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-