7712 J . Org. Chem., Vol. 66, No. 23, 2001
Bourne et al.
to the ionization source via a fused silica capillary interface
(50 µm i.d. × 50 cm length). Sample droplets were ionized at
a positive potential of 5 kV and entered the analyzer through
an interface plate and subsequently through an orifice (100-
200 µm diameter) at a potential of 80 V. Full scan mass spectra
were acquired over a range of 200-2000 Da with a scan step
size of 0.1 Da. Accurate mass measurements were acquired
on a LCT oa-TOF mass spectrometer equipped with a Z-spray
source (Manchester, U.K.). The source was generated in the
positive ion electrospray mode, with a declustering potential
of 35V. The instrument was operated at a resolution of 5000
fwhm and set to acquire over the mass range m/z 100-1000
at a speed of 5× spectra per second. Sample analysis, accurate
mass measurements and spectral interpretation were per-
formed automatically using a diversity software control.
Automated lock mass correction was performed using the
protonated molecular ion of [Leu5] enkephalin at m/z 566.2771,
C28H38N5O7. Accurate mass measurements of the final products
showed good correlation with the expected empirical chemical
formuli with mass accuracies of better then 5 ppm. TLC (thin-
layer chromatography) was performed on silica gel 60 F254
plates (Merck Art 5735). The chromatograms were viewed
under UV light and/or developed with iodine vapor. Prepara-
tive column chromatography was effected under pressure,
using for normal-phase Merck Kieselgel 60 (Merck Art 7734).
Analytical reversed-phase HPLC was run on a C18 column
(0.46 × 25 cm), and preparative reversed-phase HPLC was
run on a C-18 column (2.2 × 25 cm). Both columns were
attached to a HPLC setup fitted with a Holochrome UV
detector. Measurements were carried out at λ ) 214 nm.
Ma ter ia ls. BOC-L-amino acids, synthesis grade dimethyl-
formamide (DMF), trifluoroacetic acid (TFA), and diisopropyl-
ethylamine (DIEA) were purchased from Auspep (Parkville,
Australia). HBTU and BOP were purchased from Richelieu
Biotechnologies (Montreal, Canada). AR grade EtOAc, MeOH,
CH2Cl2, CHCl3, hexane, acetone, and HPLC-grade CH3CN
were all obtained from Laboratory Supply (Australia), HF was
purchased from CIG (Australia). Aminomethylpolystyrene
resins with a substitution value of 0.26 mmol/g were purchased
from Peptide Institute (Osaka, J apan). All other reagents were
AR grade or better and were obtained from Aldrich, Fluka, or
Bachem AG Switzerland.
CDCl3) δ 30.3, 30.6, 35.7, 36.0, 51.5, 51.5, 71.0, 71.1, 112.2,
112.4, 114.6, 114.7, 119.6, 121.2, 127.2, 127.3, 127.7, 127.8,
128.2, 128.3, 128.4, 128.6, 132.4, 134.2, 144.2, 144.2, 145.6,
145.8, 173.3, 173.3; ES-MS Mr 286.1 (calcd 286.0).
1-(3-Ben zyloxy-4-h yd r oxyp h en yl)p r op a n oic Acid a n d
1-(4-Ben zyloxy-3-h yd r oxyp h en yl)p r op a n oic Acid 5. A
solution of lithium hydroxide monohydrate (5.25 g, 125 mmol)
in water (150 mL) was added to a stirred solution of the
mixture of methyl esters (11.95 g, 41.7 mmol) in THF (150
mL). The solution was stirred overnight, and then the THF
was evaporated off and the residue was diluted with water
(150 mL) and acidified to pH 3 with 5% HCl. The mixture was
extracted with CHCl3 (3 × 350 mL), and the combined extracts
were dried (Na2SO4) and evaporated to a brown oil that
solidified on standing. This was taken up in EtOAc and passed
through a short silica column. Evaporation of the eluent gave
the product as a tan solid 5 (11.12 g, 98%): IR νmax (KBr disk)
3533, 3471, 3300-2600, 1718, 1699, 1515 cm-1; 1H NMR (300
Hz, CDCl3) δ 2.66 (m, 4H), 2.90 (t, J ) 7.6 Hz, 2H), 2.91 (t, J
) 7.7 Hz, 2H), 5.09 (s, 2H), 5.10 (s, 2H), 6.69 (dd, J ) 8.3 Hz,
2.1 Hz, 1H), 6.75 (dd, J ) 8.1, 2.0 Hz), 6.83 (d, J ) 1.9 Hz,
1H), 6.84 (d, J ) 2.0 Hz, 1H), 6.87 (d, J ) 8.4 Hz, 1H), 6.90 (d,
J ) 8.2 Hz, 1H), 7.30-7.50 (m, 10H); 13C NMR (75 MHz,
CDCl3) δ 29.9, 30.2, 35.7, 35.9, 71.1,71.2, 112.3, 112.4, 114.6,
114.7, 119.6, 121.2, 127.2, 127.3, 127.7, 127.8, 128.3, 128.4,
128.7, 132.0, 136.2, 144.3, 145.6, 145.8, 179.2, 179.2; ES-MS
Mr 272.1 (calcd 271.9).
1-[3-Ben zyloxy-4-(BOC-P h e-O)p h en yl]p r op a n oic Acid
an d 1-[4-Ben zyloxy-3-(BOC-P h e-O)ph en yl]pr opan oic Acid.
Triethylamine (1.40 mL, 1.01 g, 10.0 mmol) and ethyl chloro-
formate (0.96 mL, 1.085 g, 10.0 mmol) were added to a stirred,
chilled (-8 °C) solution of BOC-Phe-OH (2.65 g, 10.0 mmol)
in dichloromethane (20 mL). The solution was stirred for 20
min at -8 °C, during which time a precipitate formed. A
solution of a regioisomeric mixture of benzyloxy acids 5 (2.86
g, 10.0 mmol) and triethylamine (1.40 mL, 1.01 g, 10.0 mmol)
in dichloromethane (20 mL + 5 mL rinse) was then added
dropwise. The resulting solution was stirred at -5 to 0 °C for
2 h, washed with 10% citric acid and brine, dried (Na2SO4),
and evaporated to a syrup. This was dissolved in a small
amount of 1:1 ethyl acetate/petroleum ether and passed
through a short silica column. Evaporation of the eluent gave
the mixture of title carboxylic acids as a colorless syrup (4.47
g, 86%): 1H NMR (300 Hz, CDCl3) δ 2.66 (m, 4H), 2.90 (t, J )
7.6 Hz, 2H), 2.91 (t, J ) 7.7 Hz, 2H), 5.09 (s, 2H), 5.10(s, 2H),
6.69 (dd, J ) 8.3 Hz, 2.1 Hz, 1H), 6.75 (dd, J ) 8.1, 2.0 Hz),
6.83 (d, J ) 1.9 Hz, 1H), 6.84 (d, J ) 2.0 Hz, 1H), 6.87 (d, J )
Meth yl 1-(3,4-Dih yd r oxyp h en yl)p r op ion a te.47 A solu-
tion of 3,4-dihydroxyhydrocinnamic acid 4 (10.0 g, 54.9 mmol)
and concentrated H2SO4 (3 mL) in methanol (250 mL) was
heated under reflux overnight. The solvent was evaporated,
and the residue was shaken with water and extracted into
CHCl3. The combined extracts were dried (Na2SO4) and
evaporated to give the methyl ester as a pale yellow oil that
crystallized on standing (11.2 g, 100%): mp 71.9-74.1 °C (lit.47
8.4 Hz, 1H), 6.90 (d, J ) 8.2 Hz, 1H), 7.30-7.50 (m, 10H); 13
C
NMR (75 MHz, CDCl3) δ 29.9, 30.2, 35.7, 35.9, 71.1,71.2, 112.3,
112.4, 114.6, 114.7, 119.6, 121.2, 127.2, 127.3, 127.7, 127.8,
128.3, 128.4, 128.7, 132.0, 136.2, 144.3, 145.6, 145.8, 179.2,
179.2; ES-MS Mr 519.1 (calcd 519.2).
1
mp 74-76 °C); IR νmax (KBr disk) 3485, 3311, 1712 cm-1; H
NMR (300 Hz, CDCl3) δ 2.61 (t, J ) 7.5 Hz, 2H), 2.83 (t, J )
7.5 Hz, 2H), 3.69 (s, 3H), 5.40 (br s, 2H), 6.61 (dd, J ) 2.1, 8.1
1-[3-Ben zyloxy-4-(BOC-Gly-O)p h en yl]p r op a n oic Acid
an d 1-(4-Ben zyloxy-3-(BOC-Gly-O)ph en yl]pr opan oic Acid.
Triethylamine (1.40 mL, 1.01 g, 10.0 mmol) and ethyl chloro-
formate (0.96 mL, 1.085 g. 10.0 mmol) were added to a stirred,
chilled (-20 °C) solution of BOC-Gly-OH (1.75 g, 10.0 mmol)
in dichloromethane (20 mL). The solution was stirred for 20
min at -10 to -15 °C, during which time a precipitate formed.
A solution of regioisomeric mixture of benzyloxy acids 5 (2.86
g, 10.0 mmol) and triethylamine (1.40 mL, 1.01 g, 10.0 mmol)
in dichloromethane (20 mL + 5 mL rinse) was then added
dropwise. The resulting solution was stirred at -5 to 0 °C for
2 h, washed with 10% citric acid (2 × 10 mL) and brine (10
mL), dried (Na2SO4), and evaporated to a syrup. This was
dissolved in a small amount 1:1 ethyl acetate/petroleum ether
and passed through a short silica column. Evaporation of the
eluent gave the mixture of title carboxylic acids as a colorless
syrup (3.99 g, 93%): 1H NMR (300 Hz, CDCl3) 1.47 (s, 9H)
2.65 (t, J ) 6.6, 2H), 2.85-2.95 (m, 2H), 4.15-4.17 (m, 2H),
5.07 (s, 2H), 5.08-5.15 (m, 1H), 6.66-7.46 (m, 8H); 13C NMR
(75 MHz, CDCl3) 28.3, 29.6, 30.4, 35.3 35.6, 42.3, 70.7, 71.3,
80.1, 155.6, 178.0, 178.4; ES-MS Mr 430.4 (calcd 430.2).
1-[3-Ben zyloxy-4-(N-ter t-bu toxyca r bon yl)a sp a r tyloxy]-
p h en ylp r op a n oic Acid a n d 1-(4-Ben zyloxy-3-(N-ter t-bu -
Hz, 1H), 6.71 (d, J ) 2.1 Hz, 1H), 6.77 (d, J ) 8.1 Hz, 1H); 13
C
NMR (75 MHz, CDCl3) δ 30.2, 35.9, 51.9, 115.4, 120.5, 120.5,
133.2, 142.1, 143.6, 174.3; MS Mr 196.0739 (calcd 196.0736).
Meth yl 1-(3-Ben zyloxy-4-h yd r oxyp h en yl)p r op a n oa te
an d Meth yl 1-(4-Ben zyloxy-3-h ydr oxyph en yl)pr opion ate.
Benzyl bromide (6.10 mL, 8.72 g, 52.0 mmol) was added to a
stirred suspension of methyl 4-(1,2-dihydroxyphenyl)propi-
onate (10.0 g, 51.0 mmol), K2CO3 (8.45 g, 61.2 mmol), and a
catalytic amount of tetrabutylammonium iodide in DMF (250
mL). The suspension was stirred overnight under an atmo-
sphere of nitrogen. Water (1 L) and 5% HCl (150 mL) were
added, and the mixture was extracted with diethyl ether. The
combined extracts were washed with water and brine, dried
(Na2SO4), and evaporated to a brown oil that was purified by
flash chromatography (5-20% EtOAc in petroleum) to give a
1:1 mixture of the monobenzyl ethers as a colorless oil (11.5
g, 79%): IR νmax (NaCl thin film) 3446, 1732, 1592, 1514 cm -1
;
1H NMR (300 Hz, CDCl3) δ 2.60 (m, 4H), 2.87 (t, J ) 7.8 Hz,
2H), 2.89 (t, J ) 7.7 Hz, 2H), 3.67 (s, 3H) 3.68 (s, 3H) 5.08 (s,
2H), 5.09 (s, 2H), 6.67 (dd, J ) 8.2, 2.1 Hz, 1 H), 6.73 (dd, J )
8.0, 1.6 Hz, 1H), 6.81 (m, 2H), 6.82 (d, J ) 8.0 Hz, 1H), 6.88
(d, J ) 8.2 Hz, 1H), 7.30-7.50 (m, 10H); 13C NMR (75 MHz,