Trypsin-catalyzed synthesis of dipeptide containing α-aminoisobutylic acid using p- and m-(amidinomethyl)phenyl esters as acyl donor

Article abstract of DOI:10.1248/cpb.56.688

Two series of inverse substrates, p- and m-(amidinomethyl)phenyl esters derived from N-(tert-butyloxycarbonyl) amino acid, were prepared as acyl donor components for enzymatic peptide synthesis. They were found to be readily coupled with an acyl acceptor such as L-alanine p-nitroanilide to produce dipeptide. An α-aminoisobutyric acid containing dipeptide was especially obtained in satisfactory yield. Streptomyces griseus trypsin was a more efficient catalyst than the bovine trypsin. The optimum condition for the coupling reaction was studied by changing the organic solvent, pH, and acyl acceptor concentration. It was found that the enzymatic hydrolysis of the resulting product was negligible.

Full text of DOI:10.1248/cpb.56.688

688
Chem. Pharm. Bull. 56(5) 688—691 (2008)
Vol. 56, No. 5
a
Trypsin-Catalyzed Synthesis of Dipeptide Containing -Aminoisobutylic
Acid Using p- and m-(Amidinomethyl)phenyl Esters as Acyl Donor
Haruo SEKIZAKI,* Kunihiko ITOH, Akiyoshi SHIBUYA, Eiko TOYOTA, Mareshige KOJOMA, and
Kazutaka TANIZAWA
Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido; Ishikari-Tobetsu, Hokkaido 061–0293,
Japan. Received January 18, 2008; accepted March 11, 2008; published online March 11, 2008
Two series of inverse substrates, p- and m-(amidinomethyl)phenyl esters derived from N-(tert-butyloxycar-
bonyl)amino acid, were prepared as acyl donor components for enzymatic peptide synthesis. They were found to
be readily coupled with an acyl acceptor such as L-alanine p-nitroanilide to produce dipeptide. An a-amino-
isobutyric acid containing dipeptide was especially obtained in satisfactory yield. Streptomyces griseus trypsin
was a more efficient catalyst than the bovine trypsin. The optimum condition for the coupling reaction was stud-
ied by changing the organic solvent, pH, and acyl acceptor concentration. It was found that the enzymatic hy-
drolysis of the resulting product was negligible.
Key words enzymatic peptide synthesis; p-(amidinomethyl)phenyl ester; N-Boc-aminoisobutylic acid; m-(amidinomethyl)-
phenyl ester; inverse substrate; trypsin
Sterically hindered a-amino acid such as the a-amino- groups into a trypsin active site, and such acyl enzymes have
isobutylic acid (Aib, 2-methylalanine) is one of the con- proven to be useful for the peptide coupling.13,14,19—28) We
stituent of naturally occurring antibiotics.1—4) Peptides con- also reported that a,a-dialkyl amino acid p-(guanidi-
taining Aib are of interest as the model for conformational nomethyl)phenyl esters were useful substrates for enzymatic
analysis of the peptide backbone (formation of a- or 310-he- peptides synthesis containing such a hindered amino acid.21)
lices, or b-turns).5,6) However, the introduction of Aib into a Recently, we reported that preparation of new two series in-
peptide is difficult,4,7,8) because the reactivity of sterically verse substrates such as N-Boc-amino acid p- and m-(amidi-
hindered a,a-disubstituted amino acid is much lower than nomethyl)phenyl esters.29) In the present work, we investi-
that of typical a-amino acids. New chemical coupling gated trypsin-catalyzed peptide synthesis using these syn-
reagent, 2-chloro-1,3-dimethylimidazolidium hexafluo- thetic substrates as acyl donor components. Comparison was
rophosphate, (CIP), for sterically hindered a,a-disubstituted made between two trypsins of different origin (bovine and
amino acidis was developed and chemical synthesis of Streptomyces griseus (SG) trypsin) as the catalyst for peptide
Alamethicin F-30 was achieved.9)
synthesis as shown in Chart 1.
Peptide synthesis by protease-catalyzed reverse reaction
Trypsin-Catalyzed Peptide Coupling Reaction The
has been extensively studied with a variety of amino acids trypsin-catalyzed peptide coupling reaction has been studied
and peptide derivatives as coupling.10—15) The protease-cat- by using synhetic inverse substrates (1a—h, 2a—c) as acyl
alyzed peptide synthesis is superior to the chemical coupling donors (Fig. 1). The reaction of N-tert-butyloxycarbonyl-
method. The method requires less-side chain protection than L-alanine p-(amidinomethyl)phenyl ester (N-Boc-L-Ala-
the chemical coupling method. A most serious drawback of OpAM) (1a) and L-alanine p-nitroanilide (L-Ala-pNA) to
the enzymatic method, however, is the respective substrate give N-Boc-L-Ala-L-Ala-pNA (3a) was examined in dimethyl
specificity. Thus, the application of proteases for peptide syn- sulfoxide (DMSO) or N,N-dimethylformamide (DMF) as a
thesis has not been fully exploited for possible synthesis of a co-solvent. The reaction was also evaluated under the condi-
number of biologically important peptides containing D- tion where the pH of the medium was changed and the con-
amino acid or other unusual amino acid.
centration of the acyl acceptor (L-Ala-pNA) was changed.
In a previous paper, we reported that the p-amidinophenyl The coupling product was obtained in high yield with DMSO
and p-guanidinophenyl esters behave as specific substrates rather than with DMF.
for trypsin and trypsin-like enzymes such as thrombin and
Effects of DMSO and DMF concentration on coupling
plasmin, etc.16—18) In these esters the site-specific groups yields are shown in Fig. 2. Coupling yields higher than 50%
(charged amidinium and guanidinium) for the enzyme are in- were observed at the DMSO concentration range of 30—
cluded in the leaving-group portion instead of being in the 50%, and the best yield (73%) was obtained at 50% DMSO.
acyl moiety. Such a substrate was termed “inverse substrate” The DMF showed slightly different behavior from that of
by us.16) The esters provided a novel method for the specific DMSO, and the best yield (48%) was obtained at the 20%
introduction of an acyl group for a wide variety of acyl concentration. Although a high concentration of organic sol-
Chart 1
∗ To whom correspondence should be addressed. e-mail: sekizaki@hoku-iryo-u.ac.jp
© 2008 Pharmaceutical Society of Japan

May 2008
689
vent prevents the hydrolysis of the acyl enzyme, it will de- 2-morpholino-1-ethanesulfonate (MES) (50 mM, containing
crease the enzymatic activity due to the denaturation of 20 mM CaCl2), 3-morpholino-1-propanesulfonate (MOPS)
trypsin. Consequently, the coupling yield was decreased at a (50 mM, containing 20 mM CaCl2), and carbonate buffers with
concentration of organic solvents above 60%.
various pH values, as shown in Fig. 3. The pH values givin in
The effect of pH of the buffer component in the medium Fig. 3 are those of the buffer itself before mixing with or-
on the coupling yields was analyzed. DMSO was mixed with ganic co-solvent. The pH dependency of the coupling yield
for the reaction period of 24 h was determined. The yield was
greatly decreased at higher pH than 9. The observed depend-
ency was similar to that of trypsin-catalyzed hydrolysis of the
specific ester substrates as reported previously in which the
catalytic rate was increased at the higher pH and reached the
limit at around pH 9.31) The best yield (74%) was obtained
around pH8 using MOPS buffer.
The effect of acyl acceptor concentration on the coupling
yields in 50% aqueous DMSO is shown in Fig. 4. The de-
pendency can be explained to be due to the saturation of the
enzyme binding site with the acyl acceptor. The reaction
yield reached maximum (73%) at the concentration around
Fig. 1. Structure of Substrates
Fig. 2. Effect of Organic Solvent on Bovine Trypsine-Catalyzed Conden-
sation of N-Boc-L-Ala-OpAM (1a) with L-Ala-pNA
Reaction was carried out in 50 mM MOPS buffer (pH 7.0, containing 20 mM CaCl2).
Cosolvents are DMSO (ꢀ) and DMF (ꢁ) at 25 °C. Product yield was analyzed after a
reaction period of 24 h in which the coupling was completed. N-Boc-L-Ala-OpAM (1a),
1 mM; L-Ala-pNA, 20 mM; bovine trypsine, 10 mM.
Fig. 4. Effect of Acyl Acceptor Concentration on Bovine Trypsin-Cat-
alyzed Condensation of N-Boc-L-Ala-OpAM (1a) with L-Ala-pNA
Reaction was carried out in 50 mM MOPS buffer containing 50% DMSO at 25 °C.
Product yield was analyzed after a reaction period of 24 h in which the coupling was
completed. N-Boc-L-Ala-OpAM (1a), 1 mM; L-Ala-pNA, 20 mM; bovine trypsin, 10 mM.
Fig. 3. pH Dependency of Bovine Trypsin-Catalyzed Condensation of N-
Boc-L-Ala-OpAM (1a) with L-Ala-pNA
Fig. 5. Time Course Bovine Trypsin-Catalyzed Condensation of N-Boc-L-
Ala-OpAM (1a) with L-Ala-pNA
Reaction was carried out in 50% DMSO–50 mM MES buffer (pH 5.0 and 6.0, con-
taining 20 mM CaCl2), 50 mM MOPS buffer (pH 7.0 and 8.0, containing 20 mM CaCl2),
and 50 mM carbonate buffer (pH 9.0, 10.0) at 25 °C. Product yield was analyzed after a
reaction period of 24 h in which the coupling was completed. N-Boc-L-Ala-OpAM (1a),
1 mM; L-Ala-pNA, 20 mM; bovine trypsin, 10 mM.
Reaction was carried out in 50 mM MOPS buffer containing 50% DMSO at 25 °C.
Product yield was analyzed after a reaction period of 24 h in which the coupling was
completed. N-Boc-L-Ala-OpAM (1a), 1 mM; L-Ala-pNA, 20 mM; bovine trypsin, 10 mM.

690
Vol. 56, No. 5
Table 1. Yields of Bovine Trypsin and SG Trypsin-Catalyzed Peptide Synthesis with Amino Acid Ester as Acyl Donora)
Entry
No.
Acyl donor
(No.)
Reaction time
(h)
Product
(No.)
Yield
(%)b)
Enzyme
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
Bovine trypsin
Bovine trypsin
Bovine trypsin
Bovine trypsin
Bovine trypsin
Bovine trypsin
Bovine trypsin
Bovine trypsin
Bovine trypsin
Bovine trypsin
Bovine trypsin
SG trypsin
SG trypsin
SG trypsin
SG trypsin
SG trypsin
SG trypsin
SG trypsin
SG trypsin
SG trypsin
N-Boc-L-Ala-OpAM (1a)
N-Boc-D-Ala-OpAM (1b)
N-Boc-Aib-OpAM (1c)
N-Boc-Gly-OpAM (1d)
N-Boc-L-Leu-OpAM (1e)
N-Boc-D-Leu-OpAM (1f)
N-Boc-L-Phe-OpAM (1g)
N-Boc-D-Phe-OpAM (1h)
N-Boc-L-Ala-OmAM (2a)
N-Boc-D-Ala-OmAM (2b)
N-Boc-Aib-OmAM (2c)
N-Boc-L-Ala-OpAM (1a)
N-Boc-D-Ala-OpAM (1b)
N-Boc-Aib-OpAM (1c)
N-Boc-Gly-OpAM (1d)
N-Boc-L-Leu-OpAM (1e)
N-Boc-D-Leu-OpAM (1f)
N-Boc-L-Phe-OpAM (1g)
N-Boc-D-Phe-OpAM (1h)
N-Boc-L-Ala-OmAM (2a)
N-Boc-D-Ala-OmAM (2b)
N-Boc-Aib-OmAM (2c)
24
24
96
24
24
48
48
24
24
24
48
24
24
48
24
24
24
24
24
24
24
48
N-Boc-L-Ala-L-Ala-pNA (3a)
N-Boc-D-Ala-L-Ala-pNA (3b)
N-Boc-Aib-L-Ala-pNA (3c)
N-Boc-Gly-L-Ala-pNA (3d)
N-Boc-L-Leu-L-Ala-pNA (3e)
N-Boc-D-Leu-L-Ala-pNA (3f)
N-Boc-L-Phe-L-Ala-pNA (3g)
N-Boc-D-Phe-L-Ala-pNA (3h)
N-Boc-L-Ala-L-Ala-pNA (3a)
N-Boc-D-Ala-L-Ala-pNA (3b)
N-Boc-Aib-L-Ala-pNA (3c)
N-Boc-L-Ala-L-Ala-pNA (3a)
N-Boc-D-Ala-L-Ala-pNA (3b)
N-Boc-Aib-L-Ala-pNA (3c)
N-Boc-Gly-L-Ala-pNA (3d)
N-Boc-L-Leu-L-Ala-pNA (3e)
N-Boc-D-Leu-L-Ala-pNA (3f)
N-Boc-L-Phe-L-Ala-pNA (3g)
N-Boc-D-Phe-L-Ala-pNA (3h)
N-Boc-L-Ala-L-Ala-pNA (3a)
N-Boc-D-Ala-L-Ala-pNA (3b)
N-Boc-Aib-L-Ala-pNA (3c)
73
64
83
77
79
80
70
63
48
48
2
74
74
90
76
78
90
58
59
64
64
16
SG trypsin
SG trypsin
a) Conditions: acyl donor, 1 mM; acyl acceptor (L-Ala-pNA), 20 mM; bovine trypsin and SG trypsin, 10 mM, respectively; 50% DMSO-MOPS (50 mM, pH 8.0, containing
20 mM CaCl2); 25 °C. b) The values represent the mean of two runs (each value in within 2% variation).
(TLC). Bovine pancreas trypsin (EC 3.4.21.4) was purchased from Wor-
20 mM of acyl acceptor.
The time courses of the coupling reaction of N-Boc-L-Ala-
OpAM (1a) with L-Ala-pNA is shown in Fig. 5. The D-acyl
donors are an efficient substrate for the enzymatic coupling
thington Biochemical Corp. (twice crystallized, lot TRL). SG trypsin was
prepared following the reported.32,33) HPLC grade DMSO and DMF from
Kanto Chemical Co., Inc. were used. L-Ala-pNA was purchased from Pep-
tide Institute, Inc. MOPS and MES were purchased from Wako Pure Chemi-
reaction, as well as the L-acyl donors. The coupling yields cal Industries. Tricine and p-methylumbelliferyl pꢀ-guanidinobenzoate were
purchased from Merck & Co., Inc.
did not drop even after prolonged reaction period. This result
indicated that enzymatic secondary hydrolysis of the result-
ing peptides is negligible.
Synthesis of Inverse Substrates All inverse substrates were prepared
according to our previous paper.29) The results and some properties of new
inverse substrates (1d—h) are as follows:
Consequently, standard conditions for trypsin-catalyzed
peptide coupling reaction used (amidinomethyl)phenyl ester
as acyl donor component were selected as described in the
methods. The results of bovine trypsin-catalyzed coupling re-
action were compared with those of SG trypsin-catalyzed
coupling reaction as listed in Table 1. Each product com-
pletely coincides with the respective, authentic peptide sam-
ple. Coupling reaction of the p-(amidinomethyl)phenyl ester
(1a—h) is much more efficient than that of the m-(amidi-
nomethyl)phenyl ester (2a—c). In the case of N-Boc-Aib-
OpAM (1c) (Entry 3, 14), Aib containing dipeptide (3c) was
obtained in high yields with using the both catalysts. The
corresponding dipeptides were also obtained using para-
form substrates in satisfactory yield (Entry 1—8, 12—19).
N-(tert-Butoxycarbonyl)glycine p-(Amidinomethyl)phenyl Ester p-Tolu-
enesulfonic Acid Salt (1d): mp 177—179 °C (from AcOEt–hexane). IR
1
(KBr) cmꢁ1: 1753. H-NMR (DMSO-d6) d: 1.39 (9H, s), 2.28 (3H, s), 3.69
(2H, s), 3.95 (2H, d, Jꢂ5.9 Hz), 7.10—7.13 (4H, m), 7.39—7.49 (5H, m),
8.62 (2H, s), 9.12 (2H, s). 13C-NMR (DMSO-d6, 100 MHz) d: 21.29, 28.64,
37.73, 79.12, 115.98, 122.51, 125.99, 128.69, 130.51, 138.56, 145.47,
150.27, 156.50, 169.33, 169.93. Anal. Calcd for C15H21N3O4·C7H8O3S·
1/3H2O: C, 54.42; H, 6.16; N, 8.65; S, 6.60. Found: C, 54.59; H, 6.19; N,
8.24; S, 6.60.
N-(tert-Butoxycarbonyl)-L-leucine p-(Amidinomethyl)phenyl Ester p-
Toluenesulfonic Acid Salt (1e): mp 112—114 °C (from AcOEt–hexane). IR
1
(KBr) cmꢁ1: 1760. H-NMR (DMSO-d6) d: 0.89—0.94 (6H, m), 1.39 (9H,
s), 1.58—1.70 (3H, m), 2.28 (3H, s), 3.68 (2H, s), 4.17 (1H, br s), 7.07—
7.12 (4H, m), 7.39—7.49 (5H, m), 8.58 (2H, s), 9.09 (2H, s). 13C-NMR
(DMSO-d6, 100 MHz) d: 20.94, 21.42, 22.93, 24.52, 28.29, 37.37, 52.40,
122.05, 125.63, 128.35, 130.14, 138.24, 145.16, 150.05, 155.94, 168.97,
172.18. [a]D25 ꢁ31.1° (cꢂ1.0, MeOH). Anal. Calcd for C19H29N3O4·
C7H8O3S: C, 58.30; H, 6.96; N, 7.84; S, 5.99. Found: C, 58.28; H, 6.88; N,
7.68; S, 6.21.
Experimental
The melting points were measured on a Yanaco micro melting point appa-
N-(tert-Butoxycarbonyl)-D-leucine p-(Amidinomethyl)phenyl Ester p-
ratus. IR spectra were taken on a JASCO VALOR-III FT-IR spectrometer. Toluenesulfonic Acid Salt (1f): mp 177—179 °C (from AcOEt–hexane). IR
1H- and 13C-NMR spectra were recorded on a JEOL JNM-FX-400 FT NMR (KBr) cmꢁ1: 1761. H-NMR (DMSO-d6) d: 0.90—0.95 (6H, m), 1.41 (9H,
1
spectrometer. Chemical shifts are quoted in parts per million (ppm) with
s), 1.60—1.75 (3H, m), 2.30 (3H, s), 3.70 (2H, s), 4.17 (1H, br s), 7.08—
tetramethylsilane (TMS) as an internal standard. Coupling constants (J) are 7.14 (4H, m), 7.43—7.51 (5H, m), 8.30 (2H, s), 9.12 (2H, s). 13C-NMR
given in Hz. The following abbreviations are used: s, singlet; d, doublet; t, (DMSO-d6, 100 MHz) d: 19.90, 21.01, 21.41, 22.94, 24.52, 28.30, 36.10,
triplet; q, quartet; br s, broad singlet; dd, doublet of doublets; m, multiplet. 52.40, 121.42, 125.69, 128.46, 129,90, 134.72, 138.45, 144.96, 149.32,
The optical rotations were measured with a JASCO DIP-360 digital po- 155.93, 158.05, 168.61, 172.26. [a]D25 ꢃ30.6° (cꢂ1.0, MeOH). Anal. Calcd
larimeter in a 5 cm cell. The FAB-MS were taken with a JEOL JMS-HX-110 for C19H29N3O4·C7H8O3S: C, 58.10; H, 6.85; N, 7.85; S, 6.41. Found: C,
spectrometer. Flash column chromatography was performed using Silica Gel 58.28; H, 6.88; N, 7.68; S, 6.21.
60N (Kanto Chemical Co., Inc.) as a solid support in the immobile phase.
N-(tert-Butoxycarbonyl)-L-phenylalanine p-(Amidinomethyl)phenyl Ester
Kieselgel 60 F-254 plates (Merck) were used for thin-layer chromatography p-Toluenesulfonic Acid Salt (1g): A colourless amorphous powder. IR (KBr)

May 2008
691
1
cmꢁ1: 1760. H-NMR (DMSO-d6) d: 1.35 (9H, s), 2.28 (3H, s), 3.00—3.10
(2H, m), 3.68 (2H, s), 4.36—4.38 (1H, m), 6.97 (2H, d, Jꢂ8.4 Hz), 7.11
(2H, d, Jꢂ7.9 Hz), 7.23—7.35 (5H, m), 7.42 (2H, d, Jꢂ8.4 Hz), 7.48 (2H, d,
Jꢂ7.9 Hz), 7.58 (1H, d, Jꢂ7.2 Hz), 8.60 (2H, s), 9.08 (2H, s). 13C-NMR
(DMSO-d6, 100 MHz) d: 20.93, 28.25, 36.43, 37.36, 55.66, 115.68,
122.01,125.64, 126.80, 128.33, 128.48, 129.40, 130.13, 131.75, 137.29,
138.20, 145.22, 149.95, 155.74, 168.92, 171.16. [a]D25 ꢁ8.5° (cꢂ1.0,
MeOH). FAB-MS m/z: 570 (MꢃꢃH).
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N-(tert-Butoxycarbonyl)-D-phenylalanine p-(Amidinomethyl)phenyl Ester
p-Toluenesulfonic Acid Salt (1h): A colourless amorphous powder. IR
(KBr) cmꢁ1: 1761. 1H-NMR (DMSO-d6) d: 1.30 (9H, s), 2.22 (3H, s),
2.94—3.10 (2H, m), 3.62 (2H, s), 4.28—4.33 (1H, m), 6.91 (2H, d,
Jꢂ8.4 Hz), 7.06 (2H, d, Jꢂ37.9 Hz), 7.27—7.26 (5H, m), 7.36 (2H, d,
Jꢂ8.4 Hz), 7.43 (2H, d, Jꢂ7.9 Hz), 7.51 (1H, d, Jꢂ7.2 Hz), 8.54 (2H, s),
9.04 (2H, s). 13C-NMR (DMSO-d6, 100 MHz) d: 20.93, 28.26, 36.44, 37.34,
55.66, 115.68, 122.00, 125.64, 126.81, 128.34, 128.48, 129.40, 130.14,
131.77, 137.29, 138.25, 145.15, 149.97, 155.72, 168.97, 171.20. [a]D25
ꢃ7.8° (cꢂ1.0, MeOH). FAB-MS m/z: 570 (MꢃꢃH).
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Trypsin-Catalyzed Peptide Coupling Reaction A solution of 50 ml of
acyl donor (10 mM solution of inverse substrates in DMSO), 50 ml of acyl ac-
ceptor (200 mM solution of L-Ala-pNA in DMSO), 240 ml of 50 mM MOPS
buffer (containing 20 mM of CaCl2, pH 8.0) and 150 ml of DMSO were
mixed. To this mixture, 10 ml of trypsin solution (1 mM solution in 1 mM
HCl) was added and incubated at 25 °C. The progress of the coupling reac-
tion was monitored by HPLC under the following conditions: Shim-pack
CLC-ODS (M) (column i.d. 4.6ꢄ250 mm), isocratic elution at 1 ml/min,
0.1% aqueous trifluoroacetic acid/acetonitrile. An aliquot of the reaction
mixture was injected and the eluate was monitored at 310 nm (chromophore
due to the p-nitroanilide moiety). Peak identification was made by correlat-
ing the retention time with that of authentic samples which were chemically
synthesized.34—36) Observed peak areas were used for the estimation of sam-
ple concentration.
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184 (2001).
Acknowledgments This work was supported in part by a Grant-in-Aid
for High Technology Research Program from the Ministry of Education,
Culture, Sports, Science, and Technology of Japan, and by a grant from the
Japan Private School Promotion Foundation.
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528 (2002).
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