One-step C-terminal deprotection and activation of peptides with peptide amidase from stenotrophomonas maltophilia in neat organic solvent

Article abstract of DOI:10.1002/adsc.201400109

Chemoenzymatic peptide synthesis is a rapidly developing technology for cost effective peptide production on a large scale. As an alternative to the traditional C→N strategy, which employs expensive N-protected building blocks in each step, we have investigated an N→C extension route that is based on activation of a peptide C-terminal amide protecting group to the corresponding methyl ester. We found that this conversion is efficiently catalysed by Stenotrophomonas maltophilia peptide amidase in neat organic media. The system excludes the possibility of internal peptide cleavage as the enzyme lacks intrinsic protease activity. The produced peptide methyl ester was used for peptide chain extension in a kinetically controlled reaction by a thermostable protease.

Full text of DOI:10.1002/adsc.201400109

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DOI: 10.1002/adsc.201400109
One-Step C-Terminal Deprotection and Activation of Peptides
with Peptide Amidase from Stenotrophomonas maltophilia in
Neat Organic Solvent
Muhammad I. Arif,a Ana Toplak,a Wiktor Szymanski,a,b Ben L. Feringa,b
Timo Nuijens,c Peter J. L. M. Quaedflieg,c Bian Wu,a,* and Dick B. Janssena,*
a
Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen,
Nijenborgh 4, 9747 AG Groningen, The Netherlands
Fax : (+31)-50-363-4165; phone; (+31)-50-363-4209; e-mail: b.wu@rug.nl or d.b.janssen@rug.nl
Center for Systems Chemistry, Stratingh Institute for Chemistry, University of Groningen, Nijenborgh 4, 9747 AG
b
Groningen, The Netherlands
Enzypep B.V., P.O. Box 18, 6160 MD Geleen, The Netherlands
c
Received: February 7, 2014; Published online: June 20, 2014
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201400109.
Abstract: Chemoenzymatic peptide synthesis is
a rapidly developing technology for cost effective
peptide production on a large scale. As an alterna-
tive to the traditional C!N strategy, which employs
expensive N-protected building blocks in each step,
we have investigated an N!C extension route that
is based on activation of a peptide C-terminal
amide protecting group to the corresponding
methyl ester. We found that this conversion is effi-
ciently catalysed by Stenotrophomonas maltophilia
peptide amidase in neat organic media. The system
excludes the possibility of internal peptide cleavage
as the enzyme lacks intrinsic protease activity. The
produced peptide methyl ester was used for peptide
chain extension in a kinetically controlled reaction
by a thermostable protease.
offers notable advantages, including minimal need for
protection of side chains, absence of racemisation,
and environmentally friendly reaction conditions.
Chemoenzymatic peptide synthesis is usually carried
out with a protease via a kinetically controlled ap-
proach.[4] To prevent undesired oligomerisation, the
C-terminally activated amino acid or peptide used as
the acyl donor is N-terminally protected and the nu-
cleophile is an amino acid or a peptide modified by
a C-terminal amide group. After each elongation step,
a selective deprotection of one of the termini is re-
quired for further chain extension. Elongation of the
peptides in the C!N direction is highly cost-ineffi-
cient because it requires an expensive acyl donor at
each step and repeated laborious N-terminal depro-
tections. On the other hand, in the direction of N!C
peptide elongation, much cheaper amino acid or pep-
tide amides can be used. This strategy requires depro-
tection (amide removal) and re-activation (e.g., as
alkyl ester) of the product at its C-terminus. Ideally,
the deprotection and reactivation should be per-
formed simultaneously.
Keywords: chemoenzymatic synthesis; esterifica-
tion; organic solvents; peptide amidase; peptide
synthesis; peptides
Recently, such a peptide C-terminal deprotection–
reactivation was achieved by alkoxy-deamidation,
In the past decades, a large number of peptides has using the protease subtilisin A.[5] However, the inher-
been commercialised for a wide variety of applica- ent nature of the protease always presents the risk of
tions, e.g., as therapeutic agents, nutritional supple- internal peptide hydrolysis. To avoid unwanted pro-
ments, and cosmetics.[1] Despite the increasing indus- teolysis of the substrate, such reactions should employ
trial demand for bioactive peptides, cost-efficient a C-terminus selective enzyme. Peptide amidase from
large-scale peptide synthesis remains challenging.[2] In flavedo of oranges (PAF) has been used for the inter-
recent years, chemoenzymatic peptide synthesis has conversion of peptide amides into methyl esters.[6]
become a useful alternative to conventional chemical However, PAF is not stable enough in neat organic
synthesis and recombinant fermentation methods.[3] solvents and requires at least 0.5% (v/v) of water in
Compared to solid phase and solution phase chemical the reaction system. Consequently, the yield of pep-
peptide synthesis, enzyme-catalysed peptide synthesis tide ester was only moderate due to substantial hy-
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Muhammad I. Arif et al.
has not been investigated. Therefore, we decided to
explore the use of PAM as a catalyst for peptide C-
terminal alkoxy-deamidation (Scheme 1).
First, we tested the catalytic activity of PAM in
aqueous solution. The methoxy-deamidation of the
model substrate Z-Gly-Tyr-NH2 was carried out in
50 mM phosphate buffer (pH 8.0) in the presence of
methanol. With high methanol content (>85%, v/v),
Scheme 1. Peptide C-terminal alkoxy-deamidation using
PAM.
drolysis of the peptide amide. In addition, commercial the formation of Z-Gly-Tyr-OMe could be detected
utilisation of PAF is limited by the fact that the by LC/MS, albeit with low conversion rate and very
enzyme has not been cloned and isolation of the high degree of hydroxy-deamidation (hydrolysis of
enzyme from orange flavedo is a low-yield process. A the amide). A further decrease of the water content
different peptide amidase, originating from Stenotro- resulted in complete inactivation of the enzyme. Nev-
phomonas maltophilia (PAM), has been cloned, ex- ertheless, it was clear that PAM indeed possesses pro-
pressed and characterised.[7] PAM is a serine hydro- miscuous esterase activity and tolerates considerable
lase that specifically catalyses the C-terminal deami- amounts of organic solvent.
dation of peptide amides, without affecting internal
Encouraged by these initial results, we decided to
peptide bonds or amide functions in amino acid side- use methanol in neat organic solvents to prevent the
chains.[7] So far, only hydrolytic deamidation activity hydrolytic side reaction. Different organic solvents,
of this enzyme was reported and the possibility to covering a broad logP range, were tested (Table 1).
suppress hydrolysis by using a neat organic solvent We performed conversions in neat organic solvent
containing 5% (v/v) methanol and 2% (v/v) DMF,
lyophilised PAM, and molecular sieves to retain
a very low water activity during the reaction. PAM
showed activity in a variety of organic solvents, with
Table 1. Effect of organic solvents on the C-terminal me-
thoxy-deamidation.[a]
acetonitrile being the best solvent for the model reac-
tion (Table 1). Further experiments showed that the
optimal concentration of methanol was 10% (v/v)
(see the Supporting Information). Although it was
suggested that the use of apolar solvents is beneficial
for enzyme stability,[8] we did not observe a correlation
between solvent polarity and PAM in our study.
Since small molecules have been used to protect an
enzyme from deactivation during freeze-drying,[9] we
further optimised the biocatalyst preparation method.
Different lyoprotectants, including sugars, polymers,
and simple salts were tested (Table 2). Sucrose pro-
vided the highest degree of lyoprotection for PAM.
Lyophilising the enzyme with sucrose in an optimised
1:16.7 weight ratio resulted in over three-fold higher
conversion (83% conversion in 24 h, see the Support-
ing Information), although a certain degree of hydrol-
ysis of the amide substrate was observed (~10%).
Organic solvent
logP
Yield [%][b] of
Z-Gly-Tyr-OCH3
DMSO
DMF
methanol
acetonitrile
dioxane
acetone
2-propanol
THF
tert-butyl alcohol
MTBE
toluene
ꢀ1.412
ꢀ0.829
ꢀ0.69
ꢀ0.334
ꢀ0.255
ꢀ0.042
0.173
0.473
0.584
0.94
2.73
<0.5
<0.5
<0.5
40
5
17
9
10
6
28
<0.5
[a]
Reaction conditions: lyophilised PAM (0.35 mg), various
organic solvents (93%, v/v), methanol (5%, v/v), DMF
(2%, v/v), Z-Gly-Tyr-NH2 (5 mM), 3ꢁ molecular sieves
(7 mg), total volume 0.25 mL, 308C, 72 h.
[b]
Determined by HPLC.
Table 2. Effect of different excipients on PAM esterification activity in the acetonitrile/methanol co-solvent system.[a]
Excipients
Total conversion [%]
Yield [%][b] of Z-Gly-Tyr-OCH3
Yield [%][b] of Z-Gly-Tyr-OH
polyethylene glycol
sucrose
sorbitol
trehalose
potassium chloride
tris
19.5
52.1
<0.5
3.9
26.9
40.4
19.5
51.0
<0.5
3.9
26.9
33.2
<0.5
1.1
<0.5
<0.5
<0.5
7.2
[a]
Reaction conditions: PAM (0.35 mg, excipients:enzyme ratio=98:1, w/w), acetonitrile (93%, v/v), methanol (5%, v/v),
DMF (2%, v/v), Z-Gly-Tyr-NH2 (5 mM), 3ꢁ molecular sieves (7 mg), total volume 0.25 mL, 308C, 20 h.
Determined by HPLC.
[b]
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One-Step C-Terminal Deprotection and Activation of Peptides
Table 3. Alkoxy-de-amidation of Z-Gly-Tyr-NH2 with different alcohols.[a]
Alcohol
Reaction time (days)
Yield [%][b] of the peptide ester
Yield [%][b] of Z-Gly-Tyr-OH
methanol
ethanol
propanol
2-propanol
butanol
1
68.5ꢁ8.7
21.8ꢁ2.5
11.9ꢁ0.2
<0.5
1.5ꢁ1.5
11
11
11
11
11
24.8ꢁ1.5
22.8ꢁ1.2
30.0ꢁ5.2
32.4ꢁ9.6
21.2ꢁ1.2
8.4ꢁ0.5
tert-butyl alocohol
<0.5
[a]
Reaction conditions: PAM (0.35 mg, lyophilised with 5.85 mg sucrose), acetonitrile (93%, v/v), alcohol (5%, v/v), DMF
(2%, v/v), Z-Gly-Tyr-NH2 (5 mM), 3ꢁ molecular sieves (7 mg), total volume 0.25 mL, 308C.
Product identity was confirmed by LC/MS and yields were determined by HPLC.
[b]
Table 4. Methoxy-de-amidation of different amino acids and peptides with methanol.[a]
Substrate
Yield [%][b] of the peptide ester
Yield [%][b] of the hydrolytic product
Total conversion [%]
Z-Tyr-NH2
Z-Gly-NH2
68.6ꢁ0.4
70.5ꢁ0.7
73.9ꢁ4.3
75.4ꢁ0.6
43.0ꢁ0.3
67.7ꢁ4.2
42.1ꢁ5.3
65.6ꢁ3.6
58.1ꢁ1.3
23.5ꢁ4.0
7.0ꢁ0.2
4.1ꢁ0.3
4.1ꢁ0.2
5.1ꢁ0.2
4.5ꢁ0.7
8.1ꢁ3.7
9.2ꢁ0.8
9.9ꢁ3.0
7.0ꢁ1.5
3.7ꢁ0.4
75.6ꢁ0.6
74.6ꢁ0.4
78.0ꢁ4.2
80.4ꢁ0.8
47.1ꢁ0.5
75.8ꢁ7.9
51.3ꢁ6.1
75.5ꢁ6.6
65.1ꢁ0.3
27.3ꢁ4.4
Z-Gly-Tyr-NH2
Z-Gly-Leu-NH2
Z-Phe-Gly-NH2
Z-Gly-Phe-NH2
Z-Leu-Phe-NH2
Z-Pro-Gly-NH2
Z-Phe-Ala-NH2
Z-Pro-Leu-Gly-NH2
[a]
Reaction conditions: PAM (0.35 mg, lyophilised with 5.85 mg sucrose), acetonitrile (88%, v/v), alcohol (10%, v/v), DMF
(2%, v/v), Z-Gly-Tyr-NH2 (5 mM), 3ꢁ molecular sieves (7 mg), total volume 0.25 mL, 308C, 16 h.
Product identity was confirmed by LC/MS and yields were determined by HPLC.
[b]
To determine the scope of the PAM-mediated pep- performed the reaction on a 50 mg semi-preparative
tide C-terminal alkoxy-deamidation, we tested scale under optimised conditions. After 7 days, the
a range of alcohols. We observed conversion of Z- conversion of Z-Gly-Tyr-NH2 reached a value of 99%
Gly-Tyr-NH2 only if small, aliphatic, primary alcohols (95% of Z-Gly-Tyr-OMe, 4% of Z-Gly-Tyr-OH).
were used, with methanol giving the best ester yield After work-up and purification, the yield of Z-Gly-
(Table 3). This observation suggests the nucleophile Tyr-OMe was 78%. The identity and purity of the en-
binding pocket of PAM is relatively small, which is in zymatically prepared peptide ester werer confirmed
agreement with what has been observed in the PAM by HPLC and NMR analysis, and compared to the
X-ray structure.[7] In this near-anhydrous organic co- chemically prepared reference compound.
solvent system, the alcohol still needs to compete
Finally, we tested the applicability of PAM in a che-
with the remaining water for access to the enzyme moenzymatic N!C peptide elongation concept. For
active site and reaction with the covalent acyl-enzyme this two-enzyme reaction, PAM was used first for the
intermediate.[10] We observed a higher degree of hy- conversion of Z-Gly-Tyr-NH2 to the corresponding
drolysis upon increasing the length of alkyl chain of methyl ester. Subsequently, two newly described ther-
the alcohol. Likely, steric hindrance accounts for this mostable proteases DgSbt and TaqSbt[11] were used
phenomenon.
for coupling of the obtained Z-Gly-Tyr-OMe with
In an aqueous environment, PAM displayed Phe-NH2 as the nucleophile (Scheme 2). For a one-
a broad peptide substrate scope in the hydrolytic re- pot reaction, PAM was removed by centrifugation
action.[7] To explore the substrate specificity of PAM after the first reaction, and the protease DgSbt was
in a neat organic co-solvent system, we tested svereal added directly to the reaction mixture. Although the
amino acids amides and peptide amides. As shown in reaction rate was low, the yield of coupled peptide
Table 4, several substrates were efficiently converted. reached a notable value of 76%. In another experi-
To investigate the practical applicability of PAM- ment, we used purified Z-Gly-Tyr-OMe as substrate
catalysed peptide C-terminal alkoxy-deamidation, we in a subsequent TaqSbt protease-catalysed coupling
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Scheme 2. N!C peptide elongation using a combination of PAM-catalysed deprotection/activation and protease-catalysed
transacylation.
centrifugation and the clear enzyme solution was stored in
reaction, yielding 77% of Z-Gly-Tyr-Phe-NH2 product
aliquots at ꢀ208C until further use. The yield of purified
over 6 days. With the removal of protease from the
reaction mixture, the product could in principle be
used in another elongation step.
protein was ~100 mgLꢀ1 culture. For routine purposes, ali-
quots (50 mL) containing 0.35 mg enzyme were lyophilised
overnight.
In conclusion, we have established that PAM is
a useful catalyst for converting a peptide C-terminal
amide to a C-terminal ester, which can readily be
used for enzymatic peptide elongation. To the best of
our knowledge, this is the first reported application of
this class of enzyme in a near-neat organic solvent
system. Compared with the use of an enzyme isolated
from a natural source, recombinant production of
peptide amidase is convenient and more efficient.
More importantly, it forms a platform for further pro-
tein engineering aimed at developing higher activity
towards the target of interest. Considering the specif-
icity for the C-terminus of peptides, the negligible hy-
Solvent Optimisation for Methoxy-deamidation with
PAM
Samples of 0.35 mg enzyme were lyophilised in a reaction
vial. Approximately 7 mg of activated 3 ꢁ molecular sieves
were added to each reaction vial followed by addition of or-
ganic solvent (93%, v/v), methanol (5%, v/v) and Z-Gly-
Tyr-NH2 (5 mM, final concentration) as substrate. All reac-
tions contained 2% (v/v) DMF to solubilise the substrate.
Total reaction volume was 250 mL. The reaction mixtures
were incubated at 308C for 24 h with shaking at 400 rpm on
a table top incubator. For analysis, reaction vials were brief-
ly centrifuged and 5 mL samples were taken, mixed with
drolysis of internal peptide bonds and side-chain 50 mL of glacial acetic acid and analysed by HPLC (Jasco
LC-NetII/ADC) equipped with a reverse phase column
(Altech Alltima, C18, 3 mm particle size, 53 mmꢂ7 mm).
Elution was carried out in an isocratic mode with 40% ace-
tonitrile in water as eluent. The chromatograms were re-
corded at 210 nm.
amides, the broad substrate spectrum and the high
tolerance towards organic solvents, this enzyme is an
attractive addition to the toolbox of enzymes for che-
moenzymatic peptide synthesis and may provide new
possibilities for various C-terminal modifications of
peptides.
Preparative Scale Conversion of Z-Gly-Tyr-NH2 to Z-
Gly-Tyr-OCH3
A 25 mg sample of PAM was lyophilised with 417 mg of su-
crose for 48 h. To this, 2 g of 3ꢁ molecular sieves were
added followed by addition of 50 mg of Z-Gly-Tyr-NH2 dis-
solved in acetonitrile:DMF (49:1, v/v). Subsequently, metha-
nol was added to a final concentration of 10% (v/v). The
total volume of the reaction mixture was 15 mL. All solvents
used were pre-incubated with 3ꢁ molecular sieves for
30 min. The reaction was performed at 308C with shaking at
90 rpm. Approximately 50–100 mL samples were withdrawn
at 24 h intervals to monitor the progress of the reaction.
After 4 days, 1 g of 5ꢁ molecular sieves was added to
absorb the released ammonia and the reaction was further
incubated for two days. After 6 days of incubation, another
1 g of 5ꢁ molecular sieves was added to the system. At the
end of the 7th day, the suspension was centrifuged to
remove lyophilised enzyme and molecular sieves. The re-
maining supernatant was evaporated under vacuum and the
pellet was redissolved in 20 mL ethyl acetate. The ethyl ace-
tate layer was washed twice with 15 mL NaHCO3, twice
with 15 mL 0.1N HCl and twice with 15 mL brine. The or-
ganic phase was dried (Na2SO4) and concentrated under
vacuum after removing salt from the suspension. The dried
pellet was redissolved in ethylacetate:pentane (1:1, v/v) and
applied on a 2 mL silica gel column (60 ꢁ, 230–400 mesh
particle size, Aldrich) and fractions were collected. TLC was
performed to analyse the purity of fractions. TLC plates
Experimental Section
Expression and Purification of PAM
The PAM gene from S. maltophilia was obtained as codon-
optimised synthetic construct from DNA 2.0. The PAM
gene, without the N-terminal signal sequence, was further
cloned in the pET21a(+) vector (Novagen), containing a C-
terminal his-tag sequence, and transformed to E. coli Origa-
mi (DE3) for expression. Cultures were grown in 2 L of LB
medium initially at 378C and cells were induced at 308C
with 75 mM IPTG at OD600 =0.6. After 24 h, cells were col-
lected by centrifugation and a 20% (w/w) cell suspension
was prepared in 20 mM potassium phosphate buffer, pH 7.5,
containing 0.5M NaCl and 0.02M imidazole. The suspension
was sonicated and the cell-free extract was obtained by cen-
trifugation at 15000 rpm for 1 h. The cell-free extract was
applied on a 5-mL HisTrap column and proteins were col-
lected by elution with 20 mM potassium phosphate buffer,
pH 7.5, containing 0.5M NaCl and 0.2M imidazole. The pu-
rified enzyme was desalted in 20 mM potassium phosphate
buffer (pH 7.5) and further concentrated via Amicon filtra-
tion (30 kDa). The enzyme was further incubated at 308C
for up to 10 days, since we observed that the purified
enzyme slowly became more active by an as yet unknown
mechanism. After incubation, precipitates were removed by
2200
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One-Step C-Terminal Deprotection and Activation of Peptides
were visualised by immersion in cerium ammonium molyb-
date (CAM) solution followed by drying with hot air. Puri-
fied fractions (Rf =0.2) were pooled and further analysed on
HPLC for purity. The material was further dried under
vacuum and subjected to NMR analysis. 1H NMR
(400 MHz, CDCl3): d=2.96–3.04 (m, 2H, CH2CH), 3.73 (s,
3H, CH3O), 3.72–3.90 (m, 2H, Gly-CH2), 4.81–4.85 (m, 1H,
CH2CH), 5.12 (s, 2H, CH2O), 5.45 (br s, 1H, OH), 6.13 (br
molecular sieves powder (100 mgmLꢀ1). The reaction mix-
ture was shaken at 400 rpm at 608C. Samples were taken
and quenched with DMSO (1:3 v/v ratio). After 6 days, 77%
conversion was achieved. Conversions were estimated by
HPLC using the same procedure as described above and fur-
ther identified by LC/MS.
3
s, 1H, CbzNH), 6.53 (br d, 1H, NHCH), 6.67 (d, J=8.0 Hz,
2H, phenol-H), 6.90 (d, 3J=8.0 Hz, 2H, phenol-H), 7.31–
7.40 (m, 5H, Ph-H).
Acknowledgements
This project is part of Integration of Biosynthesis and Organ-
ic Synthesis program (IBOS-2; program number: 053.63.014)
funded by The Netherlands Organization for Scientific Re-
search (NWO) and Advanced Chemical Technologies for
Sustainability (ACTS). The authors thank Dr. J.-M. van der
Laan from DSM Food Specialties for helpful discussions.
Preparation of Z-Gly-Tyr-OCH3 Reference
Compound
A mixture of tyrosine methyl ester hydrochloride (1.0 mmol,
232 mg), Z-Gly (1.0 mmol, 209 mg), HOBt (1.5 mmol,
203 mg) and TEA (3.0 mmol, 418 mL) in DCM (10 mL) was
cooled in an ice-water bath. EDC (1.1 mmol, 211 mg) was
added and the cooling was removed. After 20 h, volatiles
were evaporated and the residue was redissolved in AcOEt
(30 mL). The organic solution was washed with 10% (w/v)
aqueous citric acid solution (3ꢂ20 mL), saturated aqueous
NaHCO3 (2ꢂ20 mL) and brine (20 mL), dried (MgSO4) and
the solvent was evaporated. The products were purified by
flash chromatography (silica gel, 40–63 mm, pentane/AcOEt,
1:1, v/v) to give a white powder; yield: 292 mg (75%); Rf =
0.15 (pentane/AcOEt, 1:1, v/v). 1H NMR (400 MHz,
CDCl3): d=2.96–3.04 (m, 2H, CH2CH), 3.73 (s, 3H, CH3O),
3.72–3.90 (m, 2H, Gly-CH2), 4.81–4.85 (m, 1H, CH2CH),
5.12 (s, 2H, CH2O), 5.42 (br s, 1H, OH), 5.88 (br s, 1H,
ZNH), 6.48 (br d, 1H, NHCH), 6.67 (d, 3J=8.0 Hz, 2H,
phenol-H), 6.90 (d, 3J=8.0 Hz, 2H, phenol-H), 7.31–7.40
(m, 5H, Ph-H); 13C NMR (75 MHz, CDCl3): d=37.1, 44.5,
52.7, 53.6, 67.5, 115.9, 127.0, 128.3, 128.5, 128.8, 130.5, 136.2,
155.8, 157.1, 169.6, 172.3; HR-MS (ESI+): m/z=409.1366,
calcd. for C20H22N2O6Na: 409.1370.
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Kinetic Coupling of Z-Gly-Tyr-OMe with H-Phe-NH2
using DgSbt Protease
To the amidase reaction mixture containing N-protected
acyl donor Z-Gly-Tyr-OMe (8 mmol) in acetonitrile
(1.5 mL), C-terminally protected Phe-NH2 (10 equiv.,
80 mmol) was directly added, as well as 90 mg IPREP (iso-
propyl alcohol-rinsed enzyme precipitate) containing 4 mg
of DgSbt and activated 3ꢁ crushed molecular sieves
(200 mgmLꢀ1). The reaction mixture was shaken at 400 rpm
at 608C. Samples were taken and quenched with DMSO
(1:3, v/v). After 21 days conversion to product was 76%.
Conversions were estimated using HPLC by integration of
the acyl donor starting material and the product peaks as-
suming identical response factors. Products were identified
by LC/MS.
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using TaqSbt Protease
In a parallel coupling reaction, to the purified N-protected
acyl donor Z-Gly-Tyr-OMe (28 mmol) in acetonitrile
(3 mL), C-terminally protected Phe-NH2 (10 equiv.,
280 mmol) was added, followed by 35 mg IPREP enzyme
preparation (2.2 mg of TaqSbt enzyme) and activated 3ꢁ
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Adv. Synth. Catal. 2014, 356, 2197 – 2202