Azobenzene-containing, peptidyl α-ketoesters as photobiological switches of α-chymotrypsin

Article abstract of DOI:10.1016/S0040-4020(00)00883-8

Three photoswitchable, peptidomimetic inhibitors of α-chymotrypsin have been synthesised. The compounds comprise an azobenzene, an α-ketoester and L-phenylalanine. The compounds were photoisomerised to give enriched states of the (E) and (Z) isomers and t

Full text of DOI:10.1016/S0040-4020(00)00883-8

TETRAHEDRON
Pergamon
Tetrahedron 56 (2000) 9763±9771
Azobenzene-Containing, Peptidyl a-Ketoesters as Photobiological
Switches of a-Chymotrypsin
*
Andrew J. Harvey and Andrew D. Abell
Department of Chemistry, University of Canterbury, Private Bag 4800, Christchurch, New Zealand
Received 24 April 2000; accepted 2 August 2000
AbstractÐThree photoswitchable, peptidomimetic inhibitors of a-chymotrypsin have been synthesised. The compounds comprise an
azobenzene, an a-ketoester and l-phenylalanine. The compounds were photoisomerised to give enriched states of the (E) and (Z) isomers
and these states were assayed against a-chymotrypsin. The inhibitors were shown to be moderately active with switching ability of between
two- and three-fold between the two isomer-enriched states. The behaviour of the inhibitors in solution was examined; speci®cally, their
hydration and con®gurational stability. q 2000 Elsevier Science Ltd. All rights reserved.
Introduction
switches is a peptidyl group designed to enhance binding
to a-chymotrypsin. Photoswitchable af®nity ligands of this
type, once appropriately immobilised, would provide the
basis of a novel approach to enzyme puri®cation. a-Chymo-
trypsin was chosen as the initial target enzyme because
(i) much is known about its inhibition and (ii) the general
ideas developed here should be applicable to related
proteases.
A photobiological switch is a biologically active compound
with a built-in photochromic group that allows light-
triggered switching of its biological target.1 A variety of
enzymes have been targeted with photobiological switches
either by making the environment photoresponsive, by
covalently modifying the enzyme with a photochromic
group, or by employing a photoisomerisable effector
molecule. For example, the activity of a-chymotrypsin has
been photoregulated by its immobilisation on an azo-
benzene-containing polymer.2 In another example, the
activity of papain was modi®ed by the covalent attachment
of 4-(phenylazo)benzoic acid to the enzyme.2b An inhibitor
comprising an azobenzene tethered to a simple aryl boronic
acid has been shown to afford reversible inhibition of
a-chymotrypsin.3
Results and Discussion
Peptidyl a-ketoesters are known potent inhibitors of serine
proteases.4 It has been postulated that the active site serine
of the protease reacts with the ketone carbonyl to form a
stable, but reversible, tetrahedral intermediate.5 It has also
been suggested that the peptidyl a-ketoesters bind to the
active site as a hydrate which mimics the transition state
associated with the normal catalytic mechanism of the
enzyme.6 The design of compounds 1, 2 and 3 was based
on previously reported examples of peptidyl-based-a-keto-
ester inhibitors of serine proteases.4a A benzyl group was
The photoisomerisable inhibitors described in this paper,
compounds 1, 2 and 3, incorporate an azobenzene as the
photochromic group and an a-ketoester as the inhibitory
moiety (Fig. 1). A third and novel component of these
Figure 1.
Keywords: photobiological switch; a-ketoester; inhibition; a-chymotrypsin; photoisomerisation; azobenzene; peptidomimetic.
* Corresponding author. Tel.: 164-3-3642-818; fax: 164-3-3642-110; e-mail: a.abell@chem.canterbury.ac.nz
0040±4020/00/$ - see front matter q 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(00)00883-8

9764
A. J. Harvey, A. D. Abell / Tetrahedron 56 (2000) 9763±9771
Scheme 1. (i) EDCI, DMAP, CH2Cl2, 20 h, 78%; (ii) O3, MeOH, CH2Cl2, 2788C, 10 min, 77%; (iii) Zn(BH4)2, ether, 2788C, 2 h, 8a 44%, 8b 44%; (iv) HBr,
AcOH, 20 min, 86%.
Scheme 2. (i) EDCI, HOBT, DIEA, DMF, CH2Cl2, 16 h; 12 89%; 13 39%; (ii) TEMPO, KBr, NaOCl, NaHCO3, water, CH2Cl2; 1 quant; 2 95%.
incorporated into these peptidomimetics to bind to the
primary binding pocket (S1 by the notation of Schechter
and Berger)7 of a-chymotrypsin, with the aim to correctly
orient the electrophilic carbonyl in the active site.8
in yields of 44% and 44% respectively. The absolute
con®gurations of the epimers were assigned on the basis
of literature comparison of the 1H NMR spectra and optical
rotation data.18,19
The irradiation of an azobenzene at a speci®c wavelength is
known to give a photostationary state mixture of (E) and (Z)
isomers, the composition of which is dependent on the ratio
of extinction coef®cients at that wavelength.9 The basis of
the current study was the supposition that differences in the
molecular geometry and the dipole moment of the (E) and
(Z) isomers10 would result in differential binding to
a-chymotrypsin and hence photoregulation of its activity.
Compounds 1, 2 and 3 were also designed to determine
which regioisomer of the azobenzene provides the optimal
geometry for binding to a-chymotrypsin.
A single epimer 8a was used in subsequent steps to avoid
complications that would arise from working with mixtures
of isomers. The Cbz group of 8a was removed by treatment
with HBr in acetic acid to give the amine salt 9 in 86%
yield (Scheme 1). This was then coupled with the (phenyl-
azo)benzoic acids 10 or 11,20,21 in the presence of 1-ethyl-
3-[3-(dimethylamino)propyl]carbodiimide hydrochloride
(EDCI), to give 12 (89%) and 13 (39%) respectively
(Scheme 2). The ortho isomer 16 proved more dif®cult to
synthesise, however, it was ultimately prepared in 36%
Synthesis
The key step in the synthesis of compounds 1, 2 and 3
required coupling the hydrochloride salt 9, derived from
8a (Scheme 1), with a (phenylazo)benzoic acid (Schemes
2 and 3). We initially sought an improved route to the key
intermediate 8a, and its epimer 8b, due to the complexity
and low reproducibility of existing methods11±14 for their
preparation. We have found that compounds 8 are best
prepared from Cbz-l-phenylalanine over three steps
utilising cyanoketophosphorane methodology developed
by Wasserman et al.15±17 As shown in Scheme 1, the cyano-
phosphorane 5 was acylated with Cbz-l-phenylalanine to
give the cyanoketophosphorane 6 in 78% yield. Ozonolysis
of 6, in the presence of methanol, gave the methyl a-keto-
ester 7 in 77% yield. This was followed by zinc borohydride
reduction of 7 to give the key intermediates 8a and 8b,
which were isolated separately after ¯ash chromatography
Scheme 3. (i) DCC, N-hydroxysuccinimide, THF, 08C, 16 h; (ii) 9, DIEA,
THF, 4 h, 36% over two steps; (iii) TEMPO, KBr, NaOCl, NaHCO3, water,
CH2Cl2, 88%.

A. J. Harvey, A. D. Abell / Tetrahedron 56 (2000) 9763±9771
9765
Figure 2. Hydration of compound 1.
1
yield on coupling of the N-hydroxysuccinimide ester 15
with 9 (Scheme 3). The ®nal step in the synthetic sequence
involved oxidation of the a-hydroxyl group of 12, 13 and 16
with 2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO)22,23 in
aqueous sodium hypochlorite to give 1, 2 and 3 in excellent
yields (quant, 95%, 88% respectively).
81:13:6. H NMR analysis of a solution of 1 in d3-aceto-
nitrile/D2O (2:1) showed that it existed as hydrate (75%)
and ketone (25%) after 10 min at room temperature, a
ratio that did not change over a period of 450 min. These
simple experiments suggest that the gem-diol would be
the predominant, if not the exclusive, species under the
conditions of the assay (see below).
1H NMR analysis of the azo compounds 1, 2 and 3 revealed
a predominance of the (E) isomer (typically .90%) with
traces of the (Z) isomer. It was observed that solutions of the
azobenzenes reached a photostationary state24 comprising
both (E) and (Z) isomers on exposure to sunlight or
¯uorescent light. For example, a solution of 1 in d3-aceto-
nitrile afforded a mixture (3:1 by 1H NMR spectroscopy) of
the (E) and (Z) isomers after exposure to ¯uorescent lighting
for 24 h. The formation of trace amounts of the (Z) isomers
was not a problem in the subsequent enzyme assay studies
since they were performed on photostationary states
comprising (E) and (Z) isomers (refer to Isomerisation
section), which were prepared in a standardised manner.
Stability studies
There are con¯icting reports on the epimerisation and/or
racemisation of peptidyl-a-ketoamides and peptidyl-a-
ketoesters. For instance, Harbeson et al. report that peptidyl-
a-ketoamides epimerised on silica gel, whereas Wasserman
and co-workers puri®ed these compounds by ¯ash chroma-
tography on silica gel to afford single diastereomers.15,16,22
In order to clarify this issue, studies sought to con®rm the
stability of 1 against racemisation under various conditions.
The stability of this compound on silica was investigated. A
sample of 1 with an ORD measurement of 252^58 (c 0.3,
acetonitrile) was passed down a silica column to give a
sample with a rotation of 257^58 (c 0.2, acetonitrile).
The pH-dependence of the stability of 1 was also investi-
gated by monitoring solutions of 1 in d3-acetonitrile/D2O
acidi®ed to pH 3 (2:1) and d3-acetonitrile/D2O buffered to
Hydration studies
The hydration properties of the target compounds were
brie¯y explored since this is thought to play a role in the
mechanism by which this class of compound inhibits serine
proteases. For example, it has been suggested that the `slow-
tight binding' kinetics, which are often observed with
a-ketoesters, results from a pre-binding equilibrium step
to the ketone from the hdrate.5 It has been widely reported
that a-ketoesters6 and a-ketoamides22,25 undergo hydration
in organic solutions containing water. In our case, it was
³
pH 7.8 with HEPES buffer (2:1). The resonance due to H3
of all four species (E)-1, (Z)-1, (E)-17 and (Z)-17 was
observed to decrease, due to deuterium exchange, over
30 h in the case of the pH 7.8 buffered solution but not for
the acidi®ed solution. This result is consistent with the ®nd-
ing of Harbeson et al., that a-ketoamides epimerise in
general base conditions.22 Over the time course of a typical
enzyme assay (10 min) the extent of racemisation of 1
would, however, be expected to be negligible.
²
found that a sample of 1 in d3-acetonitrile comprised (E)-1
and the gem-diol (E)-17 in a ratio of .19:1 (by H NMR).
1
After 26 h on the bench, the solution comprised (E)-1/(Z)-1/
(E)-17/(Z)-17 (Fig. 2) in the ratio 42:13:34:11, indicating
both gem-diol formation and some photoisomerism. The
solution was then diluted, dried over magnesium sulfate,
evaporated and resuspended in dry deuterochloroform. H
NMR analysis of this sample revealed a shift in the equi-
librium with (E)-1/(Z)-1/(E)-17 being present in the ratio
Isomerisation studies
Three photostationary states (PSS) were prepared for each
of the compounds 1, 2 and 3; one resulting from ambient
lighting, one from visible light and one from UV light. The
1
³
The components of the buffered D2O were at the same concentration as
the enzyme assay buffer solution, i.e. HEPES 0.1 M, calcium chloride
0.02 M, Triton X-100 0.05% w/v.
²
The d3-acetonitrile used in these experiments was not dried and
contained traces of water.

9766
A. J. Harvey, A. D. Abell / Tetrahedron 56 (2000) 9763±9771
Table 1. The inhibition constants for the inhibition of a-chymotrypsin by
the UV and ambient light PSS of 1, 2 and 3.
PSS (E):(Z)a
Percent (Z)
Ki (nM)
DKi
1 ambient
1 UV
3:1
1:3
26^5
73^5
240
130
110 mM
2-fold
2 ambient
2 UV
3:1
4:5
28^5
54^5
80
40
40 mM
2-fold
3 ambientb
3 UV
7:3
1:3
32^5
75^5
1200
360
840 mM
3-fold
a (E)/(Z) compositions determined by 1H NMR spectroscopy by integra-
tion of the methoxy resonances.
b The ambient light PSS of 3 does not provide a good approximation for
the visible light PSS of 3.
tion of the compounds 1, 2 and 3 are presented in Fig. 3. As
evidenced by the histogram, each of the compounds under-
went hydrate formation during isomerisation. For example,
the UV PSS for 3 comprised 40% hydrated species, the
visible PSS of 3 comprised 38% hydrated species and
the ambient PSS of 3 had 52% of hydrated species present.
The signi®cance of hydrate formation in these experiments
is secondary to the ratio of (E) and (Z) isomers, because
these compounds are expected to exist as the hydrate in
the aqueous conditions of the enzyme assay (see earlier
for a discussion). The photostationary states arising from
ambient light and visible light for 1 [amb 74% (E), vis
72% (E)] and 2 [amb 71% (E), vis 74% (E)] have com-
parable compositions, and the ambient PSS was used as
an approximations to the visible light PSS in the enzyme
assays for these compounds (Fig. 3). The ambient PSS for 3
is noticeably different from the visible PSS [amb 68% (E),
vis 53% (E)] and as a result does not provide a good
approximation for the visible PSS.
Figure 3. Photoisomerisation of inhibitors, 1, 2 and 3.
1
compositions of these PSS were determined by H NMR
spectroscopy in order to determine the compositions of
the mixtures that were subsequently used to inhibit
a-chymotrypsin. A solution of the compound (5 mg) in
d3-acetonitrile (150 mL) was irradiated for one hour with
light from a high pressure mercury arc lamp that was ®ltered
to allow passage of wavelengths between 330 and 370 nm
for (Z) isomer enrichment and the components of the
mixture were measured by integration of the methoxy
resonances. The visible light PSS was then obtained by
irradiation of the sample with wavelengths over 400 nm
for (E) enrichment and the mixture composition was deter-
mined. The solution was allowed to photoequilibrate under
¯uorescent lighting for 24 h and the resulting mixture
composition was measured. The results for the isomerisa-
Inhibition studies
The ambient and UV photostationary states for each of the
inhibitors were tested against a-chymotrypsin and the type
of inhibition and the inhibition constants were determined
by Dixon, modi®ed Dixon and double-reciprocal
analyses.26,27 The Dixon (plot of 1/V vs [I]) and modi®ed
Dixon (plot of [S]/V vs [I]) plot for the inhibition of
a-chymotrypsin by the ambient PSS of 2 are shown in
Fig. 4 as a representative example. Dixon and modi®ed
Dixon plots together give both the inhibition constant and
also the type of inhibition.§ It is clear from these graphs that
the ambient PSS of 2 inhibits the enzyme by a competitive
mechanism, where the Dixon plot gives a good approxi-
mation to the inhibition constant (80 nM) with a tight scatter
of line intersections in the fourth quadrant and the modi®ed
Dixon plot is composed of a set of parallel lines.
§
Competitive Inhibition. Dixon plot, Ki is given as the [I]-axis value for
the median of the line intersections in the fourth quadrant; Modi®ed Dixon
plot gives a series of parallel lines. Uncompetitive Inhibition. Dixon plot
gives a series of parallel lines; Modi®ed Dixon plot, Ki' is given as the [I]-
axis value for the median of the line intersections in the fourth quadrant.
Mixed Inhibition. Dixon plot, Ki is given as the [I]-axis value for the
median of the line intersections in the fourth quadrant; Modi®ed Dixon
plot, Ki' is given as the [I]-axis value for the median of the line intersections
in the third quadrant.
Figure 4. The Dixon and modi®ed Dixon plots for the inhibition of a-
chymotrypsin by the ambient light PSS of 2.

A. J. Harvey, A. D. Abell / Tetrahedron 56 (2000) 9763±9771
9767
For each of the inhibitors, the UV PSS gave a lower value
for the inhibition constant than the ambient PSS (see Table
1). For example the UV PSS of 1 gave Kiˆ130 nM and the
ambient PSS gave Kiˆ240 nM. These results imply that
the (Z) isomer was more active against a-chymotrypsin
than the (E) isomer in each case.k As might be expected
for a-ketoester-based inhibitors of serine proteases,4b the
type of inhibition for all of the compounds ®tted most
closely to a competitive model of inhibition. The results
in Table 1 give an indication of the structure-activity
relationship for azobenzene substitution. The meta isomer
2 was the most active (Kiamb PSS ˆ 80 nM), followed by the
para isomer 1 (Kai mb PSSˆ240 nM) with the ortho isomer 3
being the least active (Kai mb PSSˆ1200 nM). The isomer that
showed the best switching ability with respect to a-chymo-
trypsin was 3, for which there was a more than 3-fold
difference in inhibition constant between the ambient and
visible light PSS (Kai mb PSSˆ1200 nM, KiUV PSSˆ360 nM).
Spectrometer or a Micromass LCT operating in Electro-
spray (ES) mode with 50:50 acetonitrile water as solvent.
Optical rotation measurements were performed either on a
JASCO J-20C recording spectropolarimeter with a 10 mm
path length or a Perkin Elmer polarimeter Model 341 with a
100 mm path length. [a]D20 values are given in units of
g/100 ml. Absorbance measurements were made on a
Hewlett±Packard 8452A diode array spectrophotometer,
which was controlled by Hewlett±Packard 89532A
UV-VIS operating software. The plastic cuvettes (1.5 ml)
were held during the assays in a custom-made thermostatted
cell block that was set at 25.0^0.28C. Buffer, enzyme and
substrate solutions were stored during use in a water bath
thermoregulated at 258C by a Techne Tempette TE-8A
water bath thermostat. All buffer solutions were pH tested
at 258C with a custom-made digital pH meter that had been
referenced with borax solution (0.01 M) to pH 9.18. All
stock inhibitor solutions were prepared in acetonitrile
(BDH HiperSolve `Far UV' grade).
Oxidation of a-hydroxyesters with TEMPO/NaOCl.
Conclusions
General procedure22
We have prepared novel photobiological switches of
a-chymotrypsin. The inhibitors 1, 2 and 3 were designed
to target a-chymotrypsin by the incorporation of (L)-
phenylalanine to bind in the S1 subsite of the enzyme. It is
anticipated that other proteases could be targeted by simply
altering the amino acid(s) in this P1 position. The a-keto-
esters 1, 2 and 3 proved to be competitive inhibitors of
a-chymotrypsin with a magnitude of enzyme switching
between two- and three- fold by inhibition constant
measurement. The most ef®cacious switching of a-chymo-
trypsin was achieved by 3, for which the ambient PSS was a
considerably less active than the UV PSS. This result
implies that ortho substituted azobenzene provides an effec-
tive a-chymotrypsin switch in compounds of this type. The
meta isomer 2 was the most active inhibitor in either of the
photostationary states and this result suggests that meta
substitution of either (E) or (Z) azobenzene provides the
best binding with a-chymotrypsin for this type of
compound. Further work in our laboratories is being direc-
ted at the development of photobiological switches that; (i)
are more stable in the assay medium, (ii) show greater
differential activity between the two PSS and (iii) will
allow isolation and testing of pure (E) and (Z) isomers.
To TEMPO (ca. 0.1 equiv.), KBr (ca. 0.2 equiv) and water
(ca. 0.01 equiv) was added a solution of the alcohol
(1.0 equiv) in dichloromethane (ca. 0.1 M). The reaction
mixture was stirred at 08C. A buffered bleach solution was
prepared by the addition of NaHCO3 (300 mg, 3.6 mmol) to
a 5.25% aqueous sodium hypochlorite solution (9.7 ml
commercial bleach, 15.3 ml distilled water) and the result-
ing mixture was stirred until all of the solid had dissolved.
The buffered bleach solution (one part) was added dropwise
to the above reaction mixture (two parts) at 08C. After rapid
stirring for 30 min the reaction mixture was diluted with
ethyl acetate. The organic phase was separated, washed
with 0.5N aqueous HCl, saturated aqueous NaHCO3,
brine, dried over MgSO4 and concentrated in vacuo.
Where required the products were recrystallised from
ethyl acetate and petroleum ether.
(3S)-2-Oxo-4-phenyl-3-[[[4-(phenylazo)benzene]carbonyl]-
amino]butanoic acid methyl ester (1). The alcohol 12
(43 mg, 0.10 mmol) was oxidised with TEMPO/NaOCl as
described in the General procedure to give 1 (43 mg, quant)
as an orange solid: [a]2D0ˆ252^58 (c 0.29 acetonitrile); mp
138±1398C; IR (CHCl3) 1733, 1664 cm21
;
1H NMR
(CDCl3) d 7.98 (4H, m, ArH), 7.84 (2H, m, ArH), 7.55
(3H, m, ArH), 7.17±7.30 (5H, m, ArH), 6.67 (1H, d,
Jˆ4.9 Hz, NH), 5.65 (1H, m, CH), 3.91 (3H, s, CH3),
3.41 (1H, dd, Jˆ5.9, 13.7 Hz, CHA), 3.25 (1H, dd, Jˆ6.6,
13.7 Hz, CHB); 13C NMR (CDCl3) d 191.5, 166.3, 160.7,
154.6, 152.5, 134.9, 134.9, 131.6, 129.4, 129.2, 128.9,
128.0, 127.5, 123.1, 123.0, 57.1, 53.3, 36.9; HRMS (EI)
calcd for C24H21O4N3 (M1) 415.1532, found 415.1539.
Experimental
The following reagents were obtained commercially: Cbz-
(l)-phenylalanine (Sigma); cyanomethyltriphenylphos-
phonium chloride (Lancaster); EDCI (Aldrich); TEMPO
(Aldrich); Succ-Ala-Ala-Pro-Phe-4-nitroanilide and a-
nitroanilide and a-chymotrypsin (Sigma). Proton detected
NMR spectra were obtained on either a Varian Unity 3000
spectrometer or a Varian XL300 spectrometer, both operat-
ing at 300 MHz. Carbon detected NMR were obtained on
the XL300 spectrometer operating at 75 MHz. IR spectra
were obtained using a Shimadzu 8201PC series FTIR. Mass
spectrometry was performed on either a Kratos MS80 Mass
A sample of 1 (4 mg) was subjected to ¯ash chromato-
graphy eluting with dichloromethane±ethylacetate (9:1) to
give 1 (3 mg): [a]2D0ˆ257^58 (c 0.20 acetonitrile).
(3S)-2-Oxo-4-phenyl-3-[[[3-(phenylazo)benzene]car-
bonyl]amino]butanoic acid methyl ester (2). The alcohol
13 (27 mg, 65 mmol) was oxidised with TEMPO/NaOCl as
described in the General procedure to give 2 (26 mg, 95%)
k
Work is in progress to assay the pure (E) and (Z) isomers of an example
of this class of compound.

9768
A. J. Harvey, A. D. Abell / Tetrahedron 56 (2000) 9763±9771
as an orange solid: [a]2D0ˆ253^28 (c 0.67 acetonitrile); IR
(CHCl3) 1734, 1666 cm21; 1H NMR (CDCl3) d 8.21 (1H, s,
ArH), 8.07 (1H, d, Jˆ7.8 Hz, ArH), 7.94 (2H, d, Jˆ6.5 Hz,
ArH), 7.84 (1H, d, Jˆ7.5 Hz, ArH), 7.56 (4H, m, ArH),
7.17±7.33 (5H, m, ArH), 6.76 (1H, d, Jˆ6.7 Hz, NH),
5.65 (1H, m, CH), 3.90 (3H, s, CH3) 3.41 (1H, dd, Jˆ6.0,
14.0 Hz, CHA), 3.25 (1H, dd, Jˆ6.5, 14.0 Hz, CHB); 13C
NMR (CDCl3) d 191.5, 166.4, 160.7, 152.5, 152.3, 134.9,
134.3, 131.5, 129.6, 129.4, 129.2, 128.8, 127.5, 126.4,
123.0, 121.0, 57.1, 53.3, 36.8; HRMS (EI) calcd for
C24H21O4N3 (M1) 415.1532, found 415.1531.
ether±ethyl acetate (3:2), to give 7 (249 mg, 77%) as a
1
white solid: mp 68±738C; IR (®lm) 1736, 1720 cm21; H
NMR (CDCl3) d 7.20±7.38 (10H, m, ArH), 7.09 (1H, d,
Jˆ7.5 Hz, NH), 5.27 (1H, m, CH), 5.07 (2H, s, CbzCH2),
3.85 (3H, s, OCH3), 3.23 (1H, m, CHCHAPh), 3.05 (1H, m,
CHCHBPh); 13C NMR (CDCl3) d 191.8, 160.7, 155.5,
136.0, 134.7, 129.3, 128.7, 128.6, 128.5, 128.2, 127.3,
64.1, 58.1, 53.2, 37.1; HRMS (FAB) calcd for C19H20O5N
(M11)1 342.1340, found 342.1330.
(2R,3S) -and (2S,3S)-3-[[(Benzyloxy)carbonyl]amino]-2-
hydroxy-4-phenylbutanoic acid methyl esters (8a, 8b).
To a solution of 7 (30 mg, 0.10 mmol) in THF (2 ml) at
2788C was added a freshly prepared solution (0.15 M) of
zinc borohydride (1.33 ml, 0.20 mmol) in ether. The reac-
tion mixture was stirred at 2788C for 2 h before quenching
with water (10 ml), followed by ethyl acetate (10 ml). The
phases were separated and the aqueous phase was extracted
with ethyl acetate (3£15 ml). The combined organics were
washed with brine (20 ml), dried over MgSO4 and concen-
trated in vacuo to afford a cream gum. Flash chroma-
tography eluting with petroleum ether±ethyl acetate (5:1)
afforded 8a (15 mg, 44%) as a white solid: [a]2D0ˆ260^58
(c 1.1 dichloromethane), lit. [a]2D0ˆ2828 (c 0.83 metha-
nol);18a 1H NMR (CDCl3) d 7.20±7.40 (10H, m, Ar), 5.10
(1H, d, Jˆ9.8 Hz, NH), 5.04 (2H, s, CbzCH2), 4.33 (1H, q,
Jˆ9.7 Hz, CHCHOH), 4.08 (1H, br s, CHCHOH), 3.70 (3H,
s, CH3), 3.20 (1H, d, Jˆ3.0 Hz, OH), 2.92 (2H, m,
CHCH2Ph).
(3S)-2-Oxo-4-phenyl-3-[[[2-(phenylazo)benzene]carbonyl]-
amino]butanoic acid methyl ester (3). The alcohol 16
(24 mg, 57 mmol) was oxidised with TEMPO/NaOCl as
described in the General procedure to give 3 (21 mg,
88%) as an orange solid: [a]2D0ˆ116^18 (c 0.94 aceto-
1
nitrile); IR (CHCl3) 1739, 1635 cm21; H NMR (CDCl3) d
9.36 (1H, d, Jˆ5.5 Hz, NH), 8.41 (1H, m, ArH), 7.78 (1H,
m, ArH), 7.44±7.66 (7H, m, ArH), 6.95±7.09 (5H, m, ArH),
5.77 (1H, m, CH), 3.87 (3H, s, CH3), 3.37 (1H, dd, Jˆ5.9,
14.2 Hz, CHA), 3.23 (1H, dd, Jˆ6.4, 14.2 Hz, CHB); 13C
NMR (CDCl3) d 191.4, 165.2, 160.8, 152.2, 149.8, 135.1,
132.2, 132.1, 131.6, 131.3, 130.6, 129.3, 129.2, 128.3,
127.0, 123.3, 115.9, 57.6, 53.1, 36.8; HRMS (EI) calcd
for C24H21O4N3 (M1) 415.1532, found 415.1524.
(4S)-4-[[(Benzyloxy)carbonyl]amino]-3-oxo-5-phenyl-2-
triphenylphosphoranylidene-pentanenitrile (6). To a
mixture of Cbz-l-phenylalanine, 4 (0.400 g, 1.3 mmol) in
dichloromethane (14 ml) at 08C were added EDCI (0.270 g,
1.4 mmol) and DMAP (0.016 g, 0.13 mmol), followed by
the dropwise addition of a solution of cyanophosphorane 5
(0.81 g, 2.7 mmol) in dichloromethane (6 ml). The reaction
mixture was allowed to warm to rt and was stirred under
nitrogen for 20 h. The mixture was then poured over 1:1
dichloromethane/water (15 ml) and the phases were sepa-
rated. The aqueous phase was extracted with dichloro-
methane (2£5 ml) and the combined organic extracts were
washed with brine (30 ml), dried over MgSO4 and concen-
trated in vacuo. The resulting residue was puri®ed by ¯ash
chromatography, eluting with dichloromethane-ethyl
acetate (4:1), to give 6 (0.61 g, 78%) as a white solid: mp
Further elution with petroleum ether±ethyl acetate (5:3)
afforded 8b (15 mg, 44%) as a white solid: [a]2D0ˆ
213^18 (c 0.69 methanol), lit. [a]2D0ˆ268 (c 0.85
methanol);18b 1H NMR (CDCl3) d 7.20±7.40 (10H, m,
ArH), 5.22 (1H, d, Jˆ9.3 Hz, NH), 5.04 (2H, s, CbzCH2),
4.38 (1H, m, CHCHOH), 4.35 (1H, s, CHCHOH), 3.56 (3H,
s, CH3), 2.80 (2H, m, CHCH2Ph).
(2R,3S)-2-Hydroxy-4-phenyl-3-[[[4-(phenylazo)benzene]-
carbonyl]amino]butanoic acid methyl ester (12).
Compound 8a (795 mg, 2.31 mmol) was dissolved in a solu-
tion of HBr in acetic acid (33%, 1.0 ml) and the resulting
mixture was stirred at rt for 20 minutes. The addition of
ether (2 ml) caused the amine salt to precipitate. The
mixture was kept at 08C for 30 min and then ®ltered and
washed with ether (3£2 ml) to give 9 (574 mg, 86%) as a
white solid that was subsequently used without further puri-
®cation: mp 160±1618C; 1H NMR (D2O) d 7.22±7.35 (5H,
m, ArH), 4.28 (1H, d, Jˆ3.4 Hz, CHCHOH), 3.87 (1H, m,
CHCHOH), 3.64 (3H, s, CH3), 2.99 (2H, m, CH2Ph); 13C
NMR (D2O) d 172.9, 134.8, 129.4, 129.2, 127.7, 68.5, 54.2,
53.1, 35.1. To a solution of 9 (100 mg, 0.34 mmol) and 1020
(71 mg, 0.31 mmol) in 1:1 dimethylformamide/dichloro-
methane (2 ml) at rt under nitrogen were added EDCI
(78 mg, 0.41 mmol) and HOBT (63 mg, 0.47 mmol). The
reaction mixture was stirred for 5 min and DIEA (60 mL,
0.34 mmol) was added. The reaction mixture was stirred
16 h and the solution was diluted with dichloromethane
(5 ml), washed with 1N HCl (2£5 ml), saturated aqueous
NaHCO3 (5 ml), brine (5 ml), dried over MgSO4 and
concentrated in vacuo to afford 12 (127 mg, 89%) as an
101±1038C; IR (®lm) 1716 cm21 1H NMR (CDCl3) d
;
7.61±7.66 (3H, m, PPh3), 7.45±7.60 (12H, m, PPh3), 7.30
(5H, m, ArH), 7.21 (5H, m, ArH), 5.53 (1H, d, Jˆ7.8 Hz,
NH), 5.19 (1H, m, CH), 5.06 (2H, s, CbzCH2), 3.35 (1H, dd,
Jˆ4.8, 13.5 Hz, CHCHAPh), 3.08 (1H, dd, Jˆ6.8, 13.5 Hz,
CHCHBPh); 13C NMR (CDCl3) d 192.8, 155.4, 136.8,
133.6, 133.4, 133.2, 129.6, 129.2, 129.0, 128.3, 128.0,
127.7, 126.4, 122.4 (d, Jˆ92.8 Hz, CN), 120.9 (d,
Jˆ16 Hz, P-Cq), 66.2, 57.2 (d, Jˆ9 Hz, CH), 47.8 (d,
Jˆ125 Hz, PvC), 38.7; HRMS (FAB) calcd for
C37H32O3N2P (M11)1 583.2150, found 583.2145.
(3S)-3-[[(Benzyloxy)carbonyl]amino]-2-oxo-4-phenyl-
butanoic acid methyl ester (7). A solution of 6 (538 mg,
0.95 mmol) in (7:3) dichloromethane/methanol (9 ml) at
2788C, was treated with ozone at a rate of 1±2 bubbles
per second for 15 min. The solution was ¯ushed with nitro-
gen for 10 min and then allowed to warm to rt. The solution
was evaporated to dryness and the resulting residue was
puri®ed by ¯ash chromatography, eluting with petroleum
1
orange solid: mp 1768C; IR (CHCl3) 1734, 1666 cm21; H
NMR (CDCl3) d 7.95 (4H, m, ArH), 7.82 (2H, d, Jˆ8.3 Hz,

A. J. Harvey, A. D. Abell / Tetrahedron 56 (2000) 9763±9771
9769
ArH), 7.54 (3H, m, ArH), 7.24±7.36 (5H, m, ArH), 6.44
Hydration studies
(1H, d, Jˆ9.1 Hz, NH), 4.82 (1H, m, CH), 4.22 (1H, d,
Jˆ2 Hz, CHOH), 3.77 (3H, s, CH3), 3.31 (1H, d, Jˆ4 Hz,
OH), 3.05 (2H, m, CH2); 13C NMR (CDCl3) d 174.3, 166.5,
154.4, 152.5, 137.2, 135.9, 131.6, 129.4, 129.2, 128.7,
127.9, 126.9, 123.1, 122.9, 70.1, 53.4, 53.1, 38.0; Anal.
calcd for C24H23O4N3, C 69.05, H 5.55, N 10.06; found C
68.80, H 5.66, N 10.24.
A solution of 1 (5 mg) in d3-acetonitrile (150 mL) was
1
analysed by H NMR at tˆ5 min to reveal (E)-1 and (E)-
17 in a ratio of .19:1. After 26 h, (E)-1/(Z)-1/(E)-17/(Z)-17
were observed in a ratio of 42:13:34:11. Water was then
removed from the sample by diluting it with dichloro-
methane and drying over MgSO4. The solvent was concen-
trated in vacuo and the sample was redissolved in dry
1
CDCl3. The solution was examined by H NMR to reveal
(2R,3S)-2-Hydroxy-3-4-phenyl-[[[3-(phenylazo)benzene]-
carbonyl]amino]butanoic acid methyl ester (13). To a
solution of 9, prepared as described in the previous example
(52 mg, 0.18 mmol) and 1120 (37 mg, 0.16 mmol) in 1:1
dimethylformamide/dichloromethane (1.5 ml) at rt under
nitrogen were added EDCI (41 mg, 0.21 mmol) and
HOBT (24 mg, 0.18 mmol) and DIEA (43 mL, 0.24 mmol)
as described for 12. The resulting residue was puri®ed by
¯ash chromatography, eluting with dichloromethane to
afford 13 (26 mg, 39%) as an orange solid: IR (CHCl3)
(E)-1/(Z)-1/(E)-17 in the ratio 81:13:6. In an analogous
experiment the composition of a solution of 1 (5 mg) in
CD3CN/D2O (2:1, 150 mL) was monitored by H NMR
spectroscopy after 10 min to reveal a mixture of 1 and 17
in a ratio of 1:3 which was unaltered after a further 450 min.
1
Selected data for (E)-1 from the mixture: 1H NMR (CD3CN)
d 7.92±8.08 (6H, m, ArH), 7.63 (3H, m, ArH), 7.27±7.39
(5H, m, ArH), 5.32 (1H, dd, Jˆ5.4, 9.3 Hz, CH), 3.85 (3H,
s, CH3), 3.39 (1H, dd, Jˆ5.4, 14.2 Hz, CHA), 3.13 (1H, dd,
Jˆ9.3, 14.2, CHB). Selected data for (Z)-1 from the mixture:
1H NMR (CD3CN) d 7.92±8.08 (2H, m, ArH), 7.63 (3H, m,
ArH), 7.27±7.39 (5H, m, ArH), 6.89 (4H, m, H2, ArH), 5.24
(1H, m, CH), 3.81 (3H, s, CH3). Selected data for (E)-17
1
3435, 3031, 2956, 1734, 1668 cm21; H NMR (CDCl3) d
8.18 (1H, s, ArH), 8.02 (1H, d, Jˆ7.8 Hz, ArH), 7.92 (2H,
d, Jˆ8.1 Hz, ArH), 7.82 (1H, d, Jˆ7.5 Hz, ArH), 7.53 (4H,
m, ArH), 7.24±7.35 (5H, m, ArH), 6.68 (1H, m, NH),
4.86 (1H, m, CHCHOH), 4.24 (1H, s, CHOH), 3.75 (3H,
s, CH3), 3.09 (2H, m, CH2); 13C NMR (CDCl3) d 174.2,
166.5, 152.5, 152.4, 137.2, 135.2, 131.4, 129.4, 129.1,
128.7, 126.8, 125.6, 123.0, 121.2, 70.1, 53.4, 53.0, 37.9;
HRMS (EI) calcd for C24H23O4N3 (M1) 417.1689, found
417.1691.
1
from the mixture: H NMR (CD3CN) d 7.92±8.08 (6H, m,
ArH), 7.84 (2H, d, Jˆ8.8 Hz, NH), 7.63 (3H, m, ArH),
7.27±7.39 (5H, m, ArH), 4.79 (1H, dd, Jˆ3.4, 11.7 Hz,
CH), 3.78 (3H, s, CH3), 3.35 (1H, m, CHA), 3.10 (1H, m,
1
CHB). Selected data for (Z)-17 from the mixture: H NMR
(CD3CN) d 7.92±8.08 (2H, m, ArH), 7.63 (3H, m, ArH),
7.27±7.39 (5H, m, ArH), 6.89 (4H, m, ArH), 4.63 (1H, m,
CH), 3.74 (3H, s, CH3).
(2R,3S)-2-Hydroxy-4-phenyl-3-[[[2-(phenylazo)benzene]-
carbonyl]amino]butanoic acid methyl ester (16). A
solution of DCC (77 mg, 0.40 mmol) in THF (1.5 ml) at
08C was added to an ice-cold solution of acid 1421 (90 mg,
0.40 mmol) and N-hydroxysuccinimide (46 mg, 0.40 mmol)
in THF (3 ml). The reaction mixture was stirred 2 h at 08C
then stored at 48C overnight. The reaction mixture was
®ltered, washing with cold THF (2£1 ml). To the solution
was added 9 (166 mg, 0.77 mmol) and DIEA (76 mL,
0.44 mmol) and the reaction mixture was stirred 1 h at
08C and 4 h at rt. The reaction mixture was diluted with
dichloromethane (20 ml), washed with saturated aqueous
NH4Cl (20 ml), brine (20 ml), dried over MgSO4 and
concentrated in vacuo. The crude product was puri®ed by
¯ash chromatography, eluting with dichloromethane, to
afford an orange solid tentatively assigned as 15 (30 mg,
Stability studies
Solutions of 1 (5 mg) were prepared in (2:1) d3-acetonitrile/
acidi®ed D2O-pH 3 by Universal indicator paper (150 mL)
and (2:1) d3-acetonitrile/buffered D2O (150 mL). The
buffered D2O solution at pH 7.8 comprised HEPES buffer
(0.1 M), CaCl2 (0.02 M) and Triton X-100 (0.05% w/v). The
1
two solutions were monitored over a period of 30 h by H
NMR spectroscopy. The combined multiplets due to H3 of
(E)-1, (Z)-1, (E)-17 and (Z)-17 decreased by 40% over a
period of 30 h in the case of pH 7.8 solution. No change
was evident with the pH 3 sample.
Isomerisation studies
1
23%): H NMR (CDCl3) d 8.06 (3H, m, ArH), 7.75 (2H,
m, ArH), 7.48±7.59 (4H, m, ArH), 2.86 (4H, s, 2x CH2);
HRMS (ES) calcd for C17H13O4N3K (M1K)1 362.054,
found 362.055. Further elution with dichloromethane±
ethyl acetate (19:1) gave 16 (62 mg, 37%) as an orange
solid: mp 127±1308C; IR (CHCl3) 3622, 3533, 3310,
For the purposes of PSS composition measurement, solu-
tions of the inhibitors in d3-acetonitrile (typically 30 mM)
were irradiated in quartz NMR tubes for 60 min. Isomer-
enriched photostationary states were obtained by irradiation
of the solution with ®ltered light from a 200 W high
pressure mercury arc lamp. The light was ®ltered with an
Oriel 59810 ®lter to allow passage of UV light
(330,l,370 nm) for (Z) isomer enrichment. For (E)
enrichment the light was ®ltered with an Oriel 59494 ®lter
(l.400 nm) to allow the transmittance of visible light. At
500 mm from the light source the glass ®lter was held in a
cylinder, at 540 mm from the light source a water ®lter of
10 mm thickness was held and at 580 mm from the light
source the sample was held. The PSS compositions were
1743, 1656 cm21 1H NMR (CDCl3) d 9.02 (1H, d,
;
Jˆ9.0 Hz, NH), 8.43 (1H, m, ArH), 7.97 (2H, d,
Jˆ8.1 Hz, ArH), 7.76 (1H, m, ArH), 7.54 (5H, m, ArH),
7.17±7.34 (5H, m, ArH), 4.90 (1H, m, CHCHOH), 4.22
(1H, s, CHOH), 3.68 (3H, s, CH3), 3.35 (1H, br s, OH),
3.04 (2H, m, CH2); 13C NMR (CDCl3) d 174.2, 165.3,
152.3, 149.6, 137.5, 132.2, 131.8, 131.5, 131.3, 130.2,
129.3, 129.3, 128.5, 126.6, 123.4, 115.8, 70.0, 53.9, 52.8,
38.1; HRMS (EI) calcd for C24H23O4N3 (M1) 417.1689,
found 417.1692.
1
measured by H NMR spectroscopy by integration of the

9770
A. J. Harvey, A. D. Abell / Tetrahedron 56 (2000) 9763±9771
methoxy resonances (as recorded earlier) immediately after
irradiation of the solution.
Acknowledgements
We thank Bruce Clark for MS analysis, Regan Anderson
and Dr Mark Humphries for carrying out some preliminary
experiments and Dr Juliet Gerrard for assistance with the
enzyme inhibition assays. This investigation was supported
by a research grant from the New Zealand Marsden Fund.
Enzyme assay and kinetics
Buffer (HEPES) solution. HEPES (N-(2-hydroxyethyl)-
piperazine-N0-2-ethanesulfonic acid) (11.9 g, 0.05 mol),
CaCl2´6H2O (2.2 g, 0.01 mol) and Triton X-100 (0.25 g)
were dissolved in Milli-Q ionised water (400 ml) in a
500 ml volumetric ¯ask. Suf®cient 1 M NaOH solution
was added (approx. 40 ml) to reach pH 7.8 and the solution
was made up to 500 ml with more Milli-Q water.
References
1. Willner, I.; Willner, B. In Bioorganic Photochemistry:
Biological Applications of Photochemical Switches, Morrison,
H., Ed.; Wiley: New York, 1993; 2, pp 1±110.
Substrate solution. HEPES (11.9 g, 0.05 mol) and
CaCl2´6H2O (2.2 g, 0.01 mol) were dissolved in Milli-Q
ionised water (400 ml) in a 500 ml volumetric ¯ask. Suf®-
cient 1 M NaOH solution was added (approx. 40 ml) to
reach pH 7.8 and the solution was made up to 500 ml with
more Milli-Q water. N-Succinyl-(Ala)2-Pro-Phe-4-nitro-
anilide (21 mg, 34 mmol) was dissolved in a sample of
this solution (10 ml) by sonication for 10 min in cold
water. The solution was stored for up to two weeks below
08C. The concentration of the solution was determined at
2. (a) Willner, I.; Rubin, S.; Shatzmiller, R.; Zor, T. J. Am. Chem.
Soc. 1993, 115, 8690. (b) Willner, I.; Rubin, S.; Riklin, A. J. Am.
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3. Westmark, P. R.; Kelly, J. P.; Smith, B. D. J. Am. Chem. Soc.
1993, 115, 3416.
4. (a) Angelastro, M. R.; Mehdi, S.; Burkhart, J. P.; Peet, N. P.;
Bey, P. J. Med. Chem. 1990, 33, 11. (b) Peet, N. P.; Burkhart, J. P.;
Angelastro, M. R.; Giroux, E. L.; Mehdi, S.; Bey, P.; Kolb, M.;
Neises, B.; Schirlin, D. J. Med. Chem. 1990, 33, 394.
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7. Schechter, I.; Berger, A. Biochem. Biophys. Res. Commun.
1967, 27, 157.
the start of each day from its UV spectrum (e315
ˆ
14 000 Lmol21 cm21).
Enzyme solution. A stock solution of a-chymotrypsin was
prepared from a-chymotrypsin (3.03 mg, 120 nmol) and
Milli-Q water (1 ml). To a 100 ml volumetric ¯ask was
added stock solution (200 mL, 24 nmol), Triton X-100
(50 mg, 0.05% w/v) and conc. HCl (analytical grade,
10 mL, 1.2 mM) and Milli-Q water (about 50 ml). The
¯ask was shaken at length and the solution was made to
100 ml with Milli-Q water. The enzyme solutions were
prepared fresh each day.
8. Kraut, J. Annu. Rev. Biochem. 1977, 46, 331.
9. Fischer, E.; Frankel, M.; Wolovsky, R. J. Chem. Phys. 1955, 23,
1367.
10. Allmann, R. The Chemistry of the Hydrazo, Azo and Azoxy
Groups; Patai, S., Ed.; Wiley: London, 1975; pp 23±52, Part 1.
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3913.
12. Slee, D. H.; Laslo, K. L.; Elder, J. H.; Ollman, I. R.; Gustchina,
A.; Kervinen, J.; Zdanov, A.; Wlodawer, A.; Wong, C.-H. J. Am.
Chem. Soc. 1995, 117, 11867.
Inhibition of a-chymotrypsin was determined with Succ-
Ala-Ala-Pro-Phe-4-nitroanilinde as the substrate by an
assay procedure developed from the technique described
by Geiger,28 except that HEPES buffer was used instead
of TRIS buffer and that the order of addition of enzyme
and substrate was inverted. Each measurement was made
in duplicate. For the purposes of enzyme inhibition, solu-
tions of the inhibitors in d3-acetonitrile (15±90 mM) were
irradiated in quartz cuvettes for 60 min as described in the
Isomerisation under the Experimental section. Large data
sets (5£[S], 5£[I]) were obtained in duplicate for the PSS
composition resulting from ambient light. These data sets
were ®tted to the Michaelis±Menten model with double-
reciprocal, direct linear, Dixon and modi®ed Dixon plots.
13. May, B. C. H.; Abell, A. D. Synth. Commun. 1999, 29, 2515.
14. (a) Nishizawa, R.; Saino, T. J. Med. Chem. 1977, 20, 510.
(b) Herranz, R.; Castro-Pichel, J.; Vinuesa, S.; Garcia-Lopez,
M. T. J. Chem. Soc., Chem. Commun. 1989, 938. (c) Herranz,
R.; Castro-Pichel, J.; Garcia-Lopez, M. T. Synthesis 1989, 703.
(d) Matsuda, F.; Matsumoto, T.; Ohsaki, M.; Ito, Y.; Terashima,
S. Chem. Lett. 1990, 723. (e) Iizuka, K.; Kamijo, T.; Harada, H.;
Akahane, K.; Umeyama, H.; Ishida, T.; Kiso, Y. J. Med. Chem.
1990, 33, 2707. (f) Yuan, W.; Munoz, B.; Wong, C.-H.;
Haeggstrom, J. Z.; Wetterholm, A.; Samuelsson, B. J. Med.
Chem. 1993, 36, 211.
15. Wasserman, H. H.; Ho, W.-B. J. Org. Chem. 1994, 59, 4364.
16. Wasserman, H. H.; Petersen, A. K. J. Org. Chem. 1997, 62,
8972.
From these analyses the type of inhibition was determined
and the kinetic parameters KaMpp, V and Ki were calculated.
app
max
17. Paris, M.; Pothian, C.; Michalak, C.; Martinez, J.; Fehrentz,
J.-A. Tetrahedron Lett. 1998, 39, 6889.
Smaller data sets (4£[S], 3£[I]) were obtained for the UV
light photostationary states, enabling analysis by double-
18. (a) Herranz, R.; Castro-Pichel, J.; Garcia-Lopez, M. T.
Synthesis 1989, 703. (b) Vedejs, E.; Larsen, S. Org. Synth. 1984,
64, 127.
reciprocal and direct linear plots for the parameters KMapp
,
and Ki. The assumption that the visible and ambient
app
max
V
light photostationary states had the same effect on the
enzyme was tested by measuring the extent of inhibition
at two inhibitor concentrations for the ambient light PSS
with the results for the visible light PSS and it was found
that the initial rates were the same within experimental
error.
19. Herranz, R.; Castro-Pichel, J.; Vinuesa, S.; Garcia-Lopez,
M. T. J. Org. Chem. 1990, 55, 2232.
20. Byrne, C. J.; Happer, D. A. R.; Hartshorn, M. P.; Powell, H. K.
J. J. Chem. Soc., Perkin Trans. 2 1987, 11, 1649±1653.
21. Badger, G. M.; Drewer, R. J.; Lewis, G. E. Aust. J. Chem.
1964, 17, 1036.

A. J. Harvey, A. D. Abell / Tetrahedron 56 (2000) 9763±9771
9771
22. Harbeson, S. L.; Abelleira, S. M.; Akiyama, A.; Barrett III, R.;
Carroll, R. M.; Straub, J. A.; Tkacz, J. N.; Wu, C.; Musso, G. F.
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25. Ocain, T. D.; Rich, D. H. J. Med. Chem. 1992, 35, 451.
26. (a) Dixon, M. Biochem. J. 1953, 55, 170. (b) Cornish-Bowden,
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23. Harvey, A. J. Ph.D. thesis, University of Canterbury,
Christchurch, New Zealand, 2000.
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28. Geiger, R. In Methods of Enzymatic Analysis, 3rd ed.;
Bergmeyer, H. U., Ed.; Verlag Chemie: Weinheim, 1984; Vol. 5,
pp 99±129.
24. Vollmer, M. S.; Clark, T. D.; Steinem, C.; Ghadiri, M. R.
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