α-Ketoester-based photobiological switches: Synthesis, peptide chain extension and assay against α-chymotrypsin

Article abstract of DOI:10.1016/S0960-894X(01)00464-4

The design, synthesis, photoisomerism and biological testing of two peptide-based photoswitchable inhibitors of α-chymotrypsin are presented. The use of a dipeptide recognition sequence gave a 'slow-tight binding' inhibitor, while the introduction of a carbamate linker to the azobenzene gave a modest enhancement in photoswitching of enzyme activity for the photostationary state enriched in the (Z)-isomer over the (E)-isomer.

Full text of DOI:10.1016/S0960-894X(01)00464-4

Bioorganic & Medicinal Chemistry Letters 11 (2001) 2441–2444
ꢀ-Ketoester-Based Photobiological Switches: Synthesis, Peptide
Chain Extension and Assay against ꢀ-Chymotrypsin
Andrew J. Harvey and Andrew D. Abell*
Department of Chemistry, University of Canterbury, Christchurch, New Zealand
Received 30 March 2001; accepted 27 June 2001
Abstract—The design, synthesis, photoisomerism and biological testing of two peptide-based photoswitchable inhibitors of a-chy-
motrypsin are presented. The use of a dipeptide recognition sequence gave a ‘slow-tight binding’ inhibitor, while the introduction of
a carbamate linker to the azobenzene gave a modest enhancement in photoswitching of enzyme activity for the photostationary
state enriched in the (Z)-isomer over the (E)-isomer. # 2001 Elsevier Science Ltd. All rights reserved.
The activity of a biological system, such as an enzyme,
can be modulated by incorporating a photobiological
switch into its structure.1 Examplesof usch photo-
photoswitching of enzyme activity for photostationary
states enriched in either the (Z)-isomer or the (E)-isomer.
biological switches include photoisomerisable enzyme
2
In this paper, we begin to address the effect of increas-
ing the peptidic character (the enzyme binding domain)
and also the nature of the azobenzene-based substituent
(the photoswitch) on the potency7 and switchability8 of
inhibitorsof the type 1. The literature inhibitor 15
served as a template for the design of compounds 3 and
4. Unlike 1, compound 3 containsresiduesat both the
P1 and P2 positions9 [i.e., (l)-phenylalanine and (l)-leu-
cine, respectively]. Compound 4 containsa carbamate
inhibitorsand co-facto, rs
covalently modified
enzymes,3 and enzymesimmobielids on photo-
isomerisable polymers.4 Photoisomerisable inhibitors of
a-chymotrypsin have been reported in which an azo-
benzene group istethered to an inhibitory group (e.g.,
an a-keto ester as in compound 15 or a boronic acid as
in 2).6 These compounds, while lacking extended
peptide binding domains, are reported to exhibit modest
*Corresponding author. Tel.: +64-3-364-2818; fax: +64-3-364-2110; e-mail: a.abell@chem.canterbury.ac.nz
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00464-4

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A.J.Harvey, A.D.Abell / Bioorg.Med.Chem.Lett.11 (2001) 2441–2444
linker to the azobenzene photoswitch, rather than an
amide, asin 1. Thislinker representsan analogue of the
benzyloxycarbonyl (CBz) group, which hasbeen used at
the N-terminusof other a-ketoester-based serine pro-
tease inhibitors. It should also be noted that the nature
of the azobenzene substituent has been reported to
effect of the mixture on the enzyme asisthe ratio of ( E)-
to (Z)-isomers.
From the results in Table 1, it is apparent that the PSS
arising from ambient light serves as a reasonably good
approximation for the visible light PSS (l >400 nm) in
all cases studied, a trend also noted by Vollmer et al.13
It can also be seen that 1 and 3, which have the same
azobenzene substituent, gave similar E/Z ratiosunder
each of the three sets of conditions. However, 4 exhib-
ited comparatively more (E)-isomer in the ambient and
visible PSS and less (E)-isomer in the UV PSS compared
to 1 and 3. Consequently, 4 possesses the best marginal
switching between isomer-enriched states at the specified
wavelengthsof the three inhibitors.
influence the E/Z composition of photostationary states
8
(PSS) of compoundscontaining thisphotows itch.
We
now report synthesis, photoisomerisation and inhibition
studies on these new photoswitchable inhibitors of a-
chymotrypsin.
Compound 3 wasprepared from 4-(phenylazo)benzoic
acid 5 (85% yield over four steps) as detailed in Scheme 1.
The sequenece involved coupling 5 with (l)-leucine
methyl ester, followed by ester hydrolysis and sub-
sequent coupling with the amine 105 to give 8. The
hydroxyl group of 8 wasthen oxidized with TEMPO/
NaOCl5 to afford the desired a-ketoester 3 asa isngle
Next, the inhibitors 3 and 4 were assayed5 against a-
chymotrypsin using both isomer-enriched photosta-
tionary states and the results obtained were compared
to the known inhibitor 1 (see Table 2 for results on 15
and 4). Compounds 1 and 4 gave initial-rate kinetics
that were consistent with competitive inhibition,14 as
might be anticipated for compoundscomprisng an
electrophilic carbonyl moiety.15 For both these inhibi-
tors, the UV light PSS gave the lower inhibition con-
stant, indicating that the (Z)-isomer was the more active
1
diastereomer by H and 13C NMR spectroscopy. It is
noteworthy that thisreaction proceeded without evi-
dence of epimerisation at the b-carbon (a to the ketone
of 3).10 Compound 4 was synthesised via the carbamate
11, which wasitself prepared in one-pot by reaction of
the alcohol 9 with triphosgene, followed by the addition
of the amine 10. The resulting alcohol 11 wasoxi-
dised to the a-ketoester 4 using the TEMPO/NaOCl
procedure.
Separate solutions of 3 and 4, in acetonitrile-d3, were
then photoisomerised, under three sets of conditions
(see Table 1), giving isomer-enriched photostationary
Table 1. Photostationary state (PSS) compositions of the inhibitors in
acetonitrile-d3
Inhibitor
PSS composition at specified wavelengtha
(E-keto):(E-hydrate):(Z-keto):(Z-hydrate)
1
states, the compositions of which were quantified by H
NMR spectroscopy.11 Each of these acetonitrile-d3
1
330>l>370 nm
l>400 nm
Ambient
solutions was observed, by H NMR spectroscopy, to
undergo hydration of the ketone functional group to
give the corresponding gem-diol. Thisreuslted in four
species being present in each of the irradiated solutions
(see Table 1). A gem-diol derivative isknown to be
the predominant species in an aqueous solution of an
a-ketoester or a-ketoamide12 (i.e., the conditionsof an
enzyme assay). Therefore, the ratio of ketone to hydrate
in these solutions is not as important in determining the
15
3
20:7:47:26
(27:73)
18:6:56:20
(24:76)
16:6:56:22
(22:78)
52:20:20:8
(72:28)
44:30:18:8
(74:26)
75:2:23:n.o.
(77:23)
42:32:15:11
(74:26)
27:50:16:7
(77:23)
80:2:18:n.o.
(82:18)
4
aCombined E/Z in parentheses.
n.o., not observed.
Scheme 1. Reagentsand conditions: (i) ( l)-leucine methyl ester hydrochloride, EDCI, HOBT, DIEA, DMF, CH2Cl2, 16 h, 95%; (ii) LiOH, MeOH,
water, 30 min, qu; (iii) EDCI, HOBT, DIEA, DMF, CH2Cl2, 16 h, 90%; (iv) TEMPO, KBr, NaOCl, NaHCO3, water, CH2Cl2, qu; (v) 9,
(CCl3O)2CO, DIEA, CH2Cl2, 0 ꢀC to rt, then 10, DIEA, CH2Cl2, rt, 30 min, 41%.

A.J.Harvey, A.D.Abell / Bioorg.Med.Chem.Lett.11 (2001) 2441–2444
2443
Table 2. The inhibition constants for the inhibition of a-chymo-
trypsin by the UV and ambient light PSS of inhibitors 1 and 4
References and Notes
PSS E/Z
Ki (mM)
ÁKia
1. Willner, I.; Willner, B. In Bioorganic Photochemistry: Bio-
logical Applications of Photochemical Switches; Morrison, H.,
Ed.; John Wiley and Sons: New York, 1993; Vol. 2, p 1.
2. (a) Blonder, R.; Katz, E.; Willner, I.; Wray, V.; Buck-
1 ambient5
1 UV5
4 ambient
4 UV
74:26
27:73
82:18
22:78
0.24
0.13
0.77
0.29
0.11 mM (ꢁ2-fold)
0.48 mM (ꢁ3-fold)
mann, A. F. J.Am.Chem.Soc.
1997, 119, 11747. (b) West-
mark, P. R.; Kelly, J. P.; Smith, B. D. J.Am.Chem.Soc.
1993, 115, 3416.
aDifference between ambient and UV Ki values.
3. (a) Kaufman, H.; Vratsanos, S. M.; Erlanger, B. F. Science
1968, 162, 1487. (b) Willner, I.; Rubin, S. Angew.Chem,. Int.
Ed.Engl. 1996, 35, 367. (c) Willner, I.; Rubin, S.; Riklin, A. J.
Am.Chem.Soc. 1991, 113, 3321.
4. (a) Willner, I.; Rubin, S.; Shatzmiller, R.; Zor, T. J.Am.
Chem.Soc. 1993, 115, 8690. (b) Willner, I.; Rubin, S. React.
Poly. 1993, 21, 177.
5. Harvey, A. J.; Abell, A. D. Tetrahedron 2000, 56, 9763.
6. Westmark, P. R.; Kelly, J. P.; Smith, B. D. J.Am.Chem.
Soc. 1993, 115, 3416.
isomer in each case. Although the UV light PSS of 4
proved to be approximately 2-fold less active than the
UV light PSS of 1, it did exhibit more effective switching
of a-chymotrypsin than compound 1 (i.e., ꢁ3-fold). This
result is consistent with the relative E/Z compositions of
1 and 4 under ambient and UV conditions(see Table 2).
7. For a discussion on the effect of the peptide sequence on
enzyme binding and recognition, see: (a) Laskowski, M.;
Tashiro, M.; Empie, M. W.; Park, S. J.; Kato, I.; Ardelt, W.;
Wieczorek, M. In Proteinase Inhibitors: Medical and Biological
Aspects; Katunuma, N., Umezawa, H., Holzer, H., Eds.;
Springer: Berlin, 1983; p 55. (b) Schellenberger, V.; Braune,
K.; Hofmann, H.-J.; Yakubke, H.-D. Eur.J.Biochem. 1991,
199, 623. (c) Wu, Y.-T.; Hsieh, H.-P.; Chen, S.-T.; Wang, K.-
T. J.Chin.Chem.Soc. 1999, 46, 135.
8. For a discussion on the effect of azobenzene substituent on
E/Z photoisomerism, see: Ross, D. L.; Blanc, J. In Photo-
chromism; Brown, G. H., Ed.; Wiley-Interscience: New York,
1971; pp 471–556. Rau, H. In Photochromism: Molecules and
Systems; Durr, H., Bouas-Laurent, H., Eds.; Elsevier:
Amsterdam, 1990; p 165.
Inhibitor 3 wasidentified asa ‘slow-tight binder’ of a-
chymotrypsin.16 Inhibitorsof thisnature are identified
by the slow onset of inhibition in assays with no pre-
incubation of inhibitor with enzyme, but an increase in
rate over time for pre-incubated assays.17 Pre-incuba-
tion of 3 with enzyme followed by the addition of sub-
strate gave a slow initial rate, VB=90 mmolÀ1 mgÀ1
,
which stabilised to a constant rate, VS=170 mmolÀ1
mgÀ1, after about 5 min. Addition of the enzyme to a
mixture of inhibitor and substrate gave a high initial
rate, VA=490 mmolÀ1 mgÀ1, which tended downward
over time reaching a steady rate VS=170 mmolÀ1 mgÀ1
9. For a definition of thisnomenclature, ese: Schechter, I.;
Berger, A. Biochem.Biophys.Res.Commun. 1967, 27, 157.
10. TEMPO isreported to maintain chiral integrity in the
oxidation of peptidyl a-ketoamides (see ref 5). Harbeson, S. L.;
Abelleira, S. M.; Akiyama, A.; Barrett, R., III; Carroll, R. M.;
Straub, J. A.; Tkacz, J. N.; Wu, C.; Musso, G. F. J.Med.
Chem. 1994, 37, 2918.
11. A solution of the compound under study (5 mg) in
acetonitrile-d3 (150 mL) wasirradiated for 1 h with light
from a high pressure mercury arc lamp filtered to allow
passage of wavelengths between 330 and 370 nm. The 1H
NMR spectrum was recorded and the components of the
mixture were identified by integration of the OMe reso-
nances. The visible light PSS was then obtained by irradia-
tion of the sample with wavelengths over 400 nm and the
mixture composition was again determined. Finally, the
solution was allowed to photoequilibrate under fluorescent
lighting for 24 h and the resulting mixture composition was
measured. The OMe resonance for the (E)-isomers is
downfield by approx. 0.04–0.06 ppm relative to the (Z)-
isomer. The OMe resonance for the keto isomers is down-
field by approx. 0.06–0.08 ppm relative to the correspond-
ing hydrates. See ref 5 for details.
after about 5 min. The rate in the absence of inhibitor
16
wasmeausred at 950.
‘Slow-tight binding’ hasbeen
observed for a number of a-ketoester, trifluoromethylk-
etone and aldehyde inhibitors of serine proteases.15,17,18
Evidence from the trifluoromethylketone and aldehyde
inhibitors suggests that a pre-binding equilibrium from
hydrate to the putative active species, ketone, leads to
the slow onset of inhibition.19
In summary, the reported photobiological switch 1 was
used as a template for the design and synthesis of the
inhibitors 3 and 4. Methodology ispresented to incor-
porate an azobenzene switch into extended a-ketoester
peptidomimeticsusch asin
3. Inhibitor 4 displayed
improved switching action of a-chymotrypsin compared
with the template compound. This result is due, at least
in part, to the change in azobenzene substitution
between the two compounds, which led to more effec-
tive isomer enrichment of 4 by irradiation compared
with compound 1. Elaboration of 1 to give the dipeptide
mimic 3 led to the synthesis of an inhibitor of a-chy-
motrypsin that displayed ‘slow-tight binding’ inhibition.
These results provide an insight into the constraints of
shape and polarity in the binding site of azobenzene-
containing inhibitorsof a-chymotrypsin.
12. (a) Patel, D. V.; Rielly-Gauvin, K.; Ryono, D. E.; Free,
C. A.; Smith, S. A.; Petrillo, E. W. J.Med.Chem. 1993, 36,
2431. (b) Ocain, T. D.; Rich, D. H. J.Med.Chem. 1992, 35,
451.
13. Vollmer, M. S.; Clark, T. D.; Steinem, C.; Ghadiri, M. R.
Angew.Chem,. Int.Ed.Engl.
1999, 38, 1598.
14. 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 (for competitive inhibition, Ki isgiven asthe [I]-axis
value for the median of the line intersections in the fourth
Acknowledgements
This work was supported by a research grant from the
Royal Society of New Zealand Marsden Fund.

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A.J.Harvey, A.D.Abell / Bioorg.Med.Chem.Lett.11 (2001) 2441–2444
quadrant in a plot of [I] vs1/V) and double-reciprocal ana-
lyses. Large data sets were obtained in duplicate for each PSS.
See ref 5 for details.
15. Angelastro, M. R.; Mehdi, S.; Burkhart, J. P.; Peet, N. P.;
Bey, P. J.Med.Chem. 1990, 33, 11.
cussion of tight binding inhibitors, see: Cha, S. Biochem.
Pharmacol. 1975, 24, 2177.
17. 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.
16. The ambient light PSS of 3 was assayed against a-chymo-
trypsin ([I]=0.82 mM, [S]=0.2 mM, [E]=6.7 nM). For a dis-
18. Imperiali, B.; Abeles, R. H. Biochemistry 1987, 26, 4474.
19. Edwards, P. D.; Bernstein, P. R. Med.Res.Rev. 1994, 14, 127.