α-chymotrypsin-catalysed segment condensations via the kinetically controlled approach using carbamoylmethyl esters as acyl donors in organic media

Article abstract of DOI:10.1039/b108738j

The superiority of the carbamoylmethyl ester as an acyl donor for the α-chymotrypsin-catalysed segment condensations via the kinetically controlled approach is demonstrated in several model systems carried out in organic media with low water content. Furthermore, this approach is successfully applied to the construction of the Leu-enkephalin sequence via a 4 + 1 segment coupling.

Full text of DOI:10.1039/b108738j

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ꢀ-Chymotrypsin-catalysed segment condensations via the
kinetically controlled approach using carbamoylmethyl esters as
acyl donors in organic media†
1
Toshifumi Miyazawa,* Eiichi Ensatsu, Makoto Hiramatsu, Ryoji Yanagihara and Takashi
Yamada
Department of Chemistry, Faculty of Science and Engineering, Konan University,
Higashinada-ku, Kobe 658-8501, Japan
Received (in Cambridge, UK) 25th September 2001, Accepted 27th November 2001
First published as an Advance Article on the web 8th January 2002
The superiority of the carbamoylmethyl ester as an acyl donor for the α-chymotrypsin-catalysed segment
condensations via the kinetically controlled approach is demonstrated in several model systems carried out in
organic media with low water content. Furthermore, this approach is successfully applied to the construction of
the Leu-enkephalin sequence via a 4 ϩ 1 segment coupling.
Introduction
In modern peptide synthesis, a high degree of chiral integrity of
products is required.1 Stepwise elongation of a peptide chain
using urethane-protected amino acids is a method generally free
from racemisation. Much greater difficulties are encountered
during the coupling of peptide segments, because activated C-
terminal amino acid residues can be racemised easily. Never-
theless, segment condensation is indispensable for building long
peptide chains. Accordingly, a great deal of effort has been
exerted to prevent racemisation during segment couplings.2
In this regard enzymatic methodologies have attracted the
Scheme 1
attention of researchers. In particular, enzymatic peptide syn-
thesis using proteases3 is becoming recognised as an alternative
or complement to chemical synthesis of biologically active
ically controlled approach, several model coupling systems of
peptides. Besides freedom from racemisation, enzymatic
methodologies have also several other advantages: among
others, high regio- and stereoselectivity and minimal side-chain
protection. On the other hand, the following are counted as
major disadvantages: narrow substrate specificity often shown
by enzymes, and secondary hydrolysis of a growing peptide.
In previous papers,4 we have reported on the broadening of
substrate tolerance of a serine protease, α-chymotrypsin (EC
3.4.21.1), by using such activated esters as the 2,2,2-trifluoro-
ethyl (Tfe) or carbamoylmethyl (Cam) ester as acyl donors in
the kinetically controlled approach of peptide-bond formation.
Among the activated esters examined, the Cam ester proved to
have a particularly noteworthy ameliorating effect. Further-
more, the method employing this ester as the acyl donor was
applied to the amide-bond formation between an amino acid
residue and a chiral amine.5 In order to clarify the scope and
limitations we intended to apply this approach to several model
segment condensations and also to the construction of the
Leu-enkephalin sequence.6 In the present paper, we describe
the details of our relevant work.
the 2 ϩ 1 or 3 ϩ 1 type were investigated (Scheme 1). In these
segment condensations the possibility of racemisation of the
C-terminal residue of the carboxylic component and the fission
of peptide bond(s) within the carboxylic component must be
taken into consideration. Fortunately, we could resort to HPLC
analysis for the close examination of these undesirable side
reactions on the basis of our recent researches.4,5 Table 1 shows
the results of the α-chymotrypsin-catalysed couplings of several
kinds of fragments with -Leu-NH2 as a typical amino com-
ponent (molar ratio, 1 : 4).7 This Table includes also the results
obtained using the Me or Tfe ester for the purpose of com-
parison. The reaction conditions were chosen to be similar
to the dipeptide syntheses in acetonitrile containing 4%
Tris buffer (pH 7.8).4b The amounts of the donor ester, the
desired peptide and its epimer, and other possible by-products
were determined by HPLC analysis on an ODS column using
aqueous acetonitrile or MeOH containing H3PO4 as a mobile
phase. As a typical example, HPLC separation of compounds
relevant to the coupling of Z--Phe--Phe-OR (R = Me, Tfe or
Cam) with -Leu-NH2 is shown in Table 2.
The couplings of fragments bearing a C-terminal Ala residue
as carboxylic component were examined first (Table 1, entries
1–4). The reaction profile in the coupling of Z--Phe--Ala-
OCam with -Leu-NH2 is shown as an example in Fig. 1. When
the Me esters were used as acyl donors, the yields of the desired
peptides were low. The use of the Cam esters once again
resulted in a marked increase in the peptide yields. In addition,
HPLC analysis revealed that no racemisation of the -Ala
Results and discussion
Initially, in order to ascertain the usefulness of the Cam ester in
α-chymotrypsin-catalysed segment condensations via the kinet-
† Electronic supplementary information (ESI) available: elemental
analyses and HPLC separation data. See http://www.rsc.org/suppdata/
p1/b1/b108738j/
396
J. Chem. Soc., Perkin Trans. 1, 2002, 396–401
This journal is © The Royal Society of Chemistry 2002
DOI: 10.1039/b108738j

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Table 1 α-Chymotrypsin-catalysed fragment couplings with -Leu-NH2 as an amino componenta
Yield (%)
Entry
Carboxylic component
Time (min)
Peptideb
Hydrolysis productc
Other by-productsd
1
2
3
Z-Gly--Ala-OMe
Z-Gly--Ala-OCam
Z--Phe--Ala-OMe
30
30
60
300
60
120
5
5
5
5
5
8.3
86.0
1.7
1.1
12.6
0
8.7
2.1
9.8
10.7
1.1
1.2
4.7
1.0
2.4
4.5
0.9
4.2
8.1
1.6
10.6
A
B
4
Z--Phe--Ala-OCam
70.8
79.4
14.3
56.2
95.3
14.9
62.3
88.3
12.8
50.6
83.4
4.8f
39.6f
5
6
7
8
9
10
11
12
13
14
15
Z-Gly--Phe-OMe
Z-Gly--Phe-OTfe
Z-Gly--Phe-OCam
Z-Gly-Gly--Phe-OMe
Z-Gly-Gly--Phe-OTfe
Z-Gly-Gly--Phe-OCam
Z--Phe--Phe-OMe
Z--Phe--Phe-OTfe
Z--Phe--Phe-OCam
Z-Gly--Phe(2Br)-OMee
Z-Gly--Phe(2Br)-OCame
5
60
60
60
30
30
C
a A mixture of 0.05 mmol of a carboxylic component, 0.2 mmol of -Leu-NH2ؒHCl, 0.2 mmol of TEA, and 150 mg of the immobilised α-
chymotrypsin was incubated with shaking in a solvent composed of 2 ml of acetonitrile and 83 µl of Tris buffer (pH 7.8) at 30 ЊC. b Desired peptide.
c Hydrolysis product of the donor ester. d A, 1.4% of Z--Phe--Leu-NH2; B, 4.1% of Z--Phe--Leu-NH2; C, 0.5% of Z--Phe--Leu-NH2. e Using
0.1 mmol of the carboxylic component. f - Peptide.
Table 2 HPLC separation of compounds relevant to the α-chymo-
when the Me ester was used as the acyl donor, the production
of 1.4% of Z--Phe--Leu-NH2 was observed besides the
desired peptide (8.7%) after 5 h of incubation. In this case no
production of Z--Phe was detected, which indicates that
Leu-NH2 served as a much better nucleophile than water under
the present reaction conditions. In the case of the Cam ester as
the acyl donor, the production of the defective peptide was not
observed after 1 h, but it became detectable (4.1%) after
2 h together with the maximum yield of the desired peptide
(79%). The couplings of fragments bearing a C-terminal Phe
residue were next examined (Table 1, entries 5–10). As expected
from the fact that the Phe residue is one of α-chymotrypsin’s
good amino acid substrates, these couplings were rather fast
even when the Me ester was used as the acyl donor. The
Tfe ester moderately increased the coupling yield, while it
was significantly enhanced by the use of the Cam ester. No
racemisation of the -Phe residue accompanied these couplings.
The coupling of a fragment bearing the -Phe--Phe sequence
was further tried where the bond between the two Phe residues
was susceptible to enzymatic cleavage (Table 1, entries 11–13).
In fact, when the Me ester was used as the acyl donor, the
production of a small amount (0.5%) of Z--Phe--Leu-NH2
(and no formation of Z--Phe) was observed after 1 h of
incubation. The coupling yield was greatly improved by the use
of the Cam ester without either the formation of the defective
peptide or the racemisation of the C-terminal -Phe residue.
Fragment couplings of a carboxylic component bearing a
sterically demanding non-protein amino acid residue at the
C-terminal position were also examined (Table 1, entries 14
and 15). In the synthesis of dipeptides containing halogeno-
phenylalanines, the o-Br-Phe residue was found to exert an
especially deleterious effect.4a,5 The low coupling efficiency with
the Me ester as an acyl donor was significantly improved by the
use of the Cam ester. With the latter ester the peptide yield
reached a maximum (40%) within 30 min, indicating that the
Cam ester of the dipeptide carboxylic component reacted more
rapidly than that of the amino acid carboxylic component.5
Moreover, HPLC analysis revealed that starting from the
racemic substrate, the -ester reacted specifically and the
-counterpart remained unchanged, affording only the -
peptide with -Leu-NH2 as the amino component, even when
the Cam ester was employed. Thus, this approach using the
Cam ester must surely be very useful for the incorporation of
non-protein amino acids into peptides, because racemic amino
a
trypsin-catalysed coupling of Z--Phe--Phe-OR with -Leu-NH2
kЈb
Compound
Mobile phase A
Mobile phase B
Z--Phe
5.1
5.4
7.3
2.8
2.8
3.5
4.5
5.1
Z--Phe--Leu-NH2
Z--Phe--Phe-OCam
Z--Phe--Phe
Z--Phe--Phe--Leu-NH2
Z--Phe--Phe--Leu-NH2
Z--Phe--Phe-OMe
Z--Phe--Phe-OTfe
9.7
12.5
13.7
22.7
9.4
19.2
a HPLC conditions: see the Experimental section. Column temp., 40 ЊC.
Mobile phase A, 46% aq. acetonitrile containing H3PO4 (0.01 M);
mobile phase B, 54% aq. acetonitrile containing H3PO4 (0.01 M). b Ca-
pacity factor.
Fig. 1 α-Chymotrypsin-catalysed coupling of Z--Phe--Ala-OCam
with -Leu-NH2. Symbols: ᮀ, Z--Phe--Ala-OCam; ᭺, Z--Phe--
Ala--Leu-NH2; ᭝, Z--Phe--Ala; ×, Z--Phe--Leu-NH2.
residue accompanied the couplings. In entries 3 and 4, the
Phe-Ala bond was expected to be susceptible to cleavage by α-
chymotrypsin on account of its substrate specificity.8 In fact,
J. Chem. Soc., Perkin Trans. 1, 2002, 396–401
397

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acids can be directly used for coupling without resolving them
before use.
tion, the desired peptide was produced in 67% yield. The acyl
donor disappeared completely within 30 min and the peptide
yield reached to 96%. No epimeric peptide was formed and no
products attributable to the fission of the Tyr-Gly bond were
detected. However, the concomitant formation of a small
amount (4.0%) of the hydrolysis product of the donor ester was
inevitable. Next, the carboxylic component carrying the Tyr
residue with a free hydroxy group was used for the coupling
with the same amino component. In this case, the acyl donor
disappeared almost completely within 10 min and the peptide
yield reached to 95%; the yield of the hydrolysis product of
the donor ester was 5.0%. Neither racemisation of the -Phe
residue nor cleavage of the Tyr-Gly bond occurred during the
coupling. Moreover, the chromatogram of the reaction mixture
on HPLC showed no indication of the occurrence of side
reactions at the hydroxy group of the Tyr residue.
The Cam esters of N-protected peptides used in the above
experiments were prepared mainly through the EDC–HOBT-
mediated {EDC = 1-ethyl-3-[3-(dimethylamino)propyl]carbo-
diimide} coupling of an N-protected peptide with an amino
acid Cam ester. They were also prepared via the reaction of the
Cs salt of an N-protected peptide with 2-chloroacetamide in
DMF. The samples prepared by both routes were identical with
each other in terms of their physical properties including the
specific rotation (see the Experimental section). This means that
during the substitution reaction of 2-chloroacetamide with the
Cs salt no epimerisation of the peptide fragment occurs, and
thus the Cam ester necessary for the fragment condensation can
be prepared directly from the corresponding C-free compound
via its Cs salt.9
In order to demonstrate further the potential usefulness of
the Cam ester as the acyl donor in the kinetically controlled
peptide synthesis we further tried to prepare the Leu-
enkephalin sequence2b via a 4 ϩ 1 segment condensation
(Scheme 2). In this case, the Tyr-Gly bond in the carboxylic
As illustrated in the above examples, when the Cam ester
is used as a donor ester which exceptionally facilitates the
production of the desired peptide, the enzymatic cleavage of
peptide bond(s) within the carboxylic component is suppressed
completely or to a very low level. In addition, no racemisation
of the C-terminal residue accompanies the coupling. Thus,
these results indicate the usefulness of the Cam ester as an acyl
donor in the α-chymotrypsin-catalysed kinetically controlled
segment condensations.
Scheme 2
Experimental
component and the peptide formed might be cleaved and the
phenolic hydroxy group of the Tyr residue might be attacked.
First, the carboxylic component carrying the O-benzyl-Tyr
residue was allowed to couple with -Leu-NH2 (molar ratio,
1 : 4) under similar reaction conditions to those above.
Reversed-phase HPLC analysis permitted not only the quanti-
fication of the desired protected pentapeptide but also the
detection and quantification of its epimer [Boc--Tyr(Bzl)-
Gly-Gly--Phe-Leu-NH2] and other possible by-products
[i.e., Boc--Tyr(Bzl)-Gly-Gly--Phe, Boc--Tyr(Bzl) and Boc-
-Tyr(Bzl)--Leu-NH2]. These compounds were well separated
one from another by choosing appropriate analytical con-
ditions, especially by adjusting the eluent composition. The
Cam ester of the carboxylic component necessary for the
study was prepared by the following two routes: (i) the EDC–
HOBT-mediated coupling of Boc--Tyr(Bzl)-Gly-Gly with -
Phe-OCam; (ii) the substitution of 2-chloroacetamide with the
Cs salt of Boc--Tyr(Bzl)-Gly-Gly--Phe in DMF. In this case,
the sample prepared by the latter route was completely identical
with that prepared by the former route. The profile of the
coupling reaction is shown in Fig. 2. After only 5 min of incuba-
General
1H NMR spectra were obtained at 300 MHz on a Varian Unity
300 spectrometer using DMSO-d6 as a solvent with TMS as an
internal standard unless otherwise noted. Mps were determined
on a Yamato MP-21 apparatus and are uncorrected. Optical
rotations were measured using a JASCO DIP-4 digital polar-
imeter. [α]D-Values are given in units of 10Ϫ1 deg cm2 gϪ1
.
MALDI-TOF mass spectra were recorded on a PerSeptive Bio-
systems Voyager DE PRO Biospectrometry Workstation, where
α-cyano-4-hydroxycinnnamic acid was used as a matrix reagent.
All organic solvents were distilled following standard protocols
and dried over molecular sieves prior to use. Petroleum spirit
refers to the fraction with distillation range 30–70 ЊC. -Leu-
NH2ؒHCl was purchased from Kokusan Chemical Works. α-
Chymotrypsin (type II, ex bovine pancreas) was purchased
from Sigma and had a specific activity of 48 units per mg solid
with Bz-Tyr ethyl ester. It was immoblised on Celite as
described before.4a Elemental analysis data for all new com-
pounds are available as Supplementary material.†
Preparation of the Me and Tfe esters of N-protected peptides
The Me and Tfe esters of N-protected peptides were prepared
through the coupling of an N-protected amino acid or Z-Gly-
Gly with an amino acid ester hydrochloride or hydrobromide by
the EDC–HOBT method in 79–96% yield, as illustrated below
for the preparation of Z--Phe--Phe-OTfe. The Me esters were
prepared also through the treatment of N-protected peptides
with an ethereal solution of diazomethane in nearly quantita-
tive yield. The mps and [α]D-values of the following N-protected
peptide Me esters have been reported: Z-Gly--Ala-OMe,10 Z--
Phe--Ala-OMe11,12 and Z--Phe--Phe-OMe.12,13
To a stirred solution of Z--Phe (898 mg, 3.0 mmol), -Phe-
OTfeؒHBr (prepared through the debenzyloxycarbonylation
of Z--Phe-OTfe with 25% HBr in AcOH) (985 mg, 3.0 mmol),
triethylamine (TEA) (304 mg, 3.0 mmol) and HOBT (406 mg,
3.0 mmol) in DMF (12 ml) was added EDCؒHCl (579 mg,
3.0 mmol) under ice-cooling. After stirring at this temperature
for 2 h and then at ambient temperature overnight, the reaction
mixture was diluted with EtOAc, washed successively with 1 M
HCl, water, 1 M aq. NaHCO3 and brine, and dried over
Na2SO4. Evaporation of the solvent in vacuo afforded white
Fig. 2 α-Chymotrypsin-catalysed synthesis of the Leu-enkephalin
sequence via a 4 ϩ 1 segment condensation. Symbols: ᮀ, Boc--
Tyr(Bzl)-Gly-Gly--Phe-OCam; ᭺, Boc--Tyr(Bzl)-Gly-Gly--Phe-
Leu-NH2; ᭝, Boc--Tyr(Bzl)-Gly-Gly--Phe.
398
J. Chem. Soc., Perkin Trans. 1, 2002, 396–401

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crystals, which were recrystallised from EtOAc–petroleum
spirit; yield, 1.38 g (87%); mp 165–165.5 ЊC; [α]2D5 ϩ13.9 (c 1.0,
CHCl3); δH (CDCl3) 2.94–3.13 (4H, m, 2 × Phe β-CH2), 4.35–
4.51 (3H, m, Phe α-CH ϩ CH2CF3), 4.83 (1H, q, J 6.6, Phe
α-CH), 5.07 (2H, s, PhCH2O), 5.22 (1H, br, NH), 6.22 (1H,
br d, J ≈7, NH), 6.95–7.39 (15H, m, 3 × Ph).
10 mmol) in DMF (40 ml) and the mixture was stirred at 60 ЊC
overnight. The reaction mixture was partitioned between
EtOAc and water, and the aqueous phase was extracted further
with EtOAc, and the combined organic extracts were washed
successively with 1 M aq. NaHCO3 and water and dried over
Na2SO4. Evaporation of the solvent afforded white crystals,
which were recrystallised from EtOAc–petroleum spirit; yield
2.66 g (64%); mp 153–154 ЊC, [α]2D5 Ϫ15.0 (c 1.0, DMF); δH 1.34
(3H, d, J 7.2, Ala Me), 2.68–3.02 (2H, m, Phe β-CH2), 4.24–
4.32 (1H, m, Phe α-CH), 4.37 (1H, quintet, J 7.2, Ala α-CH),
4.43 (2H, s, OCH2CO), 4.92 (2H, apparent s, PhCH2O), 7.10–
7.34 (12H, m, 2 × Ph ϩ NH2), 7.52 (1H, d, J 8.1, Phe NH),
8.60 (1H, d, J 7.2, Ala NH). On the other hand, the sample
prepared through the coupling of Z--Phe with -Ala-OCam
showed mp 153.5–154 ЊC and [α]2D5 Ϫ15.4 (c 1.0, DMF).
Z-Gly-L-Phe-OMe. Oil, [α]2D5 ϩ2.7 (c 1.0, DMF); δH 2.86–3.03
(2H, m, Phe β-CH2), 3.58 (3H, s, OMe), 3.56–3.62 (2H, m, Gly
CH2), 4.47 (1H, q-like, J ≈6, Phe α-CH), 5.01 (2H, apparent s,
PhCH2O), 7.17–7.35 (10H, m, 2 × Ph), 7.39 (1H, br t, J ≈6, Gly
NH), 8.30 (1H, d, J 7.5, Phe NH).
Z-Gly-L-Phe-OTfe. Mp 85.5–86.5 ЊC (from EtOAc–
petroleum spirit), [α]2D5 ϩ28.7 (c 1.0, CHCl3); δH 2.93–3.08
(2H, m, Phe β-CH2), 3.58–3.61 (2H, m, Gly CH2), 4.54 (1H,
q-like, J ≈6, Phe α-CH), 4.71 (2H, q, J 8.7, CH2CF3), 5.00 (2H,
apparent s, PhCH2O), 7.19–7.33 (10H, m, 2 × Ph), 7.41 (1H, br
t, J ≈7, Gly NH), 8.46 (1H, d, J 7.5, Phe NH).
Z-Gly-L-Ala-OCam. Mp 135–137 ЊC (from EtOAc–
petroleum spirit), [α]2D5 Ϫ18.6 (c 1.0, DMF); δH 1.30 (3H, d,
J 7.2, Ala Me), 3.56–3.70 (2H, m, Gly CH2), 4.36 (1H, quintet,
J 7.2, Ala α-CH), 4.42 (2H, s, OCH2CO), 5.01 (2H, apparent s,
PhCH2O), 7.29–7.35 (7H, m, Ph ϩ NH2), 7.43 (1H, t, J 6.0, Gly
NH), 8.36 (1H, d, J 7.2, Ala NH).
Z-Gly-Gly-L-Phe-OMe. Mp 98–101 ЊC (from MeOH),
[α]2D5 ϩ8.0 (c 1.0, MeOH) (lit.,14 mp 98–101 ЊC); δH 2.84–3.06
(2H, m, Phe β-CH2), 3.57 (3H, s, OMe), 3.69–3.87 (4H, m,
2 × Gly CH2), 4.46 (1H, q-like, J ≈7, Phe α-CH), 5.01 (2H, s,
PhCH2O), 7.18–7.30 (10H, m, 2 × Ph), 7.49 (1H, br t, J ≈6, Gly
NH), 8.04 (1H, br t, J ≈6, Gly NH), 8.31 (1H, br d, J ≈8, Phe
NH).
Z-Gly-L-Phe-OCam. Mp 141–142.5 ЊC (from EtOAc–
petroleum spirit), [α]2D5 Ϫ9.6 (c 1.0, DMF); δH 2.88–3.17 (2H, m,
Phe β-CH2), 3.52–3.67 (2H, m, Gly CH2), 4.43 (2H, AB q, J 13,
OCH2CO), 4.55–4.62 (1H, m, Phe α-CH), 5.00 (2H, s,
PhCH2O), 7.19–7.33 (12H, m, 2 × Ph ϩ NH2), 7.41 (1H, t,
J 6.9, Gly NH), 8.35 (1H, d, J 7.8, Phe NH).
Z-Gly-Gly-L-Phe-OTfe. Mp 108–110 ЊC (from CHCl3–
petroleum spirit), [α]2D5 ϩ21.6 (c 1.0, CHCl3); δH 2.89–3.16 (2H,
m, Phe β-CH2), 3.61–3.77 (4H, m, 2 × Gly CH2), 4.42 (2H, AB
q, J 18, CH2CF3), 4.54–4.60 (1H, m, Phe α-CH), 5.00 (2H, s,
PhCH2O), 7.19–7.33 (10H, m, 2 × Ph), 7.48 (1H, br t, J ≈6, Gly
NH), 8.10 (1H, br t, J ≈6, Gly NH), 8.38 (1H, br d, J ≈7,
Phe NH).
Z-Gly-Gly-L-Phe-OCam. Mp 170–172 ЊC (from propan-2-
ol–petroleum spirit), [α]2D5 Ϫ6.8 (c 1.0, DMF); δH 2.87–3.16 (2H,
m, Phe β-CH2), 3.61–3.77 (4H, m, 2 × Gly CH2), 4.42 (2H, AB
q, J 17, OCH2CO), 4.54–4.61 (1H, m, Phe α-CH), 5.00 (2H, s,
PhCH2O), 7.19–7.34 (12H, m, 2 × Ph ϩ NH2), 7.47 (1H, br t,
J ≈6, Gly NH), 8.08 (1H, br t, J ≈6, Gly NH), 8.37 (1H, d, J 8.1,
Phe NH).
Z-L-Phe-L-Phe-OTfe. Mp 165–165.5 ЊC (from EtOAc–
petroleum spirit), [α]2D5 ϩ13.9 (c 1.0, CHCl3); δH (CDCl3) 2.94–
3.13 (4H, m, 2 × Phe β-CH2), 4.35–4.51 (3H, m, Phe α-CH ϩ
CH2CF3), 4.83 (1H, q, J 6.6, Phe α-CH), 5.07 (2H, s, PhCH2O),
5.22 (1H, br, NH), 6.22 (1H, br d, J ≈7, NH), 6.95–7.39 (15H,
m, 3 × Ph).
Z-L-Phe-L-Phe-OCam. Mp 156–158 ЊC (from EtOAc–
petroleum spirit), [α]2D5 Ϫ32.3 (c 1.0, MeOH); δH 2.63–3.19 (4H,
m, 2 × Phe β-CH2), 4.23–4.30 (1H, m, Phe α-CH), 4.42 (2H, AB
q, J 15, OCH2CO), 4.57–4.63 (1H, m, Phe α-CH), 4.91 (2H,
apparent s, PhCH2O), 7.17–7.35 (17H, m, 3 × Ph ϩ NH2), 7.49
(1H, d, J 9.0, NH), 8.35 (1H, d, J 7.5, NH).
Z-Gly-DL-Phe(2Br)-OMe. Mp 110–110.5 ЊC (from EtOAc–
petroleum spirit); δH 2.94–3.25 [2H, m, Phe(2Br) β-CH2], 3.58
(3H, s, OMe), 3.49–3.66 (2H, m, Gly CH2), 4.52–4.60 [1H, m,
Phe(2Br) α-CH], 5.01 (2H, apparent s, PhCH2O), 7.14–7.59
(11H, m, ArH), 7.38 (1H, t, J 6.9, Gly NH), 8.43 [1H, d, J 8.1,
Phe(2Br) NH].
Z-Gly-DL-Phe(2Br)-OCam. Mp 153–154.5 ЊC (from acetone–
petroleum spirit); δH 2.98–3.34 [2H, m, Phe(2Br) β-CH2], 3.52–
3.66 (2H, m, Gly CH2), 4.45 (2H, AB q, J 15, OCH2CO), 4.62–
4.71 [1H, m, Phe(2Br) α-CH], 5.01 (2H, apparent s, PhCH2O),
7.15–7.59 (12H, m, Ar ϩ Gly NH ϩ NH2), 8.47 [1H, d, J 7.5,
Phe(2Br) NH].
Preparation of N-protected peptide carbamoylmethyl esters
The Cam esters of N-protected peptides were prepared mainly
through the coupling of an N-protected peptide with an amino
acid Cam ester hydrobromide (prepared through the debenzyl-
oxycarbonylation of the N-Z-amino acid Cam ester; used in the
presence of an equimolar amount of TEA) by the EDC–HOBT
method in DMF in a similar manner to that described above
for the preparation of other esters (85–94% yield). They were
also prepared via the reaction of the Cs salt of an N-protected
peptide with 2-chloroacetamide. The samples prepared by both
routes were identical in terms of their physical properties
including the [α]D-value. The preparation of Z--Phe--Ala-
OCam by the latter route is shown below as a typical example.
To a solution of Z--Phe--Ala (3.72 g, 10 mmol) in MeOH
(60 ml) was added aq. Cs2CO3 (1.63 g, 5 mmol in 7.5 ml), and
the mixture was evaporated under reduced pressure. After
repeated evaporation to dryness with toluene, the residue was
stored over P4O10 in a vacuum desiccator. Z--Phe--Ala-OCs
thus obtained was mixed with 2-chloroacetamide (0.94 g,
Preparation of authentic N-protected peptide amides
The authentic samples of N-protected peptide amides were
prepared through the coupling of an N-protected amino acid or
Z-Gly-Gly with a dipeptide amide hydrochloride or hydro-
bromide (in the presence of an equimolar amount of TEA) by
the EDC–HOBT method in DMF (65–88% yield) as described
previously for the preparation of N-protected dipeptide
amides.4 On the other hand, the mixtures of epimers (- ϩ -
or -- ϩ --) of tripeptides Z-Xaa-Xbb-Leu-NH2 (Xaa =
Gly or Phe; Xbb = Ala or Phe) were prepared through the coup-
ling of Z-Xaa--Xbb with -Leu-NH2 (in the form of its hydro-
chloride with an equimolar amount of TEA) using EDCؒHCl
in DMF (-Xbb is prone to racemisation under these reaction
conditions). A mixture of epimers (- ϩ -) of Z-Gly-Gly-
Phe-Leu-NH2 was prepared in the same manner. Each sample
thus prepared showed only two main peaks, corresponding
to both the epimers on reversed-phase HPLC. The faster
J. Chem. Soc., Perkin Trans. 1, 2002, 396–401
399

View Article Online
eluting epimer proved to be - or -- by comparison with an
Preparation of samples related to the synthesis of the Leu-
authentic sample.
enkephalin sequence
Boc-L-Tyr(Bzl)-Gly-Gly-L-Phe-OCam. This was prepared
by the following two routes. (i) Boc--Tyr(Bzl)-Gly-Gly2b was
coupled with -Phe-OCamؒHBr (in the presence of an equi-
molar amount of TEA) by the EDC–HOBT method in DMF;
79% yield; mp 190–191 ЊC (from acetone), [α]2D5 Ϫ10.9 (c 1.0,
MeOH). Or (ii) Boc--Tyr(Bzl)-Gly-Gly--Phe2b was converted
to the Cs salt and treated with 2-chloroacetamide in DMF at
60 ЊC overnight as described above, and the crude product was
purified by recrystallisation from acetone; 65% yield; mp 190–
191 ЊC, [α]2D5 Ϫ11.2 (c 1.0, MeOH); δH 1.18 and 1.27 (9H, 2 s,
Me3CO), 2.61–3.17 (4H, m, Tyr β-CH2 ϩ Phe β-CH2), 3.69–
3.73 (4H, m, 2 × Gly CH2), 4.06–4.14 (1H, m, Tyr α-CH), 4.43
(2H, AB q, J 16, OCH2CO), 4.53–4.62 (1H, m, Phe α-CH), 5.04
(2H, AB q, J 18, PhCH2O), 6.89 and 7.16 (4H, 2 d, J 8.0, Tyr
ArH), 6.95 (1H, d, J 7.2, Tyr NH), 7.20–7.44 (12H, m, 2 × Ph ϩ
NH2), 8.05 (1H, br t, J ≈6, Gly NH), 8.21 (1H, br t, J ≈6,
Gly NH), 8.39 (1H, d, J 7.8, Phe NH).
Z-Gly-L-Ala-L-Leu-NH2. Mp 164–165 ЊC (from MeOH–
petroleum spirit), [α]2D5 Ϫ22.5 (c 1.0, DMF); δH 0.82 and 0.86
(6H, 2 d, J 6.0, Leu Me2), 1.20 (3H, d, J 6.0, Ala Me), 1.46 (2H,
t-like, J 6.6, Leu β-CH2), 1.49–1.61 (1H, m, Leu γ-CH), 3.56–
3.69 (2H, m, Gly CH2), 4.12–4.29 (2H, m, Ala α-CH ϩ Leu
α-CH), 5.01 (2H, s, PhCH2O), 6.97 and 7.13 (2H, 2 s, NH2),
7.28–7.38 (5H, m, Ph), 7.50 (1H, t, J 6.0, Gly NH), 7.79 (1H, d,
J 8.7, Leu NH), 8.09 (1H, d, J 6.0, Ala NH).
Z-L-Phe-L-Ala-L-Leu-NH2. Mp 202 ЊC (decomp.) (from
MeOH–petroleum spirit), [α]2D5 Ϫ15.8 (c 1.0, DMF); δH 0.83 and
0.86 (6H, 2 d, J 6.6, Leu Me2), 1.22 (3H, d, J 7.2, Ala Me), 1.44
(2H, t-like, J 7.2, Leu β-CH2), 1.51–1.63 (1H, m, Leu γ-CH),
2.66–3.02 (2H, m, Phe β-CH2), 4.17–4.30 (3H, m, Ala α-CH ϩ
Leu α-CH ϩ Phe α-CH), 4.92 (2H, apparent s, PhCH2O), 6.97
(1H, s, NH2), 7.16–7.34 (11H, m, 2 × Ph ϩ NH2), 7.51 (1H, d,
J 8.4, Phe NH), 7.76 (1H, d, J 8.4, Leu NH), 8.19 (1H, d, J 7.5,
Ala NH).
Boc-L-Tyr(Bzl)-Gly-Gly-L-Phe-L-Leu-NH2. This authentic
sample was prepared by the following two routes. (i) Boc--
Tyr(Bzl)-Gly-Gly was coupled with -Phe--Leu-NH2ؒHCl (in
the presence of an equimolar amount of TEA) by the EDC–
HOBT method in DMF; 51% yield. Or (ii) Boc--Tyr(Bzl)
was coupled with Gly-Gly--Phe--Leu-NH2 (prepared by
hydrogenolysis of Z-Gly-Gly--Phe--Leu-NH2 with 5% Pd/C
in MeOH) by the EDC–HOBT method in DMF; 89% yield;
mp 165.5–167 ЊC (from aq. EtOH), [α]2D5 Ϫ 15.7 (c 1.0, DMF);
δH 0.82 and 0.87 (6H, 2 d, J 6.5, Leu Me2), 1.18 and 1.27 (9H,
2 s, Me3CO), 1.42–1.63 (3H, m, Leu β-CH2 ϩ Leu γ-CH), 2.60–
2.95 (2H, m, Tyr β-CH2), 2.72–3.04 (2H, m, Phe β-CH2), 3.57–
3.7l (4H, m, 2 × Gly CH2), 4.06–4.14 (1H, m, Tyr α-CH), 4.18
(1H, apparent q, J ≈7, Leu α-CH), 4.46–4.53 (1H, m, Phe
α-CH), 5.03 (2H, s, PhCH2O), 6.89 and 7.16 (4H, 2 d, J 8.4, Tyr
ArH), 6.92 (1H, d, J ≈9, Tyr NH), 6.99 and 7.10 (2H, 2 s, NH2),
7.20–7.41 (10H, m, 2 × Ph), 7.97 (1H, d, J 8.1, Leu NH), 8.02
(1H, br t, J ≈5.5, Gly NH), 8.09 (1H, d, J 7.8, Phe NH),
8.19 (1H, br t, J ≈5.5, Gly NH); MALDI-TOF MS [Found:
m/z, 767.34. (C40H52N6O8 ϩ Na)ϩ requires m/z, 767.37].
Z-Gly-L-Phe-L-Leu-NH2. Mp 205–206.5 ЊC (from MeOH–
petroleum spirit), [α]2D5 Ϫ18.8 (c 1.0, DMF); δH 0.81 and 0.86
(6H, 2 d, J 6.3, Leu Me2), 1.43–1.61 (3H, m, Leu β-CH2 ϩ Leu
γ-CH), 2.73–3.03 (2H, m, Phe β-CH2), 3.46–3.67 (2H, m, Gly
CH2), 4.19 (1H, q-like, Leu α-CH), 4.46–4.54 (1H, m, Phe
α-CH), 4.99 (2H, apparent s, PhCH2O), 6.98 and 7.09 (2H, 2 s,
NH2), 7.15–7.34 (10H, m, 2 × Ph), 7.43 (1H, t, J 4.5, Gly NH),
7.97 (1H, d, J 8.4, Leu NH), 8.07 (1H, d, J 7.8, Phe NH).
Z-L-Phe-L-Phe-L-Leu-NH2. Mp 235–235.5 ЊC (from MeOH),
[α]2D5 Ϫ29.3 (c 1.0, DMF); δH 0.82 and 0.87 (6H, 2 d, J 6.3, Leu
Me2), 1.42–1.62 (3H, m, Leu β-CH2 ϩ Leu γ-CH), 2.61–3.08
(4H, m, 2 × Phe β-CH2), 4.19–4.24 (2H, m, Leu α-CH ϩ Phe
α-CH), 4.51–4.58 (1H, m, Phe α-CH), 4.91 (2H, apparent s,
PhCH2O), 6.99 and 7.11 (2H, 2 s, NH2), 7.19–7.32 (15H, m, 3 ×
Ph), 7.46 (1H, d, J 8.7, Leu NH), 7.97 (1H, d, J 8.4, Phe NH),
8.16 (1H, d, J 8.1, Phe NH).
Z-Gly-(L/D)-Phe(2Br)-L-Leu-NH2. (mixture of - and -
isomers). Mp 196.5–198.5 ЊC (from MeOH–diethyl ether–
petroleum spirit), [α]2D5 Ϫ29.2 (c 1.0, DMF).
Boc-L-Tyr(Bzl)-Gly-Gly-D-Phe-L-Leu-NH2. This authentic
sample was prepared through the coupling of Boc--Tyr(Bzl)-
Gly-Gly with -Phe--Leu-NH2ؒHBr (prepared through the
treatment of Boc--Phe--Leu-NH2 with 25% HBr in AcOH),
in the presence of an equimolar amount of TEA, by the EDC–
HOBT method in DMF; 45% yield; mp 196.5–198 ЊC (from
aq. EtOH), [α]2D5 Ϫ6.0 (c 1.0, DMF; MALDI-TOF MS [Found:
m/z, 767.35. (C40H52N6O8 ϩ Na)ϩ requires m/z, 767.37].
Z-Gly-Gly-L-Phe-L-Leu-NH2. Mp 195–196 ЊC (from MeOH–
petroleum spirit), [α]2D5 Ϫ18.1 (c 1.0, DMF); δH 0.81 and 0.86
(6H, 2 d, J 6.5, Leu Me2), 1.42–1.60 (3H, m, Leu β-CH2 ϩ Leu
γ-CH), 2.71–3.06 (2H, m, Phe β-CH2), 3.53–3.76 (4H, m, 2 ×
Gly CH2), 4.14–4.23 (1H, m, Leu α-CH), 4.44–4.51 (1H, m, Phe
α-CH), 5.01 (2H, s, PhCH2O), 6.96 and 7.07 (2H, 2 s, NH2),
7.13–7.34 (10H, m, 2 × Ph), 7.48 (1H, t, J 4.5, Gly NH), 7.93
(1H, d, J ≈8, Leu NH), 8.07 (1H, d, J ≈8, Phe NH), 8.08 (1H,
t-like, Gly NH).
Boc-L-Tyr(Bzl)-L-Leu-NH2. This authentic sample was pre-
pared through the coupling of Boc--Tyr(Bzl) with -Leu-NH2ؒ
HCl (in the presence of an equimolar amount of TEA) by the
EDC–HOBT method in DMF; 58% yield; mp 183–183.5 ЊC
(from CHCl3), [α]2D5 Ϫ11.0 (c 1.0, DMF); δH 0.82 and 0.86 (6H, 2
d, J 6.6, Leu Me2), 1.23 and 1.30 (9H, 2 s, Me3CO), 1.42–1.63
(3H, m, Leu β-CH2 ϩ Leu γ-CH), 2.62–2.91 (2H, m, Tyr β-
CH2), 4.02–4.11 (1H, m, Tyr α-CH), 4.20–4.27 (1H, m, Leu
α-CH), 5.04 (2H, s, PhCH2O), 6.89 and 7.15 (4H, 2 d, J 8.4, Tyr
ArH), 6.92 (1H, d, J ≈9, Tyr NH), 6.99 and 7.20 (2H, 2 s, NH2),
7.28–7.43 (5H, m, Ph), 7.81 (1H, d, J 8.1, Leu NH).
ꢀ-Chymotrypsin-catalysed segment condensations to tri- or
tetrapeptides
The preparation of Z--Phe--Phe--Leu-NH2 is described as a
typical example. A mixture of Z--Phe--Phe-OCam (25 mg,
0.05 mmol), -Leu-NH2ؒHCl (33 mg, 0.2 mmol), TEA (28 µl,
0.2 mmol) and the immobilised enzyme on Celite (150 mg,
corresponding to 4.7 mg of α-chymotrypsin) was incubated
with shaking (180 strokes minϪ1) in a solvent composed of
acetonitrile (2 ml) and 0.05 M Tris buffer (pH 7.8) (83 µl) at
30 ЊC. An aliquot (10 µl) of the reaction mixture was withdrawn
periodically, diluted with AcOH (100 µl), and analysed by
HPLC. The targeted peptide and its epimer (Z--Phe--Phe--
Leu-NH2), the remaining donor ester and possible by-products
(Z--Phe--Phe, Z--Phe and Z--Phe--Leu-NH2) were
quantified by HPLC analysis on an ODS column (see below).
Boc-L-Tyr-Gly-Gly-L-Phe. This was prepared by hydro-
genolysis of Boc--Tyr(Bzl)-Gly-Gly--Phe with 5% Pd/C in
MeOH; quantitative yield; mp 153–155 ЊC, [α]2D5 ϩ23.6 (c 1.0,
MeOH); δH 1.20 and 1.28 [9H, 2 s (15 : 85), Me3CO], 2.57–3.07
(4H, m, Tyr β-CH2 ϩ Phe β-CH2), 3.67–3.71 (4H, m, 2 × Gly
CH2), 4.03–4.10 (1H, m, Tyr α-CH), 4.32–4.39 (1H, m, Phe
α-CH), 6.62 and 7.01 (4H, 2 d, J 8.4, Tyr ArH), 6.88 (1H, d,
400
J. Chem. Soc., Perkin Trans. 1, 2002, 396–401

View Article Online
J 8.1, Tyr NH), 7.17–7.27 (5H, m, Ph), 7.94 (1H, br t, J ≈6, Gly
NH), 8.03 (1H, d, J 7.8, Phe NH), 8.14 (1H, t, J 5.4, Gly NH),
9.14 (1H, s, ArOH), 12.6 (1H, br, COOH).
flow rate, 0.1 ml minϪ1; column temperature, 30, 35 or 40 ЊC;
detection, UV at 254 nm. The epimers (- and -, or --
and --) of the resulting peptides were also separated well on
the same column by decreasing the amount of acetonitrile or
MeOH in the mobile phase. The HPLC separation of com-
pounds relevant to the α-chymotrypsin-catalysed coupling of
Z--Phe--Phe-OR with -Leu-NH2 is shown in Table 2. The
HPLC separation of compounds relevant to the synthesis of
the Leu-enkephalin sequence is also available as Supplementary
material.†
Boc-L-Tyr-Gly-Gly-L-Phe-OCam. This was prepared by
hydrogenolysis of Boc--Tyr(Bzl)-Gly-Gly--Phe-OCam with
5% Pd/C in DMF; 30% yield; mp 177–179 ЊC (from CHCl3–
EtOAc–petroleum spirit), [α]2D5 Ϫ8.8 (c 1.0, DMF); δH 1.18 and
1.27 (9H, 2 s, Me3CO), 2.56–3.17 (4H, m, Tyr β-CH2 ϩ Phe
β-CH2), 3.67–3.73 (4H, m, 2 × Gly CH2), 4.06–4.14 (1H, m,
Tyr α-CH), 4.42 (2H, AB q, J 15.5, OCH2CO), 4.53–4.60 (1H,
m, Phe α-CH), 6.61 and 7.01 (4H, 2 d, J 7.0, Tyr ArH), 6.89
(1H, d, J 7.5, Tyr NH), 7.18–7.33 (7H, m, Ph, NH2), 8.01 (1H,
br t, J ≈6, Gly NH), 8.15 (1H, br t, J ≈6, Gly NH), 8.35 (1H, d,
J ≈8, Phe NH), 9.13 (1H, s, OH). When the deprotection by
hydrogenolysis of the Bzl group from the protected Cam ester
was conducted in MeOH, Boc--Tyr-Gly-Gly--Phe-OMe was
obtained instead.
Acknowledgements
This work was supported in part by a grant from the Hirao
Taro Foundation of the Konan University Association for
Academic Research.
References and notes
Boc-L-Tyr-Gly-Gly-L-Phe-L-Leu-NH2. This was prepared by
hydrogenolysis of Boc--Tyr(Bzl)-Gly-Gly--Phe--Leu-NH2
with 5% Pd/C in MeOH; 81% yield; mp 162–164.5 ЊC (from
aq. EtOH), [α]2D5 Ϫ17.7 (c 1.0, DMF); δH 0.82 and 0.87 (6H, 2 d,
J 6.2, Leu Me2), 1.20 and 1.28 [9H, 2 s (2 : 98), Me3CO], 1.43–
1.63 (3H, m, Leu β-CH2 ϩ Leu γ-CH), 2.57–3.05 (4H, m, Tyr β-
CH2 ϩ Phe β-CH2), 3.58–3.7l (4H, m, 2 × Gly CH2), 4.03–4.10
(1H, m, Tyr α-CH), 4.19 (1H, apparent q, J ≈7, Leu α-CH),
4.47–4.54 (1H, m, Phe α-CH), 6.62 and 7.01 (4H, 2 d, J 7.8, Tyr
ArH), 6.87 (1H, d, J 8.1, Tyr NH), 6.96 and 7.08 (2H, 2 s, NH2),
7.16–7.24 (5H, m, Ph), 7.97 (1H, d, J ≈8, Leu NH), 7.98 (1H,
apparent t, Gly NH), 8.08 (1H, d, J 7.5, Phe NH), 8.15 (1H, br
t, J ≈6, Gly NH), 9.14 (1H, br, OH).
1 M. Bodanszky, Principles of Peptide Synthesis, Springer-Verlag,
Berlin, 2nd edn., 1993, pp. 9, 215.
2 We have investigated racemisation-suppressing additives in the
carbodiimide-meditated segment condensations, see (a) T.
Miyazawa, T. Otomatsu, Y. Fukui, T. Yamada and S. Kuwata, Int.
J. Pept. Protein Res., 1992, 39, 237; (b) T. Miyazawa, T. Otomatsu,
Y. Fukui, T. Yamada and S. Kuwata, Int. J. Pept. Protein Res., 1992,
39, 308.
3 For reviews, see: (a) K. Faber, Biotransformations in Organic
Chemistry, Springer-Verlag, Berlin, 4th edn., 2000, p. 370;
(b) H.-D. Jakubke, in Enzyme Catalysis in Organic Synthesis,
ed. K. Drauz and H. Waldmann, VCH, Weinheim, 1995, B.2.5,
p. 431; (c) C.-H. Wong and G. M. Whitesides, Enzymes in Synthetic
Organic Chemistry, Pergamon Press, Oxford, 1994, p. 46.
4 (a) T. Miyazawa, S. Nakajo, M. Nishikawa, K. Hamahara,
K. Imagawa, E. Ensatsu, R. Yanagihara and T. Yamada, J. Chem.
Soc., Perkin Trans. 1, 2001, 82; (b) T. Miyazawa, K. Tanaka,
E. Ensatsu, R. Yanagihara and T. Yamada, J. Chem. Soc., Perkin
Trans. 1, 2001, 87.
5 T. Miyazawa, E. Ensatsu, N. Yabuuchi, R. Yanagihara and
T. Yamada, J. Chem. Soc., Perkin Trans. 1, Preceding paper: 2002,
DOI 10.1039/b108735p.
6 Preliminary communication: T. Miyazawa, E. Ensatsu, K. Tanaka,
R. Yanagihara and T. Yamada, Chem. Lett., 1999, 1013.
7 The ameliorating effect of the Cam ester as the acyl donor
was verified in the α-chymotrypsin-catalysed couplings with a
number of -amino acid amides as amino components: see Ref. 5.
8 W. Kullmann, Enzymatic Peptide Synthesis, CRC Press, Boca Raton,
Florida, 1987.
Synthesis of the Leu-enkephalin sequence via the
ꢀ-chymotrypsin-catalysed segment condensation
The α-chymotrypsin-catalysed coupling of Boc--Tyr(Bzl)-Gly-
Gly--Phe-OCam (34 mg, 0.05 mmol) with -Leu-NH2ؒHCl
(33 mg, 0.2 mmol) in the presence of an equimolar amount of
TEA was done in the same manner as described above. The
targeted peptide and its epimer, the remaining donor ester
and possible by-products [Boc--Tyr(Bzl)-Gly-Gly--Phe, Boc-
-Tyr(Bzl) and Boc--Tyr(Bzl)--Leu-NH2] were quantified by
HPLC analysis (see below). The coupling of Boc--Tyr-Gly-
Gly--Phe-OCam with -Leu-NH2 was likewise undertaken.
9 The Cam ester was first proposed as a carboxy-protecting group for
peptide synthesis by J. Martinez, J. Laur and B. Castro, Tetrahedron,
1985, 41, 739. To the best of our knowledge, the direct preparation
of Cam esters of peptide fragments via the Cs salts has not been
reported since then.
10 J. H. Jones, B. Liberek and G. T. Young, J. Chem. Soc. C, 1967, 2371.
11 S. Goldschmidt and K. K. Gupta, Chem. Ber., 1965, 98, 2831.
12 M. A. Tilak and J. A. Hoffmann, J. Org. Chem., 1977, 42, 2098.
13 P. G. Katsoyannis, J. Gines, G. P. Schwarts and A. Cosmatos,
J. Chem. Soc., Perkin Trans. 1, 1974, 1311.
HPLC analyses
The liquid chromatograph employed was a GL Sciences PU-
610 instrument equipped with a Rheodyne 8125 sample
injector, a GL Sciences UV-620 variable-wavelength UV moni-
tor and a Shimadzu C-R6A data processor. HPLC analyses
were undertaken under the following conditions: column,
Inertsil ODS 3 (5 µm; 1.5 mm i.d. × 150 mm, GL Sciences);
mobile phase, 42–54% aq. acetonitrile containing H3PO4
(0.01 M) or 50–65% aq. MeOH containing H3PO4 (0.01 M);
14 G. Gawne, G. W. Kenner and R. C. Sheppard, J. Am. Chem. Soc.,
1969, 91, 5669.
J. Chem. Soc., Perkin Trans. 1, 2002, 396–401
401