Synthesis of (S)- and (R)-enantiomers of p-(4- hydroxybenzoyl)phenylalanine, useful photoaffinity labeling probes for peptide-protein binding sites

Article abstract of DOI:10.1016/S0957-4166(98)00229-8

A high yield, multigram synthesis of both enantiomers of p-(4- hydroxybenzoyl)phenylalanine, cross-linking probes for peptide-protein binding interactions, is reported. The synthesis was accomplished using Schollkopf chiral auxiliary reagents.

Full text of DOI:10.1016/S0957-4166(98)00229-8

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 9 (1998) 2333–2338
Synthesis of (S)- and (R)-enantiomers of p-(4-hydroxy-
benzoyl)phenylalanine, useful photoaffinity labeling probes for
peptide–protein binding sites
Abdul H. Fauq,∗ Chewki Ziani-Cherif and Elliott Richelson
Mayo Clinic Jacksonville, 4500 San Pablo Road, FL 32224, USA
Received 5 May 1998; accepted 4 June 1998
Abstract
A high yield, multigram synthesis of both enantiomers of p-(4-hydroxybenzoyl)phenylalanine, cross-linking
probes for peptide–protein binding interactions, is reported. The synthesis was accomplished using Schöllkopf
chiral auxiliary reagents. © 1998 Elsevier Science Ltd. All rights reserved.
1. Introduction
Biologically active ligands containing a photoreactive and a radiolabeling moiety have proved to
be highly valuable probes for many ligand-receptor interactions.1 These photoaffinity labeling agents,
in addition to having one or more radioactive atoms, possess a photosensitive organic azide, diazo
ester, or diazirine moiety. On exposure of the ligand-receptor complex to light, the ligand component
generates highly reactive carbene or nitrene intermediates that, in turn, can irreversibly alkylate the
receptor. This allows an investigator to elucidate the primary structure of the receptor binding sites.
A major drawback of these highly reactive probes is their non-specific attack on water molecules
and other non-target macromolecules. Following a discovery of photoactive benzophenones,2 which
on photolysis give rise to a much milder reactive intermediate carbonyl triplet diradical, Kauer et al.
reported the synthesis of a novel stable benzophenone, 4-benzoyl-L-phenylalanine (BPA).3a Though
BPA can be obtained in tritiated form,3b it was not subject to radioiodination and Edman-sequencing.
Recently, Wilson4 et al. described the synthesis and use of (ꢀ)-p-(4-hydroxybenzoyl)phenylalanine (rac)-
HBPA (1, Scheme 1), a racemic analog of BPA, which not only has the gentle attributes of BPA but
also is amenable to radioiodination. The incorporation of the rac-HBPA into a peptide nevertheless
necessitates the inconvenient separation and stereocharacterization of the two diastereomers of the
∗
Corresponding author.
0957-4166/98/$ - see front matter © 1998 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(98)00229-8

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A. H. Fauq et al. / Tetrahedron: Asymmetry 9 (1998) 2333–2338
synthetic peptide ligands. As an ongoing part of our studies for exploring the structure of the binding
pocket of the neurotensin receptor, we required incorporation of enantiomerically pure (S)-HBPA into
several novel neurotensin peptide ligands. Reported herein is a simple, high yield synthesis of pure
S- and R-enantiomers of HBPA for general use as a combined photophore and radiolabel for probing
peptide–protein interactions.
Scheme 1.
2. Results and discussion
The entire synthesis of the (S)- and (R)-HBPA was carried out in a straightforward manner using
the commercially available enantiomeric bislactim ethers 65 (Scheme 1). Thus, for the synthesis of (S)-
HBPA, p-bromobenzyl bromide (5) was coupled to the carbanion derived from the commercially avail-
able (R)-Schöllkopf reagent, (2R)-2-isopropyl-3,6-dimethoxy-2,5-dihydropyrazine (6), to give (S,R)-
bislactim ether 7a,6 which was obtained as a single diastereomer (100% de) as indicated by the NMR
and TLC analyses of the crude isolate after work-up. Halogen–metal exchange of 7a at −78°C followed
by the addition of p-benzyloxybenzaldehyde (PBB, obtained in 82% overall yield by (i) selective
benzylation of 4-hydroxybenzyl alcohol (2) to give benzyl alcohol 3, and (ii) MnO2 oxidation of 3) gave
a mixture of diastereomeric benzhydryl alcohols, which, after purification, could be readily oxidized to
the benzophenone 8a. Mild acid hydrolysis of 8a to the aminoester 9a was followed by a very rapid
saponification in commercial THF with a 1 molar excess of LiOH in H2O/THF to give O-benzylated
amino acid 10a. The remarkably efficient saponification of the ester 9a may have been due to trace
amounts of H2O2, a possible contaminant in commercial, stabilized THF. This may be a useful solvent
to use for minimizing risk of racemization in sensitive cases. The catalytic hydrogenolysis had to be
conducted in 1 molar excess of NaOH to avoid concomitant overreduction of the ketone function which
was a major problem with our use of several acidic or neutral solvent media. Thus, starting from p-
benzyloxybenzaldehyde (4) and p-bromobenzyl bromide (5), pure (S)-HBPA (1a)6 ([α]D25=−5.5 (c=2.0
mg/ml, MeOH:H2O=3:1 by volume) was obtained in an overall yield of 73%. Repetition of the above

A. H. Fauq et al. / Tetrahedron: Asymmetry 9 (1998) 2333–2338
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procedure using (S)-bislactim ether reagent (6b) in the key Schöllkopf coupling furnished pure (R)-
HBPA (1b)6 [α]D25=+5.1 (c=4.6 mg/ml, MeOH:H2O=3:1 by volume) in a similar yield. These protocols
furnished multigram lots, of both the enantiomers in a matter of days.
In summary, a highly practical synthesis of pure (L)- and (D)-HBPA (1a,b) is reported. Using this
method, it is possible to quickly make multigram quantities of these valuable photoaffinity probes. The
salient features of the synthesis are: (i) conciseness and high overall yield (73%) for both the enantiomers
of 1; (ii) essentially 100% diastereoselectivity in the key Schöllkopf coupling; (iii) crucial requirement
of a base during the debenzylation of the compounds 10 to avoid overreduction; and (iv) extremely
fast saponification of chiral aminoesters 9 using commercial, unpurified THF which might constitute a
valuable procedure in highly racemization-prone cases.
3. Experimental section
3.1. General methods
Melting points were taken with a GallenKamp instrument and are uncorrected. The column chromato-
graphic separations were performed with J. T. Baker Silica gel (230–400 mesh). THF and diethyl ether
were distilled from sodium/benzophenone ketyl. Anhydrous DMF was purchased from Aldrich Chem-
icals. Methylene chloride was distilled over P2O5. Acetonitrile was reagent grade from E. M. SCIENCE
and was used without further purification. Ethyl acetate and hexane were distilled prior to use. The purity
of all reported compounds was shown to be >95% by TLC and high field 1H NMR and 13C NMR (300
and 75 MHz Brücker instrument). Optical rotations were taken with a 241-Perkin–Elmer polarimeter
(Na lamp). IR spectra were measured with a 2020 GALAXY Series FT-IR instrument, from Mattson
Instruments. Mass spectral analyses were performed at the Mayo Clinic Rochester Mass Spectroscopy
Facility, Rochester, Minnesota. The high resolution MS and elemental analysis was performed at the
Department of Chemistry, University of Florida.
3.2. [4-(Benzyloxy)phenyl]methanol (3)
Sodium hydride (2.93 g, 73 mmol) was added in small portions to a solution of 4-hydroxybenzyl
alcohol (10 g, 80.5 mmol) in DMF (220 ml) at 0°C. The mixture was stirred for 1 h at RT before benzyl
bromide (8.68 ml, 73 mmol) was added dropwise. The reaction mixture was stirred for 6 h, then DMF
was removed by distillation under vacuum. The residue was taken up in methylene chloride (200 ml)
and washed once with water. The organic layer was dried and concentrated in vacuum. The residue was
purified by chomatography on silica gel (ethyl acetate:hexane=1:2 by volume, Rf=0.41) to give 12.86 g
(83%) of alcohol 3 as a white solid: mp 85–86°C. 1H NMR (CDCl3) δ 7.40–7.20 (m, 7H), 6.92 (d, J=6.5
Hz, 2H), 5.02 (s, 2H), 4.55 (d, J=5.0 Hz, 2H), 1.64 (t, OH); 13C NMR (CDCl3) δ 138.9, 133.4, 128.8,
128.5, 127.9, 127.4, 115.0, 114.9, 70.0, 65.0; IR (neat, cm−1) 3396, 2930.
3.3. 4-(Benzyloxy)benzaldehyde (4)
Manganese dioxide (41 g, 471.6 mmol) is added in one portion to a solution of alcohol (3) (11.17 g,
52 mmol) in methylene chloride (170 ml). The mixture was stirred at RT for 15 h, then filtered through
Celite and concentrated to produce aldehyde 4 (11 g, 99%) as a white powder: mp 70–71°C; Rf=0.79
1
(ethyl acetate:hexane=1:2 by volume); H NMR (CDCl3) δ 9.88 (s, 1H), 7.83 (d, J=8.9 Hz, 2H), 7.39

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(m, 5H), 7.08 (d, J=8.7 Hz, 2H), 5.15 (s, 2H); 13C NMR (CDCl3) δ 190.4, 163.4, 135.7, 129.8, 128.4,
128.0, 127.2, 114.8, 69.8; IR (neat, cm−1) 2744, 1684, 1601; MS (ESI) 213 (M++1).
3.4. (2S,5R)-2-(4-Bromobenzyl)-5-isopropyl-3,6-dimethoxy-2,5-dihydropyrazine (7a)
To a solution of (2R)-2-isopropyl-3,6-dimethoxy-2,5-dihydropyrazine (7.96 g, 43.2 mmol) in THF
(180 ml), is added n-BuLi (33.2 ml, 1.5 M in hexane) at −78°C dropwise. After stirring for 15 min,
a solution of the bromide 5 (11.916 g, 47.6 mmol) in THF (20 ml) is added dropwise. The resulting
mixture is stirred for 2 h at −78°C. After removal of THF in vacuo, the residue is diluted with methylene
chloride (120 ml). The organic layer is washed with 5% sulfuric acid, water, and brine, then dried and
concentrated in vacuo. Purification by chomatography on silica gel (ethyl acetate:hexane=1:7 by volume,
Rf=0.5) afforded compound 7a as a single diastereomer (100% de, 13.42 g, 88%) as a white solid:
[α]D25=+49.1 (c=6.8 mg/ml, CH2Cl2); mp 54–55°C; 1H NMR (CDCl3) δ 7.33 (d, J=8.3 Hz, 2H), 6.97
(d, J=8.3 Hz, 2H), 4.3 (dd, J=8.4, 4.7 Hz, 1H), 3.7 (s, 3H), 3.66 (s, 3H), 3.39 (t, 1H), 3.04 (d, J=4.8 Hz,
2H), 2.16 (m, 1H), 0.96 (d, J=6.8 Hz, 3H), 0.61 (d, J=6.8 Hz, 3H); 13C NMR (CDCl3) δ 163.6, 162.2,
136.2, 131.4, 130.6, 120.1, 60.1, 56.1, 52.0, 51.8, 39.1, 31.0, 18.8, 16.2; IR (neat, cm−1) 2961, 2870,
1693, 1437, 1238, 1014; MS (ESI) 353 (M++1) and 355 (M++1).
The enantiomer of 7a, i.e., (2R,5S)-2-(4-bromobenzyl)-5-isopropyl-3,6-dimethoxy-2,5-
dihydropyrazine (7b) was made in a similar manner using (2S)-2-isopropyl-3,6-dimethoxy-2,5-
dihydropyrazine (7b) as the chiral auxiliary: [α]D25=−50.3 (c=6.7 mg/ml, CH2Cl2).
3.5. [4-(Benzoyloxy)phenyl](4-{[(2S,5R)-5-isopropyl-3,6-dimethoxy-2,5-dihydro-2-pyrazinyl]methyl}-
phenyl)methanols
(Epimeric at the alcholic carbon). t-Butyllithium (28 mmol, 1.7 M in pentane) was added dropwise
at −78°C to a solution of the bromide 7a (5.82 g, 16.47 mmol) in distilled THF (350 ml). Five minutes
later, a solution of the aldehyde 4 (4.2 g, 19.8 mmol) in distilled THF (60 ml) was slowly cannulated into
the reaction mixture. The mixture was stirred for an additional 2 h at the same temperature. A saturated
ammonium chloride solution was added and the mixture was warmed to RT. The volatiles were removed
in vacuo and the residue is taken up in ethyl acetate and washed once with water. The aqueous layer
was extracted twice with ethyl acetate. The combined organic extracts were dried, concentrated, and
the residue was purified by chromatography on silica gel to furnish the desired diastereomeric alcohols
(7.468 g, 93%) as a colorless oil: [α]D25=+34.7 (c=11.8 mg/ml, CH2Cl2); 1H NMR (CDCl3) δ 7.44–7.28
(m, 5H), 7.23 (m, 4H), 7.06 (d, J=8.1 Hz, 2H), 6.92 (dt, J=2.9 Hz, J=8.7, 2.9 Hz, 2H), 5.75 (d, J=2.9 Hz,
1H), 5.03 (s, 2H), 4.29 (dd, J=8.47, 4.8 Hz, 1H), 3.70 (d, J=0.9 Hz, 3H), 3.68 (d, J=2.1 Hz, 3H), 3.26 (dd,
J=6.2, 3.2 Hz, 1H), 3.06 (d, J=4.8 Hz, 2H), 2.23 (d, J=3.7 Hz, OH), 2.12 (m, 1H), 0.93 (dd, J=6.9, 2.3
Hz, 3H), 0.60 (d, J=6.8 Hz, 3H); 13C NMR (CDCl3) δ 171.1, 163.9, 162.4, 158.2, 141.9, 138.6, 130.0,
128.5, 127.9, 127.4, 125.9, 114.7, 75.5, 70.0, 60.2, 56.5, 52.3, 52.1, 39.6, 31.1, 21.0, 19.0, 16.4, 14.2; IR
(neat, cm−1) 3368, 2961, 2870, 1695, 1240, 1016, 736; MS (ESI) 487 (M++1).
Following the above procedure, the diastereomeric mixture of alcohols of opposite configuration was
obtained in a similar yield by coupling 7b with the aldehyde 4. [α]D25=−33.4 (c=13.0 mg/ml, CH2Cl2).

A. H. Fauq et al. / Tetrahedron: Asymmetry 9 (1998) 2333–2338
2337
3.6. [4-(Benzoyloxy)phenyl](4-{[(2S,5R)-5-isopropyl-3,6-dimethoxy-2,5-dihydro-2-pyrazinyl]methyl}-
phenyl)methanone (8a)
Manganese dioxide (26.66 g, 307.15 mmol) was added in one portion at 0°C to a solution of the
benzylated alcohol (7.468 g, 15.35 mmol) in methylene chloride (250 ml). The mixture was stirred
overnight at RT, then filtered though Celite and concentrated in vacuo to yield 7.43 g (99%) of ketone 8a
as white crystals: Rf=0.56 (ethyl acetate:hexane=1:4 by volume); [α]D25=+31.8 (c=4.2 mg/ml, CH2Cl2);
mp 100–101°C; 1H NMR (CDCl3) δ 7.79 (d, J=6.8 Hz, 2H), 7.63 (d, J=8.2 Hz, 2H), 7.46–7.34 (m, 5H),
7.22 (d, J=8.2 Hz, 2H), 7.03 (d, J=6.8 Hz, 2H), 5.14 (s, 2H), 4.36 (dd, J=8.8, 5.0 Hz, 1H), 3.72 (s, 3H),
3.67 (s, 3H), 3.42 (t, J=3.5 Hz, 1H), 3.17 (d, J=5.4 Hz, 2H), 2.17 (m, 1H), 0.96 (d, J=6.9 Hz, 3H), 0.62
(d, J=6.8 Hz, 3H); 13C NMR (CDCl3) δ 195.3, 163.9, 162.1, 142.2, 136.2, 136.0, 132.3, 130.5, 129.7,
129.4, 128.6, 128.1, 127.4, 114.2, 70.1, 60.3, 56.3, 52.3, 52.1, 39.9, 31.3, 18.9, 16.4; IR (neat, cm−1
2943, 1695, 1601, 1242, 1014; MS (ESI) 485 (M++1).
)
Following the above procedure, a ketone of the opposite configuration, 8b, was obtained in a similar
yield. [α]D25=−29.4 (c=4.7 mg/ml, CH2Cl2).
3.7. Methyl (2S)-2-amino-3-{4-[4-(benzyloxy)benzoyl]phenyl}propanoate (9a)
Trifluoroacetic acid (TFA, 45.9 mmol, 0.1 N in water, 3 equiv.) is added to a solution of the compound
8a (7.43 g, 15.35 mmol) in a mixture of methylene chloride (30 ml) and acetonitrile (300 ml). The
resulting mixture was stirred at RT for 3 h. The acetonitrile and TFA were removed in vacuo to produce
a suspension of the methyl ester 9a and (D)-valine methyl ester as TFA salts in water. This suspension,
without neutralization with base, was then extracted 5 times with methylene chloride. The combined
organic phase was dried and concentrated in vacuo to give 5.91 g (99%) of the amino ester 9a as a white
powder. This procedure effectively eliminated the TFA salt of (D)-valine methyl ester which remained
25
in the aqueous phase: Rf (free amine)=0.32 (ethyl acetate); [α]D (free amine)=+5.0 (c=10.2 mg/ml,
1
MeOH); mp 102–103°C; H NMR (CDCl3, free amine) δ 7.81 (d, J=8.6 Hz, 2H), 7.72 (d, J=8.1 Hz,
2H), 7.46–7.29 (m, 5H), 7.03 (d, J=8.9 Hz, 2H), 5.12 (s, 2H), 3.78 (dd, J=7.8, 5.2 Hz, 1H), 3.73 (s, 3H),
3.15 (dd, J=13.5, 5.2 Hz, 1H), 2.95 (dd, J=13.5, 7.9 Hz, 1H), 1.54 (br, NH2); 13C NMR (CDCl3, free
amine) δ 195.0, 175.2, 162.3, 136.7, 136.0, 132.4, 130.4, 130.1, 129.1, 128.6, 128.0, 127.4, 114.4, 70.13,
55.6, 52.1, 40.9; IR (free amine, neat, cm−1) 1738, 1649, 1599, 1251; MS (ESI) 390 (M++1).
Following the above procedure, methyl ester of the opposite configuration, 9b, was obtained in a
similar yield by coupling 7b with the aldehyde 4.
3.8. (2S)-2-Amino-3{4-[4-(benzyloxy)benzoyl]phenyl}propanoic acid (10a)
Methyl (2S)-2-amino-3-{4-[4-(benzyloxy)benzoyl]phenyl}propanoate (9a) (0.83 g, 1.65 mmol) was
dissolved in 60 ml of THF. An aqueous solution of LiOH·H2O (189 mg, 4.95 mmol) in 30 ml of water
was added at RT and the mixture stirred for 4–5 min. Following neutralization of the mixture with 6 N
HCl to pH 3, the THF was evaporated in vacuo. A white precipitate of the amino acid–HCl salt appeared,
on concentration, that was cooled to 0°C for 1 h, then filtered, washed with chilled water (×2) and ether
(×3), and dried under high vacuum over P2O5 overnight (yield=674 mg, 99.6%): 1H NMR (DMSO-d6) δ
7.74 (d, J=8.7 Hz, 2H), 7.62 (d, J=7.8 Hz, 2H), 7.49–7.35 (m, 7H), 7.17 (d, J=8.7 Hz, 2H), 5.21 (s, 2H),
3.54 (dd, J=8.0, 5.0 Hz, 1H), 3.24 (dd, J=14.0, 4.23 Hz, 1H), 2.99 (dd, J=14.0, 8.0 Hz, 1H); 13C NMR
(CDCl3) δ 194.2, 168.2, 162.0, 141.9, 139.2, 136.4, 136.1, 132.1, 129.4, 128.5, 128.1, 127.9, 124.9,
114.7, 69.6, 34.4, 30.4; IR (NaCl plate, cm−1) 3431, 1741, 1599, 1249; MS (ESI) 376 (M++1).

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A. H. Fauq et al. / Tetrahedron: Asymmetry 9 (1998) 2333–2338
3.9. (S)-p-(4-Hydroxybenzoyl)phenylalanine (1a)
The benzyl group was removed by hydrogenalizing protected amino acid 10a (608 mg, 1.48 mmol
suspended in 25 ml of water) in the presence of NaOH (118 mg, 2.95 mmol) and 10% Pd–C (152 mg)
at RT and atmospheric pressure for 1.5 h in the dark. After filtration through Celite, the aqueous solution
was acidified to pH 3 with 6 N HCl. Charcoal (0.5 g) was added and the mixture was heated to 60°C for
5 min. Filtration and lyophilization gave a slightly off-colored powder which was quickly washed once
with chilled water and dried over P2O5 overnight (yield=455 mg, 95%). This product was suitable for
conversion to the fluorenylmethoxycarbonyl (Fmoc) group for the solid phase synthesis of peptides. An
analytical sample of 1a (TFA salt) was obtained by HPLC purification (RT=21.5 min on a Vydac column,
22 mm×250 mm, C-8, 10 µ particle size; gradient elution with 5% B to 100% B in 50 min at 8.0 ml flow
1
rate; UV detection at λ=254 nm): [α]D25=−5.5 (c=2.0 mg/ml, MeOH:H2O=3:1 by volume); H NMR
(as TFA salt, D2O) 7.72 (d, J=8.7 Hz, 2H), 7.69 (d, J=8.1 Hz, 2H), 7.42 (d, J=8.1 Hz, 2H), 6.93 (d, J=8.7
Hz, 2H), 4.27 (t, J=7.2 Hz, 1H), 3.39 (dd, J=14.3, 5.73 Hz, 1H), 3.73 (s, 3H), 3.26 (dd, J=14.5, 7.7 Hz,
1H); 13C NMR (H2O, as disodium salt, pH 10) δ 201.3, 186.7, 177.6, 145.0, 139.4, 137.3, 132.1, 131.6,
124.5, 121.5, 59.8, 43.3; IR (NaCl plate, cm−1) 3431, 3317, 1645, 1602, 1249; MS (ESI) 286 (M++1).
HRMS m/z calcd for C16H16NO4 (M++1) 286.1079, found 286.1079. Anal. calcd for C16H17NO5 (1a, as
monohydrate): C=63.36; H=5.65; N=4.62. Found: C=63.88; H=5.53; N=4.68.
3.10. (R)-p-(4-Hydroxybenzoyl)phenylalanine (1b)
Compound 1b was obtained by hydrogenolysis of 10b. The spectral data of 1b were identical to
1a: [α]D25=+5.1 (c=4.6 mg/ml, CH2Cl2). Anal. calcd for C16H17NO5 (1a, as monohydrate): C=63.36;
H=5.65; N=4.62. Found: C=63.60; H=5.58; N=4.67.
Acknowledgements
This work was supported by NIH (Grant no. MH27692, awarded to ER).
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