Stereospecific synthesis of a carbene-generating angiotensin II analogue for comparative photoaffinity labeling: Improved incorporation and absence of methionine selectivity

Article abstract of DOI:10.1021/jm050958a

A stereospecific convergent synthesis of N-[(9-fluorenyl)methoxycarbonyl]- p-[3-(trifluoromethyl)-3H-diazirin-3-yl]-L-phenylalanine (Fmoc-12, Fmoc-Tdf) and its incorporation into the C-terminal position of the angiotensin II (AngII) peptide to form ¹²⁵I[Sar¹,Tdf⁸]AngII ( ¹²⁵I-13) is presented. This amino acid photoprobe is a highly reactive carbene-generating diazirine phenylalanine derivative that can be used for photoaffinity labeling. Using model receptors, we compared the reactivity and the Met selectivity of 12 to that of the widely used and reputedly Met-selectivep-benzoyl-L-phenylalanine (Bpa) photoprobe. Wild-type and mutant AngII type 2 receptors, a G protein-coupled receptors, were photolabeled with ¹²⁵I-13 as well as with ¹²⁵I-[Sar¹,Bpa ⁸]AngII (¹²⁵I-14), and the respective incorporation yields were assessed. The carbene-generating 12 was more reactive toward inert residues and was not Met-selective compared to the biradical ketone-generating Bpa, allowing for more precise determination of ligand contact points in peptidergic receptors.

Full text of DOI:10.1021/jm050958a

2200
J. Med. Chem. 2006, 49, 2200-2209
Stereospecific Synthesis of a Carbene-Generating Angiotensin II Analogue for Comparative
Photoaffinity Labeling: Improved Incorporation and Absence of Methionine Selectivity
Dany Fillion, Maud Derae¨t, Brian J. Holleran, and Emanuel Escher*
Department of Pharmacology, Faculte´ de Me´decine et des Sciences de la Sante´, UniVersite´ de Sherbrooke,
Sherbrooke, Que´bec, Canada J1H 5N4
ReceiVed September 26, 2005
A stereospecific convergent synthesis of N-[(9-fluorenyl)methoxycarbonyl]-p-[3-(trifluoromethyl)-3H-diazirin-
3-yl]-L-phenylalanine (Fmoc-12, Fmoc-Tdf) and its incorporation into the C-terminal position of the
angiotensin II (AngII) peptide to form 125I[Sar1,Tdf8]AngII (125I-13) is presented. This amino acid photoprobe
is a highly reactive carbene-generating diazirine phenylalanine derivative that can be used for photoaffinity
labeling. Using model receptors, we compared the reactivity and the Met selectivity of 12 to that of the
widely used and reputedly Met-selective p-benzoyl-L-phenylalanine (Bpa) photoprobe. Wild-type and mutant
AngII type 2 receptors, a G protein-coupled receptors, were photolabeled with 125I-13 as well as with 125I-
[Sar1,Bpa8]AngII (125I-14), and the respective incorporation yields were assessed. The carbene-generating
12 was more reactive toward inert residues and was not Met-selective compared to the biradical ketone-
generating Bpa, allowing for more precise determination of ligand contact points in peptidergic receptors.
Introduction
Bpa8]AngII (14), where Bpa is introduced instead of the native
Phe in the C-terminal position of the peptide, is a neutral
antagonist of the human angiotensin II (AngII) type 1 receptor
(hAT1),16 whereas native AngII is a full agonist. This may
reduce the usefulness of Bpa in the analysis of the conforma-
tional changes associated with receptor activation. Second,
previous studies by us and others have shown that Bpa has a
pronounced selectivity for Met residues in receptors.17-22 This
reduces the precision of the photolabeling since Bpa may
selectively photolabel more distant Met residues instead of the
nearest non-Met residues. We recently exploited this unique Met
selectivity of Bpa to introduce a new method, the methionine
proximity assay (MPA), for scanning the contact areas of
peptidergic GPCRs and confirming suspected peptide-GPCR
contact points.22,23
G protein-coupled receptors (GPCRs), also known as hepta-
helical receptors, are the largest family of cell surface receptors
mediating multiple responses to a large variety of hormonal and
sensory signals.1 As common characteristic, GPCRs possess
seven transmembrane domains (TMDs) that constitute the
structural scaffold of these transmembrane proteins. Elucidating
the mechanisms by which GPCRs carry out their biological
functions requires a detailed molecular understanding of ligand-
receptor interactions leading to receptor activation and signal
transduction. The most widely used methods to probe receptor
structures include classic structure-activity relationships, com-
putational molecular modeling, site-directed mutagenesis, mag-
netic resonance spectroscopies, X-ray crystallography, and direct
labeling methods such as photoaffinity labeling.2,3 Over the past
three decades, photoaffinity labeling has emerged as an impor-
tant biochemical method for the direct mapping of peptide-
protein interactions.4-7 Following the binding of the photo-
reactive ligand within its cognate receptor, the photoprobe is
photoactivated by its conversion into a highly reactive inter-
mediate that causes covalent bonding within its immediate
molecular surroundings, presumably at its contact area within
the receptor. This method provides an insight into dynamic
systems under physiological conditions by making it possible
to analyze the exact incorporation locus of the photoprobe in
the receptor. This information may then be used in conjunction
with techniques such as computational molecular modeling
procedures8,9 to design and validate molecular models of
liganded receptors. Several recent reviews on the applications
of photoaffinity labeling for drug discovery and development
highlight this trend.10-12
To circumvent the selective photolabeling properties of Bpa,
alternative photolabeling chemical groups with different pho-
tochemical properties have to be used.24 These include nitrene-
producing arylazides and carbene-producing aryldiazirines.
Nonsubstituted arylnitrene groups, however, undergo an in-
tramolecular rearrangement leading to an electrophilic cyclic
ketenimine intermediate that may react with distant nucleophiles
due to its relatively long lifespan.25 Trifluoromethylaryldiazirine
groups have been reported to generate highly reactive carbenes
that react with virtually all 20 amino acids with no intra-
molecular rearrangements.26 However, the challenging, time-
consuming synthesis of the phenylalanine derivative p-[3-
(trifluoromethyl)-3H-diazirin-3-yl]-L-phenylalanine (12, Tdf)27-30
has until now prevented more widespread use of this photoprobe.
We present herein a new stereospecific convergent synthesis
procedure for N-[(9-fluorenyl)methoxycarbonyl]-p-[3-(tri-
fluoromethyl)-3H-diazirin-3-yl]-L-phenylalanine (Fmoc-12, Fmoc-
Tdf), its incorporation into the C-terminal position of the AngII
peptide, and the radioiodination of the resulting peptide to form
125I[Sar1,Tdf8]AngII (125I-13). To investigate the photolabeling
properties of the diazirine analogue 125I-13 and the benzophe-
none analogue 125I-14, we assessed the respective incorporation
yields on the wild-type (wt) and mutant AngII type 2 model
receptor (AT2) of human origin (hAT2). Previous studies
demonstrated that 125I-14 simultaneously photolabels Met128
Most photoaffinity labeling studies of peptidergic GPCRs
have been conducted with photoprobes containing the benzo-
phenone moiety within the phenylalanine derivative p-benzoyl-
L-phenylalanine (Bpa).13-15 However, Bpa has two important
drawbacks. First, the bulky size of the benzophenone moiety
may interfere with the ligand recognition site and/or the
biological activity of the target protein. For instance, [Sar1,-
* To whom correspondence should be addressed. Phone: (819) 564-
5346. Fax: (819) 564-5400. E-mail: Emanuel.Escher@USherbrooke.ca.
10.1021/jm050958a CCC: $33.50 © 2006 American Chemical Society
Published on Web 03/15/2006

[Sar1,Tdf8]AngII Synthesis and Photoaffinity Labeling
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7 2201
Scheme 1. Tdf Synthesisa
a Reagents and conditions: (a) n-BuLi, CF3CO2Et, Et2O, 4 h, -78 °C/rt, 81%; (b) NH2OH‚HCl, EtOH, Pyr, 2 h, reflux, 94%; (c) TsCl, Pyr, 2 h, reflux,
94%; (d) NH3, ET2O, 12-16 h, -78 °C/rt, 96%; (e) Fmoc-OSu/1,4-dioxane, Na2CO3/H2O, 24 h, 0 °C/rt, 92%; (f) DCC, t-BuOH, Cu(I)Cl, CH2Cl2, 74 h,
rt, 67%; (g) Me(PhO)3PI, N,N-DMF, 1 h, rt, 88% or ClPh2P, imidazole, I2, CH2Cl2, 1 h, 53%; (h) Zn*, Pd2(dba)3, P(o-tol)3, N,N-DMF, 3 h, rt, 55%; (i)
Ag2O, ET2O, 8 h, rt, 87%; (j) CF3CO2H, Et3Si, CH2Cl2, 3 h, rt, 81%; (k) SPPS using Wang resin, Na125I, IODO-GEN iodination reagent (1,3,4,6-tetrachloro-
3R,6R-diphenylglycoluril), 1500 Ci/mmol.
and Met138 within TDM3 on the hAT2-wt receptor.22,31 This
unique methionine environment makes AT2 an ideal model
system in which to compare reactivity and Met selectivity of
photoprobes. The AT2 receptor is a glycosylated32 peptidergic
GPCR of 363 amino acids with a protein-only molecular mass
of 41 kDA and whose pharmacological and physiological
functions are still somewhat controversial.33-36
yliodoaryl 5.38 When done at atmospheric pressure, this step
resulted in lower yields.
The chiral moiety of 12, compound 6, was orthogonally
diprotected and iodinated prior to the palladium-catalyzed
coupling reaction with 5. Protecting the NH2 group of 6 with
N-[(9-fluorenyl)methoxycarbonyloxy]succinimide resulted in a
high yield of monoprotected amino acid 7, which was used
without further purification.41 Subsequent protection of the
CO2H group of 7 using a mixture of dicyclohexylcarbodiimine,
tert-butyl alcohol, and copper(I) chloride generated a high yield
of diprotected amino acid 8.42 In our hands, this tert-butylation
method was the least expensive and most effective. Compound
8 was iodinated to form diprotected â-iodoalanine 9 through a
reaction with either methyltriphenoxyphosphonium iodide43 or
a mixture of chlorodiphenylphosphine, imidazole, and iodine,44
which resulted in high to moderate yields, respectively.
The key step of this synthesis procedure was the palladium-
catalyzed coupling reaction of the p-diaziridinyliodoaryl deriva-
tive 5 with the diprotected â-iodoalanine-derived zinc reagent
Zn-9. Compound 5 was coupled to 9 in a moderate yield reaction
using activated Zn* and tris(dibenzylideneacetone)dipalladium-
(0)/tri-o-tolylphosphine catalyst to generate the diprotected
phenylalanine diaziridine 10.45 Oxidation of 10 using silver
oxide generated a high yield of diprotected phenylalanine
diazirine 11.38,39 Last, deprotection of the CO2H group of 11
using a mixture of R,R,R-trifluoroacetic acid and triethylsilane
resulted in a good yield of Fmoc-12.45
Chemistry
Others have reported the synthesis of 12. One approach
involves the alkylation of a glycine derivative with p-[3-
(trifluoromethyl)-3-aryldiazirine]benzyl halide under basic con-
ditions followed by a wasteful enzymatic resolution of the N-acyl
amino acid.27,28 A second approach uses a noncommercial Ni
complex chiral auxiliary.29,30 We report here the stereospecific
convergent synthesis of Fmoc-12 using commercial p-diiodo-
benzene (1) and L-serine (6) as starting materials. The key step
is a palladium-catalyzed coupling reaction of a p-diaziridi-
nyliodoaryl derivative (5) with an orthogonally diprotected
â-iodoalanine organozinc reagent (Zn-9). Fmoc-12 was incor-
porated into the C-terminal position of AngII, which was
radioiodinated to form radiophotoreactive 125I-13 (Scheme 1).
The photoreactive moiety of 12, compound 5, was synthesized
in four steps according to the literature.37,38 Compound 1 was
converted into ketone 2 in good yields by reacting the
corresponding organolithium salt with ethyl R,R,R-trifluoro-
acetate and was used without further purification.37 Attempts
to synthesize 2 by reacting the organolithium reagent with
R,R,R-trifluoroacetic anhydride resulted in poor yield. Reacting
2 with hydroxylamine hydrochloride yielded the corresponding
oxime 3,38 which was subsequently, without further purification,
converted into the corresponding tosyl oxime 4 in high yields
through a reaction with p-toluenesulfonyl chloride.38,39 Mesy-
lation of 4 has been reported to be superior to tosylation since
fewer byproducts are formed.40 In the present study, the two
methods provided equivalent results. The direct reaction of 2
with hydroxylamine tosyl oxime is an alternative method for
synthesizing 4. Reacting 4 with ammonia at high pressure
generated a high yield of the corresponding pure p-diaziridin-
Fmoc-12 was incorporated into AngII by esterification to
hydroxyl-Wang resin, followed by chain elongation by standard
solid-phase peptide synthesis using the Fmoc/piperidine pro-
cedure.46 The resulting photoreactive 13 was radioiodinated47
to form radiophotoreactive 125I-13 with a specific activity of
approximately 1500 Ci/mmol.
Results and Discussion
The precision of the photolabeling process may be reduced
considerably if chemical selectivity of the photoprobe is present.
Selective photoprobes may photolabel distant reactive residues
instead of inert residues in their immediate surroundings. The

2202 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7
Fillion et al.
Figure 1. Amino acid sequence and photochemistry of (A) native AngII, (B) 125I-13, and (C) 125I-14. The photogenerated carbene of 13 is an
electrophilic electron-deficient sextet species displaying irreversible photoactivation to the singlet state.13-15 The photogenerated biradical ketone
of Bpa is an electrophilic electronic transition from an oxygen nonbonding sp2-like n-orbital to a carbonyl group antibonding π*-orbital displaying
reversible photoactivation to the triplet state.26-28
present study was thus undertaken to evaluate the Met selectivity
and reactivity of carbene-generating 12 compared to the widely
used biradical ketone-generating Bpa, two amino acid photo-
probes (Figure 1).
13 and 125I-14 photolabeled the fragment containing Met128
and Met138 within TMD3 of the hAT2 receptor. Photolabeled
Met residues are resistant to CNBr cleavage when labeled on
the γ-methylene49 and undergo slower cleavage but release a
methyl isothiocyanate derivative of the photoprobe when labeled
at the ꢀ-methyl group.17 The CNBr cleavage of the hAT2-wt
receptor photolabeled with both AngII analogues produced, for
the benzophenone analogue 125I-14, fragment Phe129-Met170
with the photoprobe attached to Met138 (lane 3, apparent
molecular mass 6.2 kDa, expected molecular mass 6.1 kDa),
fragment Cys117-Met138 with the photoprobe attached to
Met128 (lane 3, apparent molecular mass 4.2 kDa, expected
molecular mass 3.5 kDa), and the released photoprobe (lane 3,
apparent molecular mass less than 3.4 kDa, expected molecular
mass 1.1 kDa). The same fragments were also observed with
the diazirine analogue 125I-13, that is, Phe129-Met170 (lane
5, apparent molecular mass 6.2 kDa, expected molecular mass
6.1 kDa), fragment Cys117-Met138 (lane 5, apparent molecular
mass 4.2 kDa, expected molecular mass 3.5 kDa), and also the
released photoprobe (lane 5, apparent molecular mass below
3.4 kDa, expected molecular mass 1.1 kDa), but to a lower
degree than with the 125I-14 photolabeled receptor. The insertion
of 125I-13 within the sequence Phe129-Met138 would result
in a 2.2 kDa fragment, which appeared to be present (lane 5).
Finally, the CNBr cleavage of the hAT2-M128,138L double-
mutant receptor photolabeled with both AngII analogues
produced a single large fragment, Cys117-Met170 (lanes 4 and
6, apparent molecular mass 6.4 kDa, expected molecular mass
7.4 kDa), but no smaller fragments.
To investigate the reactivity and Met selectivity of diazirine
analogue 125I-13 and benzophenone analogue 125I-14, several
hAT2 mutant receptors were constructed. The endogenous
Met128 and Met138 residues in TMD3, the previously deter-
mined contacts of the C-terminal amino acid for 125I-14 in the
hAT2 receptor,22,31 were replaced either one by one or simul-
taneously by Ala residues or by more isosteric Leu residues
(Figure 2). The pharmacological parameters describing the
equilibrium binding to wild-type and mutant receptors of both
125I-13 and 125I-14 were compared. Both AngII analogues had
practically identical high binding affinities (Kd) on the wild-
type and mutant hAT2 receptors that were comparable to those
of the AngII peptide (Table 1). All the mutant receptors had
maximum binding capabilities (Bmax) that were similar to that
of the wild-type receptor. We were thus able to directly compare
the incorporation yields of these model receptors using both
AngII analogues. In general, replacing Met residues by hydro-
phobic Leu or Ala residues in helical TMDs does not perturb
the global conformation of the mutant protein and is of little
consequence to overall receptor properties and hence ligand
interactions.48
To ensure that the diazirine analogue 125I-13 photolabeled
the same hAT2 receptor regions as the benzophenone analogue
125I-14, the hAT2-wt receptor and the isosteric hAT2-M128,-
138L double-mutant receptor were photolabeled with both AngII
analogues and were then subjected to CNBr cleavage, a
C-terminal Met-specific reagent (Figure 3). The SDS-PAGE
CNBr cleavage patterns were identical, indicating that both 125I-
To assess the Met selectivity and reactivity of 12 and Bpa,
quantitative photoaffinity labeling experiments were performed
using the model receptors (Figure 4 and Table 2). Met selectivity

[Sar1,Tdf8]AngII Synthesis and Photoaffinity Labeling
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7 2203
Figure 2. (A) 2D schematic representation of the primary structure of the hAT2 receptor. Endogenous Met128 and Met138, which were mutated
for Leu and Ala, are represented. Putative sites of N-glycosylation on Asn4, Asn13, Asn24, Asn29, and Asn34 are also indicated. Rhodopsin-
homology molecular modeling according to Rihakova et al.22 showing the endogenous Met128 and Met138 residues in TMD3 interacting with both
photoactivated AngII analogues: (B) 125I-13; (C) 125I-14.
Table 1. Pharmacological Parameters of the Wild-Type and Mutant
Incorporation yield time-course experiments with both AngII
analogues on the hAT2-wt and the hAT2-M128,138L double-
hAT2 Receptorsa
Kd (nM)
125I-14
mutant receptors revealed that the incorporation yield was
optimal after 15 min of irradiation for the diazirine analogue
125I-13, but needed to be extended up to 60 min for the
benzophenone analogue 125I-14 (Figure 5). Diazirine photolysis
is irreversible and photodissociative, leading to the loss of N2
and the generation of carbene. In contrast, benzophenone
photolysis is reversible and may involve many excitation-
relaxation cycles until a favorable conformation for covalent
bonding is achieved. Indeed, prolonged irradiation appears to
increase the incorporation yield. Benzophenone-photolabeled
proteins do not necessarily reflect the principal incorporation
locus of the photoprobe since the repetitive nature of photo-
activation together with the Met selectivity may result in the
predominant photolabeling of less preferred conformations and,
consequently, skewed conclusions.
Bmax
(pmol/mg)
125I-13
AngII
hAT2-wt
hAT2-M128L
hAT2-M138L
2.1 ( 0.3 2.3 ( 0.3 0.21 ( 0.02
1.1 ( 0.1 1.2 ( 0.1 0.30 ( 0.03
1.2 ( 0.1 1.1 ( 0.1 0.32 ( 0.03
5.2 ( 0.5
3.8 ( 0.4
6.9 ( 0.6
5.2 ( 0.4
3.0 ( 0.3
6.5 ( 0.5
1.8 ( 0.2
hAT2-M128,138L 1.2 ( 0.1 1.2 ( 0.1 0.13 ( 0.01
hAT2-M128A
hAT2-M138A
1.2 ( 0.1 1.1 ( 0.1 0.11 ( 0.01
1.1 ( 0.1 1.2 ( 0.1 0.25 ( 0.02
hAT2-M128,138A 1.2 ( 0.1 1.2 ( 0.1 0.30 ( 0.03
a See the Experimental Section for details. Affinities (Kd) are presented
in nM, and maximum binding capacities (Bmax; established with 125I-13)
are presented in pmol/mg of protein in the sample. The means ( SE are
shown for three independent experiments, each with duplicate determina-
tions.
could be quantified if a significant reduction in incorporation
occurred on the hAT2-M128,138L double-mutant receptor
compared to the hAT2-wt receptor. The benzophenone analogue
125I-14 resulted in a 57% incorporation yield with the hAT2-wt
receptor compared to 5% with the M f L double-mutant
receptor. The diazirine analogue 125I-13 photolabeled the hAT2-
wt receptor and the M f L double-mutant receptor with similar
efficiencies of 24% and 21%, respectively. Both AngII ana-
logues photolabeled the single-mutant hAT2-M128L, hAT2-
M138L, hAT2-M128A, and hAT2-M138A receptors to the same
extent. The incorporation yields of the single mutants were
similar to that of the hAT2-wt receptor, although yields of the
M f A single-mutant receptors tended to be lower, but remained
well within the range of the M f L single-mutant receptors.
The higher reactivy of 12 over biradical-generating Bpa could
also be evidenced on the basis of their photolabeling efficiencies
with respect to the hAT2-M138,138A double-mutant receptor.
An incorporation yield of 6% was observed for 125I-13 compared
to less than 1% for 125I-14.
These results show, on one hand that, unlike Bpa, carbene-
generating 12 is not Met-selective. The benzophenone analogue
125I-14 has a greater than 10-fold preference of incorporation
for Met residues on the hAT2-wt receptor compared to Leu
residues on the hAT2-M128,138L double-mutant receptor,
whereas the diazirine analogue 125I-13 has the same incorpora-
tion efficiency with both receptors. On the other hand, the fact
that 125I-13 has a 4-6-fold higher photolabeling efficiency than
125I-14 with the hAT2-M128,138L and hAT2-M128,138A double-
mutant receptors indicates that 12 was much more reactive
toward non-Met residues than Bpa. The hAT2-M128,138A
double-mutant receptor reduces the close van der Waals contact
between the C-terminal position of AngII and the hAT2 receptor
in this unique microenvironment only and not elsewere. Some
studies have reported that Bpa may have an incorporation yield
of up to 90% of the specific binding capacity of the target
protein.14 These high incorporation yields may be due to the
intrinsically more reactive residues in the target protein and/or

2204 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7
Fillion et al.
Table 2. Incorporation Yields of 125I-13 and 125I-14 on Model Receptorsa
incorporation yield (%)
incorporation yield (%)
125I-13
125I-14
125I-13
125I-14
hAT2-wt
24 ( 2
21 ( 2
6 ( 1
57 ( 6
5 ( 1
<1 ( 1
46 ( 5
hAT2-M138L
hAT2-M128A
hAT2-M138A
24 ( 2
19 ( 2
20 ( 2
49 ( 5
41 ( 4
43 ( 4
hAT2-M128,138L
hAT2-M128,138A
hAT2-M128L
22 ( 2
a See the Experimental Section for details. Each AngII analogue-hAT2 receptor interaction was specific and decreased in a dose-dependent manner in
the presence of AngII (results not shown). The means ( SE are shown for five independent experiments.
Figure 3. (A) SDS-PAGE autoradiography after C-terminal Met-specific CNBr cleavage of the photolabeled 125I-13/hAT2 and 125I-14/hAT2
complexes. hAT2-wt receptor and isosteric hAT2-M128,138L double-mutant receptor transfected cell membranes were photolabeled with either
125I-13 or 125I-14, solubilized, partially purified, and submitted to CNBr cleavage. The CNBr cleavage pattern was analyzed by 16.5% T/3% C
Tris-Tricine/SDS-PAGE followed by autoradiography (see the Experimental Section for details). Key: lane 1, 125I-14/hAT2-wt - CNBr; lane 2,
125I-13/hAT2-wt - CNBr; lane 3, 125I-14/hAT2-wt + CNBr; lane 4, 125I-14/hAT2-M128,138L + CNBr; lane 5, 125I-13/hAT2-wt + CNBr; lane 6,
125I-13/hAT2-M128,138L + CNBr; lane 7, 125I-14 and 125I-13. 14C-labeled low molecular mass protein standards were run in parallel. Each AngII
analogue-hAT2 receptor interaction was specific and decreased in a dose-dependent manner in the presence of AngII (results not shown). This
figure is representative of four independent experiments. (B) 2D schematic representation of the primary structure of the hAT2 receptor showing
the CNBr-generated fragments in black closed circles.
to its Met selectivity. Several reports have also demonstrated
that the 3-(trifluoromethyl)-3-aryldiazirine group has comparable
photolabeling properties, but it is not widely used due to its
difficult synthetic access.6,7,12,50-54
The photochemical basis of the Met selectivity of benzophe-
none-containing Bpa has already been studied.55-57 Met-
containing peptides quench the triplet biradical ketone of the
excited benzophenone via a charge-transfer complex at the sulfur
atom. This mechanism seems to be effective when nonbonding
electron pairs of heteroatoms are proximal to the triplet biradical
ketone.14,15 Incorporation yields in Met are increased since this
electron transfer is more than 104-fold quicker than toward Ala
residues. Indeed, photolabeling by Bpa of the double-Met
environment in the hAT2-wt receptor resulted in the highest

[Sar1,Tdf8]AngII Synthesis and Photoaffinity Labeling
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7 2205
it allows rapid determination of the ligand-receptor contact
point when used in scanning approaches such as MPA.22,23 The
different photolabeling properties of both photoprobes are the
main reasons why photolabeling of the same membrane receptor
with different photoprobes has led to contradictory results and
conclusions. Keeping in mind that an ideal photoprobe should
efficiently photolabel residues in its immediate contact, regard-
less of the chemical nature of these residues, it is of importance
to have access to several complementary photoprobes.
Figure 4. SDS-PAGE autoradiography showing the incorporation
yields of both 125I-13 and 125I-14 in the glycosylated hAT2 receptor.
Receptor-transfected cell membranes were photolabeled with either 125I-
13 or 125I-14, solubilized, and analyzed by 7.5% Tris-glycine/SDS-
PAGE followed by autoradiography (see the Experimental Section for
details). Key: lane 1, 125I-13/hAT2-wt; lane 2, 125I-14/hAT2-wt; lane
3, 125I-13/hAT2-M128L; lane 4, 125I-14/hAT2-M128L; lane 5, 125I-13/
hAT2-M138L; lane 6, 125I-14/hAT2-M138L; lane 7, 125I-13/hAT2-M128,-
138L; lane 8, 125I-14/hAT2-M128,138L; lane 9, 125I-13/hAT2-M128A;
lane 10, 125I-14/hAT2-M128A; lane 11, 125I-13/hAT2-M138A; lane 12,
125I-14/hAT2-M138A; lane 13, 125I-13/hAT2-M128,138A; lane 14, 125I-
14/hAT2-M128,138A; lane 15, 125I-13 + 125I-14/Mock. Prestained
broad-range protein standards were run in parallel. Each AngII
analogue-hAT2 receptor interaction was specific and decreased in a
dose-dependent manner in the presence of AngII (results not shown).
The means ( SE are shown for five independent experiments.
Experimental Section
Chemistry. Starting materials and reagents were from Sigma-
Aldrich Canada Ltd. and were used without further purification
unless otherwise specified. Solvents, hydrochloric acid, acetic acid,
tert-butyl alcohol, iodine, sodium hydroxide, and silver nitrate were
from Fisher Scientific Ltd. Tris(dibenzylideneacetone)dipalladium-
(0) was from Alfa Aesar. N-[(9-Fluorenyl)methoxycarbonyloxy]-
succinimide and Wang resin (p-benzyloxybenzyl alcohol resin) were
from Novabiochem (EMD Biosciences). IODO-GEN iodination
reagent (1,3,4,6-tetrachloro-3R,6R-diphenylglycoluril) was from
Pierce Biotechnology Inc. Na125I was from Perkin-Elmer Canada
Inc. Solvents were distilled and dried in a nitrogen atmosphere
according to standard procedures.59 n-Butyllithium (1.6 M solution
in n-hexanes) was titrated according to the standard procedure.60
All reactions requiring anhydrous conditions were conducted under
a positive argon atmosphere in vacuumed flame-dried glassware
using standard syringe techniques. Analytical thin-layer chroma-
tography (TLC) was carried out on precoated (0.25 mm) Merck
silica gel 60f-254 plates. Flash chromatography was performed on
Merck silica gel 60 (230-400 mesh) using air pressure according
to the standard procedure.61 Spots were detected by fluorescence
quenching at 254 or 350 nm, treating with iodine and/or a revealing
solution (sulfuric acid-molybdic acid, cerium ammonium nitrate,
or potassium permanganate-potassium carbonate), and heating.
Reversed-phase high-pressure liquid chromatography (RP-HPLC)
purification was performed using a Waters 625 LC instrument
equipped with a Waters C-18 reversed-phase column monitored
by absorbance at 214 nm as well as by a γ-radioisotope detector.
NMR spectra were obtained using a Bruker AC-300 instrument
(300 MHz). Chemical shifts are reported in δ values (ppm) relative
to the peak for chloroform (300 MHz adjusted to 7.26 ppm for 1H
NMR and 62.5 MHz adjusted to 77.0 ppm for 13C NMR) as an
internal standard. Coupling constants (J) are in hertz. The following
abbreviations were used for the NMR spectra assignments: s )
singlet; d ) doublet; t ) triplet; q ) quartet; m ) multiplet. The
melting points of crystalline compounds were determined using a
Bu¨chi M-50 apparatus and are uncorrected. Optical rotations were
measured using a Perkin-Elmer 141 polarimeter at the sodium D
line (589 nm) with a 10.00 cm and 1.0 mL cell. The mass spectra
of amino acid derivatives were obtained using a VG Micromass
ZAB-2F instrument with electronic ionization (70 eV) and of
peptides using a Tofspec2 Micromass instrument with matrix-
assisted laser desorption ionization time of flight (MALDI-TOF).
Last, photoreactive diazirine derivatives were handled in dim yellow
light in flasks covered with aluminum foil.
Figure 5. Time-course experiments of the incorporation yields of 125I-
13 and 125I-14 on the hAT2-wt and isosteric hAT2-M128,138L double-
mutant receptors. Receptor-transfected cell membranes were photo-
labeled for 5-180 min with either 125I-13 or 125I-14, solubilized, and
analyzed by 7.5% Tris-glycine/SDS-PAGE followed by autoradiog-
raphy (see the Experimental Section for details). Each AngII analogue-
hAT2 receptor interaction was specific and decreased in a dose-
dependent manner in the presence of AngII (results not shown). The
means ( SE are shown for five independent experiments.
incorporation yield of all the model receptors. A systematic
study of photoalkylation of amino acids by benzophenone
showed that Met was the preferred target.58 In fact, efficient
hydrogen abstraction by Bpa only occurs from benzylic positions
or from R-CH to heteroatoms.15 Obviously, for both 12 and
Bpa, some chemical selectivity may arise from steric constraints
and from the chemical nature of the surrounding residues.
Conclusions
3-(p-Iodophenyl)-3-(trifluoromethyl)diaziridine (5).37,38 Com-
pound 5 was synthesized in four steps starting from 1 according to
the literature.37,38
The herein presented synthetic access to Fmoc-12 will
undoubtedly increase its heretofor quite limited use in receptor
photoaffinity labeling studies. Compared to biradical ketone-
generating Bpa, carbene-generating 12 shows an absence of Met
selectivity and an improved incorporation yield into unreactive
residues of GPCRs. Given that the TMDs of GPCRs are
extremely rich in residues containing inert aliphatic side chains,
this difference in reactivity and chemical selectivity of both 12
and Bpa is of particular importance since the absence of
methionine in the binding pocket of a receptor (e.g., hAT2-
M128,138A) can result in apparent failure to photolabel
sufficiently through a ligand containing the benzophenone
labeling moiety. Bpa will however maintain its importance since
N-[(9-Fluorenyl)methoxycarbonyl]-L-serine (7).41 Compound
7 was synthesized in one step starting from 6 according to the
literature.41
N-[(9-Fluorenyl)methoxycarbonyl]-L-serine tert-Butyl Ester
(8).42 Dicyclohexylcarbodiimide (680 mg, 3.3 mol equiv), tert-butyl
alcohol (401 µL, 4.2 mol equiv), and copper(I) chloride (7 mg,
0.07 mol equiv) were placed in an argon-purged, three-necked,
round-bottom flask fitted with a magnetic stirrer and a condenser.
The suspension was stirred at room temperature for 72 h until the
reaction had ended as judged by TLC (ethyl acetate (3)/petroleum
ether (7)). The dark green suspension was diluted in anhydrous
methylene chloride (2.0 mL, 0.5 M final), and compound 7 (327

2206 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7
Fillion et al.
mg, 1.0 mol equiv) was added. The resulting suspension was purged
with argon and was stirred at room temperature for 2 h until the
reaction had ended as judged by TLC (ethyl acetate (3)/petroleum
ether (7)). The reaction mixture was filtered, and the white
dicyclohexylurea precipitate was washed with methylene chloride
(2 × 2 mL). The filtrate was combined with the reaction mixture,
which was diluted with ethyl acetate (6 mL). The organic layer
was washed with a saturated CaCO3 solution (12 mL) and brine
(2 × 12 mL). The ethyl acetate/methylene chloride solution was
dried over anhydrous magnesium sulfate, vacuum filtered, and
evaporated under reduced pressure. The crude product was purified
by flash chromatography on silica gel using ethyl acetate/methylene
chloride as an eluant, leaving pure compound 8 as a white crystalline
solid. Yield: 67% (257 mg). Mp: 122-123 °C. 1H NMR (CDCl3,
300 MHz, δ (ppm)): 7.77 (d, J ) 7.4 Hz, 2H, H4,5-Fmoc), 7.61
resulting suspension was stirred at room temperature for 30 min
until the reaction had ended as judged by TLC (ethyl acetate (2)/
petroleum ether (8)). Compound 5 (314 mg, 1.0 mol equiv), the
catalyst tris(dibenzylideneacetone)dipalladium(0) (27 mg, 0.03 mol
equiv), and the ligand tri-o-tolylphosphine (47 mg, 0.15 mol equiv)
were quickly added to the Zn-9 suspension. The mixture was
purged with argon and stirred at room temperature for 1 h until the
reaction had ended as judged by TLC (ethyl acetate (2)/n-hexanes
(8)). The reaction mixture was filtered through a Celite pad from
which the Zn* was washed with ethyl acetate (3 × 3 mL). The
filtrate was combined with the reaction mixture, and the organic
layer was washed with a saturated sodium thiosulfate solution
(2 × 9 mL) and brine (2 × 9 mL). The ethyl acetate solution was
dried over anhydrous magnesium sulfate, vacuum filtered, and
evaporated under reduced pressure. The crude product was purified
by flash chromatography on silica gel using ethyl acetate/petroleum
ether as an eluant, leaving pure compound 10 as a dark red oil.
Yield: 55% (304 mg). Mp: 88-89 °C. 1H NMR (CDCl3, 350 MHz,
(d, J ) 7.4 Hz, 2H, H1,8-Fmoc), 7.41 (t, J ) 7.4 Hz, 2H, H3,6
-
Fmoc), 7.32 (t, J ) 7.4 Hz, 2H, H2,7-Fmoc), 5.68 (d, J ) 6.6 Hz,
1H, NH), 4.42 (d, J ) 7.0 Hz, 2H, O-CH2-Fmoc), 4.33 (m, 1H,
R-CH), 4.23 (t, J ) 6.9 Hz, 1H, CH-Fmoc), 3.94 (m, 2H, â-CH2),
2.51 (s, O-H), 1.49 (s, 9H, t-Bu).
δ (ppm)): 7.77 (d, J ) 7.5 Hz, 2H, H4,5-Fmoc), 7.55 (m, 4H, H1,8
-
Fmoc + m-H), 7.41 (t, J ) 7.3 Hz, 2H, H3,6-Fmoc), 7.31 (t, J )
7.3 Hz, 2H, H2,7-Fmoc), 7.20 (d, J ) 7.4 Hz, 2H, o-H), 5.29 (d, J
) 7.8 Hz, 1H, NH), 4.55 (∼q, J ) 6.4 Hz, 1H, R-CH), 4.47 (td, J
) 8.8, 2.0 Hz, 1H, OCHH-Fmoc), 4.34 (t, J ) 8.7 Hz, 1H, OCHH-
Fmoc), 4.20 (t, 1H, J ) 6.8 Hz, CH-Fmoc), 3.12 (d, J ) 5.6 Hz,
2H, â-CH2), 2.78 (d, J ) 8.0 Hz, 1H, NH-NH), 2.16 (d, J ) 9.0
Hz, 1H, NH-NH), 1.40 (s, 9H, t-Bu).
N-[(9-Fluorenyl)methoxycarbonyl)]-L-(â-iodo)alanine tert-
Butyl Ester (9). First Protocol.43 Compound 8 (383 mg, 1.0 mol
equiv), methyltriphenoxyphosphonium iodide (497 mg, 1.1 mol
equiv), and anhydrous N,N-dimethylformamide (2.0 mL, 0.5 M
final) were placed in an argon-purged round-bottom flask fitted
with a magnetic stirrer. The resulting solution was stirred at room
temperature for 1 h until the reaction had ended as judged by TLC
(ethyl acetate (3.0)/petroleum ether (7.0)). The reaction mixture was
diluted with ethyl acetate (4 mL), and the organic layer was washed
with distilled water (6 mL), a saturated sodium thiosulfate solution
(6 mL), and a saturated CaCO3 solution (6 mL). The ethyl acetate
solution was dried over anhydrous magnesium sulfate, vacuum
filtered, and evaporated under reduced pressure. The crude product
was purified by flash chromatography on silica gel using ethyl
acetate/methylene chloride as an eluant, leaving pure compound 9
as a slightly yellow crystalline solid. Yield: 88% (434 mg). Mp:
N-[(9-Fluorenyl)methoxycarbonyl]-p-[3-(trifluoromethyl)-3H-
diazirin-3-yl]-L-phenylalanine tert-Butyl Ester (11).38,39 Com-
pound 10 (554 mg, 1.0 mol equiv), freshly prepared silver oxide
(see below; 1.16 g, 5.0 mol equiv), and diethyl ether (4.0 mL, 0.25
M final concentration) were placed in a round-bottom flask fitted
with a magnetic stirrer and a condenser. The suspension was stirred
at room temperature for 8 h in the dark until the reaction had ended
as judged by TLC (ethyl acetate (3)/petroleum ether (7)). The
reaction mixture was diluted with diethyl ether (4 mL), dried over
anhydrous magnesium sulfate, and filtered through a Celite pad to
remove the silver, silver oxide, and magnesium sulfate, which were
washed with diethyl ether (3 × 8 mL). The filtrate was combined
with the reaction mixture, and the diethyl ether solution was
evaporated under reduced pressure, leaving crude 11 as a red oil,
which was used for the last step without further purification. The
compound may be further purified by flash chromatography on
silica gel using ethyl acetate/petroleum ether as an eluant to obtain
pure product compound 11 as a white crystalline solid. Yield: 87%.
1
81-83 °C. H NMR (CDCl3, 300 MHz, δ (ppm)): 7.77 (d, J )
7.4 Hz, 2H, H4,5-Fmoc), 7.62 (d, J ) 7.4 Hz, 2H, H1,8-Fmoc), 7.41
(t, J ) 7.1 Hz, 2H, H3,6-Fmoc), 7.33 (tt, J ) 7.4, 1.6 Hz, 2H, H2,7
-
Fmoc), 5.69 (d, J ) 7.1 Hz, 1H, NH), 4.46-4.32 (m, 3H, R-CH +
OCH2-Fmoc), 4.25 (t, 1H, J ) 7.1 Hz, CH-Fmoc), 3.61 (d, J )
3.4 Hz, 2H, â-CH2), 1.53 (s, 9H, t-Bu).
Second Protocol.44 Compound 8 (383 mg, 1.0 mol equiv) and
anhydrous methylene chloride (2.0 mL, 0.5 M final) were placed
in an argon-purged round-bottom flask fitted with a magnetic stirrer.
In the following order, chlorodiphenylphosphine (269 µL, 1.5 mol
equiv), imidazole (102 mg, 1.5 mol equiv), and iodine (380 mg,
1.5 mol equiv) were then added. The suspension was purged with
argon and was stirred at room temperature for 1 h until the reaction
had ended as judged by TLC (ethyl acetate(4.5)/n-hexanes (5.0)/
acetic acid (0.5)). The reaction mixture was diluted with methylene
chloride (4 mL), and the organic layer was washed with distilled
water (6 mL), a saturated sodium thiosulfate solution (6 mL), and
a saturated CaCO3 solution (6 mL). The methylene chloride solution
was dried over anhydrous magnesium sulfate, vacuum filtered, and
evaporated under reduced pressure. The crude product was purified
by flash chromatography on silica gel using ethyl acetate/methylene
chloride as an eluant, leaving pure compound 9 as a slightly yellow
crystalline solid. Yield: 53% (261 mg).
1
Mp: 85-86 °C. H NMR (CDCl3, 350 MHz, δ (ppm)): 7.78 (d,
J ) 7.5 Hz, 2H, H4,5-Fmoc), 7.58 (dd, 2H, J ) 1.7, 0.8 Hz, H1,8
-
Fmoc), 7.42 (t, J ) 7.4 Hz, 2H, H3,6-Fmoc), 7.32 (td, J ) 7.4, 1.1
Hz, 2H, H2,7-Fmoc), 7.12 (dd, J ) 19.8, 8.3 Hz, 4H, m-H + o-H),
5.41 (d, J ) 7.9 Hz, 1H, NH), 4.50 (m, 2H, R-CH + OCHH-Fmoc),
4.35 (∼t, J ) 8.7 Hz, 1H, OCHH-Fmoc), 4.20 (t, J ) 6.8 Hz, 1H,
CH-Fmoc), 3.10 (d, J ) 5.9 Hz, 2H, â-CH2), 1.40 (s, 9H, t-Bu).
Silver Oxide Solution. A 1 M solution of sodium hydroxide
(13 mL, 1.1 mol equiv) was added dropwise to a 1 M boiling
solution of silver nitrate (2.0 g, 1.0 mol equiv) with stirring. The
precipitated silver oxide was filtered, washed with distilled water
(15.0 mL), acetone (15.0 mL), and diethyl ether (15.0 mL), and
used immediately.
N-[(9-Fluorenyl)methoxycarbonyl]-p-[3-(trifluoromethyl)-3H-
diazirin-3-yl]-L-phenylalanine (Fmoc-12).45 Compound 11 (552
mg, 1.0 mol equiv), methylene chloride (10.0 mL, 0.1 M final),
triethylsilane (242 µL, 1.5 mol equiv), and R,R,R-trifluoroacetic
acid (3.9 mL, 50.0 mol equiv, ∼40% (v/v)) were placed in a round-
bottom flask fitted with a magnetic stirrer and a condenser. The
solution was stirred at room temperature for 3 h until the reaction
had ended as judged by TLC (ethyl acetate (1.0)/methylene chloride
(8.9)/acetic acid (0.1)). The reaction mixture was washed with water
(2 × 10 mL) and brine (2 × 10 mL). The methylene chloride
solution was dried over anhydrous magnesium sulfate, vacuum
filtered, and evaporated under reduced pressure. The crude product
was purified by flash chromatography on silica gel using ethyl
acetate (1.0)/methylene chloride (8.9)/acetic acid (0.1) as an eluant,
N-[(9-Fluorenyl)methoxycarbonyl)]-p-[3-(trifluoromethyl)-
diaziridine]-L-phenylalanine tert-Butyl Ester (10).45 Zinc dust
(325 mesh; 196 mg, 3.0 mol equiv), chlorotrimethylsilane (64 µL,
0.5 mol equiv), and anhydrous N,N-dimethylformamide (400 µL,
resulting in a 7.5 M solution) were placed in an argon-purged,
flame-dried, round-bottom flask fitted with a magnetic stirrer. The
suspension was vigorously stirred at room temperature for 30 min.
The stirring was stopped to let the Zn* decant for 1 h. The
chlorotrimethylsilane and N,N-dimethylformamide were then re-
moved using a syringe. A solution of compound 9 (493 mg, 1.0
mol equiv) in anhydrous N,N-dimethylformamide (0.5 mL, 2.0 M
final) was added dropwise to the activated anhydrous Zn*. The

[Sar1,Tdf8]AngII Synthesis and Photoaffinity Labeling
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7 2207
leaving pure compound Fmoc-12 as a white crystalline solid. An
alternative purification method involves the precipitation of the
compound dissolved in a minimum volume of methylene chloride
at room temperature by the addition of petroleum ether at 0 °C
°C in a 5% CO2 atmosphere until the density reached 6 × 106 cells/
dish. Following trypsinization, 1.2 × 106 cells were suspended in
a final volume of 10.0 mL of the same medium and grown for 24
h. The transfection conditions were performed according to the
manufacturer’s instructions. Briefly, the cells were transitorily
transfected by adding a solution of FuGENE6 transfection reagent
(6 µL) and plasmid DNA (3 µg) in 300 µL of serum-free DMEM,
all of which had been previously incubated for 30 min at room
temperature. Transfected cells were grown for an additional 36 h
at 37 °C in a 5% CO2 atmosphere. The cells were washed once
with phosphate-buffered saline (PBS; 137 mM NaCl, 0.9 mM
MgCl2, 3.5 mM KCl, 0.9 mM CaCl2, 8.7 mM Na2HPO4, and 3.5
mM NaH2PO4) and immediately stored at -80 °C until use.
Saturation Binding Assays. Cell membrane preparations and
saturation binding assays were performed essentially as previously
described.65 Frozen transfected COS-7 cells were rapidly thawed
for 1 min at 37 °C in a water bath, and 10.0 mL of ice-cold washing
buffer (5 mM MgCl2, 25 mM Trizma-Base, 100 mM NaCl; adjusted
to pH 7.4) was added to the broken cells. The cells were gently
scraped and centrifuged (500g for 15 min at 4 °C). The cell pellet
was resuspended in 12.5 mL of ice-cold binding buffer (5 mM
MgCl2, 25 mM Trizma-Base, 100 mM NaCl, 0.1% (w/v) bovine
serum albumin (BSA), and 0.01% (w/v) bacitracin; adjusted to pH
7.4), and 40 × 300 µL of the cell membrane suspension (20-40
µg of protein) was independently incubated in the dark for 60 min
at room temperature with 100.0 µL of eight different final
concentrations ranging from 5 nM (cell membrane suspensions
1-5) to 39 pM (cell membrane suspensions 36-40) of radioactive
AngII analogues diluted to 100 Ci/mmol. To each of these eight
final concentrations of radioactive AngII analogues were added
binding buffer (3 × 100 µL; total binding) and AngII (5 × 10-6
M, 2 × 100 µL; nonspecific binding) for a final volume of 500
µL. Bound radioactive AngII analogue was separated from free
radioactive AngII analogue by vacuum filtration through filters
presoaked in binding buffer for 30 min at 0 °C. The radioactivity
was evaluated by γ-radiation counting. A protein standard curve
was used to determine cellular expression levels (Bmax). The binding
curves were analyzed for affinity (Kd) using the Ligand program.66
Photoaffinity Labeling Experiments. Cell membrane prepara-
tions and photoaffinity labeling experiments were performed
essentially as previously described.65 Frozen transfected COS-7 cells
were rapidly thawed for 1 min at 37 °C in a water bath, and 10.0
mL of ice-cold washing buffer (5 mM MgCl2, 25 mM Trizma-
Base, and 100 mM NaCl; adjusted to pH 7.4) was added to the
broken cells. The cells were then gently scraped and centrifuged
(500g for 15 min at 4 °C). The cell pellet was resuspended in 500
µL of ice-cold binding buffer (5 mM MgCl2, 25 mM Trizma-Base,
100 mM NaCl, 0.1% (w/v) bovine serum albumin (BSA), and
0.01% (w/v) bacitracin; adjusted to pH 7.4). The cell membrane
suspension (800 µg to 1.6 mg of protein) was incubated in the dark
for 60 min at room temperature in the presence of 107 cpm of
radioactive AngII analogues (∼1500 Ci/mmol). The cell membranes
were centrifuged (500g for 15 min at 4 °C). The pellet was
resuspended in 500.0 µL of ice-cold washing buffer and irradiated
for 5, 15, 45, 60, 120, or 180 min on ice under filtered UV light
(365 nm; mercury vapor lamp JC-Par-38, no. 5873, Westinghouse
and Raymaster black light filters, Gates and Co. Inc.). The cell
membranes were centrifuged (2500g for 15 min at 4 °C), and the
pellet was solubilized for 1 h on ice in modified radioimmunopre-
cipitation buffer (mRIPA buffer; 0.1% (w/v) SDS (electrophoresis
grade), 0.25% (w/v) sodium deoxycholate, 1% (v/v) Nonidet P-40,
5 mM NaN3, 50 mM Trizma-HCl (pH 7.4), and 150 mM NaCl)
supplemented with Complete Protease Inhibitor Cocktail. The cell
lysate was centrifuged (2500g for 30 min at 4 °C) to remove
insoluble materials, and the supernatant was stored at -80 °C until
use.
with vigorous stirring followed by filtration. Yield: 81% (397 mg).
1
Mp: 129-130 °C. [R]25 (c ) 1.06, CHCl3): +36.70. H NMR
D
(CDCl3, 350 MHz, δ (ppm)): 7.78 (d, J ) 7.6 Hz, 2H, H4,5-Fmoc),
7.55 (d, J ) 7.1 Hz, 2H, H1,8-Fmoc), 7.42 (t, J ) 7.3 Hz, 2H,
3,6-Fmoc), 7.30 (td, J ) 7.4, 1.1 Hz, 2H, H2,7-Fmoc), 7.11 (q, J
H
) 8.3 Hz, 4H, m-H + o-H), 5.16 (d, J ) 8.0 Hz, 1H, NH), 4.68 (q,
J ) 7.5 Hz, 1H, R-CH), 4.51 (∼t, J ) 8.7 Hz, 1H, OCHH-Fmoc),
4.40 (∼t, J ) 8.7 Hz, 1H, OCHH-Fmoc), 4.20 (∼t, J ) 6.5 Hz,
1H, CH-Fmoc), 3.17 (ddd, J ) 21.0, 13.8, 5.6 Hz, 2H, â-CH2).
13C NMR (CDCl3, 75 MHz, δ (ppm)): 174.75, 155.61, 143.63,
141.35, 137.30, 129.82, 127.80, 127.07, 126.64, 124.93, 120.04,
66.93, 54.22, 47.11, 37.21, 33.76, 31.98. EI-HRMS: m/z calcd for
C26H20N3O4F3 [M + H]+ 495.1406, found [M + H]+ 495.1397 (
0.0015.
125I[Sar1,Tdf8]AngII (125I-13).46,47 Compound 12 was synthe-
sized using the standard solid-phase peptide synthesis method with
N-[(9-fluorenyl)methoxycarbonyl] (Fmoc)-protected amino acids
and Wang resin (p-benzyloxybenzyl alcohol resin). Once the
synthesis was completed and the side chain protecting group and
resin linker were simultaneously cleaved, crude compound 13 was
purified by RP-HPLC and was eluted as a single signal with a
minimum of 95% purity. Compound 13 was characterized by
MALDI-TOF-LRMS analysis as previously described46 and showed
m/z 1097 [M + H]+ (m/z calcd for [Sar1,Tdf8]AngII [M + H]+
1097). The resulting photoreactive peptides were radioiodinated
using the IODO-GEN (1,3,4,6-tetrachloro-3R,6R-diphenylglycoluril)
iodination method as described elsewere62 and as previously
described47 to form radiophotoreactive 125I-13. In brief, in the
following order, 50 mM sodium phosphate buffer (pH 5.0, 30.0
µL, 20 mM final), 10-3 M AngII analogue (10 µL, 1.0 mol equiv),
and 45 µM (5 mCi) Na125I (10.0 µL, 1 mCi, 0.045 mol equiv) were
added to an Eppendorf tube coated with IODO-GEN (20.0 µg, 4.6
mol equiv) in a final volume of 50.0 µL. The solution was incubated
at room temperature for 15 min in the dark. The reaction conditions
were optimized to form monoiodinated AngII analogues. The
reaction mixture was quenched by adding a 50 mM sodium
phosphate solution (50.0 µL) and incubated for a further 15 min.
The radioiodinated AngII analogues were purified by RP-HPLC
with a 20-40% acetonitrile gradient in 0.05% aqueous R,R,R-
trifluoroacetic acid. The specific radioactivity of the labeled peptides
was approximately 1500 Ci/mmol as determined by self-displace-
ment and saturation binding analyses.63
125I[Sar1,Bpa8]AngII (125I-14). Compound 125I-14 was synthe-
sized, purified, and characterized as previously described.46,47
Biochemistry. Oligodeoxynucleotides, bovine serum albumin
(BSA), bacitracin, and all other organic chemicals were from Sigma-
Aldrich Ltd. All inorganic chemicals were from Fisher Scientific
Ltd. pcDNA3.1 plasmid was from Invitrogen Inc. Site-directed
mutagenesis materials, FuGENE 6 transfection reagent, Nonidet
P-40, and Complete Protease Inhibitor Cocktail were from Roche
Diagnostics. Cell culture materials and 14C-labeled low molecular
mass protein standards were from Gibco BRL. Electrophoresis
materials and prestained broad-range protein standards were from
Bio-Rad Laboratories Ltd. X-ray films (BioMaxMR) were from
Kodak. Binding filters (1.0 µm grade GF/B glass microfiber,
Whatman) were from VWR International Inc.
Oligodeoxynucleotide Site-Directed Mutagenesis. Site-directed
mutagenesis was performed on the hAT2-wt receptor with the
overlap extension PCR method essentially as previously described.64
Briefly, mutant receptors were subcloned into the HindIII-XbaI
sites of the mammalian expression vector pcDNA3.1. Site-directed
mutations were then confirmed by automated DNA sequencing.
Cell Culture and Transfections. COS-7 cells were grown in
10.0 mL of Dulbecco’s modified Eagle’s medium (DMEM)
containing 2 mM L-glutamine, 10% (v/v) fetal bovine serum (FBS),
100 IU/mL penicillin, and 100 µg/mL streptomycin. The cells were
seeded into 100 mm diameter culture dishes and incubated at 37
Incorporation Yield and Incorporation Yield Time-Course
Experiments. The solubilized labeled proteins were diluted with
Laemmli loading buffer (final concentration: 0.1% (w/v) bromo-
phenol blue, 2% (w/v) SDS, 10% (v/v) glycerol, 50 mM Trizma-
HCl (pH 6.8), and 100 mM dithiothreitol (DTT)) and incubated

2208 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7
Fillion et al.
for 1 h at 37 °C. Tris-glycine/SDS-polyacrylamide gel electro-
phoresis (SDS-PAGE) was performed essentially as described
elsewere67 using 7.5% analytical gels. Running conditions and gel
fixation procedures were performed according to the manufacturer’s
instructions. To localize the labeled proteins (80-200 kDa), the
dried gel was exposed to an X-ray film with an intensifying screen.
Prestained broad-range protein standards were used to determine
the apparent molecular masses (Mr). The incorporation yield of
specific binding was calculated as follows on the same cell
membrane preparations: [photolabeled receptor/ligand complex
from Tris-glycine/SDS-PAGE (cpm)]/[specific binding (cpm)] ×
100%. The specific activities of the radioiodinated AngII analogues
did not have to be taken into account in the calculation since the
photoaffinity labeling experiments to determine the value of
photolabeled receptor/ligand complex from Tris-glycine/SDS-
PAGE (cpm) and the saturation binding assay to determine the value
of specific binding (cpm) were performed using either compound
125I-13 or compound 125I-14.
Supporting Information Available: 1H NMR, 13C NMR, and
EI-HRMS data for 12 as well as RP-HPLC and MALDI-TOF
LRMS data for compounds 13 and 14. This material is available
free of charge via the Internet at http://pubs.acs.org.
References
(1) Kristiansen, K. Molecular mechanisms of ligand binding, signaling,
and regulation within the superfamily of G-protein-coupled recep-
tors: molecular modeling and mutagenesis approaches to receptor
structure and function. Pharmacol. Ther. 2004, 103, 21-80.
(2) Wess, J. Structure-Function Analysis of G Protein-Coupled Recep-
tors. In Receptor Biochemistry and Methodology Series; Sibley, D.
R., Ed.; John Wiley & Sons: New York, 1999; p 432.
(3) Jahn, O.; Eckart, K.; Tezval, H.; Spiess, J. Characterization of
peptide-protein interactions using photoaffinity labeling and LC/
MS. Anal. Bioanal. Chem. 2004, 378, 1031-1036.
(4) Bayley, H. Photogenerated Reagents in Biochemistry and Molecular
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Biology; Work, T. S., Burdon, R. H., Eds.; Elsevier: Amsterdam/
New York/Oxford, 1983; p 188.
Partial Purification of the Photolabeled Receptor/Radioligand
Complex. Partial purification of the photolabeled receptor/radio-
ligand complex was performed essentially as previously described.65
The solubilized labeled proteins were diluted with Laemmli loading
buffer (final concentration: 0.1% (w/v) bromophenol blue, 2%
(w/v) SDS, 10% (v/v) glycerol, 50 mM Trizma-HCl (pH 6.8),
and 100 mM DTT) and incubated for 1 h at 37 °C. Tris-glycine/
SDS-PAGE was performed essentially as described elsewere67
using 7.5% preparative gels. Running conditions and gel fixation
procedures were performed according to the manufacturer’s instruc-
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exposed to an X-ray film with an intensifying screen. Prestained
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Acknowledgment. We are grateful to Marie-Reine Lefebvre
for her expert help and technical assistance for peptide synthesis
and to Dr. Pierre Deslongchamps for access to his facilities.
This work was supported by the Canadian Institutes of Health
Research. E.E. is a recipient of the J.C. Edwards Chair in
Cardiovascular Research. This paper was submitted to fulfill
the requirements of a Ph.D. thesis for D.F. at the Universite´ de
Sherbrooke.

[Sar1,Tdf8]AngII Synthesis and Photoaffinity Labeling
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