New beta-strand macrocyclic peptidomimetic analogues containing alpha-(O-, S- or NH-)aryl substituted glycine residues: synthesis, chemical and enzymatic properties.

Article abstract of DOI:10.1039/b211644h

In so much as bis-macrocyclic peptidomimetics have been recognized as high affinity substrates for HIV-1 protease, we were interested in the design and synthesis of new bis-macrocyclic bioisosteric analogues whose general structure is displayed on Fig. 2. The structures of these new analogues are characterized by the specific replacement of the methylene of the benzyl group directly attached to the N-acyl glycine residue in the original molecule 1, by its main bioisosteres, i.e. O-, S- and NH-aryl groups. Knowing that an intermediate in which an heteroatomic aryl group is directly linked to a free amine glycine residue is not stable, we developed an original synthetic pathway which involved the coupling of a specific side chain to the exocyclic carboxylic acid function, followed by an elegant oxidation-nucleophilic substitution Steglich-type reaction. Analogues 2a-d were then submitted to chemical and enzymatic hydrolysis. We demonstrated that, as expected, the specific cleavage of the exocyclic N-acyl bond led to the release of aryl moieties (phenol, thiophenol and aniline species). These chemical and enzymatic stability studies brought to light the biological potential of such macrocyclic analogues in infected cells.

Full text of DOI:10.1039/b211644h

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New ꢀ-strand macrocyclic peptidomimetic analogues containing
ꢁ-(O-, S- or NH-)aryl substituted glycine residues:
synthesis, chemical and enzymatic properties
Gilles Quéléver, Frédéric Bihel and Jean-Louis Kraus*
INSERM U-382, Developmental Biology Institute of Marseille (CNRS-INSERM-Université
de la Méditerranée- AP Marseille), Laboratoire de Chimie Biomoléculaire,
Faculté des Sciences de Luminy, case 907, 13288 Marseille Cedex 09, France
Received 27th November 2002, Accepted 20th March 2003
First published as an Advance Article on the web 7th April 2003
In so much as bis-macrocyclic peptidomimetics have been recognized as high affinity substrates for HIV-1 protease,
we were interested in the design and synthesis of new bis-macrocyclic bioisosteric analogues whose general structure
is displayed on Fig. 2. The structures of these new analogues are characterized by the specific replacement of the
methylene of the benzyl group directly attached to the N-acyl glycine residue in the original molecule 1, by its main
bioisosteres, i.e. O-, S- and NH-aryl groups. Knowing that an intermediate in which an heteroatomic aryl group is
directly linked to a free amine glycine residue is not stable, we developed an original synthetic pathway which involved
the coupling of a specific side chain to the exocyclic carboxylic acid function, followed by an elegant oxidation–
nucleophilic substitution Steglich-type reaction. Analogues 2a–d were then submitted to chemical and enzymatic
hydrolysis. We demonstrated that, as expected, the specific cleavage of the exocyclic N-acyl bond led to the release of
aryl moieties (phenol, thiophenol and aniline species). These chemical and enzymatic stability studies brought to light
the biological potential of such macrocyclic analogues in infected cells.
Introduction
Proteolytic enzymes of the four major classes (aspartyl, cysteine,
metallo and serine proteases) catalyse both intracellular and
extracellular peptide cleavages during numerous physiological
processes. They are crucial for disease propagation and their
selective inhibition appears to be a promising therapeutic route
for the treatment of diseases as various as cancers, viral infec-
tions, such as HIV, or neurodegenerative disorders.1 Recently,
Fairlie et al. provided compelling structural evidence that
aspartyl, serine, metallo and cysteine proteases all share a
common conformational requirement for recognition namely
an extended (β-strand) conformation for their active-site dir-
ected inhibitors and substrate analogues.1–7 This phenomenon
of conformational selection, which appears to be common for
proteases, can be used for the design of protease inhibitors.
Based on this conserved conformational mimicry, macrocyclic
peptidomimetics 1 (Fig. 1), designed to fix inhibitors in pro-
tease-binding conformations, have been studied.2 Taking into
account the approach proposed by Fairlie et al., we designed
and synthesized new HIV-protease substrates 2 whose general
structure is given in Fig. 2. Besides the presence of the macro-
cyclic ring, all the newly synthesized analogues include in their
structure a glycine residue substituted on the α-carbon position
by phenoxy, thiophenoxy or anilino groups. Ordinarily, a free
aminal α-substituted glycine in which the α-carbon is linked to
a nitrogen, oxygen or sulfur aryl group, is chemically unstable.
Fig. 2 General structure of new antiprotease substrates. X = O, S, NH;
R = H, alkoxy groups; RЈ = OH, ester group.
However, N-acylated α-substituted glycines of this type8,9 or
even N-acylated α-hydroxy glycines10 have been described in the
literature. Acylation of the amino group provides stabilization
of the resulting molecule by delocalising the nitrogen electrons
into the peptide bond. This synthetic approach that employs
α-substitution on a glycine residue in a peptide has already been
described by Kingsbury et al.11,12 (Fig. 3) and previously used by
our group.13,14
Our findings showed that if the exocyclic peptide bond is
more specifically cleaved by HIV-1 protease, the result would be
the release of the α-substituent. Thanks to this enzymatic
specificity, selected phenols,15 thiophenols or anilines,16 present
as α-heteroatom aryl substituents of the glycine residue could
then lead to the release of toxophoric moieties which could
affect HIV-1 protease processing and the fate of the infected
cell.
In this paper, we report the synthesis of these new analogues,
their comparative stabilities towards basic chemical hydrolysis
and the HIV-1 protease substrate properties of some of
them.
Results and discussion
We first synthesized the bis-macrocyclic substrate 1 as reported
by Fairlie et al.2 using an elegant method in which the last step
is the coupling reaction between the carboxylic acid function of
Fig. 1
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 1 6 7 6 – 1 6 8 3
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 3
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Fig. 3 Structure of α-glycine-substituted peptides and their mode of breakdown after peptidase cleavage.11–13
the characteristic peaks corresponding to the deuterated NMR
solvent, i.e. DMSO-d6.
This synthesis was performed in order to confirm that only
the exocyclic amide bond between the two macrocyclic moieties
was specifically cleaved upon incubation with HIV-1 recombin-
ant protease. This fact was proven by HPLC analysis using
both separate macrocyclic entities 3 and 4 as internal references.
In contrast, this bicyclic substrate 1 was found to be very stable
under alkaline hydrolysis even after several hours in the
presence of a large excess of sodium hydroxide.
Unfortunately, this elegant synthetic method cannot be used
for the synthesis of our new bioisosteres 2 (Fig. 2). Indeed, as
already mentioned, a free aminal glycine residue bearing a good
leaving group at the α-carbon position is chemically unstable,
spontaneously decomposing leading to the release of the
α-substituent (Fig. 3). For this reason, we cannot perform
the synthesis of both separate entities, i.e. on the one hand the
macrocyclic acid 3 and, on the other hand, the free aminal
α-substituted glycine residue, followed by the coupling reaction
between these two entities as the final reaction step.
To introduce this α-substituent at this specific position, we
were obliged to use a different synthetic pathway involving the
synthesis of a linear peptidic side-chained analogue of the
macrocyclic carboxylic acid 3. The very important feature of
this original strategy is based on the fact that this side-chain
moiety can be modified on the desired position, i.e. on the
α-carbon position linked to the macrocyclic moiety, allowing
the formation of an α-branched side-chain derivative.
For this purpose, the side-chain peptide must bear a function
that can be removed by a nucleophilic agent to perform the
bioisosteric replacement of the methylene group of the tyrosine
moiety by its bioisosteres, i.e. O, S and N atoms.
An analysis of the literature showed that α-acetoxyglycine
peptides are good substrates for this kind of nucleophilic
reactions18–21 due to the presence of the acetoxy leaving group.
According to Steglich et al.,18 a broad range of seryl peptides
can be converted to α-acetoxyglycine residues by using lead
tetraacetate as the oxidizing agent. The resulting acetylated
derivatives could then be smoothly substituted in the presence
of nucleophilic agents such as chloride,18 thioalkyl and thioaryl
derivatives,18,19 or alcohols.20
The introduction of this kind of substituent on our side-
chained peptidic macrocycle appears to be feasible using this
methodology. For this purpose, it was necessary to have a seryl
residue directly linked to the macrocyclic entity. The scaffold of
the macrocyclic entity 3 was extended on its carboxylic function
by a coupling reaction with a seryl-based dipeptide 6 as sum-
marized on Scheme 2. This side-chain was then converted to a
mixture18 of its α-acetoxyglycine diastereomers by using the
Steglich’s Pb(OAc)4 oxidation methodology. The following step
consisted of the smooth elimination of the acetoxy group by
treatment with the desired nucleophilic agent, i.e. the anilino
derivative 7 which was synthesized according to a two-step
procedure, leading to the final compound 2a, as a mixture of
Scheme 1 Synthesis of bis-macrocycle 1.2,7 (i) BOP reagent, DIEA,
DMF, 12 h; 57%.2
macrocycle 32 and the amine function of macrocycle 47 using
BOP reagent17 as coupling agent (Scheme 1).
The cyclic structures of both synthons were confirmed by 1H
NMR analysis. Due to their cyclic structures, the resulting
compounds are of a rigid shape and consequently, the
aromatic protons of the tyrosine residues of each cycle are
different resulting in a four-different signal spectrum. We can
observe this pattern on the spectrum of the protected molecule
which led to the acid synthon 3. Furthermore, the two
β-protons of the tyrosine residue were not equivalent but dis-
played two different coupling constants with their single
α-neighbouring proton: on the one hand, a syn-type coupling
constant (J = 4.6 Hz) and, on the other hand, an anti-type
coupling constant (J = 12.3 Hz). We were able to determine
the syn-type coupling constant (J = 6.3 Hz) for the second
macrocyclic N-protected species, which led to the TFA salt 4.
Unfortunately, the anti-type coupling constant overlapped with
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Scheme 2 Synthesis of new α-substituted glycine analogues 2a–d. (i)
p-tosylate.H2N-()-IleOBzl, BOP reagent, DIEA, RT, 3 h; quantitative;
(ii) TFA, CH2CI2 (1 : 1), RT, 1 h; quantitative; (iii) BOP reagent, DIEA,
THF, RT, 2 h; 79%; (iv) Pb(OAc)4, molecular sieves 4 Å, EtOAc, ∆,
overnight; (v) a) 1-amino-4-[3-[N-(tert-butoxycarbonyl)amino]propan-
1-yloxy]benzene 7, CH2CI2, Et3N, ∆, 48 h; 50% from 6; b) 4-ethoxy-
phenol, CH2CI2, Et3N, RT, 48 h, 38% from 6; c) 4-methoxythiophenol,
CH2Cl2, Et3N, RT, 48 h, 47% from 6; d) aniline, CH2Cl2, Et3N, RT,
48 h, 15% from 6.
diastereomers. Separation of the diastereomeric mixture
was not achieved since our objective was first to ascertain the
relevance of our concept.
To complete this synthetic work, we attempted the ring-
closure of the conjugate 2a obtained from the anilino derivative
7 that was supposed to lead to the exact and original bioisostere
analogue of the bis-macrocyclic substrate 1 described by Fairlie
et al.2
The terminal acidic function of compound 2a was
released by hydrogenolysis of the benzyl ester protecting
group to give the free acidic side-chain macrocycle 8. The
tert-butoxycarbonyl amino protecting group was removed
by HCl treatment of this ω-N-protected acid 8 producing
the deprotected ω-amino-acid 9 as an hydrochloride salt
(Scheme 3).
Unfortunately, we were not able to accomplish the final ring-
closure step to complete the desired synthesis despite various
attempts including BOP, DCC–HOBt22 or HATU–HOAt23,24
as coupling agents in high dilution conditions.24 The crude
materials obtained from these reactions were analyzed by MS
and LC-MS. Both reaction mixtures displayed no trace of the
desired bicyclic system but did show a main peak which was
an MS signal characteristic for the macrocyclic acid 3 (m/z 363
(M ϩ H)ϩ).
This unfortunate chemical degradation occurring during the
final ring-closure step of this synthesis is in line with our
expectations and in agreement with Kingsbury et al.11 Indeed,
the exocyclic amide bond between the macrocyclic entity and
the α-anilino substituted glycine residue was hydrolyzed, releas-
ing an unstable gem-aryl diamino species which regenerates the
Scheme 3 Attempt of synthesis of the original amino bioisoster of
bis-macrocyclic substrate 1. (i) H2 (1 atm), 10% Pd–C, THF–MeOH
(1 : 3), RT, 12 h; 89%; (ii) HCl, Et2O, RT, 6 h; 90%.
aniline derivative according to the mechanism described by
Kingsbury et al. (Fig. 3).11
This cleavage reaction was not expected during the synthesis
of the substrate but was supposed to be catalyzed by the HIV-1
protease in the infected cell to release toxophoric species able to
affect the cell viability.
As far as we could not perform the complete synthesis of the
exact bioisoster of 1, we took advantage of the previous results
to study the influence of the nature of the substitution of the
glycine residue on the release of this α-substituent.
For this purpose, we elaborated the synthesis of the novel
mono-cyclic substrates 2b–d according to the same procedure
used for the synthesis of 2a (Scheme 2). We successively substi-
tuted the α-acetoxy leaving group by an oxygen (2b), a sulfur
(2c) and a nitrogen (2d) atom. The nucleophilic agents were
carefully selected among some known toxophoric phenols,15
thiophenols and anilines16 in order to release these toxophoric
species as mentioned previously.
Basic hydrolysis assay
The unexpected hydrolytic process observed during the syn-
thetic procedure led us to study the behaviour of compound 1
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Table 1 Kinetic data for the basic hydrolysis of substrates 2a–d
Compound
–X–Ar1
λmax a/nm
t1,2 b/min
Slope/µMϪ1 minϪ1
1.2.10Ϫ7
2a
300
3528 30
2b
2c
2d
302
302
281
0.32 0.07
1.8.10Ϫ3
6.10Ϫ6
98
1
5040 60
1.7.10Ϫ7
a The suitable wavelength for each chromophoric substance was determined in the same conditions as for the hydrolysis reaction, i. e. 250 µM of the
chromophoric species in the presence of 1250 µM of aqueous sodium hydroxide in EtOH solution. b Kinetics data, i. e. half-reaction time and kinetic
constants, were calculated according to the mathematical rules for non-equimolar second-order rate reactions.
and our α-substituted glycine derivatives 2a–d towards basic
conditions. The basic hydrolysis of substrate 1 was monitored
by HPLC because of the lack of a suitable chromophoric
moiety for UV analysis. For 2a–d, we studied the release of
the chromophoric α-substituents of the side-chain glycine
residue at a suitable wavelength by UV-monitoring. The sub-
strates 2a–d were hydrolyzed in ethyl alcohol in the presence of
a large excess (5 equiv.) of aqueous sodium hydroxide. Experi-
mentally, we confirmed that these basic hydrolysis reactions
were non-equimolar second-order rate reactions. The quanti-
fication of the released chromophoric substances allowed the
calculation of the main kinetic parameters, i.e. rate constants
and half-reaction times (Table 1), according to the mathe-
matical rules concerning the kinetics of this kind of reaction.
The stability of our various substrates 2a–d and Fairlie’s bis-
macrocyclic substrate 1 towards basic hydrolysis was as
expected, dependent on the nature of the Ar1-X group:
the selected anilines that are stronger bases than the two other
oxy and thio substituents, will be weaker leaving groups.
Concerning the C–S–Ar and C–O–Ar, the polarisability
of the C–O–Ar bond is increased because of the higher
electronegativity of the oxygen atom. Therefore, the cleavage
of this bond is favoured and the rate of hydrolysis of the
phenoxy analogue 2b is enhanced compared to the thiophenoxy
derivative 2c.
Due to the presence of an electron-donating O-alkoxy sub-
stituent at the 4-position of the aniline ring in compound 2a,
the electronic density is enhanced on the nitrogen atom through
the resonance effect. Therefore, the C–NH–Ar-(4-substituted)
bond (2a) is weakened, and the leaving properties of this α-ani-
lino substituent are increased compared to the unsubstituted
anilino group (2d). The cleavage of the C–NH–Ar bond in 2a is
then favoured compared to the corresponding unsubstituted
anilino derivative 2d.
Enzymatic hydrolysis assay
Interestingly, some of these substrates lead upon enzymatic
hydrolysis processing to the specific cleavage of the exocyclic
amide bond. Indeed, 1 and 2a were incubated at 37 ЊC for
about 6 hours with the HIV-1 protease. The resulting crude
reaction mixtures were then analyzed by HPLC (1) or LC-MS
(2a).
In the first case, it was clearly demonstrated that the proteo-
lytic enzyme catalyzed specifically the complete cleavage of the
main compound with the release of the two entities, i.e. the
macrocyclic acid moiety 3 and the macrocyclic free amine entity
4. These two released species were unambiguously identified by
the use of both macrocycles 3 and 4 as internal references for
the HPLC analysis.
The effect of α-substituent groups on the rate of hydrolysis of
the exocyclic amide bond is under the control of the nucleo-
philicity constants associated with the leaving group properties
of the released moieties. Among these properties, one can list
the solvation energy of the nucleophile, the strength of its bond
to carbon, its effective size, the basicity properties of the substi-
tuent and the inductive and mesomeric effects. As far as all the
hydrolysis reactions are carried out in the same experimental
conditions of solvents and nucleophile, one can assume that
only the strength of the bond which binds the heteroatom to the
carbon at the α-position of the glycine residue is likely to affect
the rate of hydrolysis of the new analogues.
Considering the nature of the heteroatom linked to the
aliphatic carbon of the glycine residue, the phenoxy group (2b)
drastically increases the polarisability of the C–X bond
compared to the corresponding thiophenoxy group (2c). This
polarisability is far less important in the case of an anilino
substituent group (2a and 2d). Consequently, the C–NH–Ar
bond is more stable towards basic hydrolysis than the C–S–Ar
bond, which is itself far more stable than the C–O–Ar bond.
This order of stability can be related to various factors, such
as the basicity of the released species. The conjugate bases of
In the second case, as proven by a LC-MS method, 2a (m/z
797 (M ϩ H)
ϩ) was specifically processed by the enzyme into its
acidic moiety 3 (m/z 363 (M ϩ H)ϩ) and the anilino compound
7 (m/z 267 (M ϩ H)ϩ) according to the LC-MS traces of the
different compounds used for this analytical study. In this latter
case, the result confirms our expectations concerning the release
of a toxophoric anilino species, and it is also in agreement with
the results reported by Fairlie et al.2 about the substrate charac-
teristics of the macrocyclic derivatives. Indeed, due to this
specific cleavage catalyzed by the enzyme, 2a behaved as a
substrate of the HIV-1 protease.
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As demonstrated by this analysis, such reactivity can be used
to deliver toxophoric substances directly into infected cells
where it could selectively affect cell viability.
(9S,12S )-12-Carboxy-7,10-dioxo-9-isopropyl-2-oxa-8,11-diaza-
bicyclo[12.2.2]octadeca-14,16,17-triene 32
The macrocyclic carboxylic acid synthon 3 was synthesized as
summarized on Scheme 1, according to a 7-step reaction path-
way from Boc-()-tyrosine benzyl ester using the procedure
described by Fairlie et al.2 The NMR and MS analytical data
for the final compound 3 and the previous synthetic intermedi-
ates were in accordance with those described in the literature.
In conclusion, we report the synthesis of new macrocyclic
modified peptides incorporating an exocyclic glycine moiety
α-substituted by an heteroatomic-aryl group. As far as direct
introduction of such a glycine residue is not possible due to
the instability of the gem-heteroatom aryl aminal intermedi-
ate, we used an original retrosynthetic scheme which involved
the construction of the precursor by side-chain extension of
the carboxylic acid function of the first macrocyclic ring. The
ring closure of the second macrocyclic moieties of 2a was
supposed to be the final step of the total convergent synthetic
pathway of the original bioisostere analogue of the bismacro-
cyclic substrate 1 described by Fairlie et al.2 An unfortunate
chemical instability of the intermediate α-anilino substituted
glycine residue occurring during this last synthetic step
confirmed our expectations about the release of an anilino
species, as shown by the cleavage of the exocyclic amide bond
and the formation of an unstable gem-aryl diaminal species. We
further demonstrated that the α-exocyclic amide bond of this
new analogue 2a enzymatically cleaved, is responsible of the
release of the toxophoric anilino species. These results con-
firmed the substrate characteristics of this new macrocyclic
peptidomimetic containing an α-anilino substituted glycine
residue. Further interesting experiments will include studies
on the mechanism of action of this kind of macrocyclic deriv-
ative bearing α-heteroatom aryl substituted glycine moieties
to show how the release of some potent toxophoric species
could affect the HIV replication cycle in infected cells or cell
viability.
(11S,8S )-11-Amino-7,10-dioxo-8-(1-methylpropyl)-2-oxa-6,9-
diazabicyclo[11.2.2]heptadeca-13,15,16-triene trifluoroacetic
acid salt 47
The trifluoroacetic acid salt 4 was synthesized according to a
5-step reaction pathway from Boc-()-isoleucine (Scheme 1). As
previously noted for the synthesis of 3, analytical results for
each synthetic intermediate and final compound 4 were in
accordance with those described by Fairlie et al.7
Bicyclic substrate 12
The bicyclic substrate 1 was the result of the coupling reaction
between macrocyclic carboxylic acid synthon 3 and tri-
fluoroacetic acid salt 4 using BOP reagent as coupling agent
(Scheme 1). MS and NMR analytical results were in accord-
ance with those described by Fairlie et al.2
N-[(S )-Seryl]-(S )-isoleucine benzyl ester trifluoroacetic acid salt
5
Boc-()-serine (2.0 g, 9.75 mmol) was dissolved in 50 mL of
anhydrous THF with BOP reagent (4.4 g, 8.86 mmol). The reac-
tion mixture was cooled down to 0 ЊC and diisopropylethyl-
amine (1.7 mL, 8.86 mmol) was added dropwise. The solution
was allowed to warm up to room temperature, stirred for
30 minutes and cooled down to 0 ЊC. A solution of H2N-()-
IleOBzl p-tosylate salt (3.5 g, 8.86 mmol) and diisopropylethyl-
amine (3.4 mL, 17.70 mmol) in THF (10 mL) was then added
dropwise and the resulting reaction mixture was stirred at room
temperature for 3 hours. The solvent was removed under
reduced pressure and the residue was dissolved in EtOAc
(50 mL). The organic layer was washed with HCl 1 M (5 ×
20 mL), brine (20 mL), saturated NaHCO3 (5 × 20 mL) and
brine (2 × 20 mL), dried over anhydrous MgSO4, filtered and
concentrated under reduced pressure. The crude residue was
purified by flash chromatography (EtOAc–hexane 3 : 7) to give
the resulting dipeptide (3.66 g, quantitative) as a white solid. Rf
0.12 (EtOAc–hexane 3 : 7). δH (250 MHz, CDCl3) 0.62 (m, 6 H,
2 × CH3 Ile), 1.00 (m, 1 H, CHaHbγ Ile), 1.22 (m, 10 H, 3 × CH3
Boc ϩ CHaHbγ Ile), 1.70 (m, 1 H, CHβ Ile), 2.95 (m, 1 H, OH ),
3.20 (m, 2 H, CH2β Ser), 3.87 (m, 1 H, CHα Ser), 4.35 (m,
1 H, CHα Ile), 4.95 (AB, 2 H, J 12.2, –O–CH2–C6H5), 5.38
(br d, 1 H, NH ), 6.88 (m, 1 H, NH ), 7.11 (m, 5 H, –C6H5).
δC (62.9 MHz, CDCl3) 11.8, 15.8, 25.1, 28.5 (3 C), 37.7, 54.9,
57.0, 63.0, 67.3, 80.7, 128.6–128.8 (5 C), 135.5, 156.4, 171.8 (2
C). MS-ES m/z 431 (M ϩ Na)ϩ.
The previous compound (780 mg, 1.91 mmol) was dissolved
in CH2Cl2 (1.5 mL). Trifluoroacetic acid (1.5 mL, 19.10 mmol)
was added dropwise at 0 ЊC. The reaction mixture was stirred at
room temperature for 1 hour. The solvent and excess of tri-
fluoroacetic acid were removed under reduced pressure. The
resulting oil was then cooled down in the freezer for 2 hours and
allowed to warm up to room temperature to crystallize as a
white solid. The solid was triturated in hexane and filtered off
to give quantitatively the desired TFA salt 5 (845 mg). δH (250
MHz, CD3OD) 0.70 (m, 6 H, 2 × CH3 Ile), 1.05 (m, 1 H,
CHaHbγ Ile), 1.20 (m, 1 H, CHaHbγ Ile), 1.72 (m, 1 H, CHβ Ile),
3.75 (ddd ϩ m, 3 H, J 11.3 and 7.2, CH2β and CHα Ser), 4.30
(d, 1 H, J 5.5, CHα Ile), 4.97 (AB, 2 H, J 12.1, –O–CH2–C6H5),
7.17 (m, 5 H, –C6H5). δC (62.9 MHz, DMSO-d6) 11.0, 15.2,
24.5, 36.4, 54.1, 56.5, 60.3, 66.0, 128.0 (2 C), 128.1, 128.3
Experimental section
Unless otherwise noted, starting materials and reagents were
obtained from commercial suppliers and were used without
purification. All the protected amino acids and peptide coup-
ling reagents were purchased from Bachem and Neosystem.
Rec-HIV-1 protease kit was purchased from Bachem. Tetra-
hydrofuran (THF) was distilled over sodium benzophenone
ketyl immediately prior to use. Methylene dichloride (CH2Cl2)
was distilled over P2O5 just prior to use. Acetone was dried
and distilled over calcium sulfate. Ethyl acetate (EtOAc), di-
ethyl ether and dimethylformamide (DMF) were of anhydrous
quality from commercial suppliers (Aldrich, Acros, Carlo
Erba Reagents). Nuclear magnetic resonance spectra were
recorded at 250 MHz for 1H and 62.9 MHz for 13C on a Bruker
AC-250 spectrometer and at 300 MHz for 1H on a Bruker
Avance 300. Chemical shifts are expressed as δ units (part per
million) downfield from TMS (tetramethylsilane). Electro-
spray mass spectral analysis and LC-MS analysis were obtained
from Dr Drouot, Dr Maux and Miss Saint-Pé (Trophos,
Marseille, France) on a Waters Micromass ZMD spectrometer
for the ES-MS analysis by direct injection of the sample solubil-
ized in acetonitrile. The LC-MS analysis was carried out by
using a Waters model 2690 pump and a Waters C18 Symmetry
column with a two-mobile phase system (0.1% formic acid
in water and 0.1% formic acid in acetonitrile). Microanalyses
were carried out by Service Central d’Analyses du CNRS
(Venaison, France) and were within 0.4% of the theoretical
values. UV spectra for basic and enzymatic hydrolysis studies
were recorded on a Safas UV mc2 spectrophotometer (Safas,
Monaco).
Analytical thin layer chromatographies (TLC) and pre-
parative thin layer chromatographies (PLC) were performed
using silica gel plates 0.2 mm thick and 1 mm thick respectively
(60F254 Merck). Preparative flash column chromatographies
were carried out on silica gel (230–240 mesh, G60 Merck). The
melting points were not determined because of the amorphous
character of our peptides.
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(2 C), 135.6, 167.0, 170.6. MS-ES m/z 331 (M ϩ Na)ϩ and 309
(M ϩ H)ϩ.
ative 7 (2.56 g, 89%) as an yellowish solid (Found: C, 63.72; H,
8.57; N, 10.41. C14H22N2O3 requires C, 63.17; H, 8.33; N,
10.53%). Rf 0.36 (EtOAc–hexane 1 : 1). δH (250 MHz, CDCl3)
1.59 (s, 9 H, 3 × CH3 Boc), 2.07 (m, 2 H, –CH2–CH2–CH2–),
3.44 (m, 2 H, –CH2–NH-Boc), 3.58 (br s, 2 H, –NH2), 4.09
(t, 2 H, J 6.0, –O–CH2–), 4.98 (br s, 1 H, –NH-Boc), 6.79 (dd,
2 H, J 6.5 and 2.4, ArH ), 6.88 (dd, 2 H, J 6.5 and 2.4, ArH ).
δC (62.9 MHz, CDCl3) 28.6 (3 C), 29.8, 38.3, 66.8, 79.3,
115.8 (2 C), 116.5 (2 C), 140.4, 152.0, 156.2. MS-ES m/z 289 (M
ϩ Na)ϩ.
N-[N-{(9S,12S )-7,10-Dioxo-9-isopropyl-2-oxa-8,11-diaza-
bicyclo[12.2.2]octadeca-14,16,17-trien-12-ylcarbonyl}seryl]-
(S )-isoleucine benzyl ester 6
The macrocyclic acid 3 (60 mg, 0.17 mmol) was dissolved
in freshly distilled THF (1.2 mL). BOP reagent (75 mg, 0.17
mmol) was then added and the resulting mixture was cooled
down to 0 ЊC. Diisopropylethylamine (30 µL, 0.17 mmol) was
then added. The reaction mixture was allowed to warm up to
room temperature and stirred for 1 hour. The solution was
cooled down to 0 ЊC. A solution of the trifluoroacetic acid salt
5 (72 mg, 0.17 mmol) and diisopropylethylamine (60 µL, 0.34
mmol) in 0.5 mL of anhydrous THF was then added. The solu-
tion was allowed to warm up to room temperature and stirred
for 2 hours. The solvent was evaporated under reduced pressure
and the residue was dissolved in CH2Cl2 (10 mL). The organic
layer was washed successively with HCl 1 M (2 × 2 mL),
NaHCO3 5% (2 × 2 mL), brine (2 mL), dried over anhydrous
MgSO4, filtered and concentrated under reduced pressure. The
crude material was purified by flash chromatography to yield
the desired compound 6 (88 mg, 79%) as a white solid (Found:
C, 64.56; H, 7.22; N, 8.73. C35H48N4O8 requires C, 64.39; H,
7.41; N, 8.58%). Rf 0.40 (EtOAc–MeOH 95 : 5). δH (250 MHz,
CDCl3) 0.58–0.65 (m, 9 H, CH3 Val ϩ 2 × CH3 Ile), 0.80 (d,
3 H, J 6.7, CH3γ Val), 0.97–1.17 (m, 7 H, CHβ Val ϩ –(CH2)2–
CH2–C(O) ϩ CH2δ Ile), 1.52 (m, 1 H, CHβ Ile), 1.95 (br s, 2 H,
–CH2–C(O)), 2.61 (m, 1 H, OH ), 3.11 (m, 1 H, CHaHbβ Tyr),
3.42 (m, 1 H, CHaHbβ Tyr), 3.81–3.92 (m, 4 H, CHα Ser ϩ
CH2β Ser ϩ CHα Ile), 4.13 (m, 2 H, Tyr–O–CH2), 4.41 (m, 1 H,
CHα Val), 4.80–5.03 (m, 4 H, –CH2–C6H5 ϩ CHα Tyr ϩ NH ),
6.49 (m, 1 H, ArH Tyr), 6.65 (m, 1 H, ArH Tyr), 6.98 (m, 1 H,
ArH Tyr), 7.04 (m, 1 H, ArH Tyr), 7.07 (m, 5 H, –C6H5), 7.81
(br s, 1 H, NH ), 7.97 (br s, 1 H, NH ), 8.12 (br s, 1 H, NH ).
δC (62.9 MHz, CDCl3) 11.5, 14.1, 15.6, 18.6, 19.0, 20.9, 25.0,
35.2, 37.2, 49.3, 53.4, 54.4, 57.2, 58.3, 60.4, 67.2, 71.6, 72.2,
128.3–129.3 (9 C), 135.1 (2 C), 155.9, 171.2 (2 C), 171.9, 172.2,
173.0. MS-ES m/z 675 (M ϩ Na)ϩ.
[C-{[4-[3-[N-(tert-Butoxycarbonyl)amino]propan-1-yloxy]-
phenyl]amino}-N-[N{(9S,12S )-7,10-dioxo-9-isopropyl-2-
oxa-8,11-diazabicyclo[12.2.2]octadeca-14,16,17-trien-12-
ylcarbonyl}]glycyl]-(S )-isoleucine benzyl ester 2a
The seryl residue extended macrocyclic derivative 6 (88 mg,
0.14 mmol) was dissolved in anhydrous EtOAc (2 mL). Lead
tetraacetate (146 mg, 0.34 mmol) was then added as a solid with
75 mg of molecular sieves 4 Å. The reaction mixture was stirred
at 75 ЊC overnight. The reaction mixture was filtered off and
3 mL of citric acid 10% were added. The resulting solution was
stirred until it turned clear yellow. The aqueous layer was then
extracted with EtOAc (2 × 10 mL). The combined organic
layers were washed successively with citric acid 5% (3 × 10 mL),
H2O (2 × 10 mL), brine (10 mL), dried over anhydrous MgSO4,
filtered and concentrated under reduced pressure to give the
desired compound as a colourless oil. This compound was used
immediately without any further purification. This intermediate
was dissolved in anhydrous CH2Cl2 (3 mL) and the solution
was cooled down to 0 ЊC. A solution of 1-amino-4-[3-[N-(tert-
butoxycarbonyl)amino]propan-l-oxy]benzene 7 (36 mg, 0.14
mmol) and triethylamine (58 µL, 0.42 mmol) in anhydrous
CH2Cl2 (2 mL) was then added. The reaction was allowed to
warm up to room temperature then was refluxed for 48 hours.
The solvent was removed under reduced pressure and the crude
material was purified by flash chromatography to give the
desired compound 2a (60 mg, 50%) as a pale brown solid
(Found: C, 65.43; H, 7.61; N, 9.31. C48H66N6O10 requires C,
64.99; H, 7.50; N, 9.47%). Rf 0.50 (EtOAc). δH (300 MHz,
DMSO-d6) 0.58–0.70 (m, 12 H, 2 × CH3 Val ϩ 2 × CH3 Ile),
1.00–1.80 (m, 19 H, CHβ Val ϩ CHβ Ile ϩ CH2γ Ile ϩ –CH2–
CH2–NHBoc ϩ 3 × CH3 Boc ϩ –O–CH2–(CH2)2–CH2–), 2.01
(m, 2 H, –CH2–C(O)–), 2.33 (m, 1 H, CHaHbβ Tyr), 2.70 (m,
1 H, CHaHbβ Tyr), 3.00 (m, 2 H, –CH2–NHBoc), 3.58 (m, 2 H,
–CH2–O–), 3.90 (m, 2 H, –CH2–O–), 4.17 (m, 2 H, –NH–CH–
NH– ϩ CHα Val), 4.50 (m, 2 H, CHα Tyr ϩ CHα Ile), 4.91
(AB, 2 H, –CH2–C6H5), 5.54 (m, 2 H, 2 NH ), 5.82 (m, 1 H,
NH ), 6.87–6.48 (m, 8 H, ArH ), 7.07 (br s, 5 H, –C6H5), 7.54 (br
s, 1 H, NH ), 7.80 (br s, 1 H, NH ), 8.05 (br s, 1 H, NH ). δC (62.9
MHz, DMSO-d6) 11.7, 14.4, 15.9, 18.8, 19.1, 21.2, 24.3, 28.6 (3
C), 29.9, 32.0, 38.2, 38.4, 53.2, 55.6, 58.3, 60.5, 61.9, 66.0, 71.8,
73.2, 79.1, 117.5, 119.0, 128.4–129.2 (13 C), 131.8, 135.5, 140.9,
144.5, 156.2, 171.3 (2 C), 171.9, 172.2, 173.0. MS-ES m/z 909
(M ϩ Na)ϩ and 888 (M ϩ H)ϩ.
1-Amino-4-[3-[N-(tert-butoxycarbonyl)amino]propan-1-yloxy]-
benzene 7
3-[N-(tert-Butoxycarbonyl)amino]propan-1-ol (4.60 g, 26.3
mmol) was dissolved in freshly distilled THF (75 mL). p-Nitro-
phenol (3.65 g, 26.3 mmol) and triphenylphosphine (10.33 g,
39.4 mmol) were added. The reaction mixture was cooled down
to 0 ЊC and DEAD (6.20 mL, 39.4 mmol) was added drop-
wise.25 The reaction mixture was stirred at 0 ЊC for 20 minutes,
then allowed to warm up to room temperature and stirred over-
night. The solvent was removed under reduced pressure. The
residue was dissolved in CH2Cl2 (80 mL) and the organic layer
was washed with saturated NaHCO3 (4 × 60 mL). The organic
layer was dried over anhydrous MgSO4, filtered and concen-
trated. The residue was purified by flash chromatography to
yield the desired nitro derivative (7.16 g, 92%) as a yellow
solid (Found: C, 56.43; H, 6.95; N, 9.31. C14H20N2O5 requires
C, 56.78; H, 6.81; N, 9.46%). Rf 0.30 (EtOAc–hexane 3 : 7).
δH (250 MHz, CDCl3) 1.21 (s, 9 H, 3 × CH3 Boc), 1.80 (qt, 2 H,
J 6.3, –CH2–CH2–CH2–), 3.10 (q, 2 H, J 6.3, –CH2–NH-Boc),
3.90 (t, 2 H, J 6.3, –O–CH2–), 4.52 (br s, 1 H, –NH-Boc), 6.71
(dd, 2 H, J 7.1 and 2.1, ArH ), 7.96 (dd, 2 H, J 7.1 and 2.1,
ArH ). δC (62.9 MHz, CDCl3) 28.4 (3 C), 29.5, 37.6, 66.5, 79.4,
114.4 (2 C), 125.9 (2 C), 141.5, 156.1, 163.9. MS-ES m/z 319
(M ϩ Na)ϩ.
[C-(4-Ethoxyphenyl)oxy-N-[N-{(9S,12S )-7,10-dioxo-9-iso-
propyl-2-oxa-8,11-diazabicyclo[12.2.2]octadeca-14,16,17-
trien-12-ylcarbonyl}]glycyl]-(S )-isoleucine benzyl ester 2b
The title compound was synthesized according to the procedure
described previously for the synthesis of 2a, from seryl
extended macrocyclic acid 6 (100 mg, 0.15 mmol) and 4-ethoxy-
phenol (21 mg, 0.15 mmol) after 48 hours at room temperature
in CH2Cl2. 2b was isolated after chromatography as a white
solid (44 mg, 38%; Found: C, 66.15; H, 7.53; N, 7.59.
C42H54N4O9 requires C, 66.47; H, 7.17; N, 7.38%). Rf 0.52
(EtOAc). δH (300 MHz, CDCl3) 0.55–0.85 (m, 12 H, 2 × CH3
Val ϩ 2 × CH3 Ile), 1.02–1.41 (m, 11 H, CHβ Val ϩ CHβ Ile ϩ
CH2γ Ile ϩ –O–CH2–(CH2)2–CH2– ϩ –O–CH2–CH3), 2.08 (m,
2 H, –CH2–C(O)–), 2.51 (m, 1 H, CHaHbβ Tyr), 2.77 (m, 1 H,
The previous nitro compound (3.18 g, 10.8 mmol) was dis-
solved in MeOH (100 mL). 10% Pd-C (0.27 g) was then added
and the reaction mixture was stirred 6 hours under H2 atmos-
pheric pressure. The solution was then filtered on Celite and
concentrated under reduced pressure to give the aniline deriv-
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 1 6 7 6 – 1 6 8 3
1681

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CHaHbβ Tyr), 3.80 (m, 2 H, –CH2–O–), 4.32 (m, 2 H, –O–CH2–
CH3), 4.56 (m, 2 H, –NH–CH–O– ϩ CHα Val), 4.70 (m, 2 H,
CHα Tyr ϩ CHα Ile), 5.05 (m, 2 H, –CH2–C6H5), 5.32 (m, 1 H,
NH ), 5.78 (m, 1 H, NH ), 6.23 (m, 1 H, NH ), 6.50–7.00 (m,
4 H, ArH Tyr), 7.00–7.30 (m, 9 H, ArH ), 7.55 (m, 1 H, NH ).
MS-ES m/z 759 (M ϩ H)ϩ.
ArH ), 6.98 (br d, 1 H, ArH ). δC (62.9 MHz, CD3OD) 11.5,
14.7, 14.9, 18.5, 19.3, 20.0, 25.6, 28.3 (3 C), 31.3, 32.0, 38.5
(2 C), 52.9, 54.9, 58.3, 62.3, 62.5, 69.4, 74.5, 81.2, 117.4, 118.5,
129.4–131.3 (10 C), 146.0, 156.6, 171.4, 171.5, 172.3, 173.0,
174.9. MS-ES m/z 819 (M ϩ Na)ϩ and 797 (M ϩ H)ϩ.
Substrate 9
[C-(4-Methoxyphenyl)thio-N-[N-{(9S,12S )-7,10-dioxo-9-iso-
propyl-2-oxa-8,11-diazabicyclo[12.2.2]octadeca-14,16,17-trien-
12-ylcarbonyl}]glycyl]-(S )-isoleucine benzyl ester 2c
The previous N-Boc protected derivative 8 (364 mg, 0.46 mmol)
was dissolved in 15 mL of anhydrous diethyl ether. The result-
ing solution was cooled down to 0 ЊC and a solution of HCl in
diethyl ether (7.9 mL, 30% of HCl in weight) was then added
dropwise. The reaction mixture was allowed to warm up to
room temperature and was stirred for 6 hours. The reaction was
monitored by TLC (EtOAc–MeOH l : l and n-BuOH–AcOH–
H2O 12 : 3 : 5). The solvent was evaporated to yield the final
compound 9 as a brown solid (282 mg, 90%). δH (250 MHz,
CD3OD) 0.56–0.77 (m, 14 H, 2 × CH3 Ile ϩ 2 × CH3 Val ϩ
CH2β Ile), 1.00–2.00 (m, 12 H, 5 × CH2 ϩ CHβ Val ϩ CHβ Ile),
2.45 (m, 2 H, CH2β Phe), 2.90–3.40 (m, 5 H, 2 × –CH2–O– ϩ
CHα Val), 3.50 (m, 1 H, CHα Ile), 4.22 (m, 1 H, –NH–CH–
NH–), 4.54 (m, 1 H, CHα Phe), 6.58 (br d, 1 H, J 8.0, ArH ),
6.67 (br d, 1 H, J 6.2, ArH ), 6.83 (br d, 1 H, J 7.6, ArH ), 6.91
(m, 2 H, ArH aniline), 7.03 (br d, 1 H, J 7.6, ArH ), 7.16 (br d,
2 H, J 7.2, ArH ). MS-ES m/z 697 (M ϩ H)ϩ.
The title compound was synthesized according to the procedure
described previously for the synthesis of 2a, from seryl
extended macrocyclic acid 6 (100 mg, 0.15 mmol) and 4-meth-
oxythiophenol. 2c was isolated after chromatography as a pale
yellow solid (55 mg, 47%; Found: C, 65.06; H, 6.74; N, 7.53.
C41H52N4O8S requires C, 64.71; H, 6.89; N, 7.36%). Rf 0.52
(EtOAc). δH (300 MHz, CDCl3) 0.55–0.94 (m, 12 H, 2 × CH3
Val ϩ 2 × CH3 Ile), 1.07–1.43 (m, 8 H, CHβ Val ϩ CHβ lle ϩ
CH2γ Ile ϩ –O–CH2–(CH2)2–CH2–), 2.00 (m, 2 H, –CH2–
C(O)–), 2.41–2.75 (m, 2 H, CH2β Tyr), 3.02 (m, 2 H, –CH2–O–),
3.55 (s, 3 H, –O–CH3), 3.92–4.15 (m, 2 H, –NH–CH–S– ϩ CHα
Val), 4.40–4.70 (m, 2 H, CHα Tyr ϩ CHα Ile), 5.18 (m, 2 H,
–CH2–C6H5), 5.36 (m, 1 H, NH ), 5.72 (m, 1 H, NH ), 6.38 (m,
1 H, NH ), 6.57–6.95 (m, 6 H, ArH Tyr ϩ 2 × ArH ), 7.05–7.42
(m, 7 H, ArH ), 7.66 (m, 1 H, NH ). MS-ES m/z 761 (M ϩ H)ϩ.
General method for the basic hydrolysis assay
[C-Anilino-N-[N-{(9S,12S )-7,10-dioxo-9-isopropyl-2-oxa-8,11-
diazabicyclo[12.2.2]octadeca-14,16,17-trien-12-ylcarbonyl}]-
glycyl]-(S )-isoleucine benzyl ester 2d
Basic hydrolysis of a 250 µM solution of substrate 2a–d was
performed in ethyl alcohol in the presence of aqueous sodium
hydroxide (5 equiv., 1250 µM), at 26 ЊC. The chromophore
release was monitored by UV analysis at a suitable wavelength,
i.e. 300 nm for 1-amino-4-[3-[N-(tert-butoxycarbonyl)amino]-
propan-1-yloxy]benzene (2a), 302 nm for 4-ethoxyphenol (2b)
and 4-methoxythiophenol (2c), and 281 nm for aniline (2d). The
quantification curves for each chromophoric substance were
performed in the same conditions in the presence of 1250 µM
of aqueous sodium hydroxide. The observed results are
summarized in Table 1.
The title compound was synthesized according to the procedure
described previously for the synthesis of 2a, from seryl
extended macrocyclic acid 6 (104 mg, 0.16 mmol) and aniline.
2d was isolated after chromatography as a pale brown solid
(17 mg, 15%; Found: C, 67.68; H, 6.95; N, 9.73. C40H51N5O7
requires C, 67,30; H, 7,20; N, 9,81%). Rf 0.57 (EtOAc). δH (300
MHz, CDCl3) 0.66–0.88 (m, 12 H, 2 × CH3 Val ϩ 2 × CH3 Ile),
1.09–1.42 (m, 8 H, CHβ Val ϩ CHβ Ile ϩ CH2γ Ile ϩ –O–CH2–
(CH2)2–CH2–), 2.18 (m, 2 H, –CH2–C(O)–), 2.58 (m, 1 H,
CHaHbβ Tyr), 2.90 (m, 1 H, CHaHbβ Tyr), 3.07 (m, 2 H, –CH2–
O–), 4.12 (m, 2 H, –NH–CH–NH– ϩ CHα. Val), 4.63 (m, 2 H,
CHα Tyr ϩ CHα Ile), 5.12 (m, 2 H, –CH2–C6H5), 5.25 (m, 1 H,
NH ), 5.66 (m, 1 H, NH ), 6.27 (m, 1 H, NH ), 6.62–7.16 (m,
4 H, ArH Tyr), 7.24–7.35 (m, 10 H, ArH ), 7.82 (m, 1 H, NH ),
8.05 (m, 1 H, NH ). MS-ES m/z 714 (M ϩ H)ϩ.
Acknowledgements
ANRS and INSERM are greatly acknowledged for financial
support. We thank Johann Grognux for synthetic assistance
as a M.Sc. student. We thank Miss Nathalie Saint-Pé,
Dr Delphine Maux and Dr Cyrille Drouot (Trophos, Marseille,
France) for help in obtaining Mass Spectral Data. We are
indebted to Drs Françoise Gondois-Rey and Ivan Hirsch
(INSERM U372, Faculté des Sciences de Luminy, Marseille,
France), and to Miss Delphine Palet and Dr Pascale Galéa
(Trophos, Marseille, France) for their biological assistance. We
are indebted to Professor Keith Dudley (Head of the Depart-
ment of Biology, INSERM U382, Faculté des Sciences de
Luminy, Marseille, France) for revising the manuscript. Special
thanks to Cédrik Garino, Ph.D. student, for his very helpful
comments, and to Dr Nico P. M. Smit (Leids Universitair
Medish Centrum, University of Leiden, The Netherlands) who
provided us with his Ph.D thesis.
[C-{[4-[3-[N-(tert-Butoxycarbonyl)amino]propan-1-yloxy]-
phenyl]amino}-N-[N-{(9S,12S )-7,10-dioxo-9-isopropyl-2-oxa-
8,11-diazabicyclo[12.2.2]octadeca-14,16,17-trien-12-yl-
carbonyl}]glycyl]-(S )-isoleucine 8
The benzyl ester 2a (554 mg, 0.62 mmol) was dissolved in a
mixture THF–MeOH (1 : 3) (27 mL). A suspension of 10%
Pd-C (59 mg) in THF (3 mL) was then added and the reaction
mixture was stirred overnight at room temperature under H2
atmospheric pressure. The suspension was filtered and the solu-
tion was concentrated under reduced pressure. The residue was
purified by flash chromatography using a gradient of MeOH in
EtOAc. The acid 8 was obtained as a white solid (441 mg, 89%;
Found: C, 61.51; H, 7.75; N, 10.30. C41H60N6O10 requires C,
61.83; H, 7.59; N, 10.55%). Rf 0.40 (EtOAc–MeOH 2 : 1).
δH (250 MHz, CD3OD) 0.53 (m, 3 H, CH3δ Ile), 0.71 (m, 3 H,
CH3γ Ile), 0.78 (br s, 6 H, 2 × CH3 Val), 1.05 (m, 2 H, CH2γ Ile),
1.23 (m, 15 H, 3 × CH3 Boc ϩ –CH2–CH2–NHBoc ϩ –(CH2)2–
CH2–C(O)–), 1.50–1.80 (m, 2 H, CHβ Ile ϩ CHβ Val), 1.90 (m,
2 H, –CH2–C(O)–), 2.30–2.53 (m, 2 H, CH2β Tyr), 3.00 (m, 2 H,
–CH2–NHBoc), 3.66 (m, 2 H, –CH2–O), 3.90 (m, 4 H, –NH–
CH–NH– ϩ CHα Val ϩ –CH2–O–), 4.40–4.70 (m, 2 H, CHα
Tyr ϩ CHα Ile), 6.55 (m, 5 H, 1 × ArH cyclic peptide ϩ 4 ×
ArH aniline residue), 6.71 (br s, 1 H, ArH ), 6.80 (br d, 1 H,
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O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 1 6 7 6 – 1 6 8 3
1683