Racemization-free chemoenzymatic peptide synthesis enabled by the ruthenium-catalyzed synthesis of peptide enol esters via alkyne-addition and subsequent conversion using alcalase-cross-linked enzyme aggregates

Article abstract of DOI:10.1002/adsc.201200423

The C-terminal activation of peptides as prerequisite for the formation or ligation of peptide fragments is often associated with the problem of epimerization. We report that ruthenium-catalyzed alkyne addition with (+)-2,3-O-isopropylidene-2,3-dihydroxy-1,4-bis(diphenylphosphino)butane as ligand allows the racemization-free synthesis of peptide enol esters tolerating a wide range of functional groups. The transformation can be performed in a variety of different solvents addressing the solubility issues imposed by peptides with varying amino acid side chain patterns. We show that peptide enol esters with an amide motif in the enol moiety are excellent acyl donors for the peptide condensation with other peptide fragments in organic solvents using serine endopeptidase subtilisin A as catalyst. The reported combination of transition metal catalysis with enzymatic peptide ligations adds an important tool for the racemization-free synthesis and ligation of peptides which is compatible even with unprotected amino acid side chains. Copyright

Full text of DOI:10.1002/adsc.201200423

FULL PAPERS
DOI: 10.1002/adsc.201200423
Racemization-Free Chemoenzymatic Peptide Synthesis Enabled
by the Ruthenium-Catalyzed Synthesis of Peptide Enol Esters via
Alkyne-Addition and Subsequent Conversion Using Alcalase-
Cross-Linked Enzyme Aggregates
Hilmar Schrçder,a,b Gernot A. Strohmeier,b Mario Leypold,a Timo Nuijens,c
Peter J. L. M. Quaedflieg,c and Rolf Breinbauera,b,*
a
Institute of Organic Chemistry, Graz University of Technology, Stremayrgasse 9, 8010 Graz, Austria
Fax : (+43)-316-873-32402
(homepage: www.orgc.tugraz.at); e-mail: breinbauer@tugraz.at
ACIB – Austrian Centre of Industrial Biotechnology GmbH, Petersgasse 14, 8010 Graz, Austria
DSM Innovative Synthesis, P. O. Box 18, 6160 MD Geleen, The Netherlands
b
c
Received: May 14, 2012; Revised: December 7, 2012; Published online: February 28, 2013
Dedicated to Professor Herfried Griengl on the occasion of his 75th birthday.
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201200423.
Abstract: The C-terminal activation of peptides as with an amide motif in the enol moiety are excellent
prerequisite for the formation or ligation of peptide acyl donors for the peptide condensation with other
fragments is often associated with the problem of ep- peptide fragments in organic solvents using serine
imerization. We report that ruthenium-catalyzed endopeptidase subtilisin A as catalyst. The reported
alkyne addition with (+)-2,3-O-isopropylidene-2,3- combination of transition metal catalysis with enzy-
dihydroxy-1,4-bis(diphenylphosphino)butane
as matic peptide ligations adds an important tool for
ligand allows the racemization-free synthesis of pep- the racemization-free synthesis and ligation of pep-
tide enol esters tolerating a wide range of functional tides which is compatible even with unprotected
groups. The transformation can be performed in a va- amino acid side chains.
riety of different solvents addressing the solubility
issues imposed by peptides with varying amino acid Keywords: alcalase; biocatalysis; enol esters; peptide
side chain patterns. We show that peptide enol esters synthesis; ruthenium
Introduction
zymatic chemoselectivity, which allows the conversion
of non-side chain protected peptides and the absolute
In recent years, the scalable synthesis of peptides, absence of racemization. We have recently reported
ranging from simple dipeptides to oligopeptides up to in a series of publications that the serine endopepti-
50 amino acids in length, has gained significant impor- dase subtilisin A (commercially available as Alcalase)
tance, as such products have been increasingly investi- is a practical enzyme for the preparation of oligopep-
gated and marketed in the pharmaceutical, cosmetics tides.[7] One challenge we identified is the racemiza-
and nutrition industry.[1] Although several excellent tion-free preparation of activated peptide esters
methods and reagents for the assembly of peptides which are suitable substrates for this approach. In this
are available, the issue of racemization-free fragment manuscript we report about a convenient and atom-
coupling remains a problem without a general solu- economical method to prepare peptide enol esters
tion.[2] One attractive method is the chemoenzymatic without racemization using transition metal catalysis
synthesis of peptides using proteases,[3] where either and their subsequent use for enzymatic peptide syn-
suitable reaction conditions shift the equilibrium to thesis. This approach takes advantage of the so far
the coupling product,[4] or activated esters[5] or thio- rather unexplored opportunities offered by the combi-
esters[6] are used. In principle, chemoenzymatic pep- nation of homogeneous transition metal catalysis and
tide synthesis is distinguished by the advantage of en- enzymatic catalysis.[8]
Adv. Synth. Catal. 2013, 355, 1799 – 1807
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Hilmar Schrçder et al.
FULL PAPERS
Results and Discussion
nal alkyne moiety would result in the atom-economi-
cal production of enol esters, which have been recog-
Activation of a peptideꢁs C-terminal carboxylic acids nized as ideal reagents in enzymatic acylations, al-
for chemoenzymatic condensation with peptide nucle- though so far only for simple and more or less un-
ophiles has so far relied on the chemical or enzymatic functionalized acids.[10,11] These peptide enol esters
synthesis of thioesters, or the chemical synthesis of ac- would then act as substrates for peptide coupling with
tivated esters. Most of these strategies involve the ac- a serine endopeptidase (Scheme 1).
tivation of the C-terminal carbonyl group followed by
The key challenge in our approach outlined in
addition of a nucleophile – a process which is often Scheme 1 was therefore to develop a mild, racemiza-
accompanied by a certain amount of racemization. tion-free method for the synthesis of enol esters of
We[7] and others[9] could demonstrate that the activa- quite complex peptide fragments which are signifi-
tion of peptides as Cam esters (Cam=carboxamido- cantly more prone to racemization than single amino
methyl), in which the carboxyl group serves as a nucle- acids.[2d] Among several methods considered, we iden-
ophile in the reaction with the strong electrophile io- tified the reports by Watanabe,[12] Dixneuf,[13] and
doacetamide, is a viable alternative method for C-ter- Gooßen[14] for the Ru-catalyzed addition of carboxylic
minal activation. However, this method is less suitable acids to alkynes as rewarding starting points for our
for unprotected peptides featuring nucleophilic amino investigations.[15,16] Indeed, Dixneuf and Gooßen dis-
acid side chains. In order to provide a more general closed the synthesis of amino acid enol esters without
solution to this problem, we envisaged that the addi- racemization.[13,14,17] We could positively reproduce
tion of the C-terminal carboxyl group across a termi- this finding for single amino acid substrates, but we
observed substantial epimerization of the C-terminal
amino acid residue when dipeptide Z-Leu-Phe-OH
(1a) was subjected to the reported conditions (Z=
benzyloxycarbonyl). In our original plan, we would
have preferred to produce the Markovnikov-enol
ester M-3 (Scheme 1), which is accessible via Goo-
ßenꢁs method, as a substrate for the enzymatic reac-
tion, as upon conversion a less electrophilic ketone
side product would form. However, our efforts in op-
timizing the reaction conditions did not result in a sig-
nificant reduction of epimerization, probably because
the addition of a base appears to be an important
component to achieve Markovnikov-selectivity.[14]
Gratifyingly, our parallel efforts in optimization stud-
ies for the production of the anti-Markovnikov prod-
uct AM-Z-3 were much more rewarding.
We chose Z-Leu-Phe-OH (1a) as a suitable test
substrate, as Phe is known as the amino acid to be
most prone towards racemization,[18] so any successful
reagent combination for this substrate should be suit-
Scheme 1. General scheme for the preparation of peptide
able as well for peptides bearing less challenging C-
enol esters and their use in enzymatic coupling with peptide
nucleophiles.
terminal amino acid residues (Scheme 2, Figure 1,
Table 1). In an extensive screening of different types
Scheme 2. Ru-catalyzed addition of alkynes 2 to N-protected amino acids 1.
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Racemization-Free Chemoenzymatic Peptide Synthesis
Figure 1. Structures of bidentate phosphorus ligands 6–24 used for the Ru-catalyzed alkyne addition to Z-Leu-Phe-OH (1a).
of ligands in which we measured the conversion after lowed us to conclude that a strong electronic effect of
24 h using in situ prepared Ru complexes,[19] we recog- the bidentate phosphine ligands is exerted, leading to
nized that the bite angle of the bidentate phosphine an improvement of conversion by shifting towards
ligand imposes an important influence on the reac- more electron-poor phosphines (see entries 2 and 7
tion, as has been suggested before.[13c] In the homolo- or, respectively, 4 and 8, Table 1).
gous series of bis(diphenylphosphino)alkane ligands
As the solubility of peptide substrates varies with
6a–f, dppb ligand 6d showed the highest activity lead- the amino acid side chains, the question which sol-
ing to complete conversion of substrate in 24 h, and vents were most suitable for the alkyne addition re-
with a high selectivity towards the Z-anti-Markovni- quired special attention. Ideally, a set of several sol-
kov-product AM-Z-3a. Testing ligands 7–23 which vent systems should be available addressing the chal-
possess a more rigid backbone, gave mixed results, lenges associated with varying solubilities imposed by
but no improvement in comparison with dppb 6d.
different peptide sequences. For the enol ester forma-
However, 2,3-O-isopropylidene-2,3-dihydroxy-1,4- tion with isolated catalysts 26 and 27[20] derived from
bis(diphenylphosphino)butane (DIOP) ligand 24, our best ligands dppb 6d and (+)-DIOP [(+)-24], we
which can be regarded as a conformationally restrict- observed strong solvent effects on both the reaction
ed version of dppb 6d gave superior results. Interest- rate as well as on the degree of racemization
ingly, (+)-24 showed higher activity than (À)-24 indi- (Scheme 3, Table 2).[21] In general, slightly polar sol-
cating a possible cooperative interaction between the vents, such as THF, DME, chloroform, or 1,4-dioxane
chiral catalyst and the chiral substrate [89% conver- seemed to provide the best results. Surprisingly, using
sion after 3 h in THF with (+)-24 vs. 40% conversion polar protic solvents such as MeOH, EtOH and 2-
after 3 h in THF with (À)-24]. Our studies also al- propanol led to the fastest conversions and the lowest
Adv. Synth. Catal. 2013, 355, 1799 – 1807
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Table 1. Screening of different types of bidentate phos-
ACHTUNGTRENNUNGphorus ligands 6–24 in the Ru-catalyzed addition of
tion at the C-terminal Phe residue, when using
MeOH, EtOH, 1-propanol, 2-propanol or chloroform
as solvents (entries 17–20, 22, Table 2).
Notably, a solvent which leads to a high degree of
racemization, such as dichloromethane, can be inte-
grated in a suitable solvent system if 2-propanol is
added as an additive (entries 10 and 24, Table 2).
These results make us optimistic that it will be possi-
ble to identify an appropriate solvent combination for
almost any substrate.
1-hexyne (2a) to Z-Leu-Phe-OH (1a) in THF at 408C
(conc. 0.5 mmolLÀ1).
Entry Ligand[a] Conversion AM-Z-3a/AM-E-3a/M-3a[c]
[%][b]
1
2
3
4
5
6
7
8
6a
6b
6c
6d
6e
6f
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
(À)-24
(+)-24
43
18
57
97
57
43
51
77
54
22
40
5
46
15
78
18
57
10
5
93/–/6
41/–/59
29/–/71
98/–/2
84/–/16
53/–/47
91/–/9
25/–/75
93/–/7
19/–/81
78/–/22
nd
97/–/3
83/–/17
99/–/1
64/–/36
98/–/2
nd
With (+)-DIOP 24 as the best ligand and 2-propa-
nol as the best solvent, we had conditions at hand
with which we set out to explore the scope of this re-
action for different peptide substrates 1a–j and alkyne
partners 2a–d (Scheme 4, Table 3). We were pleased
that our method allowed the quantitative and racemi-
zation-free enol esterification of a great variety of
peptide substrates with several functionalized alkynes.
Notably, the method tolerates unprotected Ser and
Tyr in substrates (entries 11 and 12). Also the S-con-
taining amino acid Met was tolerated, although for
complete conversion we had to add a second amount
of catalyst (entry 15). Fortunately, no or negligible
racemization was encountered in the Ru-catalyzed
alkyne addition using (+)-DIOP 24 as ligand.
Having now established a general and efficient
access to peptide enol esters 3a–o, we were keen to
investigate their suitability in enzymatic condensation
reactions. As a model reaction, we studied the cou-
pling of Z-Leu-Phe-enol ester 3a with H-Phe-NH2
(4a) using Alcalase cross-linked enzyme aggregates
(Alcalase-CLEA) as catalyst in THF. Unfortunately,
but not unexpectedly, we observed a considerable
amount of an imine side product, which resulted from
the reaction of H-Phe-NH2 (4a) with the aldehyde re-
leased upon reaction of our substrate 3a with the
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
nd
56
17
21
8
98
99
99/–/1
78/–/22
75/–/25
nd
96/–/4
>99/–/<1
[a]
[b]
[c]
4 mol% of ligand used based on 1a.
Determined after 24 h.
Determined by reversed-phase HPLC. nd=not deter-
mined.
amounts of racemization. Interestingly, we did not ob- serine endopeptidase in the active center. In principle,
serve any side products resulting from transesterifica- one could increase the coupling yield by increasing
tion of 3a in the presence of these alcohols. With the amount of amine reagent 4a, but this would result
(+)-24 as ligand, we could produce AM-Z-3a in 85– in a waste of valuable starting material and an in-
90% isolated yield and with less than 0.2% racemiza- crease of the purification effort during work-up. We
Scheme 3. Ru-catalyzed addition of 1-hexyne (2a) to Z-Leu-Phe-OH (1a).
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Racemization-Free Chemoenzymatic Peptide Synthesis
Table 2. Screening of various solvents in the Ru-catalyzed addition of 1-hexyne (2a) to Z-Leu-Phe-OH (1a, 0.5M) at 408C.
Conversion [%] after
Entry
Catalyst
Solvent
1 h
3 h
24 h[a]
Yield [%]
d-Phe [%][b]
1
2
3
4
5
6
7
8
26
26
26
26
26
26
26
26
26
26
26
26
26
26
26
26
27
27
27
27
27
27
27
27
THF
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
26
73
70
99
9
36
70
55
55
13
15
<1
65
<1
54
64
52
25
85
98
65
51
91
91
nd
42
98
73
98
95
98[c]
97
92
36
29
1
68
nd
nd
nd
nd
nd
nd
nd
nd
nd
85
nd
nd
nd
84
nd
85
86
87
88
90
87
88
89
0.6
0.3
2-Me-THF
1,4-dioxane
DME
DMF
NMP
DMSO
sulfolane
MeCN
CH2Cl2
0.4
5.1[d]
nd
nd
nd
86
1
3.5[d]
nd
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
90
99
82
65
98[c]
nd
92
95
nd
nd
nd
99
nd
99 h]
nd
13.2[d]
2.0
chloroform
acetone
14.2[d]
0.5
methanol[f]
ethanol
0.7
0.3
1.9
2-propanol
tert-butyl alcohol[f]
methanol[f]
ethanol
1-propanol
2-propanol
MTBE/DMF (8:2)
chloroform
CH2Cl2
<0.1
<0.1[e]
<0.1[e]
<0.1[g]
0.3
66
39
72
0.2[e]
1.5[h]
0.7[i]
CH2Cl2/2-propanol (9:1)
[a]
Sum of isomers.
[b]
[c]
[d]
[e]
[f]
After 24 h reaction time and upon subsequent hydrolysis with 6N HCl.
After 18 h.
Determined after 48 h.
Determined after 6 h.
Reaction carried out at a concentration of 0.25 mmol·LÀ1.
Determined after 1 h.
[g]
[h]
[i]
Determined after 8 h.
Determined after 3 h.
reasoned that we could avoid imine formation by ad- actions. To verify this hypothesis, we tested Z-Leu-
dressing two issues. First of all, we aimed at improv- Phe enol esters 3b and 3c containing amide bonds
ing the rate of enzymatic peptide coupling, as this re- and compared them with substrate 3a and Z-Leu-
action is kinetically independent from the slower and Phe-Cam ester 3p (Scheme 5). We were gratified to
unwanted imine formation. Taking into account that see that indeed the enol esters 3b and 3c containing
Alcalase is an endoprotease recognizing amino acids an amide motif in the enol moiety performed much
at both directions in its active site, we speculated that better than our standard hexenyl ester 3a, with 3b
the introduction of amide motifs within the enol ester being even slightly better than the corresponding
moiety would allow a better recognition of our sub- Cam ester 3p (Table 4, Figure 2). The formation of
strates by the enzyme and ultimately lead to faster re- tetrapeptide Z-Leu-Phe-Phe-Phe-NH2 can be ex-
Scheme 4. Z-anti-Markovnikov Ru-catalyzed addition of alkynes 2 to peptide fragments 1.
Adv. Synth. Catal. 2013, 355, 1799 – 1807
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(h3-CH2C(Me)=
FULL PAPERS
Table 3. Racemization-free Ru-catalyzed synthesis of amino acid and peptide enol esters 3 using [Ru
CH2)2((+)-DIOP)] (27) at 408C in 2-propanol.
ACHTUNGTRENNUNG
Entry
Peptide
Alkyne
Time [h]
Conversion [%]
Yield [%]
d-AA [%][a]
1
2
3
4
5
6
7
8
Z-Leu-Phe-OH 1a
Z-Leu-Phe-OH 1a
Z-Leu-Phe-OH 1a
Z-Leu-Phe-OH 1a
Z-Leu-Ala-OH 1b
Z-Leu-Ala-OH 1b
Z-Phe-Leu-OH 1c
Z-Phe-Leu-OH 1c
Z-Phe-Leu-Ala-OH 1d
Z-Phe-Leu-Ala-OH 1d
Boc-Phe-Tyr-OH 1e
Z-Ile-Ser-OH 1f
2a
2b
2c
2d
2a
2b
2a
2b
2a
2b
2a
2a
2b
2b
2a
2a
1
2
2
24
1
1
1
1
1
1
3
24
5
2
99
88 3a
80 3b
82 3c
nd
<0.1
<0.1
<0.1
nd
0.1
0.2
0.3
0.1
0.1
0.1
0.2
0.1
0.3
0.2
0.7
<0.1
98[b]
97[b]
0
99
92 3d
89 3e
93 3f
92 3g
87 3h
86 3i
86 3j
83 3k
78 3l
83 3m
87 3n
89 3o
99[b]
99
98[b]
99
9
10
11
12
13
14
15
16
97[b]
99
97[c]
97[b]
98[b]
97[d]
99[e]
Z-Phe-Pro-OH 1g
Z-Phe-Val-OH 1h
Z-Phe-Met-OH 1i
Z-Phe-OH 1j
7
24
[a]
Amount of C-terminal racemization determined after the indicated reaction time upon hydrolysis with 6N HCl.
Bis-addition products up to 2% relative to product and starting material were detected.[22]
Ethanol used as solvent, 1.5% ethyl ester detected.
Addition of 4 mol% of catalyst after 3 h.
Catalyst 26 used.
[b]
[c]
[d]
[e]
form coupling reactions with larger amino frag-
ments.[7f] Fortunately, peptide enol esters 3b and 3e
were efficiently converted to the corresponding tetra-
or 7-mer in the presence of Alcalase and the appro-
priate amino fragment (Scheme 6, Table 5). The re-
sults indicate that the coupling protocol is fully appli-
cable with longer peptide fragments.
As a second line of thought in the effort to avoid
imine formation, we speculated that the molecular
sieves added to remove water might also serve as
a catalyst for the undesired imine formation, as its
acidic properties are well recognized.[23] Our efforts to
Scheme 5. Alcalase-catalyzed condensation of peptide enol
esters 3 with H-Phe-NH2 (4a).
plained by the high transamidation activity of Alca- replace the molecular sieves in the reaction mixture
lase in case of N-terminal phenylalanine amide resi- by other dehydrating agents such as alumina, MgSO4,
dues to catalyze a chain elongation of previously or Na2SO4 were not rewarded, as these reagents
formed 4a. In all other cases, this secondary reaction either even aggravated the imine formation or turned
was slower or even not observed.
out to be inefficient in removing residual water. For-
Based on the promising results of the kinetic study tunately, when using a Soxhlet-type set up (Figure 1
with type-3b peptide enol esters, we went on to per- in the Supporting Information), in which the molecu-
Table 4. Comparison of various esters 3 in the Alcalase-catalyzed peptide condensation with H-Phe-NH2 (4a) in THF at
508C.
Entry
Peptide ester
Conversion[%][a]
Product composition[b]
1
2
3
4
Z-Leu-Phe-OCam 3p
Z-Leu-Phe-O(Z)-hex-1-enyl 3a
Z-Leu-Phe-O(Z)-6-amino-6-oxohex-1-enyl 3b
Z-Leu-Phe-O(Z)-5-amino-5-oxopent-1-enyl 3c
98
70
99
93
87:11 (–)
63:7 (2)
91:8 (3)
85:8 (<1)
[a]
Determined after 3 h reaction time by reversed-phase HPLC.
Product composition Z-Leu-Phe-Phe-NH2 (5a):Z-Leu-Phe-Phe-Phe-NH2 (imine formation relative to starting material+
[b]
products).
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Racemization-Free Chemoenzymatic Peptide Synthesis
Figure 2. Kinetic comparison of Alcalase-catalyzed peptide coupling of various activated esters 3 with H-Phe-NH2 (4a) in
THF at 508C.
molecular sieves in imine formation (Scheme 7).
Combining these two insights allowed us to define
a reaction protocol which leads to the high-yielding
chemoenzymatic synthesis of oligopeptides. For our
test reaction, we obtained Z-Leu-Phe-Phe-NH2 (5a)
in 91% isolated yield, demonstrating that our now
racemization-free protocol toward peptide enol esters
gives us access to peptide acyl donors which can be
efficiently condensed in our Alcalase peptide coupling
platform.[7]
Conclusions
Scheme 6. Alcalase-catalyzed condensation of peptide enol
esters 3b and 3e with amino fragments (4b–e).
In conclusion, we have demonstrated that under ap-
propriate conditions the Ru-catalyzed addition of al-
kynes using (+)-DIOP as a ligand is a convenient
lar sieves did not get into contact with the amino nu- method for the racemization-free preparation of C-
cleophile and aldehyde side product anymore, while terminal peptide enol esters, which tolerates a consid-
removing water from the THF azeotrope, imine for- erable number of unprotected amino acids. These
mation was reduced to a negligible amount, confirm- peptide enol esters are excellent substrates in chemo-
ing our hypothesis about the catalytic effect of the enzymatic peptide coupling reactions using Alcalase.
Table 5. Alcalase-catalyzed peptide ligation using enol esters 3 and various amino fragments 4 at 508C in THF.
Entry Enol ester Amino fragment
Time [h] Conversion [%] Isolated yield [%][b] Purity [%][c]
1
2
3
4
5
6
3b
3b
3e
3e
3e
3e
H-Ala-Val-NH2[a] 4b
H-Leu-Phe-NH2 4c
H-Ala-Val-NH2[a] 4b
H-Gly-Phe-NH2 4d
H-Leu-Phe-NH2 4c
91
22
22
22
22
61
71
84
74
91
63
62 5b
30 5c
78 5d
41 5e
51 5f
27 5g
94
87
82
80
98
92
[a]
H-Phe-Val-Leu-Met-Gly-NH2 4e 91
[a]
[b]
[c]
Used as hydrochlorides.
Purified by preparative reversed-phase HPLC.
Determined by reversed-phase HPLC at 210 nm.
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CH-(CH3)2, -CO2-CH=CH-CH2-(CH2)2-CH3], 2.04–2.11 (m,
2H, -CH=CH-CH2-CH2-), 3.15–3.17 (m, 2H, C6H5-CH2-CH-
NH), 4.17–4.22 [m, 1H, NH-CH
4.92–4.99 [m, 2H, CO-NH-CH(CH2-Ph)-CO2-, -CO2-CH=
CH-CH2-], 5.06–5.15 (m, 3H, HN-CO2-CH2-C6H5, -NH-),
ACHTUTGNREN(NUG CH2-CHACHTUNGTREN(NGUN CH3)2)-CO-NH],
AHCTUNGTRENNUNG
3
6.51 (d, 1H, 3JH,H =7.2 Hz, -NH-), 6.97 (d, 1H, JH,H
=
6.3 Hz, CO2-CH=CH-CH2), 7.09–7.36 (m, 10H, HAr);
13C NMR (75 MHz, CDCl3): d=14.0 [CH3-(CH2)3-CH=CH-
O2C], 22.1, 22.3, 23.0, 24.2, 24.8, 31.3 (Caliphatic), 38.1 (C6H5-
CH2-CH-NH-), 41.4 [NH-CH-CH2-CH-(CH3)2], 53.2, 53.6
(2ꢂNH-CH), 67.3 (HN-CO2-CH2-C6H5), 115.8 (CO2-CH=
CH-CH2-), 127.4 (CAr), 128.2 (2ꢂCAr), 128.4 (CAr), 128.7 (2ꢂ
CAr), 128.8 (2ꢂCAr), 129.4 (2ꢂCAr), 133.6 (CO2-CH=CH-
CH2-), 135.5 (Cq,Ar), 136.3 (Cq,Ar), 156.2 (NH-CO2-CH2-
C6H5), 168.7 (Cq), 171.9 (Cq); HR-MS: m/z=517.2716, calcd.
for C29H38N2O5 [MNa]+: 517.2678; [a]2D5: À13.18 (c 0.5,
chloroform).
Scheme 7. Alcalase-catalyzed coupling of 3b and H-Phe-
NH2 (4a) under Soxhlet conditions.
Amide recognition motifs within the enol ester
moiety are recognized by the enzyme and enable
more favorable reaction rates and coupling yields.
With this universal method for making enol esters
available, by taking advantage of the power of Ru-
catalyzed alkyne addition, we have expanded the
scope of enzyme-catalyzed peptide fragment conden-
sation. We are confident that such a strategy in which
the advantages of homogeneous catalysis and bioca-
talysis are combined, will offer many more rewarding
opportunities. Activities towards this goal are current-
ly being pursued in our laboratories.
Acknowledgements
This work has been supported by the Federal Ministry of
Economy, Family and Youth (BMWFJ), the Federal Ministry
of Traffic, Innovation and Technology (BMVIT), the Styrian
Business Promotion Agency SFG, the Standortagentur Tirol
and ZIT – Technology Agency of the City of Vienna through
the COMET-Funding Program managed by the Austrian Re-
search Promotion Agency FFG. We thank Bernhard Wçlfl
for skilful assistance in experimental work.
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Experimental Section
General Procedure for Ru-Catalyzed Enol Ester
Formation of Peptides using 1-Hexyne (2a)
All experiments were carried out under argon in an oven-
dried Schlenk tube containing a magnetic stirring bar. The
vessel was charged with Z-Leu-Phe-OH (1a) (103.1 mg,
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Adv. Synth. Catal. 2013, 355, 1799 – 1807
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
asc.wiley-vch.de
1807