Metallopeptidase inhibitors of tetanus toxin: A combinatorial approach

Article abstract of DOI:10.1021/jm981066w

The bacterial protein tetanus toxin (TeNt), which belongs to the family of zinc endopeptidases, cleaves synaptobrevin, an essential synaptic protein component of the neurotransmitter exocytosis apparatus, at a single peptide bond (Gln⁷⁶-Phe

Full text of DOI:10.1021/jm981066w

J . Med. Chem. 1999, 42, 515-525
515
Meta llop ep tid a se In h ibitor s of Teta n u s Toxin : A Com bin a tor ia l Ap p r oa ch
Lo¨ıc Martin, Fabrice Cornille, Serge Turcaud, Herve´ Meudal, Bernard P. Roques,* and
Marie-Claude Fournie´-Zaluski
De´partement de Pharmacochimie Mole´culaire et Structurale, U266 INSERM, UMR 8600 CNRS, UFR des Sciences
Pharmaceutiques et Biologiques, 4, Avenue de l’Observatoire, 75270 Paris Cedex 06, France
Received J uly 31, 1998
The bacterial protein tetanus toxin (TeNt), which belongs to the family of zinc endopeptidases,
cleaves synaptobrevin, an essential synaptic protein component of the neurotransmitter
exocytosis apparatus, at a single peptide bond (Gln76-Phe77). This protease activity is a
particularly attractive target for designing potent and selective synthetic inhibitors as a possible
drug therapy for tetanus. â-Aminothiols mimicking Gln76 of synaptobrevin have been previously
shown to inhibit the tetanus neurotoxin enzymatic activity in the 35-250 µM range. These
compounds have now been modified to interact with S′ subsites of the TeNt active site, with
the aim of increasing their inhibitory potencies. Combinatorial libraries of pseudotripeptides,
containing an ethylene sulfonamide or an m-sulfonamidophenyl moiety as the P1 side chain
and natural amino acids in P1′ and P2′ positions, were synthesized. The best inhibitory activity
was observed with Tyr and His as P1′ and P2′ components, respectively. This led to new
inhibitors of TeNt with Ki values in the 3-4 µM range. These molecules are the most potent
inhibitors of TeNt described so far.
In tr od u ction
quent activation of proteolysis.15-17 This probably ex-
plains why potent inhibitors of well-known zinc metallo-
peptidases such as captopril, thiorphan, or phosphor-
amidon have a weak inhibitory activity on TeNt, in the
10 mM range in vitro18,19 or ex vivo.20,21 As no efficient
drug therapy is yet available for tetanus or botulism,
we identified the zinc-dependent metallopeptidase ac-
tivity of tetanus neurotoxin as a good target.
Tetanus neurotoxin (TeNt) is a 150-kDa protein
produced by the anaerobic bacillus Clostridium tetani,
which causes the lethal spastic paralysis of tetanus by
blockade of inhibitory neurotransmitter release at cen-
tral synapses.1,2 It is a member of the clostridial
neurotoxin family, which includes seven botulinum
neurotoxins responsible for the flaccid paralysis of
botulism.1,2 TeNt is composed of two polypeptide chains
joined by a single interchain disulfide bond.1 The heavy
chain (100 kDa) participates in binding to the nerve
terminals, internalization, retrograde axonal transport,
and membrane translocation of the toxic light chain (50
kDa) into the neuronal cytosol.1,3 The light chain, after
reduction of the disulfide bridge, is responsible for the
inhibition of neurotransmitter release.1
TeNt belongs to the thermolysin family of Zn2+
metallopeptidases that contains the HExxH consensus
sequence,4-6 in which the two histidines are involved
in zinc chelation whereas the glutamate is involved in
catalysis.7 This has been shown by site-directed mu-
tagenesis experiments8,9 and recently confirmed by the
crystal structure determination of botulinum neurotoxin
type A (BoNt/A).10 TeNt has been shown to cleave a
single 116-residue protein, VAMP/synaptobrevin, at the
Gln76-Phe77 peptide bond11 (Figure 1). This protein is
an essential component of the neuro-exocytosis appa-
ratus,12-14 and its cleavage by TeNt leads to the
blockade of inhibitory neurotransmitter release.11 Such
a narrow specificity, not common for other zinc pepti-
dases,6 has been recently explained by a cooperative
mechanism, involving the binding to TeNt of synapto-
brevin domains distal from the cleavage site for subse-
The best-described inhibitory potencies for TeNt were
obtained recently22 with glutaminethiol 1 (Ki ) 250 (
35 µM) and its sulfonamide analogues 2 (Ki ) 100 ( 5
µM)) and 3 (Ki ) 35 ( 5 µM), expected to interact with
the S1 subsite of TeNt (Figure 2A). To improve the
potency of these compounds, we report here the syn-
thesis of analogues able to interact with additional
subsites of the TeNt catalytic site. Given the critical
importance of the primary amino group in compounds
1-3 for TeNt recognition,22 these molecules were ex-
tended in order to interact with S′ subsites (Figure 2B).
To have access to such compounds, the synthesis of the
new fully protected â-amino-R-sulfanyl acids (Figure 2C)
corresponding to synthons A, B, and C was achieved.
These synthons were used in the solid-phase peptide
synthesis of pseudotripeptides. The screening of a
combinatorial library of such analogues toward TeNt
allowed the preference of the S1′ and S2′ subsites to be
determined.
Ch em istr y
Syn th esis of Syn th on s A a n d B. The commercially
available Boc-L-Gln(Trt)-OH (4a ) and the (2S)-4-(tert-
butylsulfamoyl)-2-(Cbz-amino)butanoic acid (4b) pre-
pared from the Z-L-homocystine as previously de-
scribed22 were first homologated by the Arndt-Eistert
reaction as described in Scheme 1.23 Their free acid
functions, activated as mixed anhydride derivatives,
* To whom correspondence should be addressed. Tel: (33) 01-43-
25-50-45. Fax: (33) 01-43-26-69-18.
10.1021/jm981066w CCC: $18.00 © 1999 American Chemical Society
Published on Web 01/21/1999

516 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 3
Martin et al.
F igu r e 1. (A) Schematic representation of VAMP/synaptobrevin II. TMR indicates the transmembrane region and IR the
intravesicular region. The cleavage site Gln76-Phe77 is indicated by the arrow. Domains 27-55 and 82-93 represent respectively
the acidic and basic clusters whose binding to tetanus neurotoxin is required to switch on its proteolytic activity.16 (B) Human
synaptobrevin II sequence surrounding the scissible bond. The side chains of the different synaptobrevin residues are putatively
interacting with subsites of the catalytic domain of TeNt designated S1 to S6 on the left of the cleavage bond and S1′ to S5′ on the
right.
Sch em e 1. Synthesis of Synthon A and Synthon Ba
a
(a) iBuOCOCl, NMM, CH2N2; (b) BzOAg, NEt3, MeOH; (c)
LDA, 4-methoxybenzyl 2,4-dinitrophenyl disulfide; (d) MeOH,
NaOH.
6b were hydrolyzed by 1 M NaOH to yield the final
synthons A and B, which were obtained as racemates
on the C2 carbon bearing the thiol group.
Syn th esis of Syn th on C. Scheme 2 shows the
pathway used in the synthesis of the meta-substituted
sulfonamide analogues of â-aminophenylalanine (syn-
thon C) obtained as a mixture of two diastereomers
(2SR,3S) or as a pure diastereomer (2S,3S). The com-
mercially available 3-(chlorosulfonyl)benzoic acid (7)
was transformed into the tert-butylsulfonamide deriva-
tive 8 by tert-butylamine treatment (78% yield) and then
F igu r e 2. (A) â-Aminothiols bearing carboxamide or sulfon-
transformed into the corresponding substituted benz-
amide functionalities on aliphatic or aromatic side chains
aldehyde via a reduction of the free carboxylic group
endowed with inhibitory activities against tetanus neurotoxin.
into the corresponding alcohol 925 (90% yield), subse-
(B) Extension of these â-aminothiols toward S′ subsites of
quently oxidized by the Swern method to afford the
aldehyde 1026 (96% yield). Compound 10 was then
converted by a Horner-Wadsworth-Emmons olefina-
tion27 into an acrylate intermediate 11 (85% yield). A
Michae¨l addition performed on compound 11 with the
homochiral lithium (S)-benzyl(R-methylbenzyl)amide
yielded the â-aminophenylalanine derivative (RS,3R)-
12a 28 with an enantiomeric excess > 99% (87% yield).
Hydrogenolysis under 5 atm in ethyl acetate in the
presence of Boc2O28 afforded the N-Boc-protected ethyl
ester â-aminophenylalanine analogue (3R)-13a (85%
yield). The formation of the R-carbanion of 13a with
lithium diisopropylamide (LDA) followed by treatment
with 4-methoxybenzyl 2,4-dinitrophenyl disulfide yielded
TeNt. (C) Protected synthons required for the synthesis of such
types of molecules.
were converted into the corresponding diazo ketones by
action of diazomethane. These diazo ketones underwent
the Wolff rearrangement in methanol with silver ben-
zoate as a catalyst to afford the â-amino esters 5a and
5b (84% and 95% yields, respectively). The carbanions
of these â-amino esters, formed by action of lithium
diisopropylamide, were sulfenylated by the asymmetric
4-methoxybenzyl 2,4-dinitrophenyl disulfide according
to the recent improvements reported in the synthesis
of R-sulfenylated â-amino acids24 (43% and 54% yields,
respectively). The ester functions of resulting 6a and

Metallopeptidase Inhibitors of Tetanus Toxin
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 3 517
Sch em e 2. Synthesis of Synthon Ca
F igu r e 3. (A) Synaptobrevin sequence 76QFETS in interaction
with S and S′ subsites of tetanus neurotoxin. (B) Inhibition
constants of pseudopeptides mimicking the synaptobrevin
sequence 76QF (15), 76QFE (16), 76QFET (17), and 76QFETS
(18), respectively.
a
(a) tBuNH2; (b) LiAlH4; (c) DMSO, (COCl)2, NEt3; (d) triethyl
previously described protocol35,36 (Figure 4). The Divide/
Couple/Recombine method37 was used to synthesize
these 19 sublibraries. A first amino acid mixture is
prepared by combining the 19 different amino acids
linked to the 2-chlorotrityl resin (Cys was omitted). This
homogenized mixture is then split into 19 identical
samples to which a known amino acid is coupled. This
gives rise to 19 different mixtures, each of them
containing 19 different dipeptides. The next step is the
coupling of synthon B in each sample allowing the
synthesis of 19 mixtures containing 19 couples of
diastereomers. The 19 fractions of peptidyl resin were
then cleaved from the resin by using a dichloromethane/
trifluoroethanol mixture38 (8/2). The deprotection of the
TFA-labile protecting groups was achieved by using
trifluoroacetic acid,32 and the final deprotection step
used to hydrolyze the benzylcarbamate group and the
4-methoxybenzyl group was performed with anhydrous
hydrogen fluoride.33 The 19 freeze-dried sublibraries
were soluble in water at 1 mM. Electrospray mass
spectra and quantitative amino acid analysis showed
in each case a correct mass distribution and a good
stoichiometry in the S2′ position, respectively (data not
shown).
phosphonoacetate, NaH, DME, - 50 °C; (e) lithium (S)-(R-
methylbenzyl)benzylamide, THF, -78 °C; (f) Pd(OH)2/C (1 equiv),
H2 (10 atm), Boc2O (3.7 equiv), EtOAc, rt, 24 h; (g) LDA,
4-methoxybenzyl 2,4-dinitrophenyl disulfide; (h) BBTO, CH3CN,
82 °C, 48 h.
the diastereomer (2S,3S)-14a (2S,3S/2R,3S ) 85/15).
The hydrolysis of the ethyl ester by bis(tributyltin)oxide
(BBTO) gave the synthon C (2S,3S) without further
racemization (70% yield), while its hydrolysis by 1 M
NaOH in MeOH overnight at room temperature gave a
50/50 mixture of the two diastereomers (2R,3S and
2S,3S). To obtain the two other diastereomers (2R,3R
and 2S,3R), the homochiral lithium (R)-benzyl(R-methyl-
benzyl)amide was used in the same synthetic pathway
instead of the S isomer. In addition, the 2,4-dimethoxy-
benzylthio 4-methylphenylsulfonate was used as sulf-
enylating agent to obtain 14c and 14d with a thiol
protected by a dimethoxybenzyl group.30 The HPLC
separation of both diastereomers after TFA deprotection
and before HF cleavage becomes easier in this case than
with the monomethoxybenzyl protecting group.
Syn th eses of Su bstr a te Mim ics Der ived fr om
Com p ou n d 1. The syntheses of the thiol peptide
derivatives spanning the various subsites defined as S1,
S1′, S2′, S3′, and S4′ of the tetanus toxin catalytic domain
were performed by solid-phase peptide synthesis using
synthon A, the protected amino acids corresponding to
Phe, Glu, Thr, Ser, and the Fmoc/tBu strategy on Wang/
HMP resin (Figure 3).31 Cleavage from the solid support
and deprotection of side-chain-protecting groups were
achieved by using trifluoroacetic acid in the presence
of scavengers.32 This was followed by a final HF treat-
ment33 in order to obtain the various carboxamide thiols
(15-18) which were then purified by reverse-phase
HPLC.
A second library corresponding to the parallel syn-
thesis of 19 mixtures of two diastereomers of pseudo-
tripeptides of formula (synthon B)-Tyr-AA′2x was achieved
on 2-chlorotrityl chloride resin with the same protocol
described above.
Syn th eses of In h ibitor s Der ived fr om th e Li-
br a r ies. The two diastereomers 19a and 19b (Figure
5A), issued from the best mixtures of the library (tyrosyl
in S1′ and histidyl in S2′), were purified and separated
by RP-HPLC. To assign unambiguously the absolute
configuration of the C2 carbon bearing the thiol group,
the solid-phase synthesis of the diastereomer 19b alone
was performed by successive coupling of Fmoc-protected
histidine, tyrosine, and the optically pure synthon B
(2S,3S) obtained by hydrolysis of the methyl ester of
6b by BBTO. Cleavage from the resin and side-chain
deprotection were achieved with the same protocol
described for the libraries.
Syn th esis of Com bin a tor ia l Libr a r ies of Th iol-
Con ta in in g In h ibitor s. A pseudotripeptide library,
corresponding to 19 mixtures of thiol-containing pep-
tides of formula (synthon B)-AA′1x-AA′2x, where AA′1x
and AA′2x represent the 19 different natural amino acids
(Cys omitted), was obtained by solid-phase peptide
synthesis on 2-chlorotrityl chloride resin34 by using a

518 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 3
Martin et al.
F igu r e 5. (A) Inhibition constants values of pseudotripeptides
derived from the â-aminothiol 2 (Figure 2). The different
absolute configurations of the C2 and C3 carbons of the
â-amino acid synthon are indicated. (B) Inhibition constant
values of pseudotripeptides derived from the â-aminothiol 3
(Figure 2). The different absolute configurations of the C2 and
C3 carbons of the â-amino acid synthon are indicated.
F igu r e 4. Histograms representing the percentage of inhibi-
tion obtained with 38 mixtures of pseudotripeptides derived
from the â-aminothiol 2 (Figure 1) on tetanus neurotoxin zinc
endoprotease activity. The general formula of these mixtures
of compounds is given above the histogram. (A) Each mixture
differs by a natural amino acid (except Cys) putatively
interacting in S1′ which is represented as a one-letter code on
the abscissa. A stoichiometric amount of each natural amino
acid (except Cys) is present in S2′. These mixtures were tested
at 33 µM on TeNt. (B) A same tyrosyl residue is present in
S1′. Each mixture differs by the residue present in S2′ which
corresponds to a natural amino acid (except Cys) indicated as
a one-letter code on the abscissa. These mixtures were tested
at 25 µM on TeNt.
couples of diastereomers in order to assign the absolute
configuration of the chiral centers.
Biologica l Resu lts
The various pseudopeptides 15-18 were tested on
TeNt metallopeptidase activity in vitro.39 The results
are summarized in Figure 3. As compared to glutamine-
thiol 1, which had a Ki of 250 ( 35 µM (Figure 2A), the
four compounds 15-18 exhibited increased inhibitory
potencies with Ki values in the 80-100 µM range
(Figure 3). Nevertheless, no significant difference was
observed among them. Therefore, the pseudotripeptide
was selected as a scaffold to investigate the S1′ and S2′
subsites of tetanus neurotoxin and to try increase the
inhibitory potency. These molecules represent a good
compromise in terms of size to generate structured
diversity for exploring the S′ subsites.
For this purpose, a combinatorial library of pseudo-
tripeptides derived from the sulfonamide analogue 2
was used. The results are summarized in Figure 4.
Among the different mixtures corresponding to known
amino acids putatively interacting with S1′ (Figure 4A),
a significant preference for a tyrosyl residue (52%
inhibition) was observed. Nevertheless, no great differ-
ences were observed for the other mixtures, with a 40%
inhibition for Val, Asn, Gln, Gly, and Lys and from 15%
to 35% inhibition for the others. Surprisingly, a Phe side
Finally, synthon B (2RS,3S), and synthons C (2RS,3S
and 2RS,3R) were coupled independently with the
protected dipeptide H-Tyr(tBu)-His(Trt)-NHBzl. After
deprotection by trifluoroacetic acid,32 the resulting
compounds were purified at this step by semiprepara-
tive HPLC in order to separate the couples of diaster-
eomers and treated by anhydrous hydrogen fluoride33
to afford compounds 20a , 20b (Figure 5A), 21a , 21b,
21c, and 21d (Figure 5B), respectively. To assign
unambiguously the absolute configuration of the C2
carbon bearing the thiol, the syntheses of compounds
20b, 21b, and 21c were achieved independently by
using optically pure synthon B (2S,3S), synthon C
1
(2S,3S), and synthon C (2R,3R). The H NMR chemical
shifts as well as the HPLC retention times of these
compounds were compared to those of the corresponding

Metallopeptidase Inhibitors of Tetanus Toxin
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 3 519
chain, mimicking the natural substrate synaptobrevin
in the P1′ position, was not among the best inhibitors
(30% inhibition). Although no clear preference for the
S1′ subsite of TeNt was observed, the apparent best P1′
component, Tyr, was used to develop a second library
of these pseudotripeptides possessing different natural
amino acids in S2′. This library, composed of mixtures
of two diastereomers, was screened against TeNt met-
allopeptidase activity (Figure 4B). The best residues
issued from this screening were His and Ile (60% and
55% inhibition, respectively). The other analogues dem-
onstrated an inhibitory potency in the 30-50% range
(except Gly, 15%). However, no great subsite specificity
could be defined, even if a slight preference for aromatic
or aliphatic side chains (His, Ile, Tyr, Val) was observed.
The best compound issued from both libraries, corre-
sponding to a tyrosyl residue in S1′ and a histidyl
residue in S2′, was purified and fully characterized. The
corresponding two diastereomers 19a and 19b (Figure
5A) were separated by HPLC and screened independ-
ently on TeNt proteolytic activity. The compound 19a
corresponding to the 2R,3S absolute configuration was
the best with a Ki ) 5 ( 1 µM as compared to Ki ) 15
( 2 µM for 19b.
F igu r e 6. Lineweaver-Burk plot analysis of the inhibitory
activity of compound 21c for TeNt. The experiments were
performed at 37 °C in 20 mM Hepes buffer (pH 7.4) containing
100 mM NaCl and 0.1 mM DTT. The enzymatic hydrolysis
rates of the fluorescent substrate [Pya88]S39-88 at different
concentrations (40, 50, 66, 100, 200 µM) were determined by
RP-HPLC quantitation of the appearance of the fluorescent
cleavage product [Pya88]S77-88 in the absence or presence of
different concentrations (1, 10, 33, 100 µM) of inhibitor. Each
point of the Lineweaver-Burk plot represents the mean of
three independent experiments performed in triplicate.
potencies of these compounds, they were modified to
incorporate moieties able to interact with the S′ subsites
of TeNt (Figure 2). This approach has already been used
successfully in the rational design of inhibitors of other
Zn peptidases, in particular aminopeptidases.23
Furthermore, the effect of the C-terminal substitution
was explored on these compounds by adding a C-
terminal benzamide. A slight improvement in Ki values
was observed for compounds 20a and 20b (Ki ) 3.9 ( 1
and 6.8 ( 1 µM, respectively) (Figure 5A).
The improvement of the inhibitory potencies of the
extended compounds 15-18 (Ki ) 80-100 µM), as
compared to their parent compound 1 (Ki ) 250 µM)
(Figure 2), confirmed the validity of this strategy. To
optimize the interaction of these molecules with the S1′
and S2′ subsites of TeNt, the use of combinatorial
chemistry techniques was chosen. Indeed combinatorial
chemistry40-42 has recently emerged as a powerful
method, complementary to structure-based ligand de-
sign,43,44 for the discovery and optimization of ligands
for a variety of enzymes and receptors. Such methods
reduce the need for repetitive syntheses of related
compounds as well as laborious and expensive screen-
ing. Moreover they have been already successfully used
in the design of inhibitors of zinc metallopeptidases such
as angiotensin-converting enzyme,45 matrix metallopro-
teinases,46 and zinc endopeptidases 24.1535 and 24.16.36
Thus a library of 19 mixtures of 38 pseudotripeptides,
derived from the sulfonamide analogue of â-glutamine-
thiol and containing natural amino acids, was synthe-
sized and screened against TeNt (Figure 4). The best
compound issuing from this library possessed a tyrosyl
and a histidyl residue putatively interacting with the
S1′ and S2′ subsites of TeNt, respectively. Nevertheless,
very few differences, in terms of percentage of inhibition,
were observed (Figure 4), indicating an apparent low
specificity of the TeNt subsites. This contrasts with the
importance of subsite specificity observed for other zinc
endopeptidases using the same combinatorial ap-
proach.35,36 The best compound issuing from the librar-
ies (Tyr-His) was purified, and the two diastereomers
were separated (19a and 19b). The best Ki value (5 ( 1
µM) was obtained with the compound whose absolute
configuration is 2R,3S on the sulfonamide synthon
(Figure 5A). A slight improvement was observed by
adding a C-terminal benzylamide to these molecules
especially on the 2S,3S diastereomer resulting in Ki
Finally, this tyrosyl-histidyl-benzylamide moiety was
used to obtain the corresponding compounds derived
from the m-sulfonamido-â-phenylalaninethiol 3 (Figure
5B). These molecules (21a , 21b, 21c, and 21d ) cor-
respond to the four possible diastereomers. No signifi-
cant improvement were obtained for compounds 21a
and 21b possessing S absolute configuration at the level
of the C3 carbon as compared to compounds 20a and
20b with Ki values equal to 3.0 ( 0.9 and 9.6 ( 0.9 µM,
respectively (Figure 5). Interestingly, the compounds
21c and 21d , corresponding to the R absolute configu-
ration at the level of the C3 carbon, did not demonstrate
significant differences in terms of inhibitory potency
(Ki21c ) 6.0 ( 0.9 µM and Ki21d ) 4.0 ( 0.9 µM) as
compared to those with the S configuration.
Finally, the evaluation of the kinetics of action of one
of these inhibitors was undertaken. The cleavage rate
of the fluorescent substrate [Pya88]S39-88 by TeNt39 was
determined with concentrations of substrate ranging
from 40 to 200 µM and with concentrations of compound
21c ranging from 1 to 100 µM. The Lineweaver-Burk
plot in Figure 6 represents the mean of three independ-
ent experiments performed in triplicate. This represen-
tation is particularly useful to determine a decrease of
Km and an unchanged Vmax with increasing concentra-
tions of inhibitors, which is the typical feature of a
competitive inhibitor.
Discu ssion
The inhibitory potencies recently obtained with
â-glutaminethiol 1 (Ki ) 250 µM) and m-sulfonamido-
â-phenylalaninethiol 3 (Ki ) 35 µM)22 (Figure 2A) have
shown that compounds capable of interacting with the
S1 subsite and the Zn metal atom are potent neurotoxin
inhibitors. With the aim of improving the inhibitory

520 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 3
Martin et al.
values that decrease from 15 to 6.8 µM. This tyrosyl-
histidyl-benzylamide moiety was introduced in pseudo-
tripeptides derived from the m-sulfonamido-â-phenyl-
alaninethiol 3 whose Ki was 35 µM on TeNt22 (Figure
2). The four corresponding diastereomers, obtained by
introducing the different synthons of absolute configu-
ration S or R on the C2 and C3 carbons, were synthe-
sized and unambiguously assigned. Still no tremendous
differences of activity were obtained with these com-
pounds, their Ki values ranging from 3 to 9.6 µM.
Ki values against TeNt endopeptidase activity in the
3-4 µM range. Such an inhibitory potency has never
been reached until now for any clostridial neurotoxin.
Ex vivo and in vivo studies of these compounds are now
in progress.
Exp er im en ta l Section
In h ibitor y P oten cy. Enzymatic studies were performed
using synaptobrevin [Pya88]S39-88 as a substrate according
to the protocol described by Solheihac et al.39 with slight
modifications.22
Such a lack of specificity in the stereochemistry of
residues at the level of S1 subsite is unexpected for zinc
peptidases.6,47,48 To check if our molecules were inter-
acting with the zinc atom of TeNt via the thiol group,
we performed the synthesis of an analogue of compound
21 with a hydrogen replacing the free SH. This was
achieved by coupling the corresponding acid synthon of
m-sulfonamido-â-phenylalaninethiol 13b to tyrosyl-his-
tidyl-benzylamide (Scheme 2). This compound was
devoid of any inhibitory activity even at 1 mM (data not
shown). This clearly demonstrated that the thiol group
was crucial for the activity of our compounds, probably
via zinc chelation. Furthermore, the kinetics of action
of compound 21c was evaluated. The Lineweaver-Burk
plot analysis of the experiments indicated the typical
pattern observed for inhibitors competing with the
substrate at the level of the active site.
Ch em istr y. Solvents, reagents, and protected amino acids
for peptide synthesis were obtained from Perkin-Elmer (les
Ulis, France). High-performance liquid chromatography (HPLC)
grade solvents and solvents were from SDS (Peypin, France).
Melting points of the crystallized compounds were measured
on an electrothermal apparatus and are reported uncorrected.
Flash chromatography was carried out with Merck silica gel
60 (230-400 Mesh). TLC was performed on precoated silica
gel plates (60 F254, 0.2 mm thick; Merck). Plates were devel-
oped with UV light, iodine vapor, or ninhydrin. The purity of
the final compounds was also checked by HPLC using a
reverse-phase column (Nucleosil, C18, 150 × 4.6 mm, 5 µm,
100 Å, flow rate 1 mL/min) with eluents A (H2O, TFA 0.1%
(v/v)) and B (CH3CN/H2O (7:3), TFA 0.09% (v/v)), on an Applied
Biosystem 151A HPLC apparatus. The eluted peaks were
monitored at 214 nm.
The structure of all the compounds was confirmed by 1H
NMR spectroscopy (Bru¨ker AC 270 MHz or Bru¨ker AM 400
MHz) in DMSO-d6 or CDCl3 or D2O solutions (5 × 10-3 M)
using HMDS as internal reference. The signals are described
as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet),
and br (broad). Satisfactory elemental analyses, performed at
the university of Paris VI, were obtained (C, H, N) for all
compounds. Optical rotations were mesured on a Perkin-Elmer
241 polarimeter (1.0 dm cell) for MeOH solutions at 20 °C.
{R}D values are given in units of 10-1 deg cm2 g-1. Mass
spectral analyses were performed by Quad Service (Poissy,
France) using the electrospray ionization (ESI) technique.
Abbr evia tion s: BOP, (benzotriazolyl-1-yl)oxytris(dimeth-
ylamino)phosphonium hexafluorophosphate; HATU, O-(7-aza-
benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophos-
phate; Pya, pyrenylalanine; BBTO, bis(tributyltin) oxide; TeNt,
tetanus neurotoxin; BoNt/A, botulinum neurotoxin type A.
Solid -P h a se Syn th esis of th e Libr a r y. Solid-phase syn-
theses were performed with an automated 357 Advanced
Chemtech multiple peptide synthesizer using a previously
described procedure.35,36 The synthon B was incorporated as
a building block during the solid-phase peptide synthesis.
Fully protected peptides were cleaved from 2-chlorotrityl
resin with 5 mL of a mixture of TFE and CH2Cl2 (2/8) during
4 h.38 Solutions of protected peptides were concentrated in
vacuo. Protective groups were removed by 10 mL of TFA
containing 0.5 mL of H2O and 0.25 mL of triisopropylsilane.32
Solutions of deprotected peptides were concentrated in vacuo
and freeze-dried, and a final cleavage of the thiol protecting
group (4-methoxybenzyl) and the benzyl carbamate was
achieved by using 5 mL of anhydrous HF in the presence of
m-cresol (100 µL) during 1 h at 0 °C.33 After removal of HF in
vacuo, the tripeptides were solubilized with 1.5 mL of TFA,
precipitated with ether/hexane (1/1) (25 mL), and freeze-dried
after addition of water. The correct stoichiometric content of
peptide mixture in each expected amino acid was verified by
quantitative amino acid analyses.
The difficulty in improving the affinity of inhibitors
for TeNt could be related to the mechanism of TeNt
enzymatic activity. Indeed, it has recently been dem-
onstrated that the shortest fragment of synaptobrevin
behaving as a substrate contains 50 residues (sequence
39-88).18,39,49 Thus, short synaptobrevin sequences sur-
rounding the cleavage site (G73ASQFETSA81) are nei-
ther substrates nor inhibitors of TeNt proteolytic activ-
ity.11,19 This appears to be due to the lack of N- and
C-terminal acidic and basic sequences of synaptobrevin
distal from the cleavage site.15,17 The binding of these
sequences to TeNt activates the proteolytic activity of
TeNt probably by a transconformation,16 as occurs in
allosteric-type enzymatic mechanisms. Our inhibitors,
designed to interact with the active site via zinc chela-
tion and catalytic subsite recognition, are competing
with the synaptobrevin substrate once it is already
bound to TeNt via its N- and C-terminal domains. This
is an unfavorable situation which could explain the
micromolar inhibitory potencies observed. Nevertheless,
the inhibitory potencies of our inhibitors were not
modified by addition of peptidic fragments correspond-
ing to these allosteric effectors16 in the incubation
medium (data not shown). This is not surprising because
it has been recently shown that a small synaptobrevin
sequence encompassing the cleavage site (73GASQFET-
SA81) is not cleaved by TeNt even in the presence of 1
mM S1 (27-55) and S2 (82-93) (unpublished results),
while the sequence 56-81 is cleaved.16 Thus, even
residues localized in the 56-72 sequence of synapto-
brevin are involved in the enzyme-substrate recogni-
tion, and S1 and S2 sequences of synaptobrevin are
required but not sufficient for the hydrolysis to occur.
Con str u ction of th e P ep tid e Libr a r y by Com bin a to-
r ia l Ch em istr y. The protocol employed to synthesize the
various mixtures of thiol-containing peptides of formula (syn-
thon B)-AA′1x-AA′2x, where AA′1x and AA′2x represent 19
different natural amino acids (Cys was omitted) was previously
described.35,36
Syn t h esis of (2SR,3S)-3-(Boc-a m in o)-2-[(4-m et h oxy-
ben zyl)su lfa n yl]-6-oxo(tr ityla m in o)h exa n oic Acid (Syn -
th on A). Meth yl (3S)-3-(Boc-a m in o)-6-oxo(tr ityla m in o)-
In conclusion, this study raises important points in
the understanding of tetanus neurotoxin proteolytic
activity. Moreover, the combinatorial approach reported
here led to the design of new pseudotripeptides, with

Metallopeptidase Inhibitors of Tetanus Toxin
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 3 521
h exa n oa te (5a ). Compound 5a was prepared by using a
previously described procedure.23 To a solution of compound
Boc-Gln(Trt)-OH (4a ) (4.48 g, 10 mmol) and N-methylmor-
pholine (1.2 mL, 11 mmol) in THF (50 mL) at -15 °C under
nitrogen was added, over a 5-min period, isobutyl chlorofor-
mate (1.44 mL, 11 mmol). After the mixture stirred for 20 min
at -20 °C, the N-methylmorpholine hydrochloride was re-
moved by filtration and the filter cake was washed with a
small portion of cold THF. The filtrate was treated with a cold
(-78 °C) ethereal solution of diazomethane (prepared from 4.3
g (20 mmol) of diazald and distilled). The reaction mixture was
allowed to reach room temperature and stirred for 1 h. After
THF was removed in vacuo, the crude product was solubilized
in ethyl acetate, washed with H2O, NaHCO3 (10%), and brine,
and dried with Na2SO4. The solvent was then evaporated to
afford the corresponding diazo ketone (4.45 g, 8.7 mmol). To a
solution of this diazo ketone in anhydrous MeOH (20 mL) was
added silver benzoate (119 mg, 0.52 mmol) in triethylamine
(12 mL). The solution darkened, and rapid gas evolution was
noted. After the mixture was stirred for 90 min, Celite and
decolorizing carbon was added. After stirring, the mixture was
filtered through Celite and the filtrate concentrated. The
residue was dissolved in EtOAc and washed with water, 1 N
NaHCO3, 1 N KHSO4, and brine. After drying (Na2SO4), the
solvent was removed at reduced pressure and the crude
product chromatographed (cyclohexane/CH2Cl2/AcOEt, 5/3/2)
to give 4.35 g of compound 5a as a colorless solid (84% yield):
Rf (AcOEt:CH2Cl2, 1:3) ) 0.58; 1H NMR (DMSO-d6) δ 1.32 (9H,
s, tBu), 1.25-1.37 (1H, m, CH-CH2-CH2), 1.40-1.53 (1H, m,
CH-CH2-CH2), 2.20 (2H, t, CH-CH2-CH2), 2.30 (2H, d,
CH2COOMe), 3.50 (3H, s, COOMe), 3.63-3.77 (1H, m, CH-
CH2-CH2), 6.67 (1H, d, NH-Boc), 7.08-7.22 (15H, m, Trt),
8.50 (1H, s, NH-Trt). Anal. (C31H36N2O5) C, H, N.
Met h yl (2S,3S)-3-(Boc-a m in o)-2-[(4-m et h oxyb en zyl)-
su lfa n yl]-6-oxo(tr ityla m in o)h exa n oa te (6a ). This com-
pound was prepared according to the procedure described in
Bischoff et al.24 To a solution of compound 5a (1.032 g, 2 mmol)
in tetrahydrofuran (20 mL) at -78 °C under nitrogen was
added dropwise a solution of lithium diisopropylamide (1.6 M)
(4.8 mL, 7.6 mmol) in THF/heptane/ethylbenzene. After 30 min
of reaction time, a solution of 4-methoxy-2,4-dinitrophenyl
disulfide (700 mg, 2.2 mmol) in 8 mL of THF was added, and
the solution stirred for 2 h. The reaction was stopped by adding
KHSO4 (1 N) and extracted with ethyl acetate. The organic
layer was washed by KHSO4 (1 N) and brine, dried (Na2SO4),
and then evaporated at reduced pressure. The crude product
was chromatographed (AcOEt/CH2Cl2/cyclohexane, 2/3/5) to
obtain 574 mg of compound 6a (43% yield): Rf (AcOEt:CH2Cl2:
cyclohexane, 20:30:50) ) 0.27; 1H NMR (DMSO-d6) δ 1.32 (9H,
s, tBu), 1.28-1.37 (1H, m, CH-CH2-CH2), 1.86-1.95 (1H, m,
CH-CH2-CH2), 2.10-2.24 (2H, m, CH-CH2-CH2), 3.22 (1H,
m, CH-S), 3.55 (3H, s, COOMe), 3.67 (5H, 2s, S-CH2-C6H4-
OMe), 3.70-3.78 (1H, m, CH-CH2-CH2), 6.72 (1H, d, NH-Boc),
6.79 (2H, d, H3,H5 PhOMe), 7.12 (2H, d, H2,H6 PhOMe), 7.10-
7.23 (15H, m, Trt), 8.50 (1H, s, NH-Trt); MS (EI) 153, 243,
559, 669. Anal. (C39H44N2O6S) C, H, N.
15 was obtained by purification on a semipreparative HPLC
with a C18 Vydac column (5 µM, 100 Å, 10 × 250 mm) (38%
yield): retention time ) 12.1 min (Nucleosil, C8, 150 × 4.6
mm, 5 µm, 100 Å, gradient 0-40% B in 15 min, flow rate ) 1
1
mL/min, λ ) 214 nm); purity > 99%; H NMR (DMSO-d6) δ
1.37 (1H, m, â (Gln)), 1.50 (1H, m, â (Gln)), 2.08 (2H, t, γ (Gln)),
2.82 (1H, dd, â (Phe)), 3.06 (1H, dd, â (Phe)), 3.15 (1H, m,
CHNH3+), 3.51 (1H, d, CHSH), 4.40 (1H, m, R(Phe)), 7.00 (1H,
s, CONH2), 7.13-7.22 (5H, m, Ph), 7.40 (1H, s, CONH2), 8.77
(1H, d, NH(Phe)); MS (ESI) (M + H)+ m/z ) 340.1. Anal.
(C15H21N3O4S).
N-[(2SR,3S)-3-Am in o-5-car bam oyl-2-su lfan ylpen tan oyl]-
L-p h en yla la n yl-L-glu ta m ic Acid (16). Synthon A (150 mg)
was coupled with H-Phe-Glu(tBu)-2-chlorotrityl resin using
BOP as previously described.50 After deprotection by trifluoro-
acetic acid32 and anhydrous hydrogen fluoride,33 the crude
product 16 was obtained and purified by semipreparative
HPLC on a Vydac C18 column (5 µM, 100 Å, 10 × 250 mm)
(29% yield): retention time ) 10.8 min (Nucleosil, C8, 150 ×
4.6 mm, 5 µm, 100 Å, gradient 0-40% B in 15 min, flow rate
) 1 mL/min, λ ) 214 nm); purity > 99%; 1H NMR (DMSO-d6)
δ 1.29 (1H, m, â (Gln)), 1.43 (1H, m, â (Gln)), 1.78 (1H, m, â
(Glu)), 1.84 (1H, m, â (Glu)), 2.04 (2H, t, γ (Gln)), 2.26 (2H, t,
γ (Glu)), 2.69 (1H, dd, â (Phe)), 3.03 (1H, dd, â (Phe)), 3.09
(1H, m, CHNH3+), 3.55 (1H, d, CHSH), 4.20 (1H, m, R(Glu)),
4.60 (1H, m, R(Phe)), 7.00 (1H, s, CONH2), 7.12-7.27 (5H, m,
Ph), 7.40 (1H, s, CONH2), 7.93 (3H, br s, NH3+), 8.40 (1H, d,
NH(Glu)), 8.67 (1H, d, NH(Phe)); MS (ESI) (M + H)+ m/z )
469.2. Anal. (C20H28N4O7S).
N-[(2SR,3S)-3-Am in o-5-car bam oyl-2-su lfan ylpen tan oyl]-
L-p h en yla la n yl-L-glu ta m yl-L-th r eon in e (17). Synthon A
(150 mg) was coupled with H-Phe-Glu(tBu)-Thr(tBu)-2-chlo-
rotrityl resin to give, by the procedure described for compound
16, crude product 17 which was purified by semipreparative
HPLC on C18 Vydac column (5 µM, 100 Å, 10 × 250 mm) (27%
yield): retention time ) 11.6 min (Nucleosil, C8, 150 × 4.6
mm, 5 µm, 100 Å, gradient 0-40% B in 15 min, flow rate ) 1
1
mL/min, λ ) 214 nm); purity > 99%; H NMR (DMSO-d6) δ
1.02 (3H, d, γ (Thr)), 1.28 (1H, m, â (Gln)), 1.43 (1H, m, â
(Gln)), 1.76 (1H, m, â (Glu)), 1.90 (1H, m, â (Glu)), 2.07 (2H, t,
γ (Gln)), 2.28 (2H, t, γ (Glu)), 2.68 (1H, dd, â (Phe)), 3.03 (1H,
dd, â (Phe)), 3.08 (1H, m, CHNH3+), 3.54 (1H, d, CHSH), 4.11
(1H, m, â (Thr)), 4.16 (1H, m, R (Thr)), 4.40 (1H, m, R(Glu)),
4.60 (1H, m, R(Phe)), 4.93 (1H, br s, OH), 7.00 (1H, s, CONH2),
7.10-7.20 (5H, m, Ph), 7.42 (1H, s, CONH2), 7.83 (1H, d,
NH(Thr)), 7.92 (3H, br s, NH3+), 8.30 (1H, d, NH(Glu)), 8.67
(1H, d, NH(Phe)), 12.35 (2H, br s, COOH); MS (ESI) (M + H)+
m/z ) 570.3. Anal. (C24H35N5O9S).
N-[(2SR,3S)-3-Am in o-5-car bam oyl-2-su lfan ylpen tan oyl]-
L-p h en yla la n yl-L-glu ta m yl-L-th r eon yl-L-ser in e (18). Syn-
thon A (150 mg) was coupled with H-Phe-Glu(tBu)-Thr(tBu)-
Ser(tBu)-2-chlorotrityl resin as described for compound 16.
Crude product 18 was purified by semipreparative HPLC on
C18 Vydac column (5 µM, 100 Å, 10 × 250 mm) (32% yield):
retention time ) 13.9 min (Nucleosil, C8, 150 × 4.6 mm, 5
µm, 100 Å, gradient 0-40% B in 15 min, flow rate ) 1 mL/
1
(2SR,3S)-3-(Boc-a m in o)-2-[(4-m eth oxyben zyl)su lfa n yl]-
6-oxo(tr ityla m in o)h exa n oic Acid (Syn th on A). Compound
6a (334 mg, 0.5 mmol) was treated by 1 M NaOH in MeOH/
H2O (1/1). After classical workup, the solvent was removed in
vacuo to afford 317 mg of synthon A (95% yield): mp ) 118
°C; Rf (MeOH:CH2Cl2, 10:90) ) 0.4; 1H NMR (DMSO-d6) δ 1.30
(1.39) (9H, s, tBu), 1.55-1.66 (1H, m, âCH2(Gln)), 1.80-1.93
(1H, m, âCH2(Gln)), 2.13-2.26 (2H, m, γCH2(Gln)), 3.12 (3.18)
(1H, d, CH-S), 3.68 (5H, s, CH2-C6H5-OCH3), 3.70-3.80 (1H,
m, RCH(Gln)), 3.62(3.70) (1H, d, BocNH), 6.80 (2H, d, H3,H5
PhOMe), 7.12 (2H, d, H2,H6 PhOMe), 7.10-7.25 (15 H, m, Trt),
8.48 (8.52) (1H, s, NHTrt); {R}D ) -9.4 (MeOH). Anal.
(C38H42N2O6S) C, H, N.
min, λ ) 214 nm); purity > 99%; H NMR (DMSO-d6) δ 1.04
(3H, d, γ (Thr)), 1.25 (1H, m, â (Gln)), 1.42 (1H, m, â (Gln)),
1.73 (1H, m, â (Glu)), 1.89 (1H, m, â (Glu)), 2.03 (2H, t, γ (Gln)),
2.24 (2H, t, γ (Glu)), 2.65 (1H, dd, â (Phe)), 3.03 (1H, dd, â
(Phe)), 3.04 (1H, m, CHNH3+), 3.52 (1H, d, CHSH), 3.57 (1H,
dd, â (Ser)), 3.68 (1H, dd, â (Ser)), 3.94 (1H, m, â (Thr)), 4.23
(1H, m, R (Ser)), 4.27 (1H, m, R (Thr)), 4.33 (1H, m, R(Glu)),
4.60 (1H, m, R(Phe)), 4.96 (2H, br s, OH(Ser) and OH(Thr)),
7.03 (1H, s, CONH2), 7.10-7.24 (5H, m, Ph), 7.42 (1H, s,
CONH2), 7.78 (1H, d, NH(Thr)), 7.92 (2H, br s, NH2), 7.93 (1H,
d, NH(Ser)), 8.36 (1H, d, NH(Glu)), 8.69 (1H, d, NH(Phe)),
12.35 (2H, br s, COOH); MS (ESI) (M + H)+ m/z ) 657.4. Anal.
(C27H40N6O11S).
N-[(2SR,3S)-3-Am in o-5-car bam oyl-2-su lfan ylpen tan oyl]-
L-p h en yla la n in e (15). Synthon A (100 mg) was coupled with
H-Phe-OtBu using BOP as coupling agent as previously
described.50 After classical workup and deprotection by TFA32
and anhydrous hydrogen fluoride,33 54 mg of crude product
Syn th esis of (2SR,3S)-4-(ter t-Bu tylsu lfa m oyl)-2-(Cbz-
a m in o)bu ta n oic Acid (Syn th on B). Meth yl (3S)-5-(ter t-
Bu t ylsu lfa m oyl)-3-(Cb z-a m in o)p en t a n oa t e (5b ). Com-
pound 5b was prepared from 4b (20 g, 53.7 mmol) (prepared
as previously described23) by the procedure described for 5a .

522 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 3
Martin et al.
Compound 5b (20.1 g) was obtained as a colorless solid after
flash chromatography (cyclohexane/CH2Cl2/AcOEt, 0.5/3/2)
(95% yield): mp ) 88 °C; Rf ) 0.48 (AcOEt/CH2Cl2/cyclohex-
ane, 1/1/1); 1H NMR (CDCl3) δ 1.29 (9H, s, tBu), 1.99 (2H, m,
CH2CH2SO2NH), 2.54 (2H, d, CH2COOMe), 3.05 (2H, t, CH2-
SO2NH), 3.61 (3H, s, COOMe), 4.02 (1H, m, CHCH2COOMe),
5.03 (2H, s, CH2Ph), 4.14 (1H, s, NHtBu), 5.37 (1H, d,
BzlOCONH), 7.28 (5H, m, Ph). Anal. (C18H28N2O6S) C, H, N.
Meth yl (2S,3S)-5-(ter t-Bu tylsu lfa m oyl)-3-(Cbz-a m in o)-
2-[(4-m eth oxyben zyl)su lfa n yl]p en ta n oa te (6b). Compound
6b was prepared from 5b (1.5 g, 3.75 mmol) by using the
procedure as described for 6a . The crude residue obtained was
chromatographed (AcOEt/CH2Cl2/cyclohexane, 2/3/5) to yield
1.1 g of compound 6b (54% yield) (>95% ee according to HPLC
monitoring): Rf (EtOAc/CH2Cl2/cyclohexane, 1/1/1) ) 0.57; Rf
(EtOAc/CH2Cl2/cyclohexane, 15/25/60) ) 0.15; 1H NMR (CDCl3)
δ 1.25 (9H, s, tBu), 1.86 to 2.30 (2H, m, CH-CH2-CH2), 2.96
(2H, t, CH-CH2-CH2), 3.29 (1H, 2d, CH-S), 3.64 (3H, s,
COOMe), 3.72 (5H, s, S-CH2-C6H4-OMe), 4.02 (1H, m, CH-
CH2-CH2), 4.87 (1H, s, SO2-NH), 4.95 (1H, d(AB), CH2C6H5),
5.05 (1H, d(AB), CH2C6H5), 5.50 (1H, d, NH-Z), 6.75 (2H, d,
H3,H5 PhOMe), 7.14 (2H, d, H2,H6 PhOMe), 7.37 (5H, m, Ph);
MS (ES) (M + H)+ m/z ) 539.3, (M + Na)+ 561.3. Anal.
(C26H36N2O7S2) C, H, N.
retention time ) 9.5 min (20a , 2R,3S) and 9.9 min (20b,
2S,3S) (Nucleosil, C18, 150 × 4.6 mm, 5 µm, 100 Å, gradient
20-60% B in 15 min, flow rate ) 1 mL/min, λ ) 214 nm);
purity > 99%; 1H NMR (D2O) δ 1.93-2.13 (2H, m, CH2-CH2-
SO2NH), 2.87 and 3.08 (2H, dd, â(Tyr)), 2.98 (1H, dd, â(His)),
3.17 and 3.18 (1H, dd, â(His)), 3.29 and 3.31 (2H, t, CH2-CH2-
SO2NH), 3.66 and 3.68 (1H, d, CH-SH), 3.70 and 3.72 (1H, m,
CH-CH2-CH2), 4.58 (1H, t, a(Tyr)), 4.63 (1H, t, a(His)), 6.78
and 6.82 (2H, d, H3-H5(Tyr)), 7.09 and 7.13 (2H, d, H2-
H6(Tyr)), 7.14 and 7.23 (1H, s, H4(His)), 8.48 and 8.51 (1H, s,
H2(His)); MS (ESI) (M + H)+ m/z ) 618.1. Anal. (C27H35N7O6S2).
Syn th esis of (2SR,3SR)-3-(Boc-a m in o)-3-[3-(ter t-bu tyl-
su lfam oyl)ph en yl]-2-[(4-m eth oxyben zyl)su lfan yl]pr opan o-
ic Acid (Syn th on C). 3-(ter t-Bu tylsu lfa m oyl)ben zoic Acid
(8). To a solution of 75 g (340 mmol) of 3-(chlorosulfonyl)-
benzoic acid (7) (from Aldrich) in 1.5 L of CH2Cl2 was added
at 0 °C 125 mL (1.19 mol, 0.8 M) of tBuNH2. After 2 h stirring
at room temperature, the final acid was extracted with NaOH
(1 M) (three times) and the aqueous layer acidified with HCl
(6 N). A white solid precipitated and was filtered. The filter
cake was dried over P2O5 to obtain 68.36 g of 8 which was
used without further purification (78% yield): Rf (AcOH/
1
toluene, 3/7) ) 0.45; H NMR (DMSO-d6) δ 1.05 (9H, s, tBu),
7.65 (1H, s, SO2NH), 7.65 (1H, t, Ar), 8.00 (1H, d, Ar), 8.08
(1H, d, Ar), 8.33 (1H, s, Ar). Anal. (C11H15NO4S) C, H, N.
(2SR,3S)-5-(ter t-Bu t ylsu lfa m oyl)-3-(Cb z-a m in o)-2-[(4-
m eth oxyben zyl)su lfa n yl]p en ta n oic Acid (Syn th on B).
The compound 6b (0.55 g, 1 mmol) was hydrolyzed by 3 M
NaOH in MeOH overnight at room temperature to afford 0.48
g of synthon B (91% yield): mp ) 70 °C; Rf (EtOAc/AcOH/
CH2Cl2, 30/2/70) ) 0.24 and 0.32; retention time ) 10.4 and
11.8 min (Chirose bond, C1, 250 × 4.6 mm, 5 µm, isocratic
conditions: 30% of B′′ with A′′:phosphate buffer (0.05 M, pH
N-ter t-Bu tyl-3-(h ydr oxym eth yl)ben zen esu lfon am ide (9).
A 12.94 g (50.3 mmol) portion of 3-(tert-butylsulfamoyl)benzoic
25
acid was treated with 3 molar equiv of LiAlH4 in THF to
afford 10.9 g of 9 (90% yield): Rf (AcOOH/toluene, 3/7) ) 0.24;
1H NMR (DMSO-d6) δ 1.04 (9H, s, tBu), 4.52 (2H, d, CH2OH),
5.36(1H, t, CH2OH), 7.45 (1H, s, SO2NH), 7.46 (1H, d, Ar),
7.46 (1H, d, Ar), 7.64 (1H, dd, Ar), 7.77 (1H, s, Ar). Anal.
(C11H17NO3S) C, H, N.
1
) 6); B′′:CH3CN, flow rate ) 1 mL/min); H NMR (CDCl3) δ
1.21 (9H, s, tBu), 1.79 to 1.93 (1H, m, CH-CH2-CH2), 1.96 to
2.10 (1H, m, CH-CH2-CH2), 2.97 (2H, t, CH-CH2-CH2), 3.70
(3H, s, OMe), 3.76 (2H, s, CH2-CH4-OMe), 4.04 (1H, m, CH-
CH2-CH2), 5.01 (2H, s, CH2-C6H5), 5.60 (1H, d, NH-Z), 6.72
and 6.77 (2H, d, H3,H5 PhOMe), 7.12 and 7.17 (2H, d, H2,H6
PhOMe), 7.25 (5H, br s, Ph); MS (ESI) (M + H)+ m/z ) 539.3,
561.3 (Na+) Anal. (C25H34N2O7S2) C, H, N..
N-ter t-Bu t yl-3-(oxom et h yl)b en zen esu lfon a m id e (10).
Compound 10 was synthesized by applying the Swern proce-
dure26 with slight modification. Briefly, to a solution of 4.7 mL
(53.7 mmol) of oxalyl chloride (freshly distilled) in 100 mL of
CH2Cl2 was added dropwise, at -60 °C, under N2, a solution
of 9.5 mL (134 mmol) of DMSO in 20 mL of CH2Cl2. The
reaction was stirred for 5 min, and the alcohol 9 in 4 mL of
DMSO and 30 mL of CH2Cl2 was added within 5 min. Stirring
was continued for an additional 30 min at the same temper-
ature; 31.2 mL (224 mmol) of triethylamine was then added,
and the reaction mixture was stirred for 5 min at -65 °C and
then allowed to warm to room temperature. After 1 h of
stirring, water was added and the aqueous layer was reex-
tracted with additional CH2Cl2. The organic layers were
combined, washed with KHSO4 (1 N), NaHCO3 (10%), and
brine, and dried over Na2SO4 to give after concentration in
vacuo 10.45 g of compound 10 (96% yield): Rf (AcOEt/
cyclohexane, 1/2) ) 0.32; 1H NMR (DMSO) δ 1.05 (9H, s, tBu),
7.70 (1H, s, SO2NH), 7.78 (1H, t, Ar), 8.10 (2H, d, Ar), 8.29
(1H, s, Ar), 10.05 (1H, s, CHO). Anal. (C11H15NO3S) C, H, N.
Eth yl 3-[3-(ter t-Bu tylsu lfam oyl)ph en yl]pr open oate (11).
A 20.4 mL (102 mmol) portion of triethyl phosphonoacetate
was added dropwise to a suspension of sodium hydride (3 g,
104 mmol) in dry DME (200 mL) using a previously described
protocol.27 After the mixture stirred for 30 min at 45 °C, 16.8
g (69.6 mmol) of aldehyde 10 was added rapidly, and the
mixture was stirred for 1 h at 50 °C. The reaction was poured
into 250 mL of ice-water and extracted with ethyl acetate (2
× 250 mL). The ethyl acetate solution was washed with water
and brine, dried over Na2SO4, filtered, and evaporated in
vacuo; 20.56 g of compound 11 (85% yield) was isolated as a
white solid after crystallization in ether/heptane (1/1): Rf
(AcOEt/cyclohexane, 4/6) ) 0.40; 1H NMR (DMSO) δ 1.05 (9H,
s, tBu), 1.20 (3H, t, CH3-CH2), 4.15 (2H, q, CH3-CH2), 6.65
(1H, J ) 16 Hz, d, φCHdCH), 7.50 (1H, s, SO2NH), 7.58 (1H,
t, Ar), 7.68 (1H, t, CHdCH-COOEt), 7.82 (1H, d, Ar), 7.91 (1H,
d, Ar), 8.10 (1H, s, Ar). Anal. (C15H21NO4S) C, H, N.
N-[(2SR,3S)-3-Am in o-5-su lfam oyl-2-su lfan ylpen tan oyl]-
L-tyr osyl-L-h istid in e (19a a n d 19b). To 135 mg (250 µmol)
of synthon B, 122 mg (320 µmol) of HATU, and 150 µmol of
H-Tyr(tBu)-His(Trt)-2-chlorotrityl resin in 5 mL of NMP (N-
methylpyrrolidone) was added at room temperature 200 µL
of DIEA. After 6 h of stirring and negative Kaiser test, the
peptidyl resin was successively washed with NMP and CH2Cl2.
After deprotection by trifluoroacetic acid32 and anhydrous
hydrogen fluoride,33 the crude products 19a and 19b were
purified and separated by semipreparative HPLC on a C18
Vydac column (5 µM, 100 Å, 10 × 250 mm) (22% and 17%
yields, respectively, λ ) 214 nm): purity > 99%; retention time
) 10.6 min (19a , 2R,3S) and 11.5 min (19b, 2S,3S) (Nucleosil,
C18, 150 × 4.6 mm, 5 µm, 100 Å, gradient 0-40% B in 15
min, flow rate ) 1 mL/min); 1H NMR (D2O) δ 2.12 (2H, m,
CH-CH2-CH2), 2.96 (2H, dd, â(Tyr)), 3.08 (1H, dd, â(His)), 3.18
(1H, dd, â(His)), 3.33 (2H, t, CH-CH2-CH2), 3.70 (1H, d, CH-
SH), 3.72 (1H, m, CH-CH2-CH2), 4.22 (1H, d, CH2-C6H5),
4.37 (1H, d, CH2-C6H5), 4.55 (1H, t, R(Tyr)), 4.60 (1H, t,
R(His)), 6.82 (2H, d, H3-H5(Tyr)), 7.13 (2H, d, H2-H6(Tyr)),
7.14 (1H, s, H4(His)), 7.19 (2H, d, C6H5), 7.37 (3H, m, C6H5),
8.51(1H, s, H2(His)); MS (ESI) (M + H)+ m/z ) 529.1, (M +
2H)+ 265.2. Anal. (C20H28N6O7S2).
N-[(2SR,3S)-3-Am in o-5-su lfam oyl-2-su lfan ylpen tan oyl]-
L-tyr osyl-N-ben zyl-L-h istid in a m id e (20a a n d 20b). To 300
mg (0.57 mmol) of synthon B, 260 mg (0.68 mmol) of HATU,
and 470 mg (0.68 mmol) of H-Tyr(tBu)-His(Trt)-NHBz in
CH2Cl2 (5 mL) was added 350 µL of DIEA. After stirring for 6
h at room temperature and classical workup, 460 mg of crude
protected product treated by trifluoroacetic acid32 and anhy-
drous hydrogen fluoride33 led to the mixture of 19a and 19b.
The two diastereomers were separated by semipreparative
HPLC on a C18 Vydac column (5 µM, 100 Å, 10 × 250 mm) to
yield 84 mg of 20a (18% yield) and 67 mg of 20b (14% yield):
Eth yl (3R)-3-[(S)-N-Ben zyl-1-p h en yleth yla m in o]-3-[3-
(ter t-bu tylsu lfa m oyl)p h en yl]p r op a n oa te (12a ) a n d Eth yl
(3S)-3-[(R)-N-Ben zyl-1-p h en yleth yla m in o]-3-[3-(ter t-bu -
t ylsu lfa m oyl)p h en yl]p r op a n oa t e (12b ). Both diastereo-

Metallopeptidase Inhibitors of Tetanus Toxin
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 3 523
isomers were synthesized by applying the procedure previously
described.28,51 Briefly, to a solution of 4 g (18.9 mmol) of
commercially available (S)-(-)-N-benzyl-R-methylbenzylamine
(99%) (Aldrich) in 70 mL of dry THF was added, at 0 °C, 11.8
mL (18.9 mmol) of nBuLi (1.6 M in hexane) in 30 mL of THF.
The reaction mixture was stirred for 15 min and cooled to -78
°C. A solution of 2.36 g (7.56 mmol) of 11 in 30 mL of THF
was added dropwise to the reaction mixture which was stirred
for 30 min; 30 mL of KHSO4 (1 M) was then added. After
evaporation of THF, the aqueous residue was extracted with
EtOAc. The ethyl acetate layer was extracted with KHSO4 (1
N), NaHCO3 (10%), and brine, dried over Na2SO4, and evapo-
rated in vacuo. The product was then purified by flash
chromatography on a silica gel column using AcOEt/CH2Cl2/
cyclohexane (1.5/1.5/7) as eluent to obtain 3.28 g of compound
12a (87% yield) (>99% ee according to HPLC monitoring). (R)-
(+)-N-Benzyl-R-methylbenzylamine was used to obtain 12b:
rated in vacuo; 3.11 g of 14b (59% yield) was obtained after
flash chromatography: Rf (AcOEt/cyclohexane, 1/4) ) 0.15; 1H
NMR (CDCl3) δ 1.08 (3H, t, CH2CH3), 1.12 (9H, s, tBu-NH-
SO2), 1.35 (9H, s, Boc), 3.56 (1H, m, CH-COOEt), 3.79 (6H
and 2H, s, OMe and ΦCH2S), 4.00 (2H, q, CH3-CH2), 4.51 (1H,
s, SO2NH), 5.06 (1H, m, Boc-NH-CH), 6.13 (1H, m, NH-Boc),
7.30-7.42 (3H, m, F(OMe)), 7.58-7.78 (4H, m, Ar).
(2S R ,3S )-3-(Boc-a m in o)-3-[3-(t er t -b u t ylsu lfa m oyl)-
ph en yl]-2-[(4-m eth oxyben zyl)su lfan yl]pr opan oic Acid an d
(2SR,3R)-3-(Boc-am in o)-3-[3-(ter t-bu tylsu lfam oyl)ph en yl]-
2-[(2,4-d im eth oxyben zyl)su lfa n yl]p r op a n oic Acid . To a
stirred solution of bis(tributyltin) oxide (BBTO) (5.3 mL, 10.33
mmol, 3 molar equiv) in 20 mL of CH3CN was added 2 g of
compound 14a (3.44 mmol) solubilized in 6 mL of CH3CN. The
mixture was refluxed for 48 h, and 50 mL of CH3CN was
added. The organic phase was acidified with HCl (2 N), and
the mixture was stirred for 30 min. After evaporation of
CH3CN and classical workup, the crude residue was purified
on a silica gel column using AcOEt/cyclohexane/AcOH (45/55/
2.5) as eluent; 1.3 g of synthon C (2S,3S) was thus obtained
(70% yield): retention time ) 11.8 min (2S,3S) (Chirose bond,
C1, 250 × 4.6 mm, 5 mm, isocratic at 30% of B′′ with
A′′:phosphate buffer (0.05 M, pH ) 6); B′′:CH3CN, flow rate
)1 mL/min).
1
Rf (AcOEt/cyclohexane, 3/7) ) 0.41; H NMR (CDCl3) δ 1.02
(3H, t, CH2CH3), 1.13 (9H, s, tBu), 1.20 (3H, t, CH-CH3), 2.57
(2H, m, CH2-COOEt), 3.64 (2H, d, CH2φ), 3.84-3.95 (3H, m,
CH3-CH2 and CH3-CH), 4.47 (1H, dd, CH-CH2-COOEt), 4.51
(1H, s, SO2NH), 7.11-7.35 (10H, m, 2φ), 7.39 (1H, dd, Ar), 7.51
(1H, d, Ar), 7.71 (1H, d, Ar), 7.90 (1H, s, Ar). 12a : {R}D
)
+5.6 (MeOH). Anal. (C30H38N2O4S) H, N; C: calcd, 68.94;
found, 68.44.
A 0.33 g (0.57 mmol) portion of 14a was hydrolyzed as
described for synthon A to give 310 mg of a racemic mixture
of synthon C (2SR,3S) (98% yield): Rf (CH2Cl2/MeOH/AcOOH,
96/3/1) ) 0.16; retention time ) 11.8 min (2S,3S) and 10.7
min (2R,3S) (Chirose bond, C1, 250 × 4.6 mm, 5 µm, isocratic
at 30% of B′′ with A′′:phosphate buffer (0.05 M, pH ) 6); B′′:
E t h yl (3R)-3-(Boc-a m in o)-3-[3-(ter t-b u t ylsu lfa m oyl)-
p h en yl]p r op a n oa te (13a ) a n d Eth yl (3S)-3-(Boc-a m in o)-
3-[3-(ter t-bu tylsu lfam oyl)ph en yl]pr opan oate (13b). A 3.28
g (6.28 mmol) portion of 12a (respectively 12b) and 4.8 g of
Boc2O were solubilized into 60 mL of AcOEt containing 4.4 g
of Pd(OH)2/C (20%). The reaction mixture was vigorously
stirred under 20 bar of H2 at 25 °C for 18 h. After filtration of
the catalyst on Celite, the filtrate was evaporated in vacuo
and purified on a silica gel column using AcOEt/cyclohexane
(1/3) as eluent to obtain 2.28 g of compound 13a (respectively
13b) (85% yield): Rf (AcOEt/cyclohexane, 3/7) ) 0.21; 1H NMR
(CDCl3) δ 1.13 (3H, t, CH2CH3), 1.15 (9H, s, tBu-NH-SO2-),
1.35 (9H, s, Boc), 2.76 (2H, m, CH2-COOEt), 3.99 (2H, q, CH3-
CH2), 4.60 (1H, s, SO2NH), 5.08 (1H, m, CH-CH2-COOEt), 5.58
(1H, m, NH-Boc), 7.38 (1H, dd, Ar), 7.40 (1H, d, Ar), 7.72 (1H,
d, Ar), 7.77 (1H, s, Ar). 13a : {R}D ) +24.0 (MeOH). 13b: {R}D
) -23.5 (MeOH). Anal. (C20H32N2O6S) H, N; C: calcd, 56.06;
found, 56.65.
Eth yl (2S,3S)-3-(Boc-a m in o)-3-[3-(ter t-bu tylsu lfa m oyl)-
p h en yl]-2-[(4-m eth oxyben zyl)su lfa n yl]p r op a n oa te (14a )
a n d Eth yl (2R,3R)-3-(Boc-a m in o)-3-[3-(ter t-bu tylsu lf-
am oyl)ph en yl]-2-[(2,4-dim eth oxyben zyl)su lfan yl]pr opan -
oa te (14b). A 0.5 g (1.17 mmol) portion of 13a was treated
according to the procedure described for compound 5a . The
crude residue was purified on a silica gel column, using EtOAc/
cyclohexane (1/3) as eluent, to yield 331 mg of compound 14a
(>85% ee according to HPLC monitoring) (49% yield): Rf
(AcOEt/cyclohexane, 3/7) ) 0.31; 1H NMR (CDCl3) δ 1.08 (3H,
t, CH2CH3), 1.12 (9H, s, tBu-NH-SO2-), 1.36 (9H, s, Boc), 3.40
(1H, m, CH-COOEt), 3.75 (3H and 2H, s, OMe and φ-CH2-S),
3.96 (2H, q, CH3-CH2), 4.51 (1H, s, SO2NH), 5.00 (1H, m,
BocNH-CH), 6.11 (1H, m, NH-Boc), 6.82 (2H, d, φ-OMe), 7.22
(2H, d, φ-OMe), 7.25 (1H, d, Ar), 7.30 (1H, t, Ar), 7.50 (1H, s,
Ar), 7.68 (1H, d, Ar). Anal. (C28H40N2O7S2) C, H, N.
According to a previously described protocol,30 to a solution
of 1,1,1,3,3,3-hexadimethyldisilazane (6.4 mL, 3.5 molar equiv,
30.2 mmol) in THF (70 mL) at 0 °C was added a solution of
n-butyllithium (2.5 M solution in hexane, 11 mL, 3.2 molar
equiv). After stirring at this temperature for 10 min, the
mixture was cooled to -78 °C and a solution of 3.7 g of 13b
(8.64 mmol) in 40 mL of THF was added dropwise. The stirred
mixture was maintained at 30 °C for 2 h and then cooled to
-78 °C and a solution of 2,4-dimethoxybenzylthio 4-meth-
ylphenyl sulfonate (3.7 g, 10.37 mmol) in 30 mL of THF was
added. After stirring at this temperature for 1 h, the reaction
mixture was poured into saturated aqueous ammonium chlo-
ride solution (50 mL), and the mixture obtained was extracted
with diethyl ether (2 × 300 mL). The combined extracts were
washed with water and brine, dried over Na2SO4, and evapo-
1
CH3CN, flow rate ) 1 mL/min); H NMR (CDCl3) δ 1.11 and
1.12 (9H, s, tBu-NH-SO2-), 1.30 and 1.35 (9H, s, Boc), 3.40 and
3.45 (1H, m, CH-COOEt), 3.72 and 3.75 (3H and 2H, s, OMe
and φ-CH2-S), 3.65 (1H, s, SO2NH), 5.05 and 5.32 (1H, m,
BocNH-CH), 5.37 and 6.20 (1H, m, NH-Boc), 6.75 and 6.80
(2H, d, φ-OMe), 7.12 and 7.20 (2H, d, φ-OMe), 7.30 (1H, d, Ar),
7.40 (1H, t, Ar), 7.81 and 7.70 (1H, s, Ar), 7.68 and 7.75 (1H,
d, Ar). 2S,3R: {R}D ) +29.8 (MeOH). 2S,3S: {R}D ) -29.2
(MeOH). Anal. (C26H36N2O7S2) C, H, N.
A 0.35 g (0.57 mmol) portion of 14b was saponified as
described for synthon A to give 310 mg of 2SR,3R (98% yield):
Rf (CH2Cl2/MeOH/AcOOH, 96/3/1) ) 0.16; retention time )
15.7 min (2R,3R) and 17.4 min (2S,3R) (Chirose bond, C1, 250
× 4.6 mm, 5 mm, isocratic at 30% of B with A:phosphate buffer
(0.05 M, pH ) 6); B:CH3CN, flow rate ) 1 mL/min); 1H NMR
(DMSO-d6) δ 0.98 and 1.00 (9H, s, tBu-NH-SO2-), 1.24 and 1.28
(9H, s, Boc), 3.53 and 3.63 (1H, d, CH-COOH), 3.61, 3.66, 3.68,
3.71, and 3.77 (6H and 2H, s, OMe and φ-CH2-S), 4.81 (1H,
m, BocNH-CH), 6.32 and 6.40 (1H, dd, φ-OMe), 6.40 and 6.47
(H, d, φ-OMe), 6.87 and 7.10 (1H, d, φ-OMe), 7.41-7.65 (5H,
m, Ar and NH), 7.75 and 7.80 (1H, s, Ar). Anal. (C27H38N2O8S2)
C, H, N.
N-[(2RS,3S)-3-Am in o-3-(3-su lfa m oylp h en yl)-2-su lfa n yl-
p r op a n oyl]-L-tyr osyl-N-ben zyl-L-h istid in a m id e (21a a n d
21b). To 300 mg (0.57 mmol) of synthon C (2SR,3S), 260 mg
(0.68 mmol) of HATU, 470 mg (0.68 mmol) of H-Tyr(tBu)-
His(Trt)-NHBz, and 350 µL of DIEA was added 5 mL of
CH2Cl2. After stirring for 6 h at room temperature and classical
workup, the residue was treated by trifluoroacetic acid32 to
obtain 480 mg of crude product which was purified by
semipreparative HPLC on C18 Vydac column (5 µM, 100 Å,
10 × 250 mm) in order to separate the two diastereomers:
retention time ) 12.85 and 14.35 min (Nucleosil, C18, 150 ×
4.6 mm, 5µm, 100 Å, gradient 10-90% B in 30 min, flow rate
) 1.5 mL/min).
Both diastereomers were then treated independently by
anhydrous hydrogen fluoride33 with 2% m-cresol to yield 21a
(28% yield) and 21b (20% yield): retention time ) 9.6 min
(21a ) and 10.7 min (21b) (Nucleosil, C18, 150 × 4.6 mm, 5
µm, 100 Å, gradient 10-50% B in 15 min, flow rate ) 1.5 mL/
min, λ ) 214 nm); purity > 99%.
N-[(2R,3S)-3-Am in o-3-(3-su lfa m oylp h en yl)-2-su lfa n yl-
p r op a n oyl]-L-tyr osyl-N-ben zyl-L-h istid in a m id e (21a ): re-
tention time ) 9.6 min (Nucleosil, C18, 150 × 4.6 mm, 5 µm,

524 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 3
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vesicle-associated membrane protein. Proc. Natl. Acad. Sci.
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small synaptic vesicles of rat brain. EMBO J . 1989, 8, 379-
384.
100 Å, gradient 10-50% B in 15 min, flow rate ) 1.5 mL/min,
λ ) 214 nm); purity > 99%; 1H NMR (D2O + DTT) δ 2.44 (1H,
dd, â(Tyr)), 2.63 (1H, dd, â(Tyr)), 3.07 (1H, dd, â(His)), 3.17
(1H, dd, â(His)), 3.96 (1H, d, CH-SH), 4.19 (1H, t, R(Tyr)), 4.20
(1H, d, CH2-C6H5), 4.34 (1H, d, CH2-C6H5), 4.58 (1H, t,
R(His)), 4.60 (1H, d, H2NCH), 6.69 (2H, d, H3-H5(Tyr)), 6.82
(2H, d, H2-H6(Tyr)), 7.14 (1H, s, H4(His)), 7.19 (2H, d, H2-
H6(Ar)), 7.33-7.40 (3H, m, H3-H4-H5(Ar)), 7.61 (2H, d,
PhSO2NH2), 7.84 (1H, s, PhSO2NH2), 7.96 (1H, m, PhSO2NH2),
8.51 (1H, s, H2(His)); MS (ESI) (M + H)+ m/z ) 666.2. Anal.
(C31H35N7O6S2).
N-[(2S,3S)-3-Am in o-3-(3-su lfa m oylp h en yl)-2-su lfa n yl-
p r op a n oyl]-L-tyr osyl-N-ben zyl-L-h istid in a m id e (21b): re-
tention time ) 10.7 min (Nucleosil, C18, 150 × 4.6 mm, 5 µm,
100 Å, gradient 10-50% B in 15 min, flow rate ) 1.5 mL/min,
λ ) 214 nm); purity > 99%; 1H NMR (D2O + DTT) δ 2.88 (2H,
m, â(Tyr)), 3.10 (2H, d, â(His)), 4.15 (1H, d, CH2-C6H5), 4.17
(1H, d, CH-SH), 4.36 (1H, d, CH2-C6H5), 4.50 (1H, t, R(Tyr)),
4.53 (1H, t, R(His)), 5.00 (1H, d, H2NCH), 6.77 (2H, d, H3-
H5(Tyr)), 7.01 (2H, d, H2-H6(Tyr)), 7.14 (1H, s, H4(His)), 7.16
(2H, d, H2-H6(Ar)), 7.33-7.40 (3H, m, H3-H4-H5(Ar)), 7.69
(2H, d, PhSO2NH2), 7.88 (1H, s, PhSO2NH2), 7.99 (1H, m,
PhSO2NH2), 8.46 (1H, s, H2(His)); MS (ESI) (M + H)+ m/z )
666.2. Anal. (C31H35N7O6S2).
Both diastereoisomers were synthesized by the procedure
described for compounds 21a and 21b using synthon
(2SR,3R) (23% yield for 21c and 19% yield for 21d ).
C
N-[(2R,3R)-3-Am in o-3-(3-su lfa m oylp h en yl)-2-su lfa n yl-
p r op a n oyl]-L-tyr osyl-N-ben zyl-L-h istid in a m id e (21c): re-
tention time ) 9.6 min (Nucleosil, C18, 150 × 4.6 mm, 5 µm,
100 Å, gradient 10-50% B in 15 min, flow rate ) 1.5 mL/min,
λ ) 214 nm); purity > 99%; 1H NMR (D2O + DTT) δ 2.78 (1H,
ABX, â(Tyr)), 2.88 (1H, ABX, â(Tyr)), 2.93 (2H, d, â(His)), 4.05
(1H, d, CH-SH), 4.10 (1H, d, CH2-C6H5), 4.30 (1H, d, CH2-
C6H5), 4.30 (1H, t, R(His)), 4.33 (1H, t, R(Tyr)), 4.65 (1H, d,
H2NCH), 6.76 (2H, d, H3-H5(Tyr)), 7.02 (2H, d, H2-H6(Tyr)),
7.07 (1H, s, H4(His)), 7.12 (2H, d, H2-H6(Ar)), 7.35-7.40 (3H,
m, H3-H4-H5(Ar)), 7.70 (2H, d, PhSO2NH2), 7.86 (1H, t,
PhSO2NH2), 7.96 (1H, s, PhSO2NH2), 8.45 (1H, s, H2(His)); MS
(ESI) (M + H)+ m/z ) 665.7. Anal. (C31H35N7O6S2).
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of Synaptobrevin by Tetanus Toxin Light Chain. J . Biol. Chem.
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N-[(2S,3R)-3-Am in o-3-(3-su lfa m oylp h en yl)-2-su lfa n yl-
p r op a n oyl]-L-tyr osyl-N-ben zyl-L-h istid in a m id e (21d ): re-
tention time ) 9.6 min (Nucleosil, C18, 150 × 4.6 mm, 5 µm,
100 Å, gradient 10-50% B in 15 min, flow rate ) 1.5 mL/min,
λ ) 214 nm); purity > 99%; 1H NMR (D2O + DTT) δ 2.75 (2H,
m, â(Tyr)), 3.08 (1H, d, â(His)), 3.17 (1H, d, â(His)), 4.11 (1H,
d, CH-SH), 4.22 (1H, d, CH2-C6H5), 4.35 (1H, d, CH2-C6H5),
4.38 (1H, t, R(Tyr)), 4.57 (1H, t, R(His)), 4.86 (1H, d, H2NCH),
6.74 (2H, d, H3-H5(Tyr)), 6.94 (2H, d, H2-H6(Tyr)), 7.14 (1H,
s, H4(His)), 7.19 (2H, d, H2-H6(Ar)), 7.34-7.40 (3H, m, H3-
H4-H5(Ar)), 7.54 (1H, d, PhSO2NH2), 7.65 (1H, t, PhSO2NH2),
7.87 (1H, s, PhSO2NH2), 7.98 (1H, d, PhSO2NH2), 8.50 (1H, s,
H2(His)); MS (ESI) (M + H)+ m/z ) 665.6. Anal. (C31H35N7O6S2).
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Ack n ow led gm en t. We thank Drs. V. Dive and A.
Menez from De´partement d’inge´nie´rie et d’Etudes des
prote´ines, CEA (Saclay, France), for kindly providing
their multiple peptide synthesizer facilities. We are
indebted to C. Lenoir for synthesizing the fluorescent
substrate required for the enzymatic assay, Emmanuel
Ruffet for the determinations of the Ki values of
compounds 21a -21d , and C. Anne for evaluating the
kinetics of action of these inhibitors. We also thank Dr.
J . R. Cartier from Pasteur-Me´rieux for providing the
pure TeNt dichain.
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