Photoswitch inhibitors of α-chymotrypsin - Increased substitution and peptidic character in peptidomimetic boronate esters

Article abstract of DOI:10.1039/b609320p

A series of peptidomimetic boronate esters containing the photoisomerisable azobenzene group has been synthesised and assayed against the serine protease α-chymotrypsin. Compounds with borophenylalanine inhibition groups were found to be inhibitors with m

Full text of DOI:10.1039/b609320p

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Photoswitch inhibitors of a-chymotrypsin—increased substitution and
peptidic character in peptidomimetic boronate esters†
David Pearson and Andrew D. Abell*
Received 30th June 2006, Accepted 8th August 2006
First published as an Advance Article on the web 17th August 2006
DOI: 10.1039/b609320p
A series of peptidomimetic boronate esters containing the photoisomerisable azobenzene group has
been synthesised and assayed against the serine protease a-chymotrypsin. Compounds with
borophenylalanine inhibition groups were found to be inhibitors with micromolar activity, while those
with aryl boronate inhibition groups were inactive. Selected compounds were isomerised by UV or
visible light to obtain enriched states of the (Z) or (E) isomers, respectively, and assayed. A change in
activity on photoisomerisation was observed, however some decomposition of the boronate group on
irradiation was also observed, limiting reversibility.
substrates and inhibitors7 (see notation of Schechter and Berger8).
Introduction
These compounds were assayed against a-chymotrypsin and found
The physical and chemical properties of materials can be con-
to be active micromolar inhibitors. Based on these results the
trolled at the molecular level using molecular switches that operate
disubstituted azobenzenes 9 and 10 (Fig. 2) were designed,
under a variety of conditions and in a variety of situations.
synthesised and tested as compounds suitable for incorporation
Numerous switching systems have been developed that control
into a peptide sequence and for surface attachment.
the movement or reactivity of molecules based on pH,1 light2
and electrochemical3 switches, for example. Our research aims to
control optically biological systems using photoswitchable enzyme
inhibitors. We and others have reported examples of inhibitors
Results and discussion
of serine proteases that combine an azobenzene photoswitch
with a group that binds to the enzyme of interest: for example,
boronic acid 1 was found to be 3 times less active after UV
irradiation (inhibition constant, Ki, of 41 lM vs 11 lM) to give a
predominance of the cis isomer,4 while the a-keto ester 2 was found
to be 2 times more active (Ki of 130 vs 240 nM) after irradiation to
give the cis isomer.5 In this paper, we extend this concept with the
preparation and testing of peptidomimetic-based photoswitchable
inhibitors of proteases that are amenable to incorporation into a
peptide sequence as a means to potentially increase potency and
specificity for one protease over another. These systems are also
amenable to attachment to other materials, such as metal surfaces,
polymers or nanoparticles. Photoswitch molecules attached to
such materials might form the basis for molecular computing or
reversible biosensor technologies. We also hoped to gain some
insight into those structural factors that influence the potency of
cis vs trans azobenzenes given the opposing results obtained for 1
and 2.
Further to the design of the photoswitchable inhibitors we
anticipated that potency, specificity and photoswitchability might
be further controlled by incorporating an amino acid with a
hydrophobic side chain (e.g. leucine) into the structure at P2,
this provides potential to bind to the S2 protease subsite9 (see
4, 5, 7, and 8). The design of 4 and 5 assumes that the aryl
boronic acid group occupies the S1 subsite. Compounds 3–5 were
synthesised by standard peptide coupling methods (Schemes 1–
3) and assayed against chymotrypsin using a spectrophotometric
assay. Surprisingly, these compounds were found to be inactive
against chymotrypsin up to their solubility limits (∼0.5 mM),
despite their structural similarity to reported inhibitor 1. This
suggests that the fit of 1 into the enzyme active site is significantly
enhanced by the shape and/or hydrogen bonding attributable to
the sulfonamide group. However, replacing the amide of 4 with
a sulfonamide to give 5 did not replicate this effect (see Table 1).
It is possible that the incorporation of a boronate ester into 3–5
might account for the reduction in potency relative to 1. However,
the observed >50 fold decrease in activity is not consistent with
literature, where boronate esters and their corresponding free
acids are reported to possess very similar activities.6,10 Our results
suggest that 1, although an effective photoswitch, cannot be simply
extended as a peptide analogue.
Initially, two groups of mono-substituted azobenzenes (Fig. 1)
were synthesised: those containing a simple aryl boronate (3–5)
based on literature photoswitch inhibitor 1, and a novel series
containing the boronate ester analogue of phenylalanine (6–8).6
The second series was prepared in an attempt to better target a-
chymotrypsin, where this enzyme is known to prefer aromatic
residues (e.g., the benzyl group of Phe) at the P1 position of
Better results were obtained using compounds 6–8, which
contain a boronate ester analogue of phenylalanine. Compound 6
was synthesised by coupling azobenzene 11 to the pinacol ester
of borophenylalanine 13 (Scheme 1). Difficulties were initially
encountered on attempting to perform amide coupling reactions
with 13 using normal peptide coupling reagents such as 1-ethyl-
3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDCI)
Department of Chemistry, University of Canterbury, Christchurch, New
Zealand. E-mail: andrew.abell@canterbury.ac.nz; Fax: +64 (0)33642110;
Tel: +64 (0)33642818
† Electronic supplementary information (ESI) available: NMR spectra.
See DOI: 10.1039/b609320p
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Fig. 2 Disubstituted azobenzenes.
Fig. 1 Monosubstituted azobenzenes.
Scheme 1
and benzotriazol-1-yloxytris(dimethylamino)phosphonium hex-
afluorophosphate (BOP), although the mixed anhydride coupling
method with isobutyl chloroformate gave acceptable yields. Di-
astereomers 7 and 8 were synthesised similarly from 14 and 12
in good yield (Scheme 2). Separation of the diastereomers was
problematic since they decomposed on silica. However, careful
chromatography on deactivated silica allowed separation of 7 and
8 in up to 86% de (see Table 1), although with poor recovery yields
(<10%) due to decomposition.
The 1H NMR of synthetic samples of 6, 7, and 8 revealed
them to be present almost exclusively as the E isomers (>95%).
Assay of these samples against chymotrypsin gave IC50 values of
3, 1.3 and 18 lM, respectively (see Table 1 entries vi, viii, and ix).
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Table 1 Activities of inhibitorsa
Entry
Compound
Inhibition constant/lMb
i
ii
1
2.1c
0.24c
>500
>500
>400
3.0
4.7
1.3
18
10
2 (26%, Z isomer)
iii
iv
v
vi
vii
viii
ix
x
3
4
5
6
6 (Z isomer)
7 and 8, 92 : 8 ratio
7 and 8, 7 : 93 ratio
9
10
xi
10
a >95% (E)-isomer, unless specified; b IC50, unless specified; c Ki
possible that the azobenzene group may even enhance activity by
providing either a good fit into the shallow hydrophobic S2 subsite,
or an extended mimic of a b-strand, the preferred conformation
of peptide substrates of proteases.12 This could then offset any
possible reduction in activity due to the boronic acid protection.
The configuration of compounds 7 and 8 could not be assigned
from NMR data. However, the more active isomer 7 is assigned
the (R) configuration, as this corresponds to (i) the configuration
of natural phenylalanine and (ii) that commonly found in the more
active isomers of related inhibitors.13 Interestingly, the most active
diastereomer 7 was found to have approximately twice the potency
of racemic 6. This suggests that incorporation of leucine into the
inhibitor structure does not significantly enhance binding through
interaction of leucine with the S2 subsite. It is, however, possible
that any increase in binding conferred by leucine is off-set by the
affinity of the hydrophobic azobenzene group for the S2 subsite
(as in 6) rather than the S3 subsite (as in 7).
Scheme 2
A sample of (Z) 6, obtained by chromatography from a mixture
of (E) and (Z) isomers formed from pure (E) on exposure to
ambient light conditions, was also assayed to assess the influence
of the configuration of the azobenzene on activity. This isomer
was less active (Table 1 entry vii), a result consistent with earlier
reports on the photoswitching of the related boronic acid 1, where
the (E) isomer was the more active.
The disubstituted azobenzenes 9 and 10 were synthesised based
on the successful inhibitor 6 (Scheme 4). These structures consist
of the borophenylalanine enzyme binding group, the azobenzene
photoswitch and an amino acid (glycine) at the N-terminus. The
added amino acid is intended to enhance the peptidic character
of the inhibitors in order to better mimic a natural substrate, as
well as adding extra bulk to the photoswitch with the aim of
improving photoswitching. The corresponding free amine is also
potentially useful for attachment of further substituents and for
surface attachment work.14 In addition, the Boc protecting group
of 9 allows assessment of the effect of substitution on binding
at a site well removed from the main binding and photoswitching
region of the inhibitor. Inhibitor 9 was synthesised in six steps from
commercially available 18 and previously reported 20.15 Amine 18
was oxidised by catalyst Mo(O2)2O·H2O·HMPA16 to the nitroso
derivative 19, which was then heated with 20 in HOAc to form
azobenzene 21. The Fmoc protecting group was found to cause
solubility and purification difficulties if carried into further steps.
Therefore, it was removed at this stage to give 22, and the more
Scheme 3
Encouragingly, the potency of compounds 6 and 7 is similar to the
activity of acetylborophenylalanine ityself (Ki = 2.1 lM), a well-
studied inhibitor of chymotrypsin.11 This result is perhaps better
than might have been expected, and it implies that activity is not
significantly impaired by the presence of the azobenzene group
or the protection of the boronic acid as the pinacol ester. It is
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conditions (LiOH, THF–H2O, 0 ◦C, 2 h), presumably due to the
stabilising effect of the aromatic group, however, heating and
longer reaction times afforded acid 24 in good yield. Boronate
ester 9 was synthesised by mixed anhydride coupling of 24 with
13, and 10 was formed by Boc deprotection. Assay of 9 and 10
gave identical inhibition constants (10 lM, Table 1 entries x and
xi), several times weaker than the monosubstituted parent 6, but
significantly more potent than 3–5. This suggests that the extra
substitution of the azobenzene group only slightly impairs the
‘fit’ of the inhibitor into the active site. The extra bulk of the
Boc group in 9 is tolerated, suggesting that the specificity of the
enzyme binding site does not extend further than the glycine in
these inhibitors.
The inhibitors 6 and 9 were photoisomerised and the two resul-
tant photostationary states assayed against chymotrypsin in order
to assess their efficacy as photoswitches. Good photoisomerisation
of both compounds was obtained on UV irradiation with a 500 W
mercury arc lamp through a UV filter with a narrow wavelength
band centred at 340 nm, giving rise to up to 74% (Z) isomer for
6, and 68% (Z) for 9. Assays were performed during a series of
irradiations in order to assess the impact of photoswitching and
potential decomposition. A sample of inhibitor was dissolved in
acetonitrile, irradiated with UV light, visible light (>360 nm),
then UV light, and aliquots were removed and assayed initially
and after each irradiation. For inhibitor 6 the decomposition
outweighed any photoswitching effect so that activity decreased
after each irradiation cycle. Irradiation of a sample of 6 with UV
for several hours gave a mixture that was partially purified by flash
chromatography. NMR analysis of this revealed that the pinacol
group had been lost. As this decomposition product showed no
(or very weak) activity it is assumed that the entire boronate group
was lost, or significantly altered by irradiation. Better results were
obtained for 9, and photoswitching was observed with limited
decomposition at each cycle. As shown in Fig. 3 the activity
increased from 10 lM (for sample containing <5% (Z) isomer)
to 7 lM after UV isomerisation to give 60% (Z) isomer. The
activity then decreased to 13 lM on isomerisation back to 85%
(E) isomer, and finally increased to 10 lM following a second UV
irradiation.
Fig. 3 Semi-reversible photoswitching of inhibitor 9.
Scheme 4
Conclusions
convenient Boc group was introduced by coupling to Boc-glycine,
giving 23. Deprotection of the ester was unsuccessful under normal
A new series of boronate ester inhibitors of chymotrypsin has
been developed and tested. Compounds with a disubstituted
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azobenzene core that exhibit photoswitching of enzyme activity
are presented (see compounds 9 and 10). While the magnitude
of photoswitching is not improved by increasing the substitution
and peptidic character of inhibitors, it is possible to retain
photoswitching in such extended chain inhibitors. This opens up
possibilities for such compounds to be used in useful molecular
switching applications. Some decomposition of the boronate
group was observed on irradiation, however the peptidomimetic
design of these compounds is modular and so easily modified,
to allow simple alteration of the warhead group in order to
improve stability. The amino acids used can also be easily altered,
which should allow specific photoswitch inhibitors for a variety
of proteases to be developed using these basic methods. The last
point to note is that the exact fit of the inhibitors into the active
site appears to be particularly sensitive to molecular shape or
conformation. This is evidenced by the fact that in some cases
(1 and 6) the (E) azobenzene is the more active isomer, while for
others (2 and 9) the (Z) isomer is more active. A direct correlation
does not seem to exist between the nature of the warhead or linker,
and the relative activities of the (Z) and (E) isomers. Rather, it is
dependent upon the gross structure of the inhibitor and hence the
nature of its overall ‘fit’ in the active site.
lighting conditions, i.e. glassware wrapped in foil and with the
lights turned off.
Enzyme assays
Buffer solution (Tris): Tris(hydroxymethyl)aminomethane (1.21
g), CaCl2·6H2O (0.44 g) and Triton X-100 (0.05 g) were dissolved
in Milli-Q deionised water (75 mL), adjusted to pH 7.8 with 1 M
NaOH solution and made up to 100 mL with Milli-Q water.
Substrate solution: N-succinyl–(Ala)2–Pro–Phe–4-nitroanilide
(21 mg) was dissolved in Tris buffer solution (10 mL) by
ultrasonication. The solution was stored at −18 ◦C for up to two
weeks. The concentration of the solution was determined at the
start of each day from its UV spectrum (e315 = 14 000 L mol−1 cm−1)
Enzyme solution: a stock solution was prepared from a-
chymotrypsin (15 mg) in HCl solution (10 mL, pH 3, made up
by dilution of conc. HCl with Milli-Q water). The stock solution
was stored at −18 ◦C for up to 1 month. Each day an enzyme
solution was prepared: stock solution (200 lL) and Triton X-100
(25 mg) were made up to 50 mL with Milli-Q water.
Inhibition of a-chymotrypsin was determined with Suc–Ala–
Ala–Pro–Phe–4-nitroanilide as the substrate by an assay proce-
dure developed from the technique described by Geiger,17 except
that the order of addition of enzyme and substrate was inverted,
and only one substrate concentration was used, in order to obtain
inhibition constants as IC50 rather than Ki. Briefly, inhibitors
were dissolved in acetonitrile at a series of dilutions ranging from
0.5–500 lM as appropriate for each compound. For each rate
measurement, inhibitor solution (or acetonitrile blank, 50 lL),
substrate solution (60 lL) and buffer solution (910 lL) were mixed
Experimental
General
NMR spectra were obtained on a Varian Inova spectrometer,
operating at 500 MHz for 1H NMR and at 126 MHz for 13C NMR,
or on a Varian Unity 300 spectrometer, operating at 300 MHz for
1H NMR and at 75 MHz for 13C NMR. Two-dimensional NMR
experiments including COSY and HSQC were used to assign
spectra, and were obtained on the Varian Inova spectrometer
operating at 500 MHz. Electrospray ionisation mass spectra were
detected on a Micromass LCT TOF mass spectrometer operating
in electrospray mode with 50% acetonitrile–H2O as solvent. Dry
DMF was purchased from Acros. Dry THF was distilled from
sodium or potassium, dry DCM was distilled from CaH. EtOAc,
NMM, HOAc and EtOH were distilled before use. HPLC grade
acetonitrile was purchased from BDH. All other commercial
reagents were used as received.
◦
in a cuvette, and incubated for 5 min at 25 C. Enzyme solution
(30 lL) was added, and the absorbance at 405 nm was monitored
for 5 min. An absorbance vs time plot was obtained, and the
slope used to find the initial rate. From the difference between
the initial rate and the initial rate for the acetonitrile blank, the
percent inhibition was calculated. This experiment was repeated
over as many inhibitor concentrations as required to obtain a
good straight line plot of percent inhibition vs log [inhibitor
concentration], from which the IC50 was interpolated. In the case
of the assay of photoisomerised azobenzene (Z)-isomers, assays
were performed as above except with d3-acetonitrile instead of
acetonitrile.
Photoisomerisation
Synthesis
Compounds were photoisomerised by irradiation with UV or
visible light from a 500 W mercury arc lamp. The light beam
was filtered through water to reduce heat, and through either an
Edmund optics 340 nm interference filter for UV, or a Corning
0–51 visible filter for visible light. Samples were dissolved in d3-
acetonitrile, and irradiated for 70 min with UV light, or for 30 min
with visible light. The ratio of (E) : (Z) isomers was determined
before and after irradiations by 1H NMR analysis using the
integrals of well separated peaks. The major isomer was assigned
the thermodynamically more stable (E) configuration based on
literature precedence.4,5 The minor (Z) isomer characteristically
gave rise to upfield signals for the aryl protons which were
enhanced on irradiation. 2D NMR was used to confirm the
presence of minor isomers in some representative cases. All
experiments involving photoisomerisation were carried out in dim
4-(Phenylazo)-N-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)phenyl)benzamide (3). To a mixture of 4-(phenylazo)benzoic
acid 11 (200 mg, 0.88 mmol), 3-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)benzenamine 12 (194 mg, 1 eq.) and O-
(7-azabenzotriazol-1-yl)-N,N,Nꢀ,Nꢀ-tetramethyluronium hexaflu-
orophosphate (HATU, 370 mg, 1.1 eq.) was added DMF (5 mL)
then DIEA (340 lL, 2.2 eq.). The solution was stirred for 16 h then
diluted with ethyl acetate (50 mL), washed with water (50 mL ×
2), dried over mgSO4 and concentrated. The crude product was
purified by flash chromatography, eluting with DCM followed by
1 : 9 EtOAc–DCM then 1 : 5 EtOAc–DCM, giving 3 (200 mg, 53%,
>95% E by 1H NMR) as an orange solid: mp 162–164 ◦C. Found:
C, 70.19; H, 6.04; N, 9.93, calcd for C25H26BN3O3: C 70.27, H
6.13, N 9.83%. 1H NMR (300 MHz, CDCl3) d 1.32 (12H, s,
3622 | Org. Biomol. Chem., 2006, 4, 3618–3625
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(CMe2)2), 7.37 (1H, t, J = 7.8), 7.50 (3H, m), 7.60 (1H, d, J = 7.2),
7.87–8.04 (8H, m), 8.23 (1H, s); 13C NMR (75 MHz, CDCl3) d
24.9, 84.0, 123.09, 123.10, 123.3, 126.2, 128.0, 128.7, 129.2, 131.0,
131.7, 136.5, 137.3, 152.5, 154.3, 164; m/z (ES) 428.2162, calcd
for C25H2711BN3O3 (MH+) 428.2145.
3.15 (1H, m, CHCH2), 6.96 (1H, s, NH), 7.27 (3H), 7.36 (2H, t,
J = 7.8), 7.53 (3H), 7.87 (2H, d, J = 8.5), 7.94 (4H); 13C NMR
(126 MHz, CDCl3) d 25.0, 25.3, 37.3 (CHCH2), 46.3 (CHCH2),
81.0, 122.9, 123.2, 126.3, 128.7, 128.9, 129.0, 129.2, 131.9, 140.8,
152.4, 155.2, 170.4; m/z (ES) 456.2451, calcd for C27H3111BN3O3
(MH+) 456.2458.
(S)-N-(4-Methyl-1-oxo-1-(3-(4,4,5,5-tetramethyl-1,3,2-dioxa-
borolan - 2 - yl)phenylamino)pentan-2-yl)-4-(phenyldiazenyl)benza-
mide (4). A solution of (S,E)-4-methyl-2-(4-(phenylazo)ben-
zamido)pentanoic acid 1418 (165 mg, 0.49 mmol), 12 (108 mg,
1 eq.), EDCI (130 mg, 1 eq.) and HOBt (92 mg, 1 eq.) in DCM
(5 mL) was stirred for 16 h then washed with water (50 mL), brine
(Z)-N-(2-Phenyl-1-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)ethyl)-4-(phenylazo)benzamide ((Z)-6). A solution of (E)-6
(10 mg, 0.02 mmol) in 3 : 7 EtOAc–DCM (2 mL) was exposed
to ambient indoor lighting and daylight for 2 d, giving a mixture
of (E) and (Z) 6. Separation by flash chromatography, eluting with
3 : 7 EtOAc–DCM, gave (Z)-6 (1 mg, 7%). 1H NMR (500 MHz,
CDCl3) d 1.27 (6H, s, (CMe2)2), 1.28 (6H, s, (CMe2)2), 2.76 (1H,
dd, J = 12,14), 3.03 (1H, dd, J = 14, 4.3), 3.09 (1H, m), 6.80 (3H,
m), 6.85 (2H, d, J = 8.5), 7.18 (1H, t, J = 9.3), 7.23 (5H, m), 7.33
(2H, t, J = 7.5), 7.62 (2H, d, J = 8.5).
1
(50 mL), and filtered to give 4 (113 mg, 43%, >95% E by H
NMR) as an orange solid: mp 225–227 ◦C, 1H NMR (300 MHz,
(CD3)2SO) d 1.05 (6H, t, J = 6.8, CHMe2), 1.38 (12H, s, (CMe2)2)
1.72 (1H, m), 1.80–2.00 (2H, m), 4.77 (1H, m, COCH), 7.42 (2H,
m), 7.71 (3H, m), 7.92 (1H, d, J = 6.6), 8.05 (5H, m), 8.25 (2H,
d, J = 8.4 Hz), 8.92 (1H, d, J = 7.2), 10.30 (1H, s); 13C NMR
(75 MHz, (CD3)2SO) d 21.6 (CHMe2), 23.2 (CHMe2), 24.8(2C,
(CMe2)2 and CHMe2), 41 (obscured by solvent peak, CH2), 53.2
(COCH), 83.8 (CMe2)2), 122.4, 122.9, 125.6, 128.5, 129.1, 129.4,
129.7, 132,2, 136.3, 138.8, 152.0, 153.5, 165.9, 171.5; m/z (ES)
541.2958, calcd for C31H3811BN4O4 (MH+) 541.2986.
(S)-N-(4-Methyl-1-oxo-1-(2-phenyl-1-(4,4,5,5-tetramethyl-1,3,
2-dioxaborolan-2-yl)ethylamino)pentan-2-yl)-4-(phenylazo)benza-
mide (7 and 8). A solution of 1418 (100 mg, 0.29 mmol) in THF
(4 mL) was cooled to 0 ◦C, then isobutylchloroformate (40 lL, 1
eq.) and NMM (65 lL, 2 eq.) were added. The reaction mixture
was stirred for 10 min then a solution of 136 (106 mg, 1 eq.)
in THF (4 mL) was added. The mixture was stirred for 16 h,
diluted with EtOAc (50 mL), washed with water (50 mL) then brine
(50 mL), dried over MgSO4 and concentrated. The crude material
was purified by silica column chromatography on deactivated silica
(containing 35% water w/w), eluting with 3 : 7 EtOAc–DCM to
give a mixture of 7 and 8 (125 mg, 76%) as an orange solid.
The mixture of diastereomers was further purified three times by
column chromatography to give 7 (8 mg,◦5%, 86% de, >95% E by
1H NMR)) as an orange solid: mp 55–60 C, 1H NMR (500 MHz,
CD3OD) d 0.98 (3H, d, J = 6.5, CHMe2), 1.00 (3H, d, J = 5,
CHMe2), 1.14 (12H, m, (CMe2)2), 1.68–1.87 (3H, m), 2.60 (1H,
dd, J = 9.3, 12.8), 2.80–2.88 (2H, m), 4.89 (1H, m, COCH), 7.12–
7.23 (5H, m), 7.55 (3H, m), 7.94–8.03 (6H, m), m/z (ES) 569.3307,
calcd for C33H4211BN4O4 (MH+) 569.3299; and 8 (5 mg, 3%, 86%
(S)-4-Methyl-2-(4-(phenylazo)phenylsulfonamido)-N-(3-(4,4,5,
5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)pentanamide
(5).
A mixture of 12 (50 mg, 0.23 mmol), 17 (100 mg, 1 eq.) and
HATU (95 mg, 1.1 eq.) was dissolved in DMF (5 mL) and DIEA
(87 lL, 2.2 eq.) was added. The reaction mixture was stirred
overnight then diluted with EtOAc (50 mL), washed with water
(50 mL), brine (50 mL), dried over mgSO4 and concentrated. The
crude material was purified by flash chromatography, eluting with
1 : 19 EtOAc–DCM to give 5 (◦43 mg, 28%, >95% E by 1H NMR)
as an orange solid: mp 75–80 C, 1H NMR (500 MHz, CDCl3) d
0.75 (3H, d, J = 5, CHMe2), 0.87 (3H, d, J = 5.5, CHMe2), 1.31
(12H, m, (CMe2)2), 1.54 (1H, t, J = 9), 1.65 (2H, m), 3.88 (1H,
m, COCH), 5.45 (1H, m, NH), 7.24 (1H, t, J = 7.8), 7.49–7.54
(4H, m), 7.60 (1H, s), 7.64 (1H, d, J = 7), 7.86 (1H, s), 7.90–7.96
(4H, m), 8.03 (2H, d, J = 7.5) 13C NMR (75 MHz, CDCl3) d 21.4
(CHMe2), 22.9 (CHMe2), 24.4 (CHMe2), 24.8 ((CMe2)2), 42.2
(CH2), 56.4 (COCH), 83.9 ((CMe2)2), 123.1, 123.3, 123.4, 125.9,
128.4, 128.5, 129.2, 131.1, 132.1, 136.4, 140.5, 152.3, 154.8, 169.2;
m/z (ES) 577.2665, calcd for C30H3811BN4O5S (MH+) 577.2656.
de, >95% E by H NMR)) as an orange solid: mp 60–64 ◦C,
1
1H NMR (500 MHz, CD3OD) d 0.96 (3H, d, J = 6, CHMe2),
0.99 (3H, d, J = 6.5, CHMe2), 1.10 (6H, s, (CMe2)2), 1.15 (6H, s,
(CMe2)2), 1.68 (1H, m), 1.78 (1H, m), 1.87 (1H, m), 2.62 (1H, dd,
J = 8.5, 12), 2.84 (2H, m), 4.92 (1H, dd, J = 5, 10.5), 7.17 (1H, m),
7.26 (4H, m), 7.55 (4H, m), 7.94–8.04 (6H, m); m/z (ES) 569.3279,
calcd for C33H4211BN4O4 (MH+) 569.3299.
N -(2-Phenyl-1-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
ethyl)-4-(phenylazo)benzamide (6). To a solution of 11 (190 mg,
0.84 mmol) in THF (5 mL) was added isobutylchloro-
formate (108 lL, 1 eq.) then NMM (183 lL, 2 eq.). The reaction
mixture was stirred for 10 min, then a solution of 4,4,5,5-tetra-
methyl-a-(phenylmethyl)-1,3,2-dioxaborolane-2-methanamine, tri-
fluoroacetic acid salt 136 (300 mg, 1 eq.) in THF (5 mL) was
added. The resulting mixture was stirred for 16 h, then diluted
with EtOAc (50 mL), washed with water (50 mL) then brine
(50 mL) and concentrated. The crude material was purified by
flash chromatography, eluting with 3 : 7 EtOAc–DCM to give 6
(192 mg, 51%, >95% E by 1H NMR) as an orange solid: mp 205–
206 ◦C. Found: C, 71.13; H, 6.55; N, 9.31, calcd for C27H30BN3O3:
tert-Butyl-2-oxo-2-(4-((4-(2-phenyl-1-(4,4,5,5-tetramethyl-1,3,
2-dioxaborolan-2-yl)ethylcarbamoyl)phenyl)diazenyl)benzylamino)-
ethylcarbamate (9). A solution of 24 (30 mg, 0.07 mmol) in
DMF (3 mL) was cooled in an ice bath and isobutylchloroformate
(10 lL, 1 eq.) and NMM (16 lL, 2 eq.) were added. The reaction
mixture was stirred for 10 min then a solution of 13 (26 mg, 1
eq.) in DMF (2 mL) was added. The resulting mixture was stirred
for 16 h, warming from 0 ◦C to r.t, then diluted with EtOAc
(50 mL), washed with water (50 mL × 2), brine (50 mL), dried
over MgSO4, filtered and concentrated. The crude material was
purified by silica column chromatography on deactivated silica
(containing 35% water w/w), eluting with EtOAc to give 9 (28 mg,
1
C 71.22, H 6.64, N 9.23%. H NMR (500 MHz, CDCl3) d 1.31
◦
(6H, s, CMe2)2), 1.32 (6H, s, (CMe2)2), 2.83 (1H, dd, J = 11.8
60%, >95% E by 1H NMR) as an orange solid: mp 188–191 C,
and 14.3, CHCH2), 3.08 (1H, dd, J = 4.3 and 14.3, CHCH2),
1H NMR (500 MHz, CDCl3) d 1.31 (12H, m, (CMe2)2), 1.45
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(9H, s, CMe3), 2.83 (1H, dd, J = 12,14.1, CHCH2), 3.08 (1H,
dd, J = 14.1, 3.80, CHCH2), 3.16 (1H, m, CHCH2), 3.86 (2H,
d, J = 6), 4.55 (2H, d, J = 5.5), 5.18 (1H, s, NH), 6.65 (1H, s,
NH), 7.13 (1H, s, NH), 7.28 (3H, m), 7.37 (2H, t, J = 7.8), 7.41
(2H, d, J = 8), 7.88 (6H, m); 13C NMR (75 MHz, CDCl3) d 25.0
(CMe2)2), 25.1 (CMe2)2), 28.2 (CMe3), 37.4 (CHCH2), 42.7, 44.2,
46.8 (CHCH2), 80.1 (CMe2), 80.6 (CMe2)2), 122.7, 123.3, 126.0,
127.8, 128.4, 129.0, 129.2, 140.7, 142.1, 151.4, 154.8, 156.2, 169.9,
170.5, 207.0; m/z (ES) 642.3488, calcd for C35H4511BN5O6 (MH+)
642.3463.
Ethyl 4-((4-((((9H-fluoren-9-yl)methoxy)carbonylamino)methyl)-
phenyl)diazenyl)benzoate (21). A mixture of ethyl 4-amino-
benzoate 18 (2 g, 12 mmol) and MoO5·H2O·HMPA (0.45 g, 0.1 eq.)
was dissolved in DCM (20 mL) and a solution of H2O2 (6 mL; 30%
in water) was added. The reaction mixture was stirred for 16 h then
quenched by addition of excess MgSO4, filtered and concentrated.
The crude material was purified by flash chromatography, eluting
with 1 : 9 EtOAc–DCM to obtain ethyl 4-nitrosobenzoate 19 as a
yellow solid. To this compound was added 2015 (1.2 g, 3.49 mmol)
then HOAc (25 mL). The resulting mixture was heated to 100 ◦C
for 1 h, then cooled and filtered to collect an orange precipitate.
The filtrate was diluted with DCM (100 mL), washed with sat.
NaHCO3 (100 mL × 3) then dried over MgSO4 and concentrated.
This crude material was combined with the precipitate and purified
by silica column chromatography, eluting with 1 : 19 EtOAc–DCM
4-((4-((2-Aminoacetamido)methyl)phenyl)diazenyl)-N-(2-phenyl-
1-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-ethyl)benzamide
(10). To a solution of 9 (17 mg, 0.03 mmol) in DCM (4 mL)
was added TFA (1 mL). The reaction mixture was stirred for
30 min then concentrated and the crude material purified by flash
chromatography, eluting with 1 : 3 EtOH–DCM to give 10 (14 mg,
1
to give 21 (1.345 g, 22% over◦2 steps, >95% E by H NMR) as
an orange solid: mp 191–194 C, 1H NMR (300 MHz, CDCl3) d
1.43 (3H, t, J = 7, Me), 4.24 (1H, t, J = 6.3, CH2CH), 4.40–4.53
(6H, m), 5.17 (1H, NH), 7.26–7.42 (6H, m), 7.61 (2H, d, J = 6.9),
7.77 (2H, d, J = 7.2), 7.93 (4H, m), 8.20 (2H, d, J = 8.7); 13C
NMR (75 MHz, CDCl3) d 14.3 (Me), 44.7, 47.3 (CH2CH), 61.3,
66.7, 120.0, 122.6, 123.5, 125.0, 127.1, 128.1, 130.6, 132.2, 141.4,
142.3, 143.8, 151.9, 155.0, 156.5, 166.1; m/z (ES) 506.2120, calcd
for C31H28N3O4 (MH+) 506.2080.
◦
82%, >95% E by 1H NMR) as an orange solid: mp 157–160 C,
1H NMR (500 MHz, CD3OD) d 1.15 (12H, m, (CMe2)2), 2.73
(1H, dd, J = 9.1,13.8), 2.96 (1H, dd, J = 6.4, 13.8), 2.99 (1H,
dd, J = 6.4, 9.1), 3.72 (2H, s), 4.49 (2H, s), 7.16 (1H, m), 7.27
(4H, m), 7.47 (2H, d, J = 8.1), 7.89 (2H, d, J = 8.1), 7.96 (2H,
d, J = 8.1), 8.09 (2H, d, J = 8.1), m/z (ES) 542.2965, calcd for
C30H3711BN5O4 (MH+) 542.2938.
(S)-Methyl-4-methyl-2-(4-(phenylazo)phenylsulfonamido)penta-
noate (16). A mixture of 4-(phenylazo)benzenesulfonyl chloride
1519 (773 mg, 2.75 mmol) and (S)-leucine methyl ester (500 mg, 1
eq.) was dissolved in DCM (15 mL) then DIEA (1.44 mL, 3 eq.)
was added. The reaction mixture was refluxed for 2 h, then cooled,
diluted with EtOAc (100 mL), washed with water (100 mL) then
brine (100 mL), dried over MgSO4 and concentrated. The crude
product was purified by flash chromatography, eluting with DCM
then 1 : 9 EtOAc–DCM to obtain 16 (979 mg, 91%, >95% E
by 1H NMR) as an orange solid: mp 131–134 ◦C, 1H NMR
(300 MHz, CDCl3) d 0.91 (6H, m, CHMe2), 1.53 (2H, t, J = 7.1,
CH2), 1.81 (1H, m, CHMe2), 3.45 (3H, s, OMe), 4.03 (1H, m,
COCH), 5.39 (1H, d, J = 9.6, NH), 7.54 (3H, m), 7.93–8.01 (6H,
m); 13C NMR (126 MHz, CDCl3) d 21.3, 22.7, 24.3, 42.2, 52.3,
54.4, 123.1, 123.2, 128.3, 129.2, 132.1, 141.1, 152.3, 154.6, 172.5;
m/z (ES) 390.1485, calcd for C19H24N3O4S (MH+) 390.1488.
Ethyl 4-((4-((2-(tert-butoxycarbonylamino)acetamido)methyl)-
phenyl)diazenyl)benzoate (23). To a solution of 21 (900 mg,
1.78 mmol) in DMF (12 mL) was added piperidine (3 mL). The
reaction mixture was stirred for 15 min then diluted with EtOAc
(100 mL), washed with water (100 mL) then brine (100 mL), dried
over MgSO4, filtered and concentrated in vacuo to give 22 as an
orange solid which was not purified further. To this compound
was added Boc-glycine (312 mg, 1 eq.), HATU (677 mg, 1 eq.)
then DMF (10 mL) and DIEA (620 lL, 2 eq.). The reaction
mixture was stirred for 16 h, diluted with EtOAc (100 mL) and
washed with water (100 mL) brine (100 mL), dried over MgSO4,
filtered and concentrated. The crude material was purified by silica
column chromatography, eluting with 1 : 1 EtOAc–DCM to give
23 (691 mg, 88% ov◦er 2 steps, >95% E by 1H NMR) as an orange
1
solid: mp 136–139 C, H NMR (500 MHz, CDCl3) d 1.41–1.47
(12H, CMe3 and CH2Me), 3.87 (2H, d, J = 5.5), 4.42 (2H, q,
J = 5.4), 4.57 (2H, d, J = 6.0), 5.13 (1H, br, NH), 6.56 (1H, br,
NH), 7.44 (2H, d, J = 8.0), 7.92 (2H, d, J = 8.0), 7.94 (2H, d,
J = 8.5), 8.19 (2H, d, J = 8.5); 13C NMR (75 MHz, CDCl3) d
14.3, 28.2, 42.9, 44.5, 61.2, 80.4, 122.6, 123.4, 128.2, 130.5, 132.1,
141.8, 151.8, 154.9, 156.2, 166.0, 169.6; m/z (ES) 441.2108, calcd
for C23H29N4O5 (MH+) 441.2138.
(S)-4-Methyl-2-(4-(phenylazo)phenylsulfonamido)pentanoic acid
(17). To a solution of 16 (500 mg, 1.28 mmol) in THF (20 mL)
was added a solution of LiOH (8 mL; 0.25 M in 2 : 1 THF–water).
The reaction mixture was stirred for 16 h then diluted with water
(100 mL), washed with DCM (100 mL), then acidified to pH 3
with 1 M HCl and extracted with DCM (100 mL). The organic
layer was dried over MgSO4 and concentrated. The crude material
was purified by silica column chromatography, eluting with 1 : 1
EtOAc–DCM to give 17 (3◦02 mg, 63%, >95% E by 1H NMR) as an
orange solid: mp 128–130 C, 1H NMR (300 MHz, CDCl3) d 0.83
(6H, m, CHMe2), 1.48 (2H, m, CH2), 1.74 (1H, m, CHMe2), 3.97
(1H, m, COCH), 5.18 (1H, d, J = 9.9, NH), 5.35–5.90 (1H, br s,
OH), 7.54 (3H, m), 7.96 (6H, m); 13C NMR (75 MHz, CDCl3) d
21.1, 22.7, 24.3, 41.9, 54.1, 123.2, 123.3, 128.3, 129.3, 132.2, 141.0,
152.3, 154.8, 176.6; m/z (ES) 376.1345, calcd for C18H22N3O4S
(MH+) 376.1331. Unreacted 16 (114 mg, 23%) was also recovered
from the column.
4-((4-((2-(tert-Butoxycarbonylamino)acetamido)methyl)phenyl)-
diazenyl)benzoic acid (24). To a solution of 23 (68 mg,
0.15 mmol) in THF (5 mL) was added a solution of LiOH (1 mL;
0.25 M in 2 : 1 THF–water). The reaction mixture was heated
to 50 ◦C and stirred for 16 h then concentrated. The crude
material was purified by flash chromatography, eluting with 1 : 99
HOAc–EtOAc to give 24 (55 mg, 87%, >95% E by 1H NMR) as
◦
1
an orange solid: mp > 360 C, H NMR (500 MHz, CD3OD) d
1.45 (9H, s, CMe3), 3.76 (2H, s), 4.49 (2H, s), 7.47 (2H, d, J = 8),
7.87 (4H, m), 8.08 (2H, d, J = 8); 13C NMR (75 MHz, CD3OD) d
29.0 (CMe3), 43.9, 45.1, 81.1 (CMe3), 123.4, 124.3, 129.5, 131.5,
3624 | Org. Biomol. Chem., 2006, 4, 3618–3625
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141.8, 144.0, 153.5, 155.3, 158.8, 173.1, 174.7; m/z (ES) 413.1805,
8 I. Schechter and A. Berger, Biochem. Biophys. Res. Commun., 1967, 27,
157.
calcd for C21H25N4O5 (MH+) 413.1825.
9 K. Brady and R. H. Abeles, Biochemistry, 1990, 29, 7608.
10 P. C. Weber, S.-L. Lee, F. A. Lewandowski, M. C. Schadt, C.-H. Chang
and C. A. Kettner, Biochemistry, 1995, 34, 3750.
11 D. S. Matteson, K. M. Sadhu and G. E. Lienhard, J. Am. Chem. Soc.,
1981, 103, 5241.
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The Royal Society of Chemistry 2006
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