A new tool for photoaffinity labeling studies: A partially constrained, benzophenone based, α-amino acid

Article abstract of DOI:10.1039/c003943h

The novel α-amino acid BpAib, a partially conformationally restricted analogue of the currently extensively used 3-(4-benzoylphenyl)alanine (Bpa) photoaffinity label, was synthesized, optically resolved, fully characterized, and appropriately derivatized.

Full text of DOI:10.1039/c003943h

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
A new tool for photoaffinity labeling studies: a partially constrained,
benzophenone based, a-amino acid†
Karen Wright,*a Alessandro Moretto,b Marco Crisma,b Michel Wakselman,a Jean-Paul Mazaleyrat,a
Fernando Formaggiob and Claudio Toniolo*b
Received 9th March 2010, Accepted 13th May 2010
First published as an Advance Article on the web 26th May 2010
DOI: 10.1039/c003943h
The novel a-amino acid BpAib, a partially conformationally restricted analogue of the currently
extensively used 3-(4-benzoylphenyl)alanine (Bpa) photoaffinity label, was synthesized, optically
resolved, fully characterized, and appropriately derivatized. An intermolecular photocrosslinking
experiment highlighted its regioselective reactivity towards the S-methyl side-chain group of a
Met-based dipeptide, which is closely comparable to that of Bpa.
Bpa is a flexible amino acid.17 Although it is known that
flexibility is needed for efficient crosslinking of proteins by
Introduction
benzophenone photoligands,18 for specific interactions a reduced
conformational mobility might be beneficial. In this work, we
designed, synthesized, resolved, configurationally and conforma-
tionally characterized, and intermolecularly photoreacted with a
Met-based peptide partner, the novel, main-chain and side-chain
partially restricted, chiral a-amino acid 2-amino-5-benzoyl-2,3-
dihydro-1H-indene-2-carboxylic acid (BpAib, Fig. 1), belonging
to the class of Ca-tetrasubstituted a-amino acids (strong turn
and helix promoters in peptides), the prototypes of which are
a-aminoisobutyric acid (Aib)19–21 and 1-aminocyclopentane-1-
carboxylic acid (Ac5c)21–24 (Fig. 1). A limited part of this work
has already been reported in two communications to Peptide
Symposia.25,26
Incorporation of photoreactive a-amino acids chemically or
biosynthetically into peptides or proteins has been exten-
sively exploited in biochemistry, molecular biology, and chem-
ical proteomics to map the interfaces in ligand–receptor and
protein–protein interactions.1,2 Among the a-amino acids suc-
cessfully proposed, the benzophenone-containing residue Bpa,
3-(4-benzoylphenyl)alanine (Fig. 1) is the most popular probe
for photoaffinity labeling.3–13 After photoexcitation at 350 nm,
the benzophenone chromophore of Bpa is believed to remove
by far most frequently a hydrogen atom from the side chain
of a Met residue, followed by covalent C–C bond formation
of the resulting radical pair.1–14 In addition to these literature
studies of intermolecular interactions, we recently investigated
the chemical and 3D-structures of the products arising from the
intramolecular Paterno`-Yang photocyclization in Bpa-spacer-Met
helical peptides.15,16
Results and discussion
Synthesis
The 3,4-bis(bromomethyl)benzophenone 3 (Fig. 2) was synthe-
sized in three steps from 3,4-dimethylbenzoic acid 1 by formation
of the acid chloride, using thionyl chloride, followed by Friedel–
Crafts acylation of benzene affording 2, and finally by free radical
side-chain bromination in the presence of N-bromosuccinimide
Fig. 1 Chemical structures of Bpa, BpAib, Aib and Ac5c.
aILV, UMR CNRS 8180, University of Versailles, 78035 Versailles, France.
E-mail: wright@chimie.uvsq.fr; Fax: +33 01 3925 4452; Tel: +33 01 3925
4366
bICB, Padova Unit, CNR, Department of Chemistry, University of Padova,
35131 Padova, Italy. E-mail: claudio.toniolo@unipd.it; Fax: +39 049 827
5239; Tel: +39 049 827 5247
† Electronic supplementary information (ESI) available: NMR and mass
spectra. CCDC reference numbers 746110. For ESI and crystallographic
data in CIF or other electronic format see DOI: 10.1039/c003943h
Fig. 2 Synthesis of Na-protected (blocked) BpAib. (i) SOCl2; DMF; rt.
(ii) AlCl3, benzene; DCE; rt. (iii) NBS, benzoyl peroxide; CCl4; 90 ◦C.
(iv) NC–CH2–COOEt; K2CO3; nBu4N+·HSO4 (cat.); CH3CN; 80 ◦C.
-
(v) 10 N aq. HCl; abs. EtOH; 0 ◦C to rt. (vi) Z-OSu or (Bz)2O; CH3CN;
rt. (vii) NaOH; MeOH–THF–H2O; 60 ◦C.
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(NBS) and benzoyl peroxide. bis-Alkylation of the Gly equivalent
ethyl (Et) isocyanoacetate by 3 under phase-transfer conditions,27
followed by acid hydrolysis, afforded the pentatomic cyclic,
Ca-tetrasubstituted, BpAib a-amino ester 4. Protection of the
a-amino group of 4 by Z (benzyloxycarbonyl) or Bz (benzoyl)
using the succinimidyl ester (OSu) or symmetrical anhydride, and
subsequent saponification of the ethyl esters, gave the N-acylated
a-amino acids 5 and 6.
For the separation of the BpAib enantiomers, the chiral
auxiliary H-(S)-Phe-NHChx (Chx, cyclohexyl), known to be an
effective resolving agent for Ca-tetrasubstituted a-amino acid
derivatives,28,29 was reacted with the Na-acylated (R,S)-BpAib
compounds 5 and 6 using HATU [2-(1H-7-azabenzotriazol-1-
yl)-1,1,3,3-tetramethyluronium hexafluorophosphate] and diiso-
propylethylamine (DIEA) in tetrahydrofuran (THF) to afford the
corresponding Na-acylated dipeptide alkylamides (Fig. 3).
Fig.
4
X-Ray diffraction structure of the Na-protected dipep-
tide alkylamide Z-(R)-BpAib-(S)-Phe-NHChx 8a. The intramolecular
=
C
O ◊ ◊ ◊ H–N H-bond is represented by a dashed line.
of those of an oppositely configured Ca-trisubstituted (protein)
a-amino acid. The intramolecular H-bond is weak, as the O ◊ ◊ ◊ N
34
˚
distance is 3.136(5) A.
Fig. 3 Optical resolution of (R,S)-BpAib. (i) H-(S)-Phe-NHChx, HATU,
DIEA; THF; rt. (ii) 10 M HCl, 1,4-dioxane; 100 ◦C. (iii) SOCl2; MeOH;
rt. (iv) (Boc)2O; CH3CN–CH2Cl2; rt.
Spectroscopic characterization
Using the amino acid derivative Boc-(S)-BpAib-OMe 9, we char-
acterized the spectroscopic properties of this novel benzophenone-
based residue. The near-UV absorption spectrum in MeOH
solution (Fig. 5) typically shows a very weak shoulder at about
340 nm (n → p* transition of the benzophenone chromophore),
an excellent “window” region for photoexcitation of peptides,
Chromatographic separations of the dipeptide diastereomers
afforded the Z-protected 7a and 8a, and the Bz-blocked 7b
and 8b dipeptides. The crystalline (R,S) diastereomer with Y =
Z (8a) proved to be suitable for an X-ray diffraction study (see
below). Strong acid hydrolysis of 7b, followed by esterification with
methanol (MeOH)/thionyl chloride and treatment with (Boc)2O
(di-tertbutyl-dicarbonate) gave the Boc-protected BpAib a-amino
methyl ester (S)-enantiomer 9.
3D-Structural analysis
The crystal-state 3D-structure of the Z-BpAib-(S)-Phe-NHChx
diastereomeric dipeptide analyzed (Fig. 4) unambiguously showed
that the configuration at the a-carbon of its BpAib residue is
(R), based on the known configuration of (S)-Phe. In addition,
this Z-protected dipeptide cyclohexylamide (8a) was found to
=
be folded in an intramolecularly C O ◊ ◊ ◊ H–N H-bonded b-turn
conformation,30–32 clearly induced by the BpAib residue. Indeed,
this is a typical property of Ca-tetrasubstituted a-amino acids,
originally authenticated for Aib,19–21 the prototype of this class
of conformationally-restricted compounds, and the related Ac5c
residue.21–24
The conformationally informative backbone torsion angles are
helical [j1 = -64.9(6)◦, y1 = -23.8(7)◦] for (R)-BpAib and
distorted helical [j2 = -88.6(7)◦, y2 = -5.0(7)◦] for (S)-Phe. These
values indicate that the b-turn formed is type-I. Moreover, the signs
of the helical (R)-BpAib j,y torsion angles are both negative,
characteristic of a right-handed screw sense,33 but reminiscent
Fig.
5 Near-UV absorption spectra in MeOH solution of
Boc-(S)-BpAib-OMe 9 and of the faster (11A) eluting of the two
major HPLC peaks (see Fig. 7) arising from the photoreaction.
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followed by a much more intense band near 260 nm (p → p*
transition).35,36
In the corresponding circular dichroism (CD) spectrum (Fig. 6),
the 260 nm Cotton effect is clearly visible, negative for the (S)-
enantiomer of the BpAib derivative 9. Conversely, the band near
340 nm does not seem to be optically active, at least using the
highest analyte concentration (1 mM) and the widest cell (1 cm)
available to us. Below 240 nm (far-UV region), a very intense,
negative Cotton effect dominates the CD spectrum of (S)-9.
Fig.
6
Far- and near-UV CD spectra in MeOH solution of
Boc-(S)-BpAib-OMe 9 and of the faster (11A) and slower (11B) eluting
major HPLC peaks (see Fig. 7) arising from the photoreaction.
Fig. 7 Top: chemical structures of the Boc-(S)-BpAib-OMe 9 and
Boc-(S)-Met-Aib-OMe 10 reagents and the products (11) of the photore-
action; bottom: reverse-phase HPLC profiles of the photoreaction mixture
after 25 min (I) and 60 min (II). Part III shows the HPLC profile of the
two photoreaction products after a flash chromatography purification of
the crude mixture.
Photocrosslinking experiments
To check the reactivity of the benzophenone moiety of BpAib
under the classical conditions of intermolecular photoinduced co-
valent bond formation with a Met-containing peptide, equimolar
(0.1 M) amounts of Boc-(S)-BpAib-OMe 9 and Boc-(S)-Met-Aib-
OMe17 (10) were dissolved in acetonitrile, placed in a quartz tube,
and purged of oxygen [to avoid oxidation of the (S)-Met thioether
side chain to a mixture of diastereomeric sulfoxides] by bubbling
nitrogen for 20 min. After a 25 minute irradiation at 350 nm, the
reverse-phase HPLC profile of the photoreaction mixture (Fig. 7I,
bottom) showed two newly formed, roughly equivalent peaks (11A
and 11B). After a 60 minute irradiation, the amount of the two
peaks 11 increases remarkably at the expense of the photoreactants
9 and 10 (Fig. 7II).
The mixture of the two peaks (Fig. 7III), obtained by flash-
chromatography partial purification of the crude, was submitted
to ESI-ToF mass spectrometry and NMR analyses. The com-
bined chromatographic and spectrometric results (Fig. 7 and
8, and ESI, S12†) indicated that the two compounds are the
two diastereomeric adducts originating from the regioselective
photocrosslinking of the BpAib derivative 9 to the Met e-CH3
group of the dipeptide substrate 10.
In particular, Fig. 8 clearly shows that the Met e-CH3 proton
signal occurring at about d = 2.1 ppm in the spectrum of 10 is
missing in that of the mixture of the two adducts, 11 (A + B).
Moreover, this reaction involves the formation of an additional
chiral center at the side-chain carbonyl carbon of the former
BpAib residue16,17 and consequently results in the production of
two diastereomers (Fig. 7, top). The participation of the BpAib
side-chain carbonyl group in the photoreaction was corroborated
by the observation that the band near 260 nm is missing in the
near-UV absorption spectrum of a representative diastereomeric
product (11A in Fig. 5) and in the CD spectra of both 11A and
11B as well [the relatively weak UV absorption of 11A with fine
structure centered near 275 nm is associated with its substituted
benzyl chromophores37]. Interestingly, most of the general features
(apart from the relative intensities) of the CD spectra of the
two diastereomers 11A and 11B reflect well their stereochemical
relationship. It is worth pointing out that photocrosslinking of
the BpAib derivative 9 to the Met g-CH2 group14 of dipeptide 10
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preparative column chromatography were performed on Kieselgel
F 254 and Kieselgel 60 (0.040–0.063 mm) (Merck), respectively.
The TLC profile of the Z-(RS)-BpAib-(S)-Phe-NHChx separa-
tion, 1H and 13C NMR spectra for all newly prepared compounds
and X-ray diffraction data of Z-(R)-BpAib-(S)-Phe-NHChx may
be found in the ESI.†
3,4-bis-(Bromomethyl)benzophenone 3. 3,4-Dimethylbenzo-
phenone 2 (18.0 g, 85.7 mmol) was dissolved in carbon
tetrachloride (250 mL), and NBS (29.0 g, 162.8 mmol) and
benzoyl peroxide (1.0 g) were added. The mixture was stirred
at reflux under argon for 2 h. The mixture was allowed to cool
to rt, then filtered through Celite. The filtrate was concentrated
under reduced pressure. The residue was purified by column
chromatography using ethyl acetate–cyclohexane (5 : 95) as eluent
to give the bis-bromomethyl derivative as an oil (20.8 g, 66%).
Rf 0.27 (pentane–ethyl acetate 95 : 5); dH (CDCl3) 4.68, 4.69 (2 s,
4H), 7.50 (t, J = 7.5 Hz, 3H), 7.62 (m, 1H), 7.71 (m, 1H), 7.78
(m, 3H); dC (CDCl3) 28.8, 29.2, 128.4, 129.9, 130.8, 131.0, 132.4,
132.8, 136.8, 136.9, 138.3, 140.7, 195.3. Found: C, 49.45; H, 3.42.
Calc. for C15H12Br2O: C, 48.94; H 3.28. CI-MS m/z 369 [M + H]+.
Fig.
8
400 MHz 1H NMR spectra in CDCl3 solution of
Boc-(S)-BpAib-OMe 9, Boc-(S)-Met-Aib-OMe 10, and the mixture
of the two diastereomeric products (11A and 11B) isolated after the
photoreaction.
would have created two additional chiral centers in the products
with the resulting formation of four diastereomers.
Ethyl-2-amino-5-benzoyl-2,3-dihydro-1H-indene-2-carboxylate
[H-(RS)-BpAib-OEt] 4. To a solution of 3,4-bis-(bromomethyl)-
benzophenone 3 (10.15 g, 27.6 mmol) in acetonitrile (540 mL)
was added tetrabutylammonium hydrogen sulfate (1.83 g,
5.4 mmol), potassium carbonate (22.4 g, 162.0 mmol) and ethyl
isocyanoacetate (3 mL, 25.6 mmol). The mixture was refluxed
under argon for 24 h, then filtered. The collected solids were
washed with dichloromethane and the filtrate was concentrated
under reduced pressure. The crude isonitrile obtained was
dissolved in a mixture of dichloromethane (15 mL) and ethanol
(150 mL). Concentrated hydrochloric acid (3.5 mL) was added.
The reaction mixture was stirred for 3 h at rt. The mixture was
diluted with water (100 ml) and volatiles were evaporated under
reduced pressure. The resulting mixture was further diluted
with 0.5 M aqueous hydrochloric acid and washed twice with
diethyl ether. The aqueous phase was made alkaline by the slow
addition of sodium hydrogen carbonate, and extracted with three
portions of dichloromethane. The combined dichloromethane
extracts were dried over MgSO4, filtered, and concentrated under
reduced pressure. The residue obtained was purified by column
chromatography using ethyl acetate–cyclohexane (7 : 3) as eluent
to afford the a-amino ester as a solid (2.76 g, 35% yield). Rf 0.32
(dichloromethane–MeOH 95 : 5); m.p. 58–63 ◦C; dH (CDCl3) 1.29
(t, J = 7.1 Hz, 3H), 2.94 (dd, 2H, J = 4.8, 16.6 Hz), 3.60 (dd, 2H,
J = 9.4, 16.7 Hz), 4.23 (q, J = 7.1, 14.2 Hz, 2H), 7.31 (d, J =
7.7 Hz, 1H), 7.46 (t, J = 7.7 Hz, 2H), 7.54–7.68 (m, 3H), 7.78 (d,
2H, J = 5.0 Hz). dC (CDCl3) 14.1, 45.8, 46.0, 61.5, 65.1, 124.5,
126.5, 128.2, 129.5, 129.9, 132.1, 136.6, 137.9, 140.9, 145.8, 176.2,
196.6. Found: C, 73.24; H, 6.21; N, 4.66. Calc. for C19H19NO3: C,
73.76; H, 6.19; N, 4.53%. ESI-MS m/z 310.2 [M + H]+.
Conclusions
In summary, we synthesized and resolved the two enantiomers of
BpAib, a novel main-chain and side-chain partially restricted, a-
amino acid. The configuration of one enantiomer was assigned via
X-ray diffraction analysis of a diastereomeric dipeptide. The spec-
troscopic properties of this chromophoric residue were recorded.
The usefulness of this new probe as a tool for the study of peptide–
protein interactions was demonstrated by a photocrosslinking
experiment, which showed that the regioselectivity of the BpAib
intermolecular photoreaction towards a Met-containing substrate
is closely related to that of its Bpa intramolecular counterpart.16,17
We are currently extending the application of this photolabeled
amino acid to actual peptide ◊ ◊ ◊ protein interactions under the
experimental conditions (solvent, pH, ionic strength) typically
used in biochemical studies. However, it is worth recalling that
the insertion of a turn/helix stabilizer, Ca-quaternary, a-amino
acid,19–24 such as BpAib, in a peptide sequence should be planned
carefully, to avoid a potential, undesired conformational change
of the ligand.
Experimental
Synthesis and analytical data
General methods. Melting points were measured by means
of a capillary tube immersed in an oil bath (Tottoli apparatus,
Bu¨chi) and are uncorrected. 1H NMR and 13C NMR spectra
were recorded on a Bruker WM300 spectrometer operating at
300 MHz and 77 MHz, respectively, the solvent being used as
the internal standard: CDCl3 (1H: d = 7.26 ppm; 13C: d =
77.00 ppm). Splitting patterns are abbreviated as follows: (s)
singlet, (d) doublet, (t) triplet, (q) quartet, (m) multiplet. Elemental
analyses were performed by the CNRS Service of Microanalyses
in Gif-sur-Yvette (France). Mass spectra (electrospray mode)
were recorded on a Hewlett-Packard HP5989MS spectrometer
by Mr Vincent Steinmetz (ILV, Versailles). Analytical TLC and
Z-(RS)-BpAib-OH 5. A solution of ethyl-2-amino-5-benzoyl-
2,3-dihydro-1H-indene-2-carboxylate 4 (187 mg, 0.6 mmol) in
dichloromethane (10 mL) was cooled on an ice bath and Z-OSu
(226 mg, 0.91 mmol) was added. The reaction mixture was allowed
to warm to rt and stirred for 18 h, then concentrated under
reduced pressure. The residue obtained was purified by column
chromatography using cyclohexane–ethyl acetate (7 : 3) as eluent
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to afford the intermediate protected a-amino ester. This residue
was dissolved in a mixture of tetrahydrofuran (3 mL), MeOH
(1.5 mL) and water (0.75 mL). Sodium◦hydroxide (60 mg) was
added and the mixture was stirred at 55 C for 2 h. The mixture
was allowed to cool to rt and diluted with water. Volatiles were
removed under reduced pressure. The mixture was cooled on an
ice bath and acidified by addition of 2 M aqueous hydrochloric
acid. The resulting solid was filtered and dried under vacuum
(146 mg,◦58%). Rf 0.39 (dichloromethane–methanol 90 : 10); m.
p. 66–70 C; dH (CDCl3) 3.32 (t, J = 15.4 Hz, 2H), 3.66 (t, J =
15.8 Hz, 2H), 5.03 (s, 2H), 7.37 (m, 3H), 7.24 (m, 6H), 7.44 (m,
2H), 7.56 (m, 3H), 7.73 (d, 2H, J = 7.0 Hz). dC (CDCl3) 29.7, 43.6,
67.1, 124.2, 126.1, 127.9, 128.2, 128.5, 129.6, 129.9, 132.3 132.8,
135.7, 136.7, 137.7, 140.3, 145.2, 196.6. Found: C, 72.36; H, 5.37;
N, 3.27. Calc. for C25H21NO5: C, 72.27; H, 5.09; N, 3.37. ESI-MS
m/z 438.1 [M + Na]+.
Z-(S)-BpAib-(S)-Phe-NHChx 7a. Rf 0.45 (cyclohexane–ethyl
acetate 1 : 1); m.p. 81–85 ◦C; dH (CDCl3) 1.05–1.33 (m, 5H), 1.57–
1.85 (m, 5H), 2.92–3.32 (m, 5H), 3.69–3.87 (m, 2H), 4.66 (m, 1H),
4.99 (m, 2H), 5.78 (s, 1H), 6.45 (d, 1H, J = 7.9 Hz), 6.61 (d, 1H,
J = 8.1 Hz), 7.14 (d, 1H, J = 6.9 Hz), 7.21–7.34 (m, 10H), 7.47
(m, 3H), 7.57 (m, 3H), 7.75 (d, 2H, J = 7.0 Hz); dC (CDCl3) 24.9,
25.4, 32.6, 32.8, 37.5, 42.5, 43.8, 48.5, 54.2, 67.4, 67.7, 72.6, 124.6,
126.1, 126.9, 128.0, 128.3, 128.5, 128.6, 128.7, 129.1, 129.9, 132.4,
135.4, 136.7, 136.8, 137.6, 139.2, 145.6, 155.6, 168.6, 169.3, 171.6,
25
196.6; [a]589 = +36 (c 0.27, MeOH); Found: C, 73.33; H, 6.85;
N, 6.15. Calc. for C40H41N3O5·0.5H2O: C, 73.59; H, 6.48; N 6.43.
ESI-MS m/z 666.4 [M + Na]+.
Z-(R)-BpAib-(S)-Phe-NHChx 8a. Rf 0.37 (cyclohexane–ethyl
acetate 1 : 1); m.p. 127–130 ◦C; dH (CDCl3) 1.03–1.21 (m, 5H),
1.49–1.71 (m, 5H), 2.92–3.35 (m, 5H), 3.68 (m, 2H), 4.60 (m, 1H),
4.94 (m, 2H), 5.56 (s, 1H), 6.24 (d, 1H, J = 8.0 Hz), 6.60 (d, 1H,
J = 8.1 Hz), 7.07–7.34 (m, 12H), 7.39 (m, 2H), 7.52 (m, 3H), 7.67
(d, 2H, J = 7.0 Hz); dC (CDCl3) 24.9, 25.4, 32.6, 32.8, 37.5, 42.5,
43.8, 48.4, 54.2, 67.3, 67.6, 72.6, 124.3, 126.4, 126.9, 128.0, 128.5,
128.6, 129.2, 129.6, 129.9, 132.4, 135.4, 136.7, 137.6, 140.6, 143.9,
Bz-(RS)-BpAib-OH 6.
benzoyl-2,3-dihydro-1H-indene-2-carboxylate
A
solution of ethyl-2-amino-5-
(5.51 g,
4
17.8 mmol) in acetonitrile (150 mL) was cooled on an ice
bath and benzoic anhydride (8.1 g, 35.7 mmol) was added. The
reaction mixture was allowed to warm to rt and stirred for
48 h, then concentrated under reduced pressure. The residue
was taken up in dichloromethane and washed twice with 2 M
aqueous sodium hydroxide and once with water. The organic
phase was dried over MgSO4, filtered, and concentrated under
reduced pressure. The residue obtained was purified by column
chromatography using dichloromethane–isopropanol (98 : 2) as
eluent to afford the intermediate protected a-amino ester. This
residue was dissolved in a mixture of tetrahydrofuran (50 mL),
MeOH (20 mL) and water (5 mL). Sodium hydroxide (1.20 g) was
added and the mixture was stirred at 55 ◦C for 90 min. The mixture
was allowed to cool to room temperature and diluted with water.
Volatiles were removed under reduced pressure. The mixture was
cooled on an ice bath and acidified by addition of 2 M aqueous
hydrochloric acid. The resulting solid was filtered and dried
under vacuum (4.73 g, 69%). Rf 0.57 (dichloromethane–methanol
90 : 10); m.p. 176–179 ◦C; dH (CD3OD) 3.51 (dd, 2H, J = 9.1,
17.7 Hz), 3.76 (dd, 2H, J = 17.5, 16.7 Hz), 7.37 (m, 3H), 7.47 (m,
3H), 7.59 (m, 3H), 7.73 (m, 4H). dC (CD3OD) 44.1, 44.3, 67.6,
125.5, 127.1, 128.5, 129.4, 129.5, 130.4, 130.9, 132.8, 133.7, 135.2,
137.8, 139.1, 142.2, 147.6, 170.7, 176.5, 198.8. Found: C, 71.53;
H, 5.04; N, 3.47. Calc. for C24H19NO4·H2O: C, 71.44; H, 5.25; N,
3.47. ESI-MS m/z 408.3 [M + Na]+, 793.5 [2M + Na]+.
25
155.5, 169.3, 171.6, 196.5 [a]589 = -2 (c 0.26, MeOH); Found: C,
74.58; H, 6.58; N, 6.51. Calc. for C40H41N3O5: C, 74.62; H, 6.42;
N, 6.53. ESI-MS m/z 666.4 [M + Na]+.
Bz-(RS)-BpAib-(S)-Phe-NHChx (7b and 8b). A suspension
of Bz-(RS)-BpAib-OH 6 (2.02 g, 5.24 mmol) and HCl·H-(S)-
Phe-NHChx (1.81 g, 6.40 mmol) in tetrahydrofuran (70 mL)
was cooled on an ice bath. HATU (2.39 g, 6.28 mmol) and
DIEA (2.5 mL) were added. The reaction mixture was allowed
to warm to rt and stirred for 24 h. The mixture was concentrated
under reduced pressure and the resulting residue was taken up
in dichloromethane. The solution was washed twice with 0.5 M
aqueous hydrochloric acid, once with water, and once with
saturated aqueous NaHCO3 solution. The organic phase was dried
over MgSO4, filtered, and concentrated under reduced pressure.
The residue obtained was purified by column chromatography
using cyclohexane–ethyl acetate (6 : 4) as eluent affording first
Bz-(S)-BpAib-(S)-Phe-NHChx 7b (1.176 g, 36%), then Bz-(R)-
BpAib-(S)-Phe-NHChx 8b (1.184 g, 37%).
Bz-(S)-BpAib-(S)-Phe-NHChx 7b. Rf 0.32 (cyclohexane–ethyl
acetate 1 : 1); m.p. 125–127 ◦C; dH (CDCl3) 1.06–1.29 (m, 5H),
1.57–1.86 (m, 5H), 3.03 (m, 2H), 3.39 (m, 3H), 3.68 (m, 1H), 3.93
(d, 1H, J = 17.5 Hz), 4.72 (dd, 1H, J = 6.9, 8.1 Hz), 6.66 (d, 1H,
J = 8.1 Hz), 6.79 (d, 1H, J = 8.4 Hz), 7.05–7.15 (m, 5H), 7.20–7.26
(m, 2H), 7.32–7.63 (m, 8H), 7.65–7.74 (m, 4H); dC (CDCl3) 24.8,
24.9, 25.4, 32.6, 32.7, 37.2, 42.5, 43.6, 48.6, 53.9, 67.8, 124.6, 125.9,
Z-(RS)-BpAib-(S)-Phe-NHChx (7a and 8a). A suspension of
Z-(RS)-BpAib-OH 5 (231 mg, 0.56 mmol) and HCl·H-(S)-Phe-
NHChx (188 mg, 0.67 mmol) in tetrahydrofuran (7 mL) was
cooled on an ice bath. HATU (254 mg, 0.67 mmol) and DIEA
(0.25 mL) were added. The reaction mixture was allowed to
warm to rt and stirred for 48 h. The mixture was concentrated
under reduced pressure and the resulting residue was taken up
in dichloromethane. The solution was washed twice with 0.5 M
aqueous hydrochloric acid, once with water, and once with
saturated aqueous NaHCO3 solution. The organic phase was dried
over MgSO4, filtered, and concentrated under reduced pressure.
The residue obtained was purified by column chromatography
using cyclohexane–ethyl acetate (1 : 1) as eluent affording first Z-
(S)-BpAib-(S)-Phe-NHChx 7a (119 mg, 33%), then Z-(R)-BpAib-
(S)-Phe-NHChx 8a (122 mg, 34%).
126.9, 127.2, 128.3, 128.5, 129.1, 129.9, 132.2, 132.5, 132.8, 136.5,
25
136.7, 137.5, 139.5, 146.1, 168.1, 169.5, 171.8, 196.9; [a]589
=
+98 (c 0.5, CH2Cl2); Found: C, 75.28; H, 6.47; N 6.73. Calc. for
C39H39N3O4·0.5H2O: C, 75.21; H, 6.47; N, 6.75. ESI-MS m/z 636.5
[M + Na]+.
Bz-(R)-BpAib-(S)-Phe-NHChx 8b. Rf 0.22 (cyclohexane–
ethyl acetate 1 : 1); m.p. 108–111 ◦C; dH (CDCl3) 1.01–1.33 (m,
5H), 1.55–1.81 (m, 5H), 3.04 (m, 2H), 3.41 (m, 3H), 3.64 (m, 1H),
3.79 (d, 1H, J = 16.9 Hz), 4.64 (dd, 1H, J = 6.6, 7.9 Hz), 6.51 (d,
1H, J = 8.1 Hz), 6.69 (d, 1H, J = 8.1 Hz), 7.05–7.16 (m, 5H), 7.21–
7.26 (m, 2H), 7.31–7.61 (m, 8H), 7.63–7.78 (m, 4H); dC (CDCl3)
24.8, 24.9, 25.5, 32.6, 32.8, 37.5, 42.7, 43.7, 48.6, 53.4, 54.3, 67.7,
124.4, 126.4, 126.9, 127.2, 128.3, 128.5, 128.6, 129.2, 129.5, 129.9,
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132.3, 132.4, 133.1, 136.6, 137.1, 137.7, 140.9, 144.3, 168.0, 169.3,
5 D. Fillion, M. Derae¨t, B. J. Holleran and E. Escher, J. Med. Chem.,
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25
171.9, 196.5; [a]589 = -11 (c 0.5, CH2Cl2); Found: C, 74.06; H,
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and J. M. Becker, Biochemistry, 2008, 47, 5638–5648.
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(Pept. Sci.), 2001, 60, 396–419.
6.59; N, 6.56. Calc. for C39H39N3O4·H2O: C, 74.14; H, 6.54; N,
6.65. ESI-MS m/z 636.5 [M + Na]+.
Boc-(S)-BpAib-OMe 9. Bz-(S)-BpAib-(S)-Phe-NHChx 7b
(629 mg, 1.03 mmol) was dissolved in dioxane (15 mL) and
10 M hydrochloric acid (15 mL) was added. The mixture was
heated at reflux for 72 h and concentrated under reduced pressure.
MeOH was added to the residue and the mixture was concentrated
under reduced pressure three times. The resulting brown oil was
dissolved in MeOH (5 mL) and the solution cooled on an ice
bath. Thionyl chloride (0.5 mL) was added. The reaction mixture
was allowed to warm to rt and stirred for 18 h. The mixture was
concentrated under reduced pressure. MeOH was added to the
residue and the mixture was concentrated under reduced pressure
repeatedly. The residue was dissolved in MeOH and the solution
made basic by addition of aqueous ammonia (28%). The mixture
was concentrated under reduced pressure and the residue was
filtered through silica gel, eluting with dichloromethane–MeOH
(95 : 5). The resulting residue was dissolved in acetonitrile (5 mL)
and dichloromethane (2 mL), and (Boc)2O (327 mg, 1.5 mmol)
was added. The mixture was stirred at rt for 24 h. The mixture was
concentrated under reduced pressure and the residue obtained
was purified by column chromatography using ethyl acetate–
cyclohexane (3 : 7) as eluent to afford the product as a solid
(106 mg, 26% yield). Rf 0.31 (ethyl acetate–cyclohexane 3 : 7);
m.p. 168–170 ◦C; dH (CDCl3) 1.40 (s, 9H), 3.29 (m, 2H), 3.65
(m, 2H), 3.74 (s, 3H), 5.38 (bs, 1H), 7.27 (d, 1H, J = 7.7 Hz),
7.44 (t, J = 7.7 Hz, 2H), 7.53–7.63 (m, 3H), 7.75 (d, 2H, J = 6.9
Hz). dC (CDCl3) 28.1, 43.6, 52.6, 65.8, 80.2, 124.2, 126.0, 128.1,
129.5, 129.8, 132.2, 136.6, 137.7, 140.2, 145.2, 154.9, 173.8, 196.4.
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25
[a]589 = +21 (c 0.5, CH2Cl2); Found: C, 68.57, H, 6.52; N 3.33.
Calc. for C23H25NO5·0.5H2O: C, 68.29; H, 6.48; N, 3.46. ESI-MS
m/z 418.1 [M + Na]+.
24 C. Toniolo, M. Crisma, F. Formaggio, E. Benedetti, A. Santini, R.
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Photocrosslinking experiments
25 K. Wright, M. Wakselman, J.-P. Mazaleyrat, A. Moretto, M. Crisma, F.
Formaggio, and C. Toniolo, in Peptides 2008, ed. H. Lankinen, Finnish
Peptide Society, Helsinki, 2008, p. 54.
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563–580.
An equimolar 0.1 M solution of 9 and 10 in CH3CN was placed
in a 100 mL Suprasil quartz tube, purged with dry nitrogen
for 20 min and sealed with a rubber cap. The reaction vessel
was then placed in a Rayonet multilamp photochemical reactor
(Southern New England Ultra Violet Company, Branford, CT),
equipped with 12 ¥ 350 nm lamps, and irradiated in parallel for the
desired time (up to 60 min). The progress of the photocrosslinking
reaction was monitored by means of an Agilent 1200 series
HPLC (Agilent Technologies, Santa Clara, CA), equipped with a
Phenomenex (Torrance, CA) C18 analytical column (UV detection
at 220 nm). Binary gradients of H2O–CH3CN, containing 0.05%
trifluoroacetic acid, were used for elution at a flow rate of
1 mL min-1.
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3286 | Org. Biomol. Chem., 2010, 8, 3281–3286
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