The stability of pseudopeptides bearing sulfoximines as chiral backbone modifying element towards Proteinase K

Article abstract of DOI:10.1016/S0960-894X(03)00697-8

Incorporation of sulfoximines as backbone modifying element results in two new pseudopeptide bonds which display enhanced (bond A) and strongly reduced reactivity (bond B) towards hydrolysis by Proteinase K.

Full text of DOI:10.1016/S0960-894X(03)00697-8

Bioorganic & Medicinal Chemistry Letters 13 (2003) 3207–3211
The Stability of Pseudopeptides Bearing Sulfoximines as Chiral
Backbone Modifying Element towards Proteinase K
Carsten Bolm,* Dirk Muller, Christian Dalhoff, Christian P. R. Hackenberger
and Elmar Weinhold*
Institut fur Organische Chemie, RWTH Aachen, Prof.-Pirlet-Str. 1, D-52056 Aachen, Germany
¨
Received 28 April 2003; revised 10 June 2003; accepted 24 June 2003
Dedicated to Professor Dr. M. T. Reetz on occasion of his 60th birthday
Abstract—Incorporation of sulfoximines as backbone modifying element results in two new pseudopeptide bonds which display
enhanced (bond A) and strongly reduced reactivity (bond B) towards hydrolysis by Proteinase K.
# 2003 Elsevier Ltd. All rights reserved.
In recent years, peptides have extensively been modified
in order to find new therapeutic agents for pharmaceu-
tical applications.1 Such modifications are required
because natural peptides are often excreted rapidly or
do not pass biological membranes resulting in a poor
bioavailability.2 Furthermore, replacing some (or even
all) elements of the peptide backbone by unnatural
linkages can enhance the stability against enzymatic
amide bond cleavage.3 An additional advantage of
those peptide alternations stems from the fact that they
often lead to significant stabilization of secondary
structures.1,2a,4
of secondary structures induced by the sulfonamide
group are known.13
Recently, we introduced sulfoximines as building blocks
for pseudopeptides14 and demonstrated that bis(sulfox-
imines) favor turn formation.15 In this context it is also
noteworthy, that several sulfoximines are highly biolo-
gically active leading to interesting pharmaceutical
applications. Examples include buthionine sulfoximine
(BSO), an additive in anti-cancer drugs,16 and carboxy-
peptidase A inhibitors.17
Pseudopeptides with the sulfoximine-containing core
fragment I can be regarded as structural analogues of b-
amino acid- and sulfonamide-derived pseudopeptides II
and III, respectively (Fig. 1). In this paper we describe
the first peptidase cleavage experiments of pseudo-
tripeptides containing such sulfoximine-based core I.
In that respect, b-amino acids are particularly interest-
ing. Their oligomerization leads to b-peptides which
adopt stable well-defined secondary structures5 and
cannot be cleaved by peptidases6 such as Pronase and
Proteinase K.7 Furthermore, some compounds were
found to exhibit, among others, antimicrobial activity
directly related to conformational stability.8
We initiated our study by synthesizing pseudotripep-
tides 3 (Scheme 1).18 After carboxylation of sulfox-
imines 1, the resulting pseudodipeptide ammonium salts
Another important modifying element is the sulfon-
amide moiety.9 This group mimics the tetrahedral
intermediate formed during peptide bond cleavage,10
and certain compounds containing this modifying ele-
ment are active as peptidase inhibitors.11 Moreover,
sulfonamide peptidomimetics reveal increased stability
towards enzymatic degeneration,12 and several examples
*Corresponding authors. Tel.: +49-241-809-4675; fax: +49-241-809-
2391; e-mail: carsten.bolm@oc.rwth-aachen.de (C. Bolm); fax: +49-241-
809-2528; e-mail: elmar.weinhold@oc.rwth-aachen.de (E. Weinhold).
Figure 1. Comparison of pseudopeptide fragments containing a sul-
foximine I, a b-amino acid II and a sulfonamide III.
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00697-8

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C. Bolm et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3207–3211
Scheme 1. Syntheses of pseudotripeptides (S)-3 and (R)-3: (a) nBuLi (1.0 equiv), cyclohexylisopropylamine (1.0 equiv), THF, CO2, ꢀ78 ꢁC then
ꢁ
ꢁ
.
25 C; (b) H-Phe-OBn HCl (1.0 equiv), DCC (1.05 equiv), DMAP (0.12 equiv), CH2Cl2, 0 C, 12 h; (c) Pd/C (15 mol %), H2, ethyl acetate, 24 h;
(d) TFA/CH2Cl2 (1:4), 4 h; yield after additional HPLC purification.
First NMR studies of sulfoximine-containing pseudo-
peptides 2 demonstrated that the 13C NMR resonances
of the carbonyl groups adjacent to the sulfoximine
nitrogen are significantly different to those of the amide
carbonyls in the molecules (located in the b-position of
the sulfur atom). Thus, whereas the former give signals
at about 180 ppm, the latter resonate at 160 ppm (Fig. 2).
Both values are different to the amide carbonyl signals
in the Phe trimer (H-Phe-Phe-Phe-OH), which give sig-
nals at about 170 ppm. Apparently, all carbonyl groups
differ significantly in their electronic nature and conse-
quently we expected distinct chemical properties, which
we hoped to reveal in enzymatic cleavage studies.
Scheme 2. Synthesis of pseudopeptide (S)-6 (for a–d see the legend to
Scheme 1).
For those enzymatic fragmentation reactions diastereo-
mers (S)-3 and (R)-3 were treated with Proteinase K.
(not shown) were coupled with C-terminal protected
phenylalanine using conventional DCC activation
affording 2 in good yields. Deprotection of 2 at the C-
and N-terminus by hydrogenation and subsequent TFA
treatment, respectively, gave pseudotripeptides H-Phe-
(S)-SulfCO-Phe-OH [(S)-3] and H-Phe-(R)-SulfCO-Phe-
OH [(R)-3].
This enzyme is a non-specific serine protease of the
subtilisin family, which was isolated from the mould
Tritirachium album limber.20 It readily hydrolyzes pro-
teins and has a low preference for peptide bond cleavage
after aromatic and aliphatic amino acid residues.
First, pseudotripeptide H-Phe-(S)-SulfCO-Phe-OH [(S)-
3] was incubated with Proteinase K, and the cleavage
reaction was analyzed by reversed-phase HPLC and
integration of the UV signal (210 nm) resulting from the
starting material.21 The fragmentation of (S)-3 followed
first-order kinetics (Fig. 3) with an observed rate con-
stant kobs of 3.75 hꢀ1 (Table 1, entry 1). No cleavage
was detected in a control experiment in which Protei-
nase K was omitted. This result was compared with a
In addition, pseudodipeptide (S)-6 was synthesized
(Scheme 2). Similar to the syntheses of compounds 3
BOC-protected sulfoximine (S)-419 was carboxylated
and then coupled with phenylalanine benzyl ester to give
protected pseudodipeptide (S)-5. C- and N-terminal
deprotection of (S)-5 yielded pseudodipeptide H-(S)-
SulfCO-Phe-OH [(S)-6].
Figure 2. Characteristic 13C NMR carbonyl resonances in sulfoximine-pseudopeptides (S)-2 und (R)-2 with two pseudopeptide bonds A and B.

C. Bolm et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3207–3211
3209
cleavage of the analogous natural tripeptide H-Phe-Phe-
Phe-OH by Proteinase K. As can be seen from Table 1
(entry 2) H-Phe-Phe-Phe-OH was cleaved somewhat
slower than sulfoximine-containing pseudotripeptide
(S)-3.
Most interestingly, the only products formed from the
pseudo-tripeptide (S)-3 (retention time 24.3 min) were
phenylalanine (retention time 14.2 min) and the pseu-
dodipeptide H-(S)-SulfCO-Phe-OH [(S)-6] (retention
time 15.4–16.0 min, verified by co-injection with syn-
thetic material).21 The alternative product H-Phe-(S)-
Sulf-H [(S)-7], synthesized by deprotection of (S)-1 with
TFA, retention time 27.8 min, which should be formed
spontaneously by decarboxylation of the primary pro-
duct, was not observed (Scheme 3). Thus, cleavage
occurred exclusively at the pseudopeptide bond A and
not at the pseudopeptide bond B (Scheme 3).22
Figure 3. Kinetic analysis of substrate cleavage by Proteinase K. The
ln of the substrate concentration at time t (ct) divided by the concen-
tration at time zero (co) is plotted against the reaction time. *=(S)-3
(304 mM) and Proteinase K (3 mAU mLꢀ1); *=(R)-3 (304 mM) and
Proteinase K (3 mAU mLꢀ1); &=H-Phe-Phe-Phe-OH (218 mM) and
Proteinase K (1.5 mAU mLꢀ1). One mAU (AU=Active Unit) is
defined as the amount of Proteinase K required to release folin-posi-
tive amino acids and peptides corresponding to 1 mmol of tyrosine
from hemoglobin at 37 ꢁC in 1 min.
In order to test the stereoselectivity of Proteinase K for
the P10 residue (the residue after the cleavage site), we
investigated the cleavage of pseudotripeptide H-Phe-
(R)-SulfCO-Phe-OH [(R)-3]. In (R)-3 the configuration
at sulfur is inverted compared to (S)-3, and (R)-3 can be
regarded as a tripeptide analogue with an internal
Table 1. Observed rate constants kobs for the cleavage of sulfoximine-containing pseudopeptides and natural peptides by Proteinase K
a
c
Entry
Compd
[Substrate]
(mM)
kobs
(hꢀ1
t1/2
(min)
[Enzyme]b
(mAU mLꢀ1
)
krel
)
1
2
3
4
5
(S)-3
H-Phe-Phe-Phe-OH
(R)-3
H-Phe-Phe-OH
(S)-6
304
218
304
262
262
3.75ꢂ0.36
1.11ꢂ0.07
2.72ꢂ0.10
0.06ꢂ0.01
No cleavage
11.1
37.5
15.3
693.1
3.0
1.5
3.0
30.0
300.0
1
0.59
0.73
0.002
aErrors are given as standard deviations.
bAU, Active Unit; see the note to Figure 3 for definition.
cDefined as kobs divided by the enzyme concentration relative to entry 1.
Scheme 3. Bonds in H-Phe-(S)-SulfCO-Phe-OH [(S)-3] that could be cleaved by Proteinase K. Only cleavage of the pseudopeptide bond A with the
formation of H-(S)-SulfCO-Phe-OH [(S)-6] was observed.

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C. Bolm et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3207–3211
unnatural b-d-amino acid analogue. Again cleavage
occurred exclusively at the pseudopeptide bond A. The
observed first order rate constant kobs=2.72 hꢀ1 (Table
1, entry 3) was similar to that observed with the dia-
stereoisomer (S)-3. This result can be rationalized based
on the three-dimensional structure of Proteinase K in
complex with a cleaved hexapeptide.23 It seems as if
both pseudotripeptides, (S)-3 and (R)-3, could bind
equally well after side-chain rotations on the surface of
Proteinase K, and we assume that the primary struc-
tural determinate for cleavage of our pseudotripeptides
is the N-terminal Phe residue (P1 residue).
Perspectives in Medicinal Chemistry; Testa, B., Kyburz, E.,
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Based on those results and in the light of the 13C NMR
data, we anticipated that the sulfoximine moiety should
stabilize the amide bonds connecting the b-carbonyl
group of the sulfoximine sulfur with the natural N-
bound a-amino acid. This was tested in cleavage
experiments of pseudodipeptide H-(S)-SulfCO-Phe-OH
[(S)-6] in comparison with those of natural dipeptide H-
Phe-Phe-OH. The latter was cleaved very slowly (Table
1, entry 4), which is in line with the results by Kraus et
al.,24 who suggested that small peptides were bad sub-
strates for Proteinase K. With pseudodipeptide (S)-6,
however, no cleavage was observed at all even in the
presence of a 10-fold higher concentration of Proteinase
K (Table 1, entry 5).
Taken together, our results demonstrate that the sul-
foximine-pseudopeptide modification in (S)-6 [and in
(S)-3 or (R)-3] stabilizes the following pseudopeptide
bond B against enzymatic cleavage. Furthermore it is
apparent that pseudopeptide bond A is readily cleaved
by the enzyme. These observations make this class of
pseudopeptides interesting candidates for a prodrug
approach. For example, a released free N–H-sulfox-
imine moiety is very important in sulfoximine contain-
ing active compounds like methionine sulfoximine
(MSO). MSO is a selective inhibitor of g-glutamylcys-
teine synthetase and its free N–H group is phosphory-
lated by the enzyme.25 Currently further investigations
towards an understanding of the properties of the sul-
foximine-pseudopeptide bonds A and B are in progress.
10. Radkiewicz, J. L.; McAllister, M. A.; Goldstein, E.;
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1997, 38, 1169. (b) Bolm, C.; Moll, G.; Kahmann, J. D. Chem.
Eur. J. 2001, 7, 1118.
15. Bolm, C.; Muller, D.; Hackenberger, C. P. R. Org. Lett.
2002, 4, 893.
16. Muldoon, L. L.; Walker-Rosenfeld, L. S. L.; Hale, C.;
Purcell, S. E.; Bennett, L. C.; Neuwelt, E. A. J. Pharmacol.
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4467. (b) Mock, W. L.; Zhang, J. Z. J. Biol. Chem. 1991, 266,
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18. The given absolute configurations refer to those at sulfur
only. All natural a-amino acids have S-configuration. The
structure for the abbreviation (S)-SulfCO is shown in Figure 1
(solid frame in I). The abbreviation Sulf stands for SulfCO
without the carbonyl moiety.
Acknowledgements
This work was supported by the Fonds der Chemischen
Industrie and the Deutsche Forschungsgemeinschaft
(DFG) within the Collaborative Research Center (SFB)
380 ‘Asymmetric Synthesis by Chemical and Biological
Methods’. C.D. acknowledges DFG for a scholarship
within the Graduiertenkolleg GK 440, and C.P.R.H. is
grateful to the Fonds der Chemischen Industrie and the
BMBF for a pre-doctoral stipend. We also thank Dr.
Juan Rodriguez-Dehli for inspiring discussions.
References and Notes
1. Fletcher, M. D.; Campbell, M. M. Chem. Rev. 1998, 98, 763.
2. (a) Giannis, A.; Kolter, T. Angew. Chem. 1993, 105, 1303;
Angew. Chem., Int. Ed. Engl. 1993, 32, 1244. (b) Davis, S. S. In
19. Boc-Sulf-H [(S)-4] was synthesized according to litera-
ture14a and is the reaction product of phenyl methyl sulfox-
imine with (Boc)2O and KOtBu.

C. Bolm et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3207–3211
3211
20. Ebeling, W.; Hennrich, N. M.; Klockow, M.; Orth, H. D.;
Lang, H. Eur. J. Biochem. 1974, 47, 91.
mL minꢀ1 and detected by UV-absorption at 210 nm. The
following retention times tR were observed: H-Phe-OH (14.2
min), (S)-6 (15.4–16.0 min), H-Phe-Phe-OH (18.5–19.2 min),
(R)-3 (20.7 min), H-Phe-Phe-Phe-OH (22.9 min) (S)-3 (24.3
min), and (S)-7 (27.8 min).
22. Commonly, N-acyl sulfoximines are rather stable. The
conditions for their selective cleavage are currently under
investigation. Bolm, C.; Hackenberger, C. P. R.; Raabe, G. To
be published.
23. Betzel, C.; Singh, T. P.; Visanji, M.; Peters, K.; Fittkau,
S.; Saenger, W.; Wilson, K. S. J. Biol. Chem. 1993, 268, 15854.
24. Kraus, E.; Kitz, H. H.; Femler, U. F. Hoppe-Seyler’s Z.
Physiol. Chem. 1976, 357, 233.
25. Richman, P. G.; Orlowski, M.; Meister, A. J. Biol. Chem.
1973, 248, 6684.
21. Experimental: Compounds 1–7 were prepared according
to literature procedures for analogous substrates.14 Proteinase
K fragmentation assay: The cleavage reactions were performed
in a buffer (1 mL, 30 mM Tris–HCl, 1 mM CaCl2, pH 8.0)
containing the pseudopeptides or peptides (see Table 1 for
concentrations) and Proteinase K (Qiagen Proteinase K, see
Table 1 for concentrations) at 37 ꢁC. Aliquots were withdrawn
after different incubation times, frozen in liquid nitrogen and
stored at ꢀ20 ꢁC before RP-HPLC analysis. Samples (60 mL)
were thawed and directly injected onto a C-18 column (Pron-
tosil 120-5-C18-AQ-5.0 mm, 250 mmꢃ4.6 mm, Bischoff).
Compounds were eluted with water (0–5 min) followed by
linear gradient to 70% CH3CN (5–30 min) at a flow rate of 1