Synthesis and evaluation of new elastase substrates as tripartate prodrugs

Article abstract of DOI:10.1002/1521-4184(200209)335:7<325::AID-ARDP325>3.0.CO;2-W

Compounds consisting of a peptide sequence, 4-aminobutyric acid or 5-aminovaleric acid, respectively, as spacer units, and benzyl alcohol as a model leaving group have been prepared and tested for their ability to serve as substrates for porcine pancreatic elastase and human polymorphnuclear elastase. Those compounds containing an Ala-Ala-Ala sequence served as effective substrates for both enzymes. Hydrolytic cleavage, however, occurred exclusively at the ester bond, not at the peptide-spacer bond. In order to direct the cleavage site from the ester to the amide bond the peptide sequence was varied. We introduced a proline residue in P₃ (in relation to the ester bond) which is known to prevent the cleavage of a substrate by elastase. No hydrolysis, however, either of the ester bond or of the peptide-spacer bond, was found for these compounds.

Full text of DOI:10.1002/1521-4184(200209)335:7<325::AID-ARDP325>3.0.CO;2-W

Arch. Pharm. Pharm. Med. Chem. 2002, 7, 325–330
New Elastase Substrates 325
Karin Achilles
Synthesis and Evaluation of New Elastase
Substrates as Tripartate Prodrugs
Institute of Pharmacy,
Ernst-Moritz-Arndt University
Greifswald,
Pharmaceutical/Medicinal
Chemistry, Greifswald,
Germany
Compounds consisting of a peptide sequence, 4-aminobutyric acid or 5-aminova-
leric acid, respectively, as spacer units, and benzyl alcohol as a model leaving group
have been prepared and tested for their ability to serve as substrates for porcine
pancreatic elastase and human polymorphnuclear elastase. Those compounds
containing an Ala-Ala-Ala sequence served as effective substrates for both en-
zymes. Hydrolytic cleavage, however, occurred exclusively at the ester bond, not at
the peptide-spacer bond. In order to direct the cleavage site from the ester to the
amide bond the peptide sequence was varied.We introduced a proline residue in P3
(in relation to the ester bond) which is known to prevent the cleavage of a substrate
by elastase.No hydrolysis, however, either of the ester bond or of the peptide-spacer
bond, was found for these compounds.
Keywords: Tripartate prodrugs; Intramolecular cyclisation; Substrates; Elastase
Received: January 17, 2002 [FP667]
tate prodrugs, consisting of a protease recognition se-
quence as the carrier unit, a spacer unit, and the at-
Introduction
A severe problem in current cancer chemotherapy is the
lack of selectivity of anticancer drugs toward malignant
cells.To increase the selectivity of these drugs for tumour
cells, the evaluation of prodrugs, non-toxic compounds
activated selectively in the vicinity of the tumour (ideally
due to tumour-selective activation mechanisms), is an
attractive drug-design approach. Recently a number of
tumour-specific properties have been identified [1]. The
occurrence of elevated levels of various enzymes, in par-
ticular proteases, at the tumour-site is the focus of ongo-
ing interest of many researchers.Work has been report-
ed on the development of prodrugs activated by tumour-
associated proteases, among them prodrugs activated
by the serine proteases plasmin [2] and chymotrypsin [3]
and the cysteine protease cathepsin B [4]. The occur-
rence of significantly increased levels of the serine pro-
tease polymorphnuclear elastase (PMN-E) in a number
of malignant diseases such as lung [5], breast [6], and
colon cancer [7], but also in inflammatory diseases such
as rheumatoid arthritis is known [8].However, little atten-
tion until now has been paid to the development of PMN-
E-sensitive prodrugs.
tached drug.The introduction of a spacer prevents steric
hindrance during the release of the drug [9], which could
be a problem if the drug is directly attached to the carrier
unit (as in bipartate prodrugs). A requirement for the
spacer is that it will release the drug spontaneously if the
carrier-spacer bond is cleaved. As spacer units com-
pounds are often used that take advantage of intramo-
lecular cyclisation reactions: While unmasking the
nucleophile by enzymatic cleavage of the carrier-spacer
bond, the cyclisation reaction will be initiated to release
the attached drug (Scheme 1) [10]; several examples for
the successful implementation of this strategy have
been reported [11].An alternative approach is the devel-
opment of drug delivery systems based on self-immola-
tive spacers such as 4-aminobenzyl alcohol and its
derivatives [9].
Prodrugs need to fulfil two main criteria: the cleavage
should be selective and the release of the attached drug
has to be ensured. An attractive approach utilises tripar-
Correspondence: Karin Achilles, Institute of Pharmacy,
Ernst-Moritz-Arndt University Greifswald, Pharmaceutical/
Medicinal Chemistry, Friedrich-Ludwig-Jahn-Str. 17, D-17487
Greifswald, Germany. Phone: +49 (0)3834 864851, Fax:
+49 (0)3834 864874, e-mail: achilles@mail.uni-greifswald.de.
Scheme 1. Activation mechanism of tripartate pro-
dudrugs.
© 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
0365-6233/07/0325 $ 17.50+.50/0

326 Achilles
Arch. Pharm. Pharm. Med. Chem. 2002, 335, 325–330
The suitability of 4-aminobutyric acid (Abu) as spacer
unit has been shown: Unblocked N-terminally, 4-ami-
nobutyric acid (Abu) undergoes intramolecular cyclisa-
tion leading to a γ-lactam by releasing the drug [12]. A
similar intramolecular cyclisation leading to a δ-lactam
can be assumed for 5-aminovaleric acid (Ava). Herein
the synthesis and stability of tripartate compounds con-
sisting of peptides as carrier units, Abu and Ava as spac-
er units, and benzyl alcohol as a model leaving group are
reported.
gated by incubation in the presence of purified porcine
pancreatic elastase (PPE) or human polymorphnuclear
elastase (PMN-E), respectively (TRIS, 0.1 M, pH 8.0).
PPE is commonly used as a model enzyme for PMN-E,
since both enzymes show only slight differences in sub-
strate specificity:Both cleave peptides behind amino ac-
ids with small aliphatic side chains.Whereas PPE has a
substrate specificity for alanine, PMN-E is specific for va-
line in P1 [13, 14]. The enzyme concentrations used in
this assay (0.375 µg/mL PMN-E or 0.75 µg/mL PPE, re-
spectively) have been optimised to allow convenient
analysis under experimental conditions.
Results and discussion
The investigation of the hydrolysis showed that only
compounds 5 and 6 were hydrolysed. Although release
of the benzyl alcohol was observed (Figure 1), there was
no evidence for the cyclisation reaction, because the ex-
pected tripeptide was never detected. On the contrary,
only the formation of BOC-Ala-Ala-Ala-Abu(OH) or
BOC-Ala-Ala-Ala-Ava(OH), respectively, was observed
via HPLC (Table 1). The hydrolysis was very rapid and
occurred specifically in the presence of the active en-
zyme. Without enzyme and with enzyme denatured by
heating no hydrolysis was observed. No degradation
was found for the compounds containing only one
alanine residue or an alanine-alanine dipeptide and the
spacer group (compounds 1–4). A problem in the study
of the hydrolysis of small peptide substrates is, as shown
above, that these substrates can bind misaligned to the
enzyme. Since hydrolysis of the ester bond is much fast-
er than the hydrolysis of the peptide bond with which it
competes, only products resulting from the cleavage of
the ester bond can be observed. While proline and/or
leucine are the preferred residues at the P2 site, a proline
Standard peptide synthesis protocols were applied for
the preparation of the required compounds. The step-
wise synthesis of the tripeptide-spacer units from the C-
to the N-terminus was unsuccessful because the attach-
ment of the last amino acid residue was not possible with
this synthetic strategy. Alternatively, the tripeptides were
synthesised (DCC/NHS), the C-terminal protective
group removed by hydrogenolysis, and the resulting free
acids subsequently coupled with the spacer units by us-
ing DPPA (DMF, 0 °C) as the coupling reagent (Scheme
2). The synthesis was unsuccessful with other coupling
reagents (for example DCC or EDC). The synthesis of
compounds 5–10 was achieved in acceptable yields by
fragment condensation of the appropriate tripeptide with
the spacer units. The compounds were purified by col-
umn chromatography to elemental analysis purity.
The analysis of possible cleavage products was per-
formed by RP-HPLC. The cleavage products and their
retention times are summarised inTable 1.The enzymat-
ic cleavage of the synthesised compounds was investi-
Scheme 2. Synthesis of compounds 1–10.

Arch. Pharm. Pharm. Med. Chem. 2002, 335, 325–330
New Elastase Substrates 327
residue at the P3 site renders the cleavage of a substrate
by elastase impossible [15]. Therefore we assumed a
shift from the ester to the peptide bond by introducing a
proline residue at the P2 site of the peptide sequence
(which is thus the P3 site in relation to the ester bond;
Scheme 3).
residue in P2 could be that, in contrast to the alanine me-
thyl group, the i-propyl group of the valine residue of
compounds 7–10 does not fit into the S2 pocket of the en-
zyme. Since Abu in P1 is known to be accepted from
PPE, another explanation for the misaligned binding
could be that Abu (or Ava, respectively) fits into the S1
pocket but not into the SЈ1 pocket of the enzyme [16].
However, no hydrolysis was found for compounds 7–10,
either at the ester bond or at the amide bond during the
course of the experiment. A possible explanation for the
exclusive hydrolysis of those compounds with an alanine
None of the investigated compounds showed inhibitory
activity toward PPE or PMN-E, as could be shown by the
releasing rate of p-nitroaniline from the commercially
available substrate Ac-Ala-Ala-Ala-pNA.
The kinetics of the enzymatically catalysed cleavage of
the benzyl ester were monitored by recording progress
curves. In contrast to the measurement of initial rates,
where several measurements at different substrate con-
centrations are required to obtain the kinetic parameters
Km and Vmax, a progress curve contains all the informa-
tion necessary for the calculation of Km andVmax, assum-
ing the starting substrate concentration to be high
enough to cover the entire range of substrate concentra-
tions.The Michaelis constant Km could be obtained from
a linearised form of the integrated Michaelis-Menten re-
lationship [17]:
Scheme 3. Possible cleavage sites (⇓).
(A0 – At)/t = Vmax – Km ln (A0/At)/t
The rate of enzymatically catalysed hydrolysis was mon-
itored by HPLC at λ = 215 nm.
A difficulty with these investigations, however, is that the
substrate concentration decreased too fast, which
makes it difficult to fulfil the assumption of steady-state
conditions and leads to a shift of the first order kinetics to
a higher order kinetics during the progress of hydrolysis.
Possible solutions to this problem are either to decrease
the concentration of the enzyme or to increase the con-
centration of the substrate. In this case, the concentra-
Figure 1. HPLC chromatogram (KH2PO4, 20 mM, pH
4.0/acetonitrile = 1/1) of the degradation of 6 catalysed
by PMN-E.At time 0, PMN-E was added;at various times
aliquots were withdrawn and immediately loaded onto
the HPLC column.
Table 1. Retention times of compounds 5 and 6 and possible cleavage products.
Compds
KH2PO4, 20 mM,
pH 4.0/ACN (9/1)
KH2PO4 20 mM,
pH 4.0/ACN (1/1)
Benzyl alcohol
BOC-Ala-Ala-Ala(OH)
BOC-Ala-Ala-Ala-Abu(OH)
BOC-Ala-Ala-Ala-Ava(OH)
5
6
13.20 min
6.08 min
8.64 min
8.36 min
–
4.13 min
–
–
–
6.40 min
5.49 min
a
–
a
– : not detected under these conditions.

328 Achilles
Arch. Pharm. Pharm. Med. Chem. 2002, 335, 325–330
tion of the substrate could not be increased due to its
poor solubility. Due to the instability of the enzyme, the
enzyme levels could also not be lowered.Thus the calcu-
lation of Km was not possible in every case and half-lives
were used for the evaluation (Table 2); only a few time
points are required for this purpose. Determination of
Interestingly, both 5 and 6 showed equal half-lives in the
presence of PMN-E, while compound 6 showed a sub-
stantially shorter half-live than 5 in the presence of PPE
(Figure 2).
Although the tripartate prodrug concept could not be re-
alised in this work, very effective substrates for elastase
have been designed, which could be considered as bi-
partate prodrugs. The specificity of activation by
elastase could be a problem with these compounds,
since the presence of other hydrolases could also lead to
ester hydrolysis. In our current research we are investi-
gating the efficacy and the specificity of compounds 5
and 6 in cell culture assays.With the intention of directing
the enzymatically catalysed hydrolysis to the desired
cleavage site, two approaches can be applied:a bulky al-
cohol could be used for esterification or the more labile
ester bond could be replaced by an amide bond.
V
max was not undertaken here because with the available
data a very long extrapolation to the y-axis would have
been necessary.
Table 2. Km values and half-lives of the investigated com-
pounds.
Compds
Km [mM]
Half-life [min]
PMN-E
PPE
PMN-E
PPE
5
6
n.d.a
2.09
0.22
n.d.
16.5
17.3
36.8
6.9
Acknowledgements
The author would like to thank Dr. J. Maus and Prof. P.J.
Bednarski for valuable comments on the manuscript.
Financial support by the Fonds der Chemischen In-
dustrie is gratefully acknowledged.
a
n.d.: determination of Km not possible.
Experimental
General
Mp:not corrected, Linström apparatus.– IR spectra (cm–1):Per-
kin-Elmer 1600 series FTIR; in KBr. – NMR spectra: Bruker
DPX 200 (200 MHz);δ in ppm;1H values from spectra in CDCl3;
standard reference for all spectra was TMS (δ = 0 ppm). – Ele-
mental analysis:Perkin-Elmer elemental analyser 2400 CHN.–
Chromatography:CC:Merck silica gel 60 (7734);tlc:Merck Alu-
folien, silica gel 60 F254. – HPLC: Merck Hitachi, LaChrom;
Pump: L-7100; Detector: DAD L-7450.
Dry dimethyl formamide was purchased commercially. Unless
noted otherwise, moisture- and air-sensitive reactions were
conducted under a dry nitrogen atmosphere. All amino acids
used in this work are L-amino acids. PMN-E (20927) and PPE
(20929) were purchased from Serva.
Abbreviation: Abu: 4-aminobutyric acid; Ava: 5-aminovaleric
acid; BOC: tert-butyloxycarbonyl; DCC: dicyclohexyl carbodi-
imide; DCM: dichloromethane; DMF: dimethyl formamide;
DPPA: diphenyl phosphorazidate; EDC: N-(3-dimethylamino-
propyl)-NЈ-ethylcarbodiimide hydrochloride; NHS: N-hydroxy-
succinimide ester; PMN-E: polymorphnuclear elastase; PPE:
porcine pancreatic elastase; TEA: triethyl amine; TOS: 4-tolu-
enesulfonic acid.
Stepwise synthesis of peptide-spacer units
1.One equivalent of the N-protected amino acid, the C-terminal
component (1.2 eq), TEA (1.2 eq), and NHS (2.1 eq) were dis-
solved in dry DCM and the solution was cooled to 0 °C. DCC
(1.1 eq) was added and the mixture stirred at r.t. overnight.The
precipitated urea was filtered off and the solvent was removed.
The residue was taken up in ethyl acetate and the solution was
Figure 2.(A) Plot of time versus ln (At/A0) used in the cal-
culation of half-lives. (B) Plot of ln (A0/At)/t versus (A0 –
At)/t used in the calculation of Km for the hydrolysis of 5
catalysed by PPE (r = 0.956).

Arch. Pharm. Pharm. Med. Chem. 2002, 335, 325–330
New Elastase Substrates 329
Benzyl N-BOC-alanylalanylalanyl-4-aminobutyrate (5)
cooled to –20 °C to complete the precipitation of the by-product
dicyclohexylurea. The precipitate was filtered off and the sol-
vent removed in vacuo. The residue was used directly for the
next step.
From 300 mg (0.90 mmol) BOC-alanylalanylalanine, 342 mg
(0.94 mmol) benzyl 4-aminobutyrate*TOS, 0.3 mL (1.89 mmol)
TEA, and 272 mg (0.99 mmol) DPPA.Yield: 60 mg (0.12 mmol,
1
13 %) of a colourless solid. – Mp: 133 °C. – H NMR: δ = 1.36
Benzyl N-BOC-alanyl-4-aminobutyrate (1)
[3d, 9 H, CH3 (Ala)], 1.44 (s, 9 H, t-Bu), 1.89 (m, 2 H, CH2), 2.44
(m, 2 H, CH2), 3.31 (m, 2 H, CH2), 4.03 (dq, J = 7.2 Hz, 1 H,
α-H), 4.30 (dq, J = 7.2 Hz, 1 H, α-H), 4.49 (dq, J = 7.2 Hz, 1 H,
α-H), 5.03 (m, 1 H, NH), 5.10 (s, 2 H, CH2), 6.64 (m, 1 H, NH),
6.87 (m, 1 H, NH), 7.35 (m, 5 H, Ar-H; 1 H, NH). – IR: ν = 3275,
3068, 2978, 2932, 1737, 1668, 1633, 1547, 1454, 1365,
1166 cm–1. – C25H38N4O7 (506.60). C, H, N.
From 2.5 g (13.2 mmol) BOC-alanine, 5.8 g (15.8 mmol) benzyl
4-aminobutyrate*TOS, 2.2 mL (15.8 mmol) TEA, 3.2
g
(27.7 mmol) NHS, and 3.0 g (14.5 mmol) DCC. Yield: 4.5 g
(12.3 mmol, 95 %) of a viscous liquid. – [α]2D0 = –10.1° (2.11 %,
EtOH).– 1H NMR:δ = 1.24 (d, J = 7.0 Hz, 3 H, CH3), 1.34 (s, 9 H,
t-Bu), 1.78 (m, 2 H, CH2), 2.32 (m, 2 H, CH2), 3.19 (m, 2 H, CH2),
4.08 (m, 1 H, α-H), 5.02 (s, 2 H, CH2), 5.36 (m, 1 H, NH), 6.85
(m, 1 H, NH), 7.25 (m, 5 H, Ar-H). – IR: ν = 3324, 2978, 2934,
1732, 1660, 1538, 1454, 1167 cm–1. – C19H28N2O5 (364.44)
Benzyl N-BOC-alanylalanylalanyl-5-aminovaleriate (6)
From 300 mg (0.90 mmol) BOC-alanylalanylalanine, 355 mg
(0.94 mmol) benzyl 5-aminovaleriate*TOS, 0.3 mL (1.89 mmol)
TEA, and 272 mg (0.99 mmol) DPPA.Yield: 68 mg (0.13 mmol,
15 %) of colourless solid. – Mp: 132 °C. – 1H NMR: δ = 1.38 [3d,
9 H, CH3 (Ala)], 1.43 (s, 9 H, t-Bu), 1.61 (m, 4 H, CH2), 2.38 (m,
2 H, CH2), 3.26 (m, 2 H, CH2), 4.30 (m, 1 H, α-H), 4.59 (m, 2 H,
α-H), 5.09 (s, 2 H, CH2), 5.73 (m, 1 H, NH), 7.23 (m, 1 H, NH),
7.59 (m, 1 H, NH), 7.34 (m, 5 H, Ar-H; 1 H, NH). – IR: ν = 3320,
2981, 1743, 1676, 1639, 1517, 1168 cm–1. – C26H40N4O7
(520.63). C, H, N.
Benzyl N-BOC-alanyl-5-aminovaleriate (2)
From 2.5 g (13.2 mmol) BOC-alanine, 6.0 g (15.8 mmol) benzyl
5-aminovaleriate*TOS, 2.2 mL (15.8 mmol) TEA, 3.2
g
(27.7 mmol) NHS and 3.0 g (14.5 mmol) DCC.Yield:4.0 g (10.6
mmol, 81 %) of a viscous liquid.– [α]2D0 = –13.8° (2.17 %, EtOH).
– 1H NMR: δ = 1.24 (d, J = 7.0 Hz, 3 H, CH3), 1.34 (s, 9 H, t-Bu),
1.50 (m, 4 H, CH2), 2.32 (m, 2 H, CH2), 3.19 (m, 2 H, CH2), 4.07
(m, 1 H, α-H), 5.02 (s, 2 H, CH2), 5.34 (m, 1 H, NH), 5.71 (m,
1 H, NH), 7.25 (m, 5 H, Ar-H). – IR:ν = 3398, 2874, 1730, 1672,
1520, 1455, 1368, 1172 cm–1. – C20H30N2O5 (378.47)
Benzyl N-BOC-alanylalanylvalyl-4-aminobutyrate (7)
2. The compounds obtained under 1. were deprotected N-ter-
minally with TFA/DCM (1/1). After stirring for 2 h at r.t. the sol-
vent was removed in vacuo.The coupling of the second amino
acid followed the procedure described under 1.
From 400 mg (1.01 mmol) BOC-alanylalanylvaline, 385 mg
(1.05 mmol) benzyl 4-aminobutyrate*TOS, 0.3 mL (2.12 mmol)
TEA, and 306 mg (1.11 mmol) DPPA.Yield:312 mg (0.58 mmol,
5 8%) of a colourless solid. – Mp: 153 °C. – 1H NMR: δ = 0.91,
0.93 [d, J = 6.7 Hz, 3 H, CH3(Val)], 1.34 [d, J = 7.5 Hz, 6 H,
CH3(Ala)], 1.42 (s, 9 H, t-Bu), 1.80 (m, 2 H, CH2), 2.12 [hept., J =
6.9 Hz, 1 H, β-H(Val)], 2.39 (m, 2 H, CH2), 3.30 (m, 2 H, CH2),
4.39 [m, 2 H, α-H(Ala,Val)], 4.64 [dq, J = 7.2 Hz, 1 H, α-H(Ala)],
5.10 (s, 2 H, CH2), 5.12 (m, 1 H, NH), 5.53 (d, J = 7.0 Hz, 1 H,
N-H), 7.35 (m, 5 H, Ar-H;1 H, N-H), 7.78 (m, 1 H, N-H).– IR:ν =
3284, 3069, 2973, 2933, 1737, 1668, 1634, 1543, 1454, 1365,
1251, 1167 cm–1. – C27H42N4O7 (534.66). C, H, N (*0.5 H2O).
Benzyl N-BOC-alanylalanyl-4-aminobutyrate (3)
From 1.9 g (10.2 mmol) BOC-alanine, 4.5 g (12.3 mmol) 1,
1.7 mL (12.3 mmol) TEA, 2.5 g (21.4 mmol) NHS and 2.3 g
(11.2 mmol) DCC. Yield: 4.0 g (9.2 mmol, 75 %) of a viscous
liquid.– [α]2D0 = –16.3° (2 %, MeOH).– 1H NMR:δ = 1.35, 1.36 [d,
J = 7.0 Hz, 3 H, CH3(Ala)], 1.45 (s, 9 H, t-Bu), 1.86 (m, 2 H, CH2),
2.40 (m, 2 H, CH2), 3.28 (m, 2 H, CH2), 4.11 (dq, J = 7.0 Hz, 1 H,
α-H), 4.42 (dq, J = 7.0 Hz, 1 H, α-H), 5.00 (d, J = 7.0 Hz, 1 H,
N-H), 5.11 (s, 2 H, CH2), 6.67 (m,2 H, N-H), 7.35 (m, 5 H, Ar-H).
– IR: ν = 3324, 2979, 1732, 1650, 1530, 1454, 1370, 1246,
1168 cm–1. – C22H33N3O6 (435.52)
Benzyl N-BOC-alanylalanylvalyl-5-aminovaleriate (8)
From 400 mg (1.01 mmol) BOC-alanylalanylvaline, 399 mg
(1.05 mmol) benzyl 5-aminovaleriate*TOS, 0.3 mL (2.12 mmol)
TEA and 306 mg (1.11 mmol) DPPA.Yield:293 mg (0.53 mmol,
53 %) of a colourless solid.– Mp:151 °C.– 1H NMR:δ = 0.93 [m,
6 H, CH3(Val)], 1.33 [m, 6 H, CH3(Ala)], 1.42 (s, 9 H, t-Bu), 1.64
(m, 4 H, CH2), 2.06 [m, 1 H, β-H(Val)], 2.37 (m, 2 H, CH2), 3.16,
3.39 (m, 1 H, CH2), 4.41, 4.79 [m, 1 H, α-H(Ala)], 4.54 [m, 1 H,
α-H(Val)], 5.09 (s, 2 H, CH2), 5.80 (d, J = 7.6 Hz, 1 H, N-H), 7.33
(m, 5 H, Ar-H), 7.52, 7.60, 8.08 (m, 1 H, N-H). – IR: ν = 3306,
2970, 1735, 1671, 1634, 1538, 1454, 1365, 1237, 1166 cm–1.–
C28H44N4O7 (548.69). C, H, N (*0.5 H2O).
Benzyl N-BOC-alanylalanyl-5-aminovaleriate (4)
From 1.7 g (8.8 mmol) BOC-alanine, 4.0 g (10.6 mmol) 2,
1.5 mL (10.6 mmol) TEA, 2.1 g (18.5 mmol) NHS and 2.0 g
(9.7 mmol) DCC. Yield: 2.8 g (6.2 mmol, 59 %) of a viscous
liquid. – [α]2D0 = –20.0° (2 %, EtOH). – 1H NMR:δ = 1.36, 1.37 [d,
J = 7.2 Hz, 3 H, CH3(Ala)], 1.45 (s, 9 H, t-Bu), 1.54 (m, 4 H, CH2),
2.38 (m, 2 H, CH2), 3.23 (m, 2 H, CH2), 4.10 (dq, J = 7.2 Hz, 1 H,
α-H), 4.41 (dq, J = 7.0 Hz, 1 H, α-H), 4.93 (d, J = 7.2 Hz, 1 H,
N-H), 5.11 (s, 2 H, CH2), 6.48 (m, 1 H, N-H), 6.61 (d, J = 7.2 Hz,
1 H, N-H), 7.35 (m, 5 H, Ar-H).– IR:ν = 3291, 2933, 1732, 1649,
1537, 1167 cm–1. – C23H35N3O6 (449.55)
Benzyl N-BOC-alanylprolylvalyl-4-aminobutyrate (9)
From 275 mg (0.71 mmol) BOC-alanylprolylvaline, 271 mg
(0.74 mmol) benzyl 4-aminobutyrate*TOS, 0.2 mL (1.50 mmol)
TEA, and 216 mg (0.78 mmol) DPPA.Yield:210 mg (0.37 mmol,
General procedure for the coupling of BOC-protected tripep-
tides with benzyl 4-aminobutyrate and benzyl 5-aminovaleriate
1
53 %) of a colourless solid. – Mp: 149 °C. – H NMR: δ = 0.92
One equivalent of the appropriate BOC-protected tripeptide
and 1.04 eq of the C-protected spacer were dissolved in 20 mL
of dry DMF. The solution was cooled to 0 °C. 2.1 eq TEA and
1.1 eq DPPA were added and the mixture was stirred for 10 h at
0 °C and then at r.t. overnight. Ethyl acetate (50 mL) was added
and the organic layer was washed with water (3 × 20 mL), sat.
NaHCO3-solution (2 × 20 mL) and brine (1 × 20 mL).The organ-
ic layer was dried over Na2SO4 and evaporated to dryness in
vacuo. The residue was purified by cc (ethyl acetate).
[2d, J = 7.3 Hz, 6 H, CH3(Val)], 1.34 [d, J = 6.8 Hz, 3 H,
CH3(Ala)], 1.43 (s, 9 H, t-Bu), 1.83 (m, 2 H, CH2), 1.93 [m, 3 H,
3Ј-H, 4Ј-Hs(Pro)], 2.34 (hept, J = 7.1 Hz, 1 H, β-H(Val); m, 2 H,
CH2; m, 1 H, 3Ј-H(Pro)], 3.30 (m, 2 H, CH2), 3.61 [m, 2 H, 5Ј-
H(Pro)], 4.23 [m, 1 H, α-H(Val)], 4.50 [dq, 1 H, α-H(Ala)], 4.64
[m, 1 H, α-H(Pro)], 5.12 (s, 2 H, CH2), 5.39 (d, J = 7.6 Hz, 1 H,
N-H), 6.44 (m, 1 H, N-H), 7.05 (d, J = 8.5 Hz, 1 H, N-H), 7.35 (m,
5 H, Ar-H).– IR:ν = 3310, 2963, 2932, 2876, 1742, 1679, 1641,
1540 cm–1. – C29H44N4O7 (560.69). C, H, N.

330 Achilles
Arch. Pharm. Pharm. Med. Chem. 2002, 335, 325–330
[5] J.-I.Yamashita, K. Tashiro, S. Yoneda, K. Kawahara, T.
Shirakusa, Chest 1996, 109, 1328–1334; J.-I.Yamashita,
M. Ogawa, M. Abe, N. Hayashi, Y. Kurusu, V. Kawahara, T.
Shirakusa, Chest 1997, 111, 885–890.
Benzyl N-BOC-alanylprolylvalyl-5-aminovaleriate (10)
From 275 mg (0.71 mmol) BOC-alanylprolylvaline, 282 mg
(0.74 mmol) benzyl 5-aminovaleriate*TOS, 0.2 mL (1.50 mmol)
TEA, and 216 mg (0.78 mmol) DPPA.Yield:214 mg (0.37 mmol,
53 %) of a colourless solid. – Mp:97 °C. – 1H NMR:δ = 0.89 [2d,
J = 7.1 Hz, 6 H, CH3(Val)], 1.34 [d, J = 6.8 Hz, 3 H, CH3(Ala)],
1.43 (s, 9 H, t-Bu), 1.64 (m, 4 H, CH2), 2.04 [m, 3 H, 3Ј-H, 4Ј-
Hs(Pro)], 2.35 [m, 1 H, β-H(Val);m, 2 H, CH2;m, 1 H, 3Ј-H(Pro)],
3.25 (m, 2 H, CH2), 3.62 [m, 2 H, 5Ј-H(Pro)], 4.21 [m, 1 H,
α-H(Val)], 4.46 [dq, 1 H, α-H(Ala)], 4.63 [m, 1 H, α-H(Pro)], 5.11
(s, 2 H, CH2), 5.43 [d, J = 6.8 Hz, 1 H, N-H(Ala)], 6.27 (m, 1 H,
N-H), 7.03 [d, J = 8.5 Hz, 1 H, N-H(Val)], 7.35 (m, 5 H, Ar-H). –
IR:ν = 3310, 2961, 1741, 1679, 1641, 1540, 1253, 1164 cm–1.–
C30H46N4O7 (574.72). C, H, N (*0.5 H2O).
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Incubation of the compounds with PPE or PMN-E, respectively,
and HPLC-experiments
[9] P. L. Carl, P. K. Chakravarty, J. A. Katzenellenbogen, J.
Med. Chem. 1981, 24, 489–480.
The respective compound was dissolved in acetonitrile and di-
luted with tris-buffer [0.1 M, pH 8.0; final concentration ace-
tonitrile: 10 %; final concentrations of compounds [mM] (reten-
tion time, solvent 1): 1: 0.64 (10.63 min); 2: 0.82 (12.53 min); 3:
0.75 (8.35 min); 4: 0.84 (9.68 min); 5: 0.85 (6.40 min); 6: 0.87
(5.49 min); 7: 0.53 (8.52 min); 8: 0.53 (9.33 min); 9: 0.94
(12.96 min); 10: 0.73 (11.85 min)].The assays were performed
at room temperature with enzyme concentrations of
0.75 µg/mL (PPE) and 0.375 µg/mL (PMN-E) (stock solution of
enzymes were prepared in 1 mM acetic acid). At various times
20 µL aliquots were withdrawn and immediately analysed by
HPLC (detection wavelength: 215 nm; column: cc 250/4 Nucle-
osil 100-5 C18 HD; oven temperature: 30 °C; flow: 0.7 mL/min;
mobile phase: KH2PO4 (20 mM, pH 4.0)/acetonitrile [either 1/1
(solvent 1) or 95/5 (solvent 2)]. All assays were run at least in
duplicate.
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[15] R. C. Thompson, E. R. Blout, Biochemistry 1973, 12,
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