Subtilisin-catalyzed synthesis of amino acid and peptide esters. Application in a two-step enzymatic ligation strategy.

Article abstract of DOI:10.1021/ol0167614

We describe an efficient enzymatic approach to the synthesis of amino acid and peptide esters. The serine protease subtilisin Carlsberg (EC 3.4.21.62) was found to efficiently catalyze the specific formation of C(alpha)-carboxyl 3-hydroxypropyl or 4-hydro

Full text of DOI:10.1021/ol0167614

ORGANIC
LETTERS
2001
Vol. 3, No. 26
4157-4159
Subtilisin-Catalyzed Synthesis of Amino
Acid and Peptide Esters. Application in
a Two-Step Enzymatic Ligation Strategy
Chuan-Fa Liu† and James P. Tam*
Department of Microbiology and Immunology, A-5116, MCN,
NashVille, Tennessee 37232-2363
tamjp@ctrVax.Vanderbilt.edu
Received September 16, 2001
ABSTRACT
We describe an efficient enzymatic approach to the synthesis of amino acid and peptide esters. The serine protease subtilisin Carlsberg (EC
r
3.4.21.62) was found to efficiently catalyze the specific formation of C -carboxyl 3-hydroxypropyl or 4-hydroxybutyl esters of certain Boc-
amino acids and peptides in high-content 1,3-propanediol or 1,4-butanediol solution, with substrate specificity parallel to that of the normal
hydrolytic reaction. This approach can be coupled with kinetic-control reverse proteolysis in a two-step enzymatic peptide ligation scheme.
Amino acid and peptidyl CR-esters are important and versatile
intermediates in organic and peptide synthesis. Such esters
are widely used for temporary protection of the R-COOH
groups,1 and more importantly they can also be transformed
to a variety of functionalities2 such as amide, hydrazide, or
aldehyde for further transformation to form more complex
structures. A particularly prominent application of these esters
resides in their use as acyl donors in kinetically controlled
enzymatic peptide synthesis.3 Simple amino acid esters are
traditionally prepared by chemical means through either
strong acid catalysis or intervention of a dehydration agent.
At the same time, special resins can be developed for
preparing peptide esters by solid-phase synthesis.4,2c-d,3f-g
Here, we report an alternative strategy of using a simple and
mild enzymatic method for the preparation of amino acid or
peptide CR-esters. Although serine or cysteine proteases are
very efficient in catalyzing the hydrolysis of peptide CR-
esters, more so than in the hydrolysis of the respective
peptide bonds, the reverse esterase activity of such proteases
for ester synthesis has seldom been exploited. One pioneer
work was Kise’s investigation on the use of R-chymotrypsin
and subtilisin for the esterification, with poor to moderate
efficiency, of simple NR-protected aromatic amino acids in
high-content ethanol solution.5 We found that the bacterial
serine protease subtilisin Carlsberg retained high catalytical
† Current address: Amgen Inc., AC-5A, 4000 Nelson Rd., Longmont,
CO 80503.
(1) Bodanszky, M. Principle of Peptide Synthesis; Springer-Verlag:
Berlin, 1984; pp 69-79.
(2) (a) Strachan, R. G.; Paleveda, W. J., Jr.; Nutt, R. F.; Vitali, R. A.;
Veber, D. F.; Dickinson, M. J.; Garsky, V.; Deak, J. E.; Walton, E., Jenkins,
S. R.; Holly, F. W.; Hirschmann, R. J. Am. Chem. Soc. 1969, 91, 503. (b)
Merrifield, R. B. AdV. Enzymol. 1969, 32, 221. (c) Liu, C. F.; Tam, J. P. J.
Am. Chem. Soc. 1994, 116, 4149. (d) Liu, C. F.; Tam, J. P. Proc. Natl.
Acad. Sci. U.S.A. 1994, 91, 6584.
(3) (a) Morihara, K.; Oka, T. Biochem. J. 1977, 163, 531. (b) Mitin, Y.
V.; Zapevalova, N. P.; Gorbunova, E. Y. Int. J. Peptide Protein Res. 1984,
23, 528. (c) Nakatsuka, T.; Sasaki, T.; Kaiser, E. T. J. Am. Chem. Soc.
1987, 109, 3808. (d) Margolin, A. L.; Klibanov, A. M. J. Am. Chem. Soc.
1987, 109, 3802. (e) Barbas, C. F., III; Matos, J. R.; West, B.; Wong, C.-
H. J. Am. Chem. Soc. 1988, 110, 5162. (f) Abrahmsen, L.; Tom, J.; Burnier,
J.; Butcher, K. A.; Kossiakoff, A.; Wells, J. A. Biochemistry 1991, 30, 4151.
(g) Jackson, D. Y.; Burnier, J.; Quan, C.; Stanley, M.; Tom, J.; Wells, J.
A. Science 1994, 266, 243. For reviews on enzymatic peptide synthesis in
general, see: (h) Fruton, J. S. AdV. Enzymol. 1982, 53, 239. (i) Schellen-
berger, V.; Jakubke, H. D. Angew. Chem., Int. Ed. Engl. 1991, 30, 1437.
(j) Wong, C.-H. Trends Biotechnol. 1992, 10, 378.
(4) Merrifield, R. B. J. Am. Chem. Soc. 1963, 85, 2149.
(5) (a) Kise, H.; Shirato, H. Tetrahedron Lett. 1985, 26, 6081. (b) Kise,
H. Bioorgan. Chem. 1990, 18, 107.
10.1021/ol0167614 CCC: $20.00 © 2001 American Chemical Society
Published on Web 11/29/2001

activity in two aliphatic diols, 1,3-propanediol and 1,4-
butanediol. In the presence of this enzyme, many Boc-amino
acids and unprotected peptides, when solubilized in 1,3-
propanediol or 1,4-butanediol containing 1-2.5% H2O, were
selectively esterified at their CR-COOHs to the corresponding
diol mono-esters in excellent yields (Table 1, see also
Supporting Information for more examples).
Many unprotected peptides were found to be readily
soluble in these two diols,6 and their esterification proceeded
smoothly without significant cleavage (through hydrolysis
or alcoholysis) of the internal peptide bonds (Figure 1). In
Table 1. Subtilisin-Catalyzed Esterification of Boc-amino
Acids and Peptides
substrate
R1CO-OH
alcohol
R2-OH
time
(h)
yielda
(%)
ester
MSb
no.
1
2
3
Boc-F-OH
Boc-F-OH
Boc-F-OH
HOPrOH
HOBuOH
EtOH
10
10
10
48
24
9
39
24
60
2.5
3
85
90
22
65
0
91
94
95
86
90
92
66
80
324
338
294
4
5
6
7
8
Boc-D-F-OH
Boc-Y-OH
Boc-M-OH
Boc-L-OH
Ac-FA-OH
neurotensin
xenopsin
HOPrOH
HOPrOH
HOPrOH
HOPrOH
HOPrOH
HOPrOH
HOBuOH
HOBuOH
HOBuOH
340
308
290
337
Figure 1. HPLC monotoring of the esterification of xenopsin (3
h) and endothelin (72 h) in 1,4-butanediol. (A) Peak 1, xenopsin;
peak 2, xenopsin-OBuOH. (B) Peak 1, endothelin; peak 2, endo-
thelin-OBuOH. HPLC was run on a Shimadzu system with a Vydac
C18 analytical column (0.46 × 25 cm, 5 µm) at a flow rate of 1.5
mL/min, using linear gradient of 20-55% for 35 min of buffer B
(60% CH3CN in H2O, 0.04%TFA) in buffer A (5% CH3CN in H2O,
0.045% TFA).
9
1731.3
1166.2
1198.4
2565.5
10
11
12
neuropeptide
endothelin
3
72
a Yields were calculated from HPLC. b [M + H]+ mass data were
obtained with FAB-MS for entries 1-8 and MALDI-MS for entries 9-12.
Amino acids in entries 1-8 are represented by the single-letter code.
HOPrOH: 1,3-propanediol. HOBuOH: 1,4-butanediol. Water content in
the reaction was 2.5% for entries 1-7 and 2% for entries 8-12. Enzyme/
sustrate molar ratio was 1/500. Amino acid concentration was 50 mM.
Peptides were used at 20 mM (10 mM for endothelin). Neorotensin: pGlu-
Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu-OH. Xenopsin: H-Phe-
His-Pro-Lys-Arg-Pro-Trp-Ile-Leu-OH. Neuropeptide (The Head Activator):
pGlu-Pro-Pro-Gly-Gly-Ser-Lys-Val-Ile-Leu-Phe-OH. Endothelin: H-Cys-
Ser-Cys-Ser-Ser-Leu-Met-Asp-Lys-Glu-Cys-Val-Tyr-Phe-Cys-His-Leu-Asp-
Ile-Ile-Trp-OH (1-4 and 2-3 disulfide). Peptides 9-12 were used in TFA
salt forms.
fact, certain peptides (Table 1, entries 9-11) displayed even
higher reactivity than simple amino acids. In general, 1,3-
propanediol is a better solvent for these peptides, but 1,4-
butanediol has a practical advantage of generating ester
products that are more separable by HPLC from the starting
materials. Substrate solubility is clearly one of the factors
that would limit the use of this method, especially when
larger, hydrophobic peptides are concerned. One should note
that the peptides examined in this report are not particularly
large in size. The general use of this methodology is also
limited by the substrate specificity of subtilisin, which
requires certain preferred amino acids at the P1 position. In
fact, nonpreferred substrates such as Boc-Val-OH and Boc-
Gly-OH were not esterified after 5 days of reaction (see
Supporting Information).
The enzymatic activity of subtilisin in the two diols was
found to be much higher than in ethanol when the reactions
of Boc-Phe-OH in these three alcohols were compared. In
addition, the reaction was stereospecific, as Boc-D-Phe-OH
was totally unreactive (Table 1, entry 4). Subtilisin retained
its broad substrate specificity of favoring amino acids with
aromatic, long aliphatic, or neutral side chains at the P1
position. The esterification was also regiospecific at the
C-terminal R-COOH for the tested peptides, with the side
chain carboxylic groups of Asp or Glu unaffected. This was
confirmed by enzymatic digestion of the esterified Glu/Asp-
containing peptides using a specific endopeptidase, V8, which
can only hydrolyze a Glu/Asp-Xxx peptide bond. For
example, for the peptide neurotensin (Table 1, entry 9) which
contains a Glu at position 4, V8 treatment of its ester product
in 50 mM phosphate buffer (pH 7.8) gave two fragments
corresponding respectively to pGluLeuTyrGlu-OH and H-
AsnLysProTyrIleLeu-OPrOH as confirmed by MS analysis.
Similar results were obtained with V8 digestion of endothelin
hydroxybutyl ester (Table 1, entry 12).
A plausible explanation for the catalytic activity of
subtilisin in these nearly pure diols is found in nonaqueous
enzymology.7 It has been postulated that as long as an
enzyme can retain a layer or so of water molecules on its
surface for maintaining its catalytically active conformation,
(6) The solubility of a peptide in the two diols was found to depend on
its hydrophilicity. In general, hydrophilic peptides are more soluble in these
diols than hydrophobic ones. Poorly soluble hydrophobic peptides gave poor
results in esterification.
(7) (a) Zaks, A.; Klibanov, A. M. Proc. Natl. Acad. Sci. U.S.A. 1985,
82, 3192. (b) Zaks, A.; Klibanov, A. M. J. Biol. Chem. 1988, 263, 8017.
(c) For reviews, see: Klibanov, A. M. Trends Biochem. Sci. 1989, 14, 141,
and references therein.
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Org. Lett., Vol. 3, No. 26, 2001

Table 2. Protease-Catalyzed Aminolysis of Esters for Peptide Ligation
product [M + H]+
no.
acyl donor (mM)
acyl acceptor (mM)
enzymea
time (min)
yieldb (%)
calcd
foundc
1
2
3
Ac-Phe-Ala-OPrOH (25)
Ac-Phe-Ala-OPrOH (25)
neuropeptide-OBuOH (2)
H-Leu-NH2 (200)
FI-6 (30)
RF-9 (6)
papaind
10
20
10 (h)
95f
66f
80
390.2
1028.2
2221.0
390.0
1028.0
2222.5
papaind
subtiligasee
a Enzymes were used at a 1/500 molar ratio to acyl donors. b Yields were calculated from HPLC on the basis of acyl donors. c Mass spectral data were
obtained with FAB-MS for entry 1 and MALDI-MS for entries 2-3. d Reaction was run in 60% acetonitrile in 0.3 M Triethanolamine buffer (pH 8.5) and
ein aqueous Tricine buffer (0.1M, pH 8.0) containing 2 mM DTT at 37 °C. f In these two cases, ester hydrolysis accounted for the rest of percentage. FI-6:
H-Phe-Val-Phe-Asn-Lys-Ile-OH. RF-9: H-Arg-Tyr-Gln-Ala-Glu-Val-Ser-Leu-Phe-OH.
the remaining bulk aqueous milieu is replaceable with an
organic solvent. Thus, in our proposed system, subtilisin
captures the essential amount of water after distribution of
the 1-2.5% of H2O between the hydrophilic enzyme surface
and the diol solvent. The yield of esterification was decreased
when higher water content was used, due to a shift of the
reaction equilibrium to the reactant direction (Table 1,
reaction scheme). These diol systems effectively lowered the
amidase activity of subtilisin, since no significant cleavage
of the internal peptide bonds was observed in our peptide
examples which contain one or more susceptible peptide
bonds to subtilisin under normal hydrolytic conditions. This
is in general agreement with previous observations that a
high content of organic solvents such as DMF or DMSO
diminishes or suppresses the amidase activity of serine or
cysteine proteases while retaining their esterase activity at a
significant level.3e
A number of applications can be envisioned for these
particular ester products. For instance, after the oxidation of
the free hydroxyl to an aldehyde or carboxyl, the amino acid
esters may find their use as new chiral synthons in organic
synthesis. As part of our efforts in developing peptide ligation
strategies, we envisioned that this enzymatic esterification
method could be coupled with kinetic-control enzymatic
aminolysis of peptide esters in a two-step enzymatic peptide
ligation scheme depicted in the abstract graphic. We tested
the feasibility of this strategy by using some of these esters
as acyl donors for enzymatic peptide synthesis. As shown
in Table 2, several peptides were prepared in satisfactory
yields through catalysis of different proteases. A particularly
noteworthy point was the use of subtiligase, a DNA-
recombinant engineered subtilisin designed for enzymatic
segment ligation,3g in the synthesis of a 20-residue peptide
using a 11-mer peptide ester (from Table 1, entry 11)
prepared by the above method as the acyl donor. In this
example, ligation took place smoothly for 10 h in a normal
aqueous buffer at relatively low molar concentrations of both
components and no significant hydrolysis of the ester linkage
or peptide bond was observed. Our two-step enzymatic
ligation scheme is conceptually similar to a method reported
by Hwang et al. who have utilized a chemical method to
activate the R-carboxylic group to 5(4H) oxazolone in place
of carboxyl esters as activated acyl donors for enzymatic
peptide synthesis.8 These enzymatic approaches represent an
important supplement to the currently available chemical
ligation methods.9,10
In conclusion, we have shown that certain Boc-protected
amino acids and peptides can be selectively esterified by
making use of the reverse esterase activity of subtilisin, even
in the presence of other unprotected functionalities including
the equally reactive side chain carboxylic groups. In addition,
such esters are useful substrates as acyl donors for kinetically
controlled enzymatic peptide synthesis. Our proposed race-
mization-free esterification system provides a useful alterna-
tive to the conventional chemical methods for ester synthesis,
which usually require harsh and stringent reaction conditions.
These results may stimulate further research interest in the
design and development of environmentally benign processes
for the manufacturing of agriculturally or therapeutically
important products.
Acknowledgment. We thank Dr. James A. Wells for his
generous gift of subtiligase from Genentech, Inc. We also
thank Ms. Chang Rao for spectral analysis of some of the
Boc-amino acid esters. This work was supported in part by
NIH Grant CA36544.
Supporting Information Available: Detailed experi-
1
mental procedures, HPLC profiles, and H NMR data of
certain Boc-amino acid esters. This material is available free
of charge via the Internet at http://pubs.acs.org.
OL0167614
(8) Hwang, B. K.; Gu, Q.-M.; Sih, C. J. Am. Chem. Soc. 1993, 115,
7912.
(9) For a comprehemsive review on chemical peptide ligation methods,
see: Coltant, D. M. Tetrahedron 2000, 56, 3449.
(10) Nilsson, B. L.; Kiessling L. L.; Raines, R. T. Org. Lett. 2001, 3, 9.
Org. Lett., Vol. 3, No. 26, 2001
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