Synthesis and inhibition properties of a series of pyranose derivatives towards a Zn-metalloproteinase from Saccharomonospora canescens

Article abstract of DOI:10.1016/j.carres.2010.07.038

The Zn-proteinase, isolated from Saccharomonospora canescens (NPS), shares many common features with thermolysin, but considerable differences are also evident, as far as the substrate recognition site is concerned. In substrates of general structure AcylAlaAlaPhe 4NA, this neutral proteinase cleaves only the arylamide bond (non-typical activity of Zn-proteinases), while thermolysin attacks the peptide bond Ala-Phe. Phosphoramidon is a powerful tight binding inhibitor for thermolysin and significantly less specific towards NPS. The K_i-values (65 μM for NPS vs 0.034 μM for thermolysin) differ nearly 2000-folds. This implies significant differences in the specificity of the corresponding subsites. The carbohydrate moiety is supposed to accommodate in the S₁-subsite and the series of arabinopyranosides and glucopyranosides (12 compounds), which are assayed as inhibitors in a model system: NPS with SucAlaAlaPhe4NA as a substrate could be considered as mapping the S₁-subsite of NPS. Members of the series with an additional ring (3,4-epithio, 3,4-anhydro-derivatives) turned out to be reasonably good competitive inhibitors (K_i ≈ 0.1-0.2 mM are of the same order as the K_i value for phosphoramidon). The structure of these compounds (8, 9, 11 and 12) seems to fit the size of the S₁-subsite and due to an appropriately oriented OH-group in addition, to protect the active site Zn²⁺.

Full text of DOI:10.1016/j.carres.2010.07.038

Carbohydrate Research 345 (2010) 2343–2347
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis and inhibition properties of a series of pyranose derivatives towards a
Zn-metalloproteinase from Saccharomonospora canescens
Pavlina Dolashka-Angelova a, , Raid J. Abdel-Jalil b, Raed A. Al-Qawasmeh c, Nicolina Stambolieva a,
⇑
⇑
,
Wolfgang Voelter d
a Institute of Organic Chemistry, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
b Chemistry Department, Faculty of Science, Hashemite University, Jordan
c Department of Chemistry, The University of Jordan, Amman 11942, Jordan
d Interfacultary Institute of Biochemistry, University of Tübingen, Hoppe-Seyler-Strasse 4, D-72076 Tübingen, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 May 2010
Received in revised form 16 July 2010
Accepted 23 July 2010
Available online 29 July 2010
The Zn-proteinase, isolated from Saccharomonospora canescens (NPS), shares many common features with
thermolysin, but considerable differences are also evident, as far as the substrate recognition site is con-
cerned. In substrates of general structure AcylAlaAlaPhe 4NA, this neutral proteinase cleaves only the
arylamide bond (non-typical activity of Zn-proteinases), while thermolysin attacks the peptide bond
Ala-Phe. Phosphoramidon is a powerful tight binding inhibitor for thermolysin and significantly less spe-
cific towards NPS. The Ki-values (65 lM for NPS vs 0.034 lM for thermolysin) differ nearly 2000-folds.
Keywords:
This implies significant differences in the specificity of the corresponding subsites. The carbohydrate moi-
ety is supposed to accommodate in the S1-subsite and the series of arabinopyranosides and glucopyrano-
sides (12 compounds), which are assayed as inhibitors in a model system: NPS with SucAlaAlaPhe4NA as
a substrate could be considered as mapping the S1-subsite of NPS. Members of the series with an addi-
tional ring (3,4-epithio, 3,4-anhydro-derivatives) turned out to be reasonably good competitive inhibitors
(Ki ꢀ 0.1–0.2 mM are of the same order as the Ki value for phosphoramidon). The structure of these com-
pounds (8, 9, 11 and 12) seems to fit the size of the S1-subsite and due to an appropriately oriented OH-
Carbohydrate inhibitors
Neutral Zn-proteinase
S1-Subsite
group in addition, to protect the active site Zn2+
.
Ó 2010 Published by Elsevier Ltd.
1. Introduction
lar matrix. They also have a number of non-traditional roles in
processing factors related to cell growth/proliferation and inflam-
mation. There are 23 human and 23 mouse matrix metalloprotein-
ases, most of which share orthology among most vertebrates;
other examples have been found in invertebrates and plants.5
The interest in Zn-metalloproteinases from microorganisms is
due to their diverse biological functions and promising ideas for
practical application.1 They are thought to play a role in many dis-
ease states, including arthritis, vascular disease, lung injury, wound
repair, cancer and various neurodegenerative disorders. This family
includes a growing number of biologically important enzymes
which are attractive targets for rational drug design.
There is a lot of evidence for resemblance of the catalytic sites of
most Zn-proteinases, accompanied with a considerable diversity in
the substrate recognition sites. The special features of the zinc-
binding environment of carboxypeptidase A were examined, focus-
ing on the geometrical considerations. The results of these studies
suggest that the zinc ion is important for both the binding and the
catalytic activation of the substrate as well as for stabilization of
the tetrahedral reaction intermediate.6 The chemical mechanism
of Zn-proteinase-catalyzed hydrolysis of peptide bonds has been
elusive and is still under debate.
Zinc proteinases (EC 3.4.24) have been recognized as a distinct
class of proteolytic enzymes in which at least one ion of zinc is in-
volved directly in catalysis.1
In recent years, the number of identified zinc proteinases has
dramatically increased. A classification has been proposed on the
basis of unique, repeated amino acid sequences, so called ‘signa-
tures’ and also significant group similarities in their crystallo-
graphic structures.2,3 Three groups of zinc metalloproteases were
found to be encoded in Trypanosoma brucei genome, each of which
contains ꢁ30% amino acid identity with the major surface protease
of Leishmania.4
A very big group of zinc proteases includes matrix metallopro-
teinases which degrade most of the components of the extracellu-
Abbreviations: NPS, neutral metalloproteinase from strain 5 of Saccharomonos-
pora canescens; 4-NA, 4-nitroanilide; Suc, succinyl; Ant, anthraniloyl; FA,
furylacryloyl.
⇑
Corresponding authors. Tel.: +359 2 722297; fax: +359 2 700225 (P.D.-A.).
E-mail addresses: pda54@abv.bg (P. Dolashka-Angelova), nstambol@orgchm.
bas.bg (N. Stambolieva).
0008-6215/$ - see front matter Ó 2010 Published by Elsevier Ltd.
doi:10.1016/j.carres.2010.07.038

2344
P. Dolashka-Angelova et al. / Carbohydrate Research 345 (2010) 2343–2347
Therefore, mechanistic studies on new members of Zn2+-pro-
oxygens chelates Zn2+ in the active site, displacing a structural
water molecule; (2) the leucyl side chain of the inhibitor binds in
the hydrophobic pocket S11 (predilection to Leu, Ile, Phe as P11 resi-
due, (3) the Trp residue binds in what appears to be a secondary,
less specific hydrophobic site P12. Simultaneously a hydrogen bond
is formed between the indole nitrogen and the protein backbone at
Asn 111. The sugar moiety is supposed to accommodate at the S1
subsite, it makes several contacts with the enzyme11 and probably
makes some contribution to the free energy of binding. It is sup-
posed that a intermolecular hydrogen bond (2.4 Å) between the
carboxyl terminal of phosphoramidon and the C(2) hydroxyl of
the sugar is formed. This hydrogen bond may help ‘lock’ the inhib-
itor in a conformation optimal for binding to the enzyme.
teinases are expected to be an additional source of information
for this class. The recently isolated Zn-proteinase from Saccharo-
monospora canescens (NPS) was purified to homogeneity.7 The
intriguing properties of this enzyme are its high thermostability
and the non-typical for this class arylamidase activity. This paper
is a logic extension of the studies on this enzyme7 in order to ad-
dress it to some known Zn-proteinase family. We report in this pa-
per on the comparative inhibition activities between NPS and
thermolysin for the known inhibitor phosphoramidon.8 New clas-
ses of carbohydrate inhibitors were synthesized and assayed to-
wards NPS.
In the light of the mode of phosphoramidon binding to Zn-pro-
teinases, the series of compounds (Table 2) are assayed as NPS
inhibitors and they could be considered as mapping the S1 subsite.
The values of Ki are an indication of the degree of structural fit of
the corresponding structure to the S1 subsite and also how much
2. Results and discussion
The enzyme Saccharomospora canescens sp. novus is a thermo-
stable neutral Zn-proteinase with a molecular mass of 33 kDa, con-
sisting of a single polypeptide chain. The N-terminal sequence of
NPS, including 26 amino acid residues, has no homology with other
bacterial neutral proteinases such as themolysin and carboxypep-
tidase A. NPS is active towards casein. It is capable of hydrolyzing
an anilide bond (a single cleavage) in peptide 4-nitroanilides of
corresponding amino acid sequences, which is unusual for the
great majority of the Zn-proteinases.9,3 Thermolysin, a 34.6 kDa
protease, isolated from Bac. thermoproteolyticus, being the most
thoroughly studied Zn-metalloproteinase was used as an enzyme
for comparison with the recently reported NPS. The data in Table
1 show that the two enzymes differ in the type of cleaved bond,
recognized in the structure of the tripeptide nitroanilide sub-
strates. Thermolysin attacks Ala-Phe peptide bond, while NPS
causes only one cleavage at the nitroanilide bond, based on the
HPLC analysis of the cleaved products of the substrates AntAlaAla-
Phe4NA and SucAlaAlaPhe4NA. The arylamidase activity is typical
for serine- and thiol-proteinases.10 Common for a number of Zn-
it hampers the alignment of the substrate to the active site Zn2+
.
The series of carbohydrate compounds inhibited NPS in a compet-
itive manner. Using Dixon plot of inhibition of NPS with inhibitor
(9), the Ki value was calculated to be 1.5 mM. Based on the Ki val-
ues, the series could be divided into two groups: the first with Ki
>1 mM and the second with Ki <1 mM. The common structural fea-
ture of the first group (compds 1, 2, 3, 6 and 10) is the presence of a
number of OH groups (substituted or non-substituted), the pre-
ferred conformation being the ‘boat conformation’. Maybe there
is a hindrance for the appropriate accommodation of these com-
pounds on the enzyme surface. The second group (compds 7, 8,
9, 11 and 12) is characterized with a presence of a second ring at-
tached to the pyranose structure in positions 3 and 4 (3,4-epithio
or 3,4-anhydro) and an OH group in position 2. They are of less
flexible conformation. It sounds logic to ascribe the better inhibi-
tion constants to a better fit of the second group compounds to
the S1 enzyme cavity as well as to an ability to protect in some
degree the active site Zn2+. The Ki-value of compound 4, lacking a
second ring, implies a favourable role of the CH2OH group serving
as a ‘handle’ providing additional contacts with the active site.
Thermolysin, a 34.6 kDa protease, being the most thoroughly
studied Zn-metalloproteinase, was included in our comparative
kinetic studies.
proteinases inhibitor phosphoramidon N-(
hydroxylphosphinyl)- -leucyl- -tryptophan is an inhibitor for both
the compared proteinases. It is a powerful competitive (tight
binding) inhibitor with a Ki-value of 0.034
M for thermolysin.11
Significantly weaker inhibitor is phosphoramidon for NPS, with a
2000-fold higher Ki-value (Ki = 65 M). Nevertheless, we assume
a-L-rhamnopyranosyl-
L
L
l
l
an analogy in the binding mode of phosphoramidon to both en-
zymes. The chelation of the active site zinc ion by phosphoryl
group and a hydrophobic interaction between the side chain of
the P11-amino acid and the S11 subsite of the enzyme (nomenclature
of Schechter, Berger)12 have been considered as the primary factors
responsible for the effective inhibition. The crystallographic stud-
ies of the complex of thermolysin-phosphoramidon11 have sug-
gested the following mode of binding: (1) one of the phosphite
Using thermolysin isolated from Bac. Thermoproteolyticus in
conditions analogous to the NPS-experiments, for comparison of
enzyme kinetics with NPS, when assayed with compound (9) as
an effector, the second ring was not affected at all. This implies a
less importance of the S1–P1 interactions in the overall recognition
process of the corresponding effectors in the case of thermolysin. It
is known that in thermolysin, Zn2+ is tetra-coordinated, but there
Table 1
Kinetic data for NPS- and thermolysin-catalyzed hydrolysis of N-acyl-tripeptide-nitroanilides
c
c
c
Enzymatic
Enzyme
Reaction
Substrate
Km
(l
M)
kcat (sꢂ1
)
kcat/Km (Mꢂ1 sꢂ1
)
Ki (lM)
Ref.
4
NPS
NPS
AntAAF;4NA
SucAAF;4NA
80
98
4.0
1.1
51,250
11,220
NPS
SucAAF;4NA + phosph.
AntAA;F4NA
SucAA;F4NA
FA-G;L-NH2
FA-G;L-NH2 + phosph.
65.0b
0.034
Thermolysind
Thermolysine
Thermolysin
Thermolysin
4950
2700
9460
17
18
Ki values for phosphoramidon inhibition of the corresponding enzymatic reactions.
a
;Bond cleaved, proved by HPLC analysis of the reaction mixture.
Ki value, determined using Dixon plot.
The values of the kinetic parameters are evaluated with SD <10%.
b
c
d
The hydrolysis of AntAA;F4NA was followed fluorimetrically in the conditions of pseudo-first order kinetics [S] ꢃ Km
.
e
The hydrolysis of SucAA;F4NA was followed by HPLC kinetic experiment.

Table 2
Structure and inhibition constants (Ki) of a series pyranose derivative tested against NPS
Chemical structure
Name
Ki (mM) Chemical structure
Name
Ki (mM)
O
O
7
O
HO
1
Benzyl
a
-D
-arabino-pyranoside
2.80
Benzyl 2,3-anhydro-
a-
D-lyxopyranoside
0.10
OBn
HO
OBn
HO
OH
O
O
HO
HO
HO
8
OH
2
Benzyl b-
L
-arabino-pyranoside
3.50
Benzyl 3,4-dideoxy-a-D-glycero-pent-3-epopyranoside 1.15
OBn
OBn
HO
O
HO
O
9
OH
HO
3
4
Benzyl 2-O-p-tosyl-b-
L
-arabinopyranoside
2.80
Benzyl 3,4-dideoxy-3,4-epithio-a-D-arabinopyranoside 0.12
OBn
OBn
S
OTs
O
O
OAc
10
11
S
OAc
OBn
OBn
OBn
1,2,3,4-Tetra-O-acetyl-b-
D-glucopyranose
0.26
0.90
Benzyl 3,4-didesoxy-3,4-epithio-b-L-arabinopyranoside 0.10
HO
AcO
AcO
OAc
O
O
O
OAc
5
2-Acetamido-2-deoxy-3,4,6-tri-O-acetyl-b-
D
-glucopyranosyl chloride
Benzyl 2,3-anhydro-
a
-
D
-ribopyranoside
1.50
0.12
HO
Cl
AcO
O
O
NHAc
AcO
HO
O
OAc
SOPh
6
12
Phenyl 2-Acetamido-2-deoxy-3,4,6-tri-O-acetyl-1-thiophenyl-b-
D
-glucopyranosyl-sulfoxide 1.90
Benzyl 2,3-anhydro-b-L-lyxopyranoside
AcO
NHAc
The inhibition constants (Ki) are determined by the method of Dixon (substrate: SucAlaAlaPhe pNA (100–700
405 nm (
405 = 9600 Mꢂ1 cmꢂ1). Standard deviation was less than 10%.
lM), NPS (0.1 lM), phosphate buffer: (0.1 M, pH 6.8, 2% DMSO), 25 °C. The hydrolysis of the substrate was followed at
e

2346
P. Dolashka-Angelova et al. / Carbohydrate Research 345 (2010) 2343–2347
are no data available about NPS. Therefore, we could not discussion
probability for difference in the coordination number of the active
site Zn2+ and the properties of the enzymes.
syl-b-L-arabinopyranoside and 100 ml of 90% aqueous acetic acid,
as described in Ref. 14. Yield 8.7 g (91%), mp 119–120 °C (MeOH/
H2O), ½a 2D4
ꢅ
+130 (c 1, CHCl3); Ref. 17: mp 118–120 °C (MeOH/
+134 (c 1, CHCl3).
The experimental data reveal a significant difference between
NPS and thermolysin as far as the substrate recognition site is con-
cerned. It is hard to say which of the factors are (binding interac-
H2O), ½a 2D4
ꢅ
1,2,3,4-Tetra-O-acetyl-b-D-glucopyranose (4) was purchased
from Sigma Aldrich Chemie GmbH, Germany.
tions, catalytic mechanism and differently coordinated Zn2+
)
2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-a-D-glucopyranosyl
responsible for the different behaviour of the enzymes. On the ba-
sis of these experimental data, the design of a more potent NPS-
inhibitor could be consider as a structure that combines the best
‘sugar moiety’ (compounds 9 and 12) with an appropriate P01-
residue.
chloride (5) was prepared from 22.1 g (0.1 mol) of 2-acetamido-2-
deoxy- -glucose, 100 ml of acetic anhydride and the mixture was
D
saturated with hydrogen chloride (0 °C) and kept (25 °C, three
days) as described in Ref. 15. Yield 25.6 g (70%), mp = 120–
123 °C; ½a 2D4
ꢅ
+100.7 (c 0.9, CH2Cl2); Ref. 18: mp 126–127 °C.
-glucopy-
2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-1-thio-b-
D
ranoside phenyl (R,S)-sulfoxide (6) was prepared from phenyl 2-
acetamido-3,4,6-tri-O-acetyl-2-deoxy-1-thio-b- -glucopyranoside
3. Experimental
D
and meta-chloroperbenzoic acid in dichloromethane. Yield 1.94 g
3.1. Purchased chemicals
(85%); mp = 156–158 °C, ½a D24
ꢅ
+18.13 (c = 0.23, CH2Cl2).
-lyxopyranoside (7) was prepared from
Benzyl 2,3-anhydro-
a-
D
SucAlaAlaPhe4NA, a substrate for subtilisins and chymotrypsin-
3.75 g (10.5 mmol) of benzyl 2,3-anhydro-4-O-triflyl-b-
L
-ribopy-
like proteases and 3-(2-furylacryloyl)-glycyl-
gla)—a substrate for thermolysin were from BACHEM Chemicals
(Heidelberg, Germany). N-( -rhamno-pyranosylphosphono)- -leu-
cyl- -tryptophan Disodium salt (phosphoramidon disodium salt)—
L-leucine amide (Fa-
ranoside and 10 mmol of tetrabutyl-ammonium nitrate in DMF
as described in Ref. 16. Yield 1.68 g (72%), mp 65 °C (EE /PE), ½a D24
ꢅ
a
L
+60 (c 1, CHCl3); Ref. 19: mp 65–66 °C (PE), ½a D24
ꢅ
+60.1 (c 1, CHCl3).
L
Benzyl 3,4-dideoxy-a-D-glycero-pent-3-enopyranoside (8) was
a highly specific, naturally occurring thermolysin inhibitor was
from Fluka. Thermolysin with specific activity 40 units/mg lyophil-
izate (37 °C, casein as a substrate) was from Boehringer Mannheim.
All other chemicals were of analytical grade.
synthesized from 2.48 g (10 mmol) of benzyl 3,4-dideoxy-2-O-
acetyl-a-D-glyceropent-3-enopyranoside. Yield 1.82 g (90%),
mp = 85 °C, ½a 2D4
ꢅ
+141.5 (c 0.21, CH2Cl2).
Benzyl 3,4-dideoxy-3,4-epithio-a-D-arabinopyranoside (9) was
3.2. The enzyme neutral proteinase from S. canescens sp. Novus
synthesized from 0.177 g (0.5 mmol) of benzyl 4-O-triflyl-2,3-
anhydro-b- -ribopyranoside and 0.38 g (10 mmol) of lithium alu-
L
NPS was isolated from Bulgarian salt soils7 using a Sephadex G-
100 column (3.5 ꢄ 100 cm), equilibrated and eluted with a 10 mM
phosphate buffer pH 7.0. The second step of the purification proce-
dure was ion exchange chromatography on a DEAE 52 cellulose
column (3.0 ꢄ 10 cm), equilibrated with 10 mM phosphate buffer,
pH 7.0 and eluted under conditions of a linear gradient (0–
0.06 M NaCl) for 10 h and further elution with 0.2 M NaCl. The final
step of purification was ion exchange HPLC on a POROS HQ/M
4.6 ꢄ 100 mm column (Per Septive Biosystems, Germany) using a
linear gradient (0–0.4 M NaCl). The samples, containing NPS, were
pooled together, desalted on a Sephadex G-25 column, concen-
trated on an ultrafiltration membrane and lyophilized.
minium hydride in 25 ml EE as described in Ref. 20. Yield 0.113 g
(95%), mp 101–103 °C (EE/PE), ½a D24
ꢅ
+ 49.9 (c 0.4, CHCl3).
-arabinopyranoside (10)
Benzyl 3,4-didesoxy-3,4-epithio-b-
L
was synthesized from 0.177 g (0.5 mmol) of benzyl 4-O-triflyl-
2,3-anhydro-a-D-ribopyranoside as described in Ref. 20. Yield
0.109 g (92%), mp 92–95 °C (EE/PE), ½a D24
ꢅ
+130.0 (c 0.1, CHCl3).
Benzyl 2,3-anhydro-a-D-ribopyranoside (11) was prepared
from 51.46 g (0.13 mol) of benzyl 2-O-p-tosyl-a-D-arabinopyrano-
side, 950 ml of absolute methanol and 3.6 g (0.15 mol) of sodium
as described in Ref. 11. Yield 20.88 g (72%), mp 96–97 °C (EE/PE),
½
a 2D4
EtOAc).
ꢅ
+190 (c 1, EE); Ref. 21: mp 94–96 °C (PE), ½a D24
ꢅ
+202 (c 1,
Benzyl 2,3-anhydro-b-
3.75 g (10.5 mmol) of benzyl 4-O-triflyl-2,3-anhydro-
L
-lyxopyranoside (12) was prepared from
3.3. Substrate
a
-D
-ribopy-
ranoside and 10 mmol of tetrabutyl-ammonium nitrate in DMF
AntAlaAlaPhe4Na, the best tripeptide 4-nitroanilide substrate
for subtilisins with possibility for two independent methods of
analysis of the enzyme-catalyzed hydrolysis of the arylamide bond
was synthesized according to Ref. 13.
as described in Ref. 13. Yield 1.6 g (68%), mp 82–83 °C (EE/PE),
½
a 2D4
CHCl3).
ꢅ
+68 (c 1, CHCl3); Ref. 22: mp 83–85 °C (PE), ½a D24
ꢅ
+68.6 (c 1,
3.5. Kinetic studies
3.4. Synthesis of pyranose derivatives
The enzyme-catalyzed (NPS and thermolysin) hydrolysis of the
substrates AntAAF4NA and SucAAF4NA was studied in 50 mM phos-
phate buffer pH 7.0, 50 mM CaCl2, 4% DMF at 25 °C. The bond cleaved
was identified by HPLC analysis of aliquots, withdrawn from the
reaction mixture at indicated time intervals, under the following
The following pyranose derivatives were synthesized for studies
of the inhibitory effect on NPS.
Benzyl
a
-
D
-arabinopyranoside (1) was prepared from 11.2 g
-arabinopyranoside,
(20 mmol) of benzyl 2,3,4-tri-O-benzoyl-
a-D
25 ml of absolute MeOH and 2.5 g (10-mmol) of sodium as de-
conditions: WATERS equipment, RP-8/5 lm cartridge, elution with
scribed in Ref. 14. Yield 47 g (96.1%), mp 138–139 °C (EtOH), ½a D24
ꢅ
a gradient from 3.5% B to 60% B over 38 min at a flow rate of 1 ml/
min. Solvent A was 0.1% trifluoroacetic acid in water and solvent B
was 0.075% trifluoroacetic acid in acetonitrile. Elution was moni-
tored at 220 nm and peak areas were determined by integration.
The arylamide bond cleavage was monitored at 410 nm
+12 (c 1, H2O); Ref. 15: mp 140–141 °C (EtOH), ½a D24
ꢅ
+12.3 (c 1,
H2O).
Benzyl b-
(33 mmol) -arabinose and 25 ml freshly distilled benzyl alcohol
L
-arabinopyranoside (2) was prepared from 5 g
L
(e
= 9400 Mꢂ1 cmꢂ1) with a Shimadzu UV-3000 spectrophotome-
as described in Ref. 14. Yield 5.2 g (64%), mp 170–172 °C (EtOH/
H2O), ½a 2D4
ꢅ
+215 (c 0.2, H2O); Ref. 16: mp 168–171 °C (EtOH/
+ 206 (c 0.3, H2O).
ter. The
the effect of the DMF present. The substrate concentration range
was 50–600 M. Thermolysin-catalyzed hydrolysis of peptide
bond Ala-Phe in the substrate structure AntAAF4NA was followed
e value was determined by own experiment, considering
H2O), ½a 2D4
ꢅ
Benzyl 2-O-p-tosyl-b-L-arabinopyranoside (3) was prepared
l
from 107 g (24 mmol) of benzyl 3,4-O-isopropylidene-2-O-p-to-

P. Dolashka-Angelova et al. / Carbohydrate Research 345 (2010) 2343–2347
2347
by a Perkin–Elmer spectrofluorimeter. The fluorescence pseudo-
References
first order traces at 420 nm (excitation at 340 nm), due to the re-
leased AntAlaAlaOH were used to derive the kinetic parameter
kcat/Km. The substrate concentration was kept low (1–10 lM) to
1. Ohta, Y.; Ogura, Y.; Wada, A. J. Biol. Chem. 1966, 241, 5919–5925.
2. Hooper, H. M. FEBS Lett. 1994, 354, 1–6.
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avoid the problems caused by the ‘inner filter effect’. The fluores-
cence changes were transformed into product concentration
changes using a calibration curve plotting fluorescence versus con-
centration of the corresponding fluorophore solution. Thermoly-
sin-catalyzed hydrolysis of peptide bond Ala-Phe in the substrate
SucAAF4NA was followed by HPLC analysis of the withdrawn sam-
ples from the reaction mixture at indicated time intervals. The
kinetic parameters were derived from initial rates (10% or less of
substrate hydrolysis) using non-linear regression data analysis
computer programme Enzfitter.23 Thermolysin-catalyzed hydroly-
sis of Fagla was studied in Ref. 24. Traces at 345 nm under the con-
ditions of pseudo-first order ([S] ꢃ Km) have been recorded. The
substrate concentration was kept low because of the high back-
ground of the substrate, so only the value of the specific constant
kcat/Km could be estimated. The inhibition of thermolysin by phos-
phoramidon was studied in Ref. 25 using the formalism of Hender-
son26 in a model system with Fagla as substrate. This approach is
valuable for the case of tight binding inhibitors ([I] ꢁ [E]). The inhi-
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Acknowledgements
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Dr. Pavlina Dolashka thanks DFG (Deutsche Forschungsgeme-
inschaft) for granting a scholarship and Professor I. Pojarlieff for
discussion of the results.