Asymmetric reduction of (Z)-α,β-dehydrotryptophanyl-containing biological peptides with sulfur functionality. Influence of substrate on stereoselectivity

Article abstract of DOI:10.1016/S0040-4020(00)00329-X

Two (Z)-α,β-dehydropentapeptides, Boc-βAla-Δ(Z) Trp-Met-Asp-Phe-NH₂ 1 and Boc-AlaΔ(Z)-Trp-NLeu-Asp-Phe-NH₂ 2, have been synthesized by enzymatic α,β-dehydrogenation, and then submitted to rhodium-catalysed asymmetric hydrogenation. The influence of the S-atom on the rate and stereoselectivity of the reduction have been investigated. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4020(00)00329-X

TETRAHEDRON
Pergamon
Tetrahedron 56 (2000) 4473±4477
Asymmetric Reduction of (Z)-a,b-Dehydrotryptophanyl-
containing Biological Peptides with Sulfur Functionality.
In¯uence of Substrate on Stereoselectivity
,²
*
Akli Hammadi, Hubert Lam, Muriel Gondry, Andre Menez and Roger Genet
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CEA/Saclay, Departement d'Ingenierie et d'Etudes des Proteines, F91191 Gif-sur-Yvette, France
Received 24 February 2000; accepted 26 April 2000
AbstractÐTwo (Z)-a,b-dehydropentapeptides, Boc-bAla-DZ Trp-Met-Asp-Phe-NH2 1 and Boc-bAlaDZ-Trp-NLeu-Asp-Phe-NH2 2, have
been synthesized by enzymatic a,b-dehydrogenation, and then submitted to rhodium-catalysed asymmetric hydrogenation. The in¯uence of
the S-atom on the rate and stereoselectivity of the reduction have been investigated. q 2000 Elsevier Science Ltd. All rights reserved.
Introduction
stereoselective labelling of biological peptides. We also
found that the asymmetric reduction of Boc-bAla-DZTrp-
Met-Asp-Phe-NH2 1 led to low diastereomeric excesses
suggesting that a preferential conformation of the peptide
in solution or a peculiar electronic environment due to the
amino acid side chains could exert a great in¯uence on the
formation of the complex with the catalyst and, hence, on
the stereoselectivity of the reaction. One of the questions
arising was thus to determine whether the transition state
could be sensible to electronic as well as steric factors
induced by the substrate structure. Or more precisely;
How the sulfur atom in the methionyl residue (Met) could
in¯uence the enantioselectivity of the asymmetric reduc-
tion?
Asymmetric hydrogenation of a,b-dehydroamino acids
with chiral rhodium catalysts has been widely studied lead-
ing to high enantioselectivities (e.e. up to 95%).1 An
obvious development of homogeneous hydrogenation has
been the asymmetric reduction of more complex prochiral
substrates such as a,b-dehydrodipeptides,2 building blocks
for synthesis of enkephalins,3 and a,b-dehydroenkephalin
analogs, which were realized with very high catalytic
activity and stereoselectivity.4 In spite of this success,
examples of an asymmetric reduction of a a,b-dehydro-
amino acid with sulfur functionality, which are wide-spread
among biological peptides, are rarely mentioned.5 The well-
known af®nity of rhodium atom to sulfur atom6 as well as
the fact that the sulfur compounds can induce poisoning of
homogeneous RhI-diphosphine catalysts,7 could explain the
reluctance to apply the methodology to this kind of
substrates.
The aim of the present work is thus to provide experimental
evidence for this assumption. Replacement of Met in 1 by its
steric isostere Norleucine (Nleu), which is commonly used
in the literature,9 offers a convenient and powerful tool to
probe the effects of the methionyl residue on the rate and
stereoselectivity of the reduction reaction. For comparison
purpose, Boc-bAla-DZTrp-Met-Asp-Phe-NH2 1 ((DZTrp2-
In our previous study8 we reported about the asymmetric
deuteration of two biological pentapeptide hormones, the
N-terminal fragment of the gonadotropin releasing-hormone
(LH-RH1±5) of sequence pGlu-His-Trp-Ser-Tyr and penta-
gastrin (Boc-bAla-Trp-Met-Asp-Phe-NH2), an active frag-
ment of the gastrin. These experiments demonstrate that the
asymmetric reduction of (Z)-a,b-dehydrotryptophanyl-
containing peptides produced by (l)-tryptophan 20,30-
oxidase (LTO) is an ef®cient approach to the regio- and
Met3)-PG) and Boc-bAla-DZTrp-NLeu-Asp-Phe-NH2
2
((DZTrp2-NLeu3)-PG) were tested here as substrates in the
rhodium-catalyzed asymmetric reduction.
Results and Discussion
The (DZTrp2-Met3)-PG 1 and (DZTrp2-NLeu3)-PG 2 were
synthesized by enzymatic a,b-dehydrogenation of Boc-
bAla-Trp-Met-Asp-Phe-NH2 and Boc-bAla-Trp-NLeu-
Asp-Phe-NH2, respectively (Scheme 1). The (Z)-a,b-dehy-
drogenation reaction was realized by S-tryptophan 20,30-
oxidase (LTO) from Chromobacterium violaceum (ATCC
12472) which is highly speci®c for (S)-Trp residues.10 The
Keywords: asymmetric reduction; a,b-dehydropeptide; rhodium catalyst;
stereoselectivity.
* Corresponding author. Tel.: 133-1-69-086308; fax: 133-1-69-085753;
e-mail: akli.hammadi@cea.fr
Present address: CEA/Saclay, Institut National des Sciences et Tech-
²
Â
niques Nucleaires, Unite d'Enseignement Radioprotection, Biologie et
Medecine, F91191 Gif-sur-Yvette, France.
Â
Â
0040±4020/00/$ - see front matter q 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(00)00329-X

4474
A. Hammadi et al. / Tetrahedron 56 (2000) 4473±4477
Scheme 1.
a,b-dehydropentapeptides 1 and 2 were obtained in quanti-
tative yield and puri®ed by HPLC.
In the presence of the Rh/(R,R)-dipamp catalyst, the hydro-
genation of 1 in methanol at room temperature under an
initial hydrogen pressure of 5 atm. gave Boc-bAla-(S)Trp-
Met-Asp-Phe-NH2 1a in low diastereoselectivity (26% d.e.,
entry 1). Regarding the diastereoselectivity dependency on
hydrogen pressure, it is interesting to observe that an
increase in pressure from 5 to 15 atm. in the presence of
(R,R)-dipamp abolished the stereoselectivity (entries 1 and
2).
Asymmetric micro-hydrogenation under pressure of 1 and 2
(6 mmol) were carried out in methanol by using cationic
complexes [Rh (R,R)-dpcb (COD)]1 PF62,11 [Rh (S,S)-diop
(COD)]1 PF26 ,12 [Rh (R,R)-dipamp (COD)]1 BF24 ,13 and
[Rh (S,S)-cydiop (COD)]1 PF26 14 (Scheme 2). The main
results are listed in Table 1.
Scheme 2.

A. Hammadi et al. / Tetrahedron 56 (2000) 4473±4477
4475
Table 1. Asymmetric reduction of (DZTrp2-Met3)-pentagastrin 1 and (DZTrp2-NLeu3)-pentagastrin 2 catalysed by chiral rhodium complexes ([substrate]/
[Rh]ˆ1.5; [substrate]ˆ0.006 M in methanol. Reaction performed at room temperature)
Entry
Substrate
Catalyst (ligand)a
Pressure (atm)
Time (h)
Yield (%)
S/Rb
d.e. (%)
1
2
3
4
5
6
7
8
9
10
1
1
1
1
1
2
2
2
2
2
[Rh/(R,R)-dipamp]
[Rh/(R,R)-dipamp]
[Rh/(S,S)-diop]
[Rh/(R,R)-dpcb]
[Rh/(R,R)-cydiop]
[Rh/(R,R)-dipamp]
[Rh/(R,R)-dipamp]
[Rh/(S,S)-diop]
5
15
5
5
5
5
11
5
5
5
20
20
20
20
18
20
20
20
24
90
69
80
73
68
99
99
99
75
99
94
63/37
26
0
50/50
28/72
10/90
44
80
32
99
99
36
76
14
34/66
99.5/0.5
99.5/0.5
68/32
88/12
43/57
[Rh/(R,R)-dpcb]
[Rh/(R,R)-cydiop]
a The ligands are: (R,R)-dipamp: 1,2-bis[(2-methoxyphenyl)phosphino]-ethane; (S,S)-diop: 4,5-bis[diphenylphosphino]-2,2-dimethyl-1,3-dioxolane; (R,R)-
dpcb: trans-1,2-bis[diphenylphosphinomethyl]-cyclobutane.
b Measured by HPLC. The con®guration of the new asymmetric center was assigned by HPLC as compared to reference compounds.
However, the results obtained with 2 were quite different.
Indeed, using the cationic complex [Rh (R,R)-dipamp
(COD)]1 BF24 under a hydrogen pressure of 5 atm, higher
chiral recognition and quantitative reduction were found
(99% d.e., 99% yield, entry 6). In this latter case, in contrast
to 1, the stereoselectivity showed no dependency at all in the
range of 5±11 atm. (99% d.e., entries 6 and 7). This ®rst set
of data suggests that the steric and electronic character of
S-atom in 1 also has signi®cant in¯uence upon the stereo-
selectivity and the relative rate of the reaction.
dropeptides 1 and 2 were performed with achiral catalysts,
[Rh/dppb] and PdO (Table 2).
In contrast to the low selectivities obtained by hydro-
genation of linear (Z)-a,b-dehydropeptides over hetero-
geneous catalysts (0±20% d.e.)15, we showed that the
reduction of 1 catalysed by PdO could be performed
under a hydrogen pressure of 15 atm. with an acceptable
diastereoselectivity (52% d.e., entry 2). Studying the reduc-
tion of 2, then we observed that no asymmetric induction
arose from the utilization of PdO under a pressure of 5 atm.
(entry 4). Furthermore, the hydrogenation of the (Z)-a,b-
dehydropeptide 1 occured under a pressure of 15 atm. in
order to avoid a slow reaction. These results indicate that
the presence of (S)-methionine in the peptide could be an
important factor affecting both the reduction rate and the
stereoselectivity. A pausible explanation could be that a
signi®cant attractive interaction between sulfur in (S)-
methionine moiety and PdO, could increase the rigidity of
the (Z)-a,b-dehydropentapeptide and, hence, improve
optical yields.
Moreover, the stereoselectivities exhibited in the reduction
of 1 using (S,S)-diop and (R,R)-dpcb as chiral ligands were
moderate (44 and 80% d.e., respectively, in favor of the
R-tryptophan isomer, entries 3 and 4), and the direction of
asymmetric inductions were opposite to that observed for 2
(36 and 76% d.e., respectively, in favor of the S-tryptophan
isomer, entries 8 and 9). Together, these results con®rmed
the determining effect of methionyl residue on the stereo-
selectivity of reduction. The S-atom could possibly serve as
an additional rhodium ligand and, hence, affect the mode of
the enantioface selection of ole®nic moiety by the chiral Rh
complex.
We then proceeded to the reduction of (Z)-a,b-dehydropep-
tides 1 and 2 in the presence of an achiral rhodium complexe
([Rh/dppb]) and showed in both cases a clear, although
opposite, asymmetric induction: 26% in favour of the
(R)-tryptophan isomer with 1 (entry 1) and 6% in favour
of the (S)-tryptophan isomer with 2 (entry 3). The ®nding of
a more stereoselective hydrogenation of 1 than of 2 makes
the suggestion of special coordination of sulfur to rhodium
which modify the course of the reaction. Futhermore, the
lack of selectivity achieved in the reduction of 2 could be
interpreted by the exclusive coordination of the N-acyl-(Z)-
a,b-dehydrotryptophan moiety with the rhodium complex.
However, the asymmetric reduction of 1 and 2 catalyzed by
[Rh/(R,R)-cydiop] afforded in both cases ((R)-Trp)-penta-
gastrine in modest diastereoselectivity (32 and 14%, respec-
tively, entries 5 and 10) whatever the structure of the
a,b-dehydropentapeptide, suggesting that the double
asymmetric induction depends on catalyst-substrate combi-
nation.
In order to identify the simple asymmetric induction caused
by the methionyl residue, hydrogenations of (Z)-a,b-dehy-
Table 2. Asymmetric reduction of (DZTrp 2-Met3)-pentagastrin 1 and (DZTrp2-NLeu3)-pentagastrin 2 catalyzed by PdO or [Rh/dppb] (All reactions were run
with 6 mmol of substrate and 4.5 mg of PdO or 4 mmol of [Rh/dppb] in methanol. [Substrate]/[Rh]ˆ1.5; [substrate]ˆ0.006 M in methanol. Reaction performed
at room temperature)
Entry
Substrate
Catalyst
Pressure (atm)
Time (h)
Yield (%)
S/Ra
d.e. (%)
1
2
3
4
1
1
2
2
[Rh/dppb]
PdO
[Rh/dppb]
PdO
5
15
5
24
20
43
20
85
80
85
95
37/63
24/76
53/47
50/50
26
52
6
5
0
a Measured by HPLC. The con®guration of the new asymmetric center was assigned by HPLC as compared to reference compounds.

4476
A. Hammadi et al. / Tetrahedron 56 (2000) 4473±4477
In this case, the rest of the molecule is located in the outer
sphere of the coordination site. This may be the reason why
a low-asymmetric induction was observed with 2 (entry 3).
Enzymatically prepared 1 and 2
(l)-Tryptophan 20,30-oxidase was puri®ed from C.
Violaceum (ATCC 12472) according to Genet and cowork-
ers.10 The enzymatically prepared a,b-dehydrotrypto-
phanyl-peptides 1 and 2 were obtained by incubation of
the corresponding pentapeptides (Boc-bAla-Trp-Met-Asp-
Phe-NH2 and Boc-bAla-Trp-NLeu-Asp-Phe-NH2, respec-
tively, 10 mg, 1 mM) with LTO (2.6 nM), in 50 mM succi-
nate buffer, pH 5.6, containing 20 mg ml21 catalase, for 20 h
at 308C. The a,b-dehydrogenation reactions were followed
spectrophotometrically by monitoring the UV-spectrum of 1
and 2. The a,b-dehydropeptides 1 and 2 showed character-
istic absorption peaks at lmaxˆ337 nm and lmaxˆ338 nm,
respectively.
Conclusion
In order to ascertain the putative role of the methionyl resi-
due in the asymmetric reduction of the (DZTrp2-Met3)-
pentagastrine 1, the hydrogenation of two (Z)-a,b-dehydro-
pentapeptides 1 and 2 were performed with achiral and
chiral catalysts. We showed that a (Z)-a,b-dehydropeptide
with sulfur functionality can be asymmetrically hydro-
genated in the presence of homogeneous RhI-diphosphine
complexes or PdO without poisoning these catalysts.
However, the presence of (S)-methionine in the (DTrp)-
pentagastrine 1 appears to have a strong in¯uence upon
the way of asymmetric induction, and the effects on the
stereoselectivity of the reaction remain unpredictable.
Indeed, the asymmetric reduction of Boc-bAla-DZTrp-
Met-Asp-Phe-NH2 1 catalysed by [Rh (R,R)-dipamp
(COD)]1 BF24 led to low diastereomeric excesses (0±26%
d.e. in favor of the S-tryptophan isomer), whereas the asym-
metric hydrogenation of Boc-bAla-DZTrp-NLeu-Asp-
Phe-NH2 2 with the same catalytic system proceeded with
extremely high stereoselectivity (up to 99%).
Chromatographic procedure for the puri®cation of 1
and 2
The dehydropentapeptides were puri®ed on reverse-phase
chromatography (Vydac 18 C column; eluent, 35% aceto-
nitril in water; ¯ow rate, 1 ml min21; UV detection at 280
and 330 nm). Retention times were: 12.1 min for 1 and
34.1 min for 2. Evaporation of the solvent afforded 7.6 mg
(75%) of 1 and 8.1 mg (80%) of 2 as a yellow oil. The
dehydropeptides 1 and 2 were then characterized by mass
spectrometry (m/zˆ767, MH1 and m/zˆ749, MH1, respec-
tively).
The results obtained with 2 constituted a quasi-theoretical
example for the stereoselective hydrogenation of dehydro-
peptides, suggesting that asymmetric induction is mainly
determined by only steric repulsion between the substrate
and the ligand coordinated to the rhodium species. In
contrast, the asymmetric hydrogenation of (DZTrp2-Met3)-
pentagastrine 1 involves electronic interaction between
rhodium and sulfur in methionine moiety as well as steric
interactions between the chiral diphosphine and the
substrate. Furthermore, the asymmetric induction arises
from the electronic environment due to S-atom rather than
the catalyst structure.
Preparation of the in situ neutral complex [Rh/dppb]
[Rh Cl dppb] was prepared in situ, by reaction of [Rh Cl
(COD)]2 with diphosphine in degassed methanol. Typically,
[Rh Cl (COD)]2 (2 mmol) and dppb (4 mmol) were
dissolved in 1 ml of methanol under argon. The solution
was then stirred for 30 min. The complex thus obtained
was ready to use for asymmetric hydrogenation and was
introduced into the hydrogenation ¯ask by means of a
syringe. Any contact with air was avoided.
In order to improve the regio- and stereoselective reduction
(hydrogenation, deuteration or tritiation) of biological
peptides of higher molecular weight, the effects of hetero-
atoms, especially sulfur, on the stereoselectivity of the
reaction should be taken into account.
Hydrogenation, general procedure
The high pressure reductions are run using an automatic gas
transfer unit16 supplied with a liquid helium cryostat and
®tted with an inlet for introduction of the solvent. This
cryostat is used to bring the hydrogen at the solid state (at
4 K) in a thin capillary tube. The 1 ml reactor is connected
close to the capillary tube so that the hydrogen can be
compressed by heating in a very small volume (ca 1 ml).
Using this equipment, it is possible to rise the gas pressure to
about 30 atm.
Experimental
General experimental procedures
HPLC analyses were performed on a Waters 600 chromato-
graphic system (Vidac 18C column 250£4.6 mm2) supplied
with a Waters 996 photodiode array detector. Ultraviolet
spectra were recorded with a Beckman DU-70 spectro-
photometer. Products were analyzed either by chemical
ionization-mass spectrometry (Nermag R 10-10). Methanol
was freshly distilled, dried following literature method and
stored under argon. All experiments with organometallic
elements were performed using standard Schlenck tech-
niques.
The solution of appropriate catalyst (4 mmol) in degassed
methanol (1 ml) was added under argon to the dehydro-
pentapeptide (6 mmol) in the hydrogenation ¯ask which
was connected to the reduction apparatus. The argon atmos-
phere was replaced with hydrogen. The reductions were run
under the reaction conditions given in Tables 1 and 2. The
solvent was removed under reduced pressure. Conversion
rates were determined by HPLC analysis.

A. Hammadi et al. / Tetrahedron 56 (2000) 4473±4477
4477
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