An efficient synthesis of argifin: A natural product chitinase inhibitor with chemotherapeutic potential

Article abstract of DOI:10.1016/j.bmcl.2005.07.068

The first synthesis of the cyclopentapeptide family 18 chitinase inhibitor argifin has been achieved by a combination of solid phase and solution chemistry. Synthetic argifin is a nanomolar inhibitor of chitinase B1 from Aspergillus fumigatus and the high-resolution X-ray structure of the synthesized material in complex with the same enzyme is identical to that previously obtained for the natural product.

Full text of DOI:10.1016/j.bmcl.2005.07.068

Bioorganic & Medicinal Chemistry Letters 15 (2005) 4717–4721
An efficient synthesis of argifin: A natural product
chitinase inhibitor with chemotherapeutic potential
Mark J. Dixon, Ole A. Andersen, Daan M. F. van Aalten and Ian M. Eggleston*
Division of Biological Chemistry and Molecular Microbiology, School of Life Sciences, University of Dundee, Dundee DD1 5EH, UK
Received 27 May 2005; revised 21 July 2005; accepted 26 July 2005
Available online 8 September 2005
Abstract—The first synthesis of the cyclopentapeptide family 18 chitinase inhibitor argifin has been achieved by a combination of
solid phase and solution chemistry. Synthetic argifin is a nanomolar inhibitor of chitinase B1 from Aspergillus fumigatus and the
high-resolution X-ray structure of the synthesized material in complex with the same enzyme is identical to that previously obtained
for the natural product.
ꢀ 2005 Elsevier Ltd. All rights reserved.
The glycopolymer chitin, a homopolymer of b(1,4)-
linked N-acetyl-D-glucosamine residues, is a key constit-
uent of many organisms that are pathogenic to humans.
Chitin is the main structural component of the cell wall
of fungi1 and the exoskeletons of insects,2 and is also
found in the eggshells of nematodes.3 Since these organ-
isms depend on the ability to hydrolyse chitin at key-
points in their lifecycles, inhibitors of chitinases are of
considerable interest as potential fungicides and insecti-
cides.4 Furthermore, although chitin is not a component
of mammalian cells, recent studies have shown that
mammalian chitinases may be implicated in the allergic
response associated with asthma and related diseases,5
as well as glycolipid storage disorders, such as GaucherÕs
disease.6
inhibitors that are more synthetically accessible and
amenable to rational structure-based optimisation.9
Argifin (1, Fig. 1), originally isolated from a Gliocladium
fungal culture,9c–e is a cyclopentapeptide composed of
two b-linked Asp residues, an N-methyl Phe residue, a
D-Ala residue and an unusual N-methyl carbamoyl-de-
rivatised Arg residue. Although 1 shows low micromolar
inhibition of several family 18 chitinases,9c,10 no synthe-
sis has so far been achieved, thus preventing a more de-
tailed evaluation of the potential of such peptide-based
inhibitors. In this paper, we therefore describe the first
preparation of 1 and its characterisation against a repre-
sentative family 18 chitinase.
Our synthetic approach to 1 first involved Fmoc-based
solid-phase synthesis of the orthogonally protected line-
ar pentapeptide 2 (Scheme 1). Peptide 2 was assembled
Chitin metabolism is a very attractive target for drug de-
sign, but until recently relatively few effective inhibitors
of chitinases were known. The pseudotrisaccharide nat-
ural product allosamidin7 has been most widely studied
and is a potent inhibitor of many chitinases of glycosyl
hydrolase family 18 that contains enzymes from mam-
mals, insects, nematodes and fungi.8 However, the syn-
thesis of allosamidin is complex,8 which limits its
availability as a biological tool and potential as a drug
lead, and thus has prompted the search for other novel
O
H
N
O
HO
NH
O
NH
O
HN
N
N
N
H
H
H
O
O
N
N
HO
H
O
O
Keywords: Natural product; Chitinase inhibitor; Cyclic peptide; Solid
phase synthesis.
1
*
Corresponding author. Tel.: +44 1382 344319; fax: +44 1382
345517; e-mail: i.m.eggleston@dundee.ac.uk
Figure 1. Argifin 1.
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.07.068

4718
M. J. Dixon et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4717–4721
the sensitive Asp residue to a single cycle of Fmoc syn-
2-chlorotrityl polystyrene resin
(i) 46%
thesis, while the use of tert-butyl rather than benzyl ester
protection was expected to significantly reduce base-
induced aspartimide formation.13
H-βAsp(But)-D-Ala-Arg(Pmc)-MePhe-βAsp(But)-OH
2
The cyclisation of 2 to the fully protected cyclic penta-
peptide 3 proceeded remarkably efficiently in dilute
DCM solution using PyBOP activation and DIPEA as
base. Optimum results were obtained with a single
equivalent of PyBOP and a final peptide concentration
of 1 mM giving an isolated yield of 96% after a simple
extractive work-up. Under these conditions, HPLC
and ESMS analysis revealed almost quantitative forma-
tion of 3 after 16 h, with no evidence of oligomeric side
products (Fig. 2). The remarkably efficient cyclisation
presumably stems from the judicious choice of linear
precursor sequence that maximises the possibility of
favourable ÔprecyclisedÕ conformations by placing po-
tential turn-inducing residues (MePhe, D-Ala) in the sec-
ond and fourth positions of the sequence, respectively.14
(ii) 95%
3
βAsp(But)-D-Ala-Arg(Pmc)-MePhe-βAsp(But)
(iii) 28%
4
p-D-Ala-Arg-MePhe-βAsp
βAs
(iv) 65%
1
Scheme 1. Reagents and conditions:15 (i) Fmoc solid-phase peptide
synthesis; (ii) PyBOP, DIPEA, DCM, 16 h; (iii) TFA/thioanisole/
DCM/H2O (16:2:1:1), 2 h; (iv) N-Succinimidyl-N-methylcarbamate
(3 equiv), DBU (6 equiv), DMF, 40 ꢁC, 2 h.
To complete the synthesis, removal of the Pmc and tert-
butyl protecting groups from 3 was first effected using a
cleavage cocktail of TFA/thioanisole/DCM/water
(16:2:1:1) for 2 h. HPLC analysis of the crude reaction
product revealed several impurities and the rather low
isolated yield (28%) of this step therefore partly reflects
difficulties experienced in the HPLC purification of 4.
Introduction of the N-methyl carbamoyl group onto
the Nx position of the Arg side chain was then achieved
by the reaction of 4 with 3 equiv of N-succinimidyl-N-
methylcarbamate in DMF at 40 ꢁC with DBU as base.
This gave 1 in 65% yield after HPLC isolation and in
17% overall yield from linear peptide 2. No diacylated
or isomeric products were observed. The identity of 1
was confirmed by ESMS (calcd mass 676.3049 Da,
observed mass 676.3048 Da), and 1H and 13C NMR
spectra, which were identical to those reported by Arai
et al.9d for the natural product.15
on 2-chlorotrityl polystyrene resin using 2 equiv of
Fmoc-protected amino acids and PyBOP activation,11
except for the coupling of Fmoc-Arg(Pmc)-OH to the
hindered MePhe residue that required the use of Py-
BrOP.12 Cleavage from the resin with 1% TFA/DCM
gave 2 in 46% yield based on the original resin loading.
The choice of Asp protecting group was found to be
critical in the assembly of the linear precursor; in preli-
minary studies with the alternative sequence H-D-Ala-
Arg(Pmc)-MePhe-bAsp(OBn)-bAsp(OBn)-OH, HPLC
and ESMS analysis revealed an essentially quantitative
formation of a product resulting from aspartimide for-
mation at the non-terminal Asp residue. The subsequent
choice of 2 as linear precursor limited the exposure of
Figure 2. HPLC traces of linear partially protected peptide 2 (lower trace) and crude cyclised peptide 3 (upper trace).18

M. J. Dixon et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4717–4721
4719
lights the importance of the derivatised Arg residue for
binding of 1 to family 18 chitinases and hence the inhib-
itory potency of the natural product.
As a further verification of the correct structure and ste-
reochemistry of synthetic 1, a complex with AfChiB1
was obtained by soaking AfChiB1 crystals in a solution
of 1, as described previously.17 Diffraction data on a
˚
soaked crystal were collected to 1.95 A resolution, and
the unbiased FoꢀFc map was inspected together with
the partially refined structure, starting from the previ-
ously published AfChiB1–1 complex (PDB entry
1W9V).10 This shows that synthetic 1 is identical in both
structure and conformation to the AfChiB1–natural
product complex previously described by Rao et al.
(Fig. 4).10 As 1 binds to AfChiB1 mainly through active
site residues that are strongly conserved in all family 18
chitinases, most notably through interactions of the N-
methyl carbamoyl moiety with the Glu, Asp, and Trp
side chains highlighted in Figure 4, it therefore offers a
very attractive new scaffold for the development of novel
inhibitors of this class of enzymes.
Figure 3. Lineweaver–Burk plots of synthetic 1 measured against
AfChiB1 at different concentrations of the inhibitor: no inhibitor (s);
40 nM (d); 80 nM (h); 120 nM (j). A fit of all the data against a
competitive inhibition model resulted in a Ki of 17 1 nM with a Vmax
of 2.7 0.1 nM/s and a Km of 10.4 1.1 lM.
In summary, the first total synthesis of the natural prod-
uct family 18 chitinase inhibitor argifin has been achieved
utilising a combination of solid- and solution-phase
chemistry, and the synthetic compound has been shown
to be a low nanomolar, competitive inhibitor of a repre-
sentative family 18 chitinase. The synthesis is significantly
simpler than currently available routes to other potent
family 18 chitinase inhibitors and lends itself to the rapid
generation of analogues via straightforward solid-phase
peptide synthesis. Efficient access to 1 and related com-
pounds should greatly facilitate further investigation into
both the role of family 18 chitinases in human conditions
(such as asthma and GaucherÕs disease), as well as the
development of new drugs against a range of human
pathogens. Further studies on the synthesis and screening
The inhibitory properties of synthetic 1 against the
secreted chitinase B1 from Aspergillus fumigatus (Af-
ChiB1) were also investigated using a fluorometric assay
with 4-methylumbelliferyl-b-D-N,N0-diacetylchitobiose
as substrate.16 Steady-state kinetic measurements in
the presence of increasing amounts of synthetic 1 show
that it inhibits the enzyme competitively with a Ki of
17 nM (Fig. 3). Significantly, and in accordance with
X-ray structure data (see below), when 4, which differs
from 1 only in the absence of the N-methyl carbamoyl
moiety, was evaluated against AfChiB1, no inhibition
was observed up to a concentration of 1 mM. This high-
Figure 4. Stereo image of the partially refined crystal structure of AfChiB1 (grey ribbon and sticks with grey carbon atoms) in complex with synthetic
1 (sticks with magenta carbon atoms, model extracted from the previously published AfChiB1–argifin complex, PDB entry 1W9V).10 Unbiased
jFoj ꢀ jFcj, /calcd electron density maps are shown in cyan, contoured at 2.5r.

4720
M. J. Dixon et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4717–4721
15. Experimental conditions, general information: NMR spec-
tra were acquired on a Bruker Avance DPX 300 spec-
of analogues of this natural product are currently being
pursued and will be reported in due course.
1
trometer, operating at 300 MHz for H and 75 MHz for
13C. All coupling constants (J values) were measured in
hertz. Low-resolution ES mass spectra were recorded on
an ABI Mariner TOF Electrospray Ionisation mass
spectrometer in the University of Dundee Post-Genomics
and Molecular Interactions Centre. High-resolution ES
mass spectra were recorded at the EPSRC National Mass
Spectrometry Service (University of Wales, Swansea).
Analytical RP-HPLC was performed on a Dionex HPLC
system equipped with a Dionex Acclaim 3 lm C-18
(150 · 4.6 mm) column with a flow rate of 1 mL/min.
Semi-preparative RP-HPLC was performed on a Dionex
HPLC system equipped with a Phenomenex Gemini 5 lm
C-18 (250 · 10 mm) column with a flow rate of 2.5 mL/
min. Mobile phase A was 0.1% TFA in water, mobile
phase B was 0.1% TFA in acetonitrile. Gradient 1 was
T = 0 min, B = 5%; T = 10 min, B = 95%; T = 15 min,
Acknowledgments
This work was supported by a project grant from the
Wellcome Trust (to IME and DvA). DvA is supported
by a Wellcome Trust Senior Research Fellowship and
the EMBO Young Investigator Programme. We thank
the EPSRC National Mass Spectrometry Service for
accurate mass analysis.
References and notes
1. Cabib, E.; Silverman, S. J.; Shaw, J. A. J. Gen. Microbiol.
1992, 138, 97.
B = 95%; T = 15.1 min, B = 5%. Gradient
2
was
2. Merzendorfer, H.; Zimoch, L. J. Exp. Biol. 2003, 206,
4393.
T = 0 min, B = 5%; T = 20 min, B = 60%; T = 22 min,
B = 95%; T = 27 min, B = 95%; T = 27.1, B = 5%;
T = 32 min, B = 5%. Gradient 3 was T = 0 min, B = 5%;
T = 30 min, B = 30%; T = 32 min, B = 95%; T = 37 min,
B = 95%; T = 37.1 min, B = 5%; T = 42 min, B = 5%.
Synthesis of 2: Loading of the resin was achieved by
treating 2-chlorotrityl polystyrene resin (300 mg,
0.51 mmol) with a solution of Fmoc-Asp-OBut (2 equiv)
and DIPEA (8 equiv) in DCM for 90 min. Fmoc depro-
tection was achieved by treatment with piperidine/DMF
(v/v, 1:4) for 4 · 3 min, except in the final deprotection
step where HOBt (0.1 M) was also included. Peptide
couplings were performed using Fmoc-amino acid
(2 equiv), PyBOP (1.9 equiv), HOBt (2 equiv) and DIPEA
(4 equiv) in DCM/DMF (v/v, 3:1) for 90 min, except in the
case of coupling to MePhe when Fmoc-Arg(Pmc)-OH
(2 equiv), PyBrOP (2 equiv) and DIPEA (4 equiv) in
DCM/DMF (v/v, 3:1) for 3 · 90 min were used. Cleavage
from the resin was achieved by treatment with TFA/DCM
(v/v, 1:99) for 10 · 2 min. The cleavage solutions were
immediately neutralised by addition to pyridine/MeOH
(v/v, 1:9). Solid-phase reactions were monitored by use
of a qualitative Kaiser test19 for the detection of primary
amines and the chloranil test20 for detection of secondary
amines. The crude peptide was isolated by precipitation
from cold water, collected by centrifugation, dissolved in
DCM (20 mL), washed with water (20 mL), dried
(MgSO4) and concentrated in vacuo to yield a white solid
(238 mg, 0.24 mmol, 46%). RP-HPLC (analytical, gradient
1, k = 220 nm): tR = 9.5 min (100%). MS (ES+) m/z: 508.3
(100%) [M+2H]2+, 1015.6 (30%) [M+H]+.
Synthesis of 3: Peptide 2 (113 mg, 0.11 mmol) and PyBOP
(60 mg, 0.11 mmol) were dissolved in DCM (110 mL), and
the solution was adjusted to pH 8 by the addition of
DIPEA and stirred for 16 h. The reaction mixture was
concentrated in vacuo, the residue taken into EtOAc (20
mL) and washed with 5% aq citric acid (15 mL), 5% aq
NaHCO3 (3· 15 mL), satd aq NaHCO3 (3· 15 mL), dried
(MgSO4) and concentrated in vacuo to yield a white solid
(107 mg, 0.11 mmol, 96%). RP-HPLC (analytical, gradient
1, k = 220 nm): tR = 10.6 min (100%). MS (ES+) m/z:
997.7 (100%) [M+H]+.
Synthesis of 4: Peptide 3 (107 mg, 0.11 mmol) was
dissolved in TFA/thioanisole/DCM/H2O (16:2:1:1, 5 mL)
and stirred for 2 h. The reaction mixture was concentrated
in vacuo, the resulting residue was dissolved in TFA and
precipitated by a dropwise addition to ice-cold Et2O. The
precipitate was collected by centrifugation and purified by
semi-preparative HPLC (gradient 3, tR = 23.1 min) to
3. (a) Fuhrman, J. A.; Piessens, W. F. Mol. Biochem.
Parasitol. 1985, 17, 93; (b) Araujo, A. C.; Souto-Padron,
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4. (a) Hollis, T.; Monzingo, A. F.; Bortone, K.; Ernst, S.;
Cox, R.; Robertus, J. D. Protein Sci. 2000, 9, 544; (b)
Sandor, E.; Pusztahelyi, T.; Karaffa, L.; Karanyi, Z.;
Pocsi, I.; Biro, S.; Szentirmai, A.; Pocsi, I. FEMS
Microbiol. Lett. 1998, 164, 231; (c) Sakuda, S.; Isogai,
A.; Matsumoto, S.; Suzuki, A. J. Antibiot. 1987, 40, 296.
5. Zhu, Z.; Zheng, T.; Homer, R. J.; Kim, Y. K.; Chen, N.
Y.; Cohn, L.; Hamid, Q.; Elias, J. A. Science 2004, 304,
1678.
6. Hollak, C. E.; van Weely, S.; van Oers, M. H.; Aerts, J. M.
F. G. J. Clin. Invest. 1994, 93, 1288.
7. Sakuda, S.; Isogai, A.; Matsumoto, S.; Suzuki, A.; Koseki,
K. Tetrahedron Lett. 1986, 27, 2475.
8. Berecibar, A.; Grandjean, C.; Siriwardena, A. Chem. Rev.
1999, 99, 779.
9. For example: Diketopiperazines: (a) Izumida, H.; Imam-
ura, N.; Sano, H. J. Antibiot. 1996, 49, 76; (b) Houston, D.
R.; Eggleston, I.; Synstad, B.; Eijsink, V. G. H.; van
Aalten, D. M. F. Biochem. J. 2002, 368, 23; Cyclic
pentapeptides: (c) Omura, S.; Arai, N.; Yamaguchi, Y.;
Masuma, R.; Iwai, Y.; Namikoshi, M.; Turberg, A.;
Kolbl, H.; Shiomi, K. J. Antibiot. 2000, 53, 603; (d) Arai,
N.; Shiomi, K.; Iwai, Y.; Omura, S. J. Antibiot. 2000, 53,
609; (e) Shiomi, K.; Arai, N.; Iwai, Y.; Turberg, A.; Kolbl,
K.; Omura, S. Tetrahedron Lett. 2000, 41, 2141; (f) Arai,
N.; Shiomi, K.; Yamaguchi, Y.; Masuma, R.; Iwai, Y.;
Turberg, A.; Kolbl, H.; Omura, S. Chem. Pharm. Bull.
2000, 48, 1442; Brominated tyrosine derivatives: (g)
Tabudravu, J. N.; Eijsink, V. G.; Gooday, G. W.; Jaspars,
M.; Komander, D.; Legg, M.; Synstad, B.; van Aalten, D.
M. F. Bioorg. Med. Chem. 2002, 10, 1123; Complex
alkaloids: (h) Kato, T.; Shizuri, Y.; Izumida, H.; Yokoy-
ama, A.; Endo, M. Tetrahedron Lett. 1995, 36, 2133.
10. Rao, F. V.; Houston, D. R.; Boot, R. G.; Aerts, J. M. F.
G.; Hodkinson, M.; Adams, D. J.; Shiomi, K.; Omura, S.;
van Aalten, D. M. F. Chem. Biol. 2005, 12, 65.
11. Coste, J.; Le Nguyen, D.; Castro, B. Tetrahedron Lett.
1990, 31, 205.
´
12. Frerot, E.; Coste, J.; Pantaloni, A.; Dufour, M.-N.; Jouin,
P. Tetrahedron 1991, 47, 259.
13. Nicolas, E.; Pedroso, E.; Giralt, E. Tetrahedron Lett. 1989,
30, 497.
14. Humphrey, J. M.; Chamberlain, A. R. Chem. Rev. 1997,
97, 2243.

M. J. Dixon et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4717–4721
4721
yield a white solid (20 mg, 0.03 mmol, 28%). RP-HPLC
(analytical, gradient 2, k = 220 nm): tR = 7.9 min (100%).
MS (ES+) m/z: 619.4 (100%) [M+H]+.
addition of 25 lL of 3 M glycine–NaOH, pH 10.3, the
fluorescence of the liberated 4-methylumbelliferone was
quantified using a Flx 800 microtitreplate fluorescence
reader (Bio-Tek Instruments Inc.), with excitation and
emission wavelengths of 360 nm and 460 nm, respective-
ly, using 40 nm slits. Experiments were performed in
triplicate. Production of 4-methylumbelliferone was lin-
ear with time for the incubation period used, and less
than 10% of available substrate was hydrolysed. The
mode of action was determined by plotting the data as
Lineweaver–Burk plots, and by fitting all data to the
standard competitive inhibition equation with GraFitꢂ
software.21
Synthesis of 1: Peptide 4 (20 mg, 0.03 mmol) and DBU
(29 lL, 0.19 mmol) were dissolved in DMF (600 lL) and
stirred at 40 ꢁC. A solution of N-succinimidyl-N-methylc-
arbamate (17 mg, 0.1 mmol) in DMF (100 lL) was added
and the reaction mixture was stirred at 40 ꢁC for 2 h. The
crude product was purified by semi-preparative HPLC
(gradient 3, tR = 26.7 min) to yield the natural product as
the TFA salt as a white solid (14 mg, 0.02 mmol, 65%).
d
H (300 MHz, D2O): 8.59 (1H, d, J 5, Asp NH), 8.33 (1H,
d, 6.5, Arg a-NH), 7.38–7.21 (5H, m, MePhe
J
2 · dCH,2 · eCH, fCH), 5.12 (1H, dd, J 11, 3, MePhe
aCH), 4.73 (1H, m, Asp aCH), 4.52 (1H, dd, J 12, 2, Asp
aCH), 4.31 (1H, m, Arg aCH), 4.15 (1H, q, J 7, Ala aCH),
3.18–2.89 (6H, m, MePhe bCH2, Arg dCH2, Asp bCH2)
2.87 (3H, s, MePhe NCH3), 2.81 (1H, m, Asp bCHH), 2.75
(3H, s, MeCbmCH3), 2.50 (1H, t, J 12, Asp bCHH), 1.40
(1H, m, Arg cCHH), 1.30 (3H, d, J 7, Ala bCH3), 1.25–
1.04 (2H, m, Arg cCHH, Arg bCHH), ꢀ0.31 (1H, m, Arg
bCHH). dC (75 MHz, D2O): 175.2 (C), 174.2 (C), 171.4
(C), 171.1 (C), 170.2 (C), 155.0 (C), 153.5 (C), 137.3 (C),
129.6 (CH), 129.0 (CH), 127.2 (CH), 62.2 (CH), 50.7
(CH), 50.6 (CH), 49.5 (CH), 48.6 (CH), 40.6 (CH2), 37.8
(CH2), 35.0 (CH2), 33.2 (CH2), 29.8 (CH3), 26.5 (CH2),
25.9 (CH3), 23.9 (CH2), 16.7 (CH3). RP-HPLC (analytical,
gradient 2, k = 220 nm): tR = 9.1 min (100%). MS (ES+)
m/z: 676.4 (100%) [M+H]+. HRMS (ES+) C29H42N9O10
(calcd) 676.3049; (found) 676.3048 [M+H]+.
17. AfChiB1 was expressed, purified and crystallised, as
previously described.10 Crystals used for soaking experi-
ments were washed three times in 0.1 M sodium citrate,
pH 5.5, and 1.4 M Li2SO4, and thereafter soaked for 3 h in
mother liquor containing 1 mM 1. Crystals were cryopro-
tected by a 10 s immersion in 3 M Li2SO4 and frozen in a
nitrogen cryostream for data collection. Data were
˚
collected on a rotating anode (1.95 A resolution with
97% overall completeness and an overall Rmerge of 0.063),
processed with the HKL suite of programs22 and partially
refined with CNS23 to an R-factor of 0.201 (Rfree = 0.224).
Images were generated with PyMol (www.pymol.org).
18. HPLC conditions: see Ref. 15, gradient 1.
19. Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I.
Anal. Biochem. 1970, 34, 595.
20. Vojkovsky, T. Peptide Res. 1995, 8, 236.
21. Leatherbarrow R. J, GraFit version 5, Horley, UK:
Erithacus Software Ltd., 2001.
16. AfChiB1 inhibition was studied using the fluorogenic
substrate 4-methylumbelliferyl-b-D-N,N0-diacetylchitobi-
ose (Sigma), as described previously.10 Briefly, in a final
volume of 50 lL, 2 nM of enzyme was incubated with
5–30 lM substrate in McIlvain buffer (100 mM citric
acid, 200 mM sodium phosphate, pH 5.5) containing
0.1 mg/ml BSA, for 10 min at 37 ꢁC in the presence of
different concentrations of the inhibitor. After the
22. Otwinowski, Z.; Minor, W. Methods Enzymol. 1997, 267,
307.
23. Brunger, A. T.; Adams, P. D.; Clore, G. M.; DeLano, W.
L.; Gros, P.; Grosse-Kunstleve, R. W.; Jiang, J. S.;
Kuszewski, J.; Nilges, M.; Pannu, N. S.; Read, R. J.; Rice,
L. M.; Simonson, T.; Warren, G. L. Acta Crystallogr.,
Sect. D 1998, 54, 905.