. 1984; 81: 7718–7722.
33 Huang K, Chan SJ, Hua Q, Chu Y, Wang R, Klaproth B, Jia W,
Whittaker J, De Meyts P, Nakagawa SH, Steiner DF, Katsoyannis PG,
Weiss MA. The A-chain of insulin contacts the insert domain of the
insulin receptor. J. Biol. Chem. 2007; 282: 35337–35349.
34 Arslan T, Mamaev SV, Mamaeva NV, Hecht SM. Structurally modified
firefly luciferase. Effects of amino acid substitution at position 286.
J. Am. Chem. Soc. 1997; 119: 10877–10887.
c
J. Pept. Sci. 2010; 16: 563–567 Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/psc