Improving the carboxyamidomethyl ester for subtilisin A-catalysed peptide synthesis

Article abstract of DOI:10.1039/c2ob25662b

A series of novel glycine esters was evaluated for efficiency in subtilisin A-CLEA-catalysed peptide synthesis. The reactivity of the easily accessible carboxyamidomethyl (Cam) ester was further enhanced by elongating it with an amino acid residue, thereby creating more recognition space for subtilisin A.

Full text of DOI:10.1039/c2ob25662b

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PAPER
Improving the carboxyamidomethyl ester for subtilisin A-catalysed peptide
synthesis†
Roseri J. A. C. de Beer,‡a Timo Nuijens,‡b Lotte Wiermans,b Peter J. L. M. Quaedfliegb and
Floris P. J. T. Rutjes*a
Received 2nd April 2012, Accepted 2nd July 2012
DOI: 10.1039/c2ob25662b
A series of novel glycine esters was evaluated for efficiency in subtilisin A-CLEA-catalysed peptide
synthesis. The reactivity of the easily accessible carboxyamidomethyl (Cam) ester was further enhanced
by elongating it with an amino acid residue, thereby creating more recognition space for subtilisin A.
condensation to make a peptide of 20 amino acids. The major
drawback of chemical fragment condensation is that upon acti-
Introduction
A large number of peptides have been introduced into the market
as therapeutics or prodrugs,1 and an even increasing number is
in clinical trials. Additionally, peptides have found applications
as nutritional additives or as cosmetic ingredients.2 Despite this
demand for peptides, their production on a large scale remains
expensive and time consuming.3 Common peptide synthesis
approaches include fermentation, solid-phase or solution-phase
chemical peptide synthesis, and chemoenzymatic peptide syn-
thesis.4 Currently, the fermentative approach is only well feasible
for very large peptides (>50 amino acid residues) and proteins
containing natural fragments and requires a large development
effort for each individual target product. Solution-phase chemi-
cal peptide synthesis is most commonly used for the synthesis of
small peptides containing two to ten amino acid residues. Solid-
phase peptide synthesis (SPPS)5 is the most commonly applied
method for medium-sized and long peptides (10–50 amino acid
residues). However, peptides longer than 10–15 amino acids are
not cost-efficiently synthesised on the solid phase because they
tend to form tertiary structures (by so-called “hydrophobic col-
lapse”) making peptide elongation troublesome so that a large
excess of reagents and amino acid building blocks is needed.6
Additionally the purification of the final product is often cost-
inefficient due to the presence of significant amounts of peptides
of similar length. Therefore, peptides longer than 10 amino acids
are often produced using a combination of solid-phase synthesis
of protected oligopeptide fragments which are subsequently
vation of the C-terminal amino acid residue racemisation occurs,
except when C-terminal Gly or Pro residues are used. Therefore,
the chemical fragment condensation strategy is usually limited to
using C-terminally activated Gly and Pro residues, or one has to
deal with a difficult purification due to the formation of un-
desired diastereoisomers.
Chemoenzymatic peptide synthesis has been examined by
several groups during the past decades and proved to be suitable
for certain short peptide sequences up to five amino acid resi-
dues.4 The application of proteases as a coupling reagent is a
promising alternative since functionalised amino acid side chains
do not require protection and, most importantly, C-terminal race-
misation is completely absent during fragment assembly, which
is beneficial for the characterisation and purification of the final
peptide.
There are two approaches to performing enzymatic peptide
synthesis, the thermodynamically and the kinetically controlled
approach.7 In thermodynamically controlled peptide synthesis,
an N-terminal protected acyl donor having a free C-terminal car-
boxylic acid function reacts with a C-terminal protected amino
acid nucleophile, resulting in the formation of the peptide bond
and expulsion of one water molecule. Thermodynamically con-
trolled peptide coupling is, however, rather slow and the thermo-
dynamic equilibrium between the product and starting materials
needs to be shifted into the synthetic direction, for example by
product precipitation, water withdrawal, or by using organic
solvents, to obtain a high product yield. This is in contrast to
kinetically controlled peptide synthesis, in which an N-terminal
protected and C-terminal activated amino acid or peptide ester
reacts preferentially with a C-terminal protected acyl acceptor,
usually giving a higher product yield in a shorter reaction time.
The high selectivity of proteases restricts the number of amino
acids that will be recognised. Therefore, coupling of different
peptide sequences using one single enzyme remains challenging.
To remedy these limitations, designed acyl donors with an ester
chemically condensed in solution, e.g. by
a 10 + 10
aInstitute for Molecules and Materials, Radboud University Nijmegen,
Heyendaalseweg 135, NL-6525 AJ Nijmegen, The Netherlands.
E-mail: f.rutjes@science.ru.nl
bDSM Innovative Synthesis B.V., P.O. Box 18, NL-6160 MD Geleen,
The Netherlands
†Electronic supplementary information (ESI) available. See DOI:
10.1039/c2ob25662b
‡Both authors contributed equally.
This journal is © The Royal Society of Chemistry 2012
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Table 1 Relative activity of various Cbz-Gly-OR esters in Alcalase-
CLEA-catalysed peptide coupling with H-Phe-NH2
moiety that is specifically recognised by the enzyme, among
others, guanidinophenyl (Gp),8 carboxamidomethyl (Cam)9 or
2,2,2-trifluoroethyl (Tfe)10 esters, have been used. In the pres-
ence of these activated esters, protease-mediated couplings of
weak nucleophiles and non-proteinogenic amino acid residues
have become feasible.11 Although these active esters broaden the
scope of enzymatic peptide synthesis, their preparation is not
straightforward12 since highly reactive reagents are required to
couple the poorly nucleophilic alcohol derivatives, which
increases the risk of racemisation.13 In fact, the coupling reagents
used are often identical to those used for chemical peptide bond
formation.14 A few solid-phase methodologies have been
reported for the synthesis of peptide Gp esters, but these strate-
gies are complicated and require the use of resins which are not
available on an industrial scale.15
Recently, we reported the use of amino acid and peptide Cam-
esters in subtilisin A-catalysed peptide synthesis in anhydrous
organic solvents.16 Subtilisin A, a serine endoprotease, is indust-
rially available in the form of Alcalase. Although the enzyme
has a slight preference for large uncharged hydrophobic residues
in both the P1 and the P1′ positions,17 a broad range of substrates
is accepted and it can therefore be applied to the synthesis of
numerous peptides.18 It was shown that the use of Cam esters
broadened the scope of Alcalase-catalysed peptide synthesis
even further and that the coupling of very challenging substrates
became feasible in high yields without any hydrolytic side reac-
tions. However, coupling reactions sometimes remained rather
slow and a relatively large amount of enzyme was required,
especially when longer peptide fragments were to be coupled.
Clearly, there is room for improvement of the acyl donor ester
for Alcalase-catalysed peptide synthesis.
Entry
1
R =
Relative activity
100
1
2
2
3
95
70
3
4
5
6
4
5
6
68
60
34
7
7
30
8
9
8
9
31
28
In this paper we compare various new active esters for Alca-
lase-catalysed peptide synthesis in organic solvents, including
Cam esters that are elongated with any of the proteinogenic
amino acid residues. These elongated Cam esters could be con-
veniently synthesised in high yield and purity using specifically
designed solid-phase peptide synthesis techniques with readily
available starting materials and resins.
10
10
23
11
12
11
12
11
9
Results and discussion
To test the coupling efficiencies in Alcalase-catalysed dipeptide
synthesis, with H-Phe-NH2 functioning as the nucleophile, a
library of Cbz-Gly-OR esters was synthesised. The library was
synthesised using previously described chemical techniques and
was originally designed to identify improved activating esters for
papain.19 Alcalase cross-linked enzyme aggregates (CLEAs)20
were used for convenient handling and workup. A solvent
mixture of dimethylformamide–tetrahydrofuran (DMF–THF
1 : 9, v/v) ensured good solubility of all starting materials. Mo-
lecular sieves were added to remove water from the reaction
mixture, to prevent any hydrolytic side reactions. The results of
the screening are given in Table 1.
13
14
13
14
8
8
15
16
17
15
16
17
7
4
4
Clearly, the substituted phenyl esters (entries 1–3) are the
most active species for the Alcalase-CLEA-catalysed peptide
coupling, followed by the Tfe and Cam ester derivatives (entries
4 and 5). This might be explained by their strong electron-with-
drawing properties, which render the carbonyl more susceptible
to nucleophilic attack by the active site serine. In addition, it also
enhances the leaving group ability of the corresponding alcohols.
The drawbacks of these phenyl esters are that, due to their high
activation level, they are relatively difficult to synthesise, show
6768 | Org. Biomol. Chem., 2012, 10, 6767–6775
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Fig. 1 Similarity of the natural endopeptidase peptide cleavage site
with the Cam- and Cam-Xxx-NH2 ester.
spontaneous peptide coupling and other side reactions. The
Cam-ester (entry 5), which is chemically21 and enzymatically16
much easier to synthesise and does not give any spontaneous
peptide coupling or racemisation, also displays very good
activity. This activity cannot only be explained by its electron-
withdrawing properties. It is believed that the amide group of the
Cam ester moiety binds to the enzyme via a hydrogen bond in
the same fashion as an amide of a peptide backbone binds when
it is recognised and cleaved by an endoprotease (Fig. 1A). Wells
et al. already showed for subtiligase, a mutant of subtilisin BPN
wherein the active site serine is replaced by a cysteine, that the
Cam ester activation (Fig. 1B) could be improved by elongating
it with an amino acid amide, thereby creating additional binding
interactions with the enzyme (Fig. 1C).22
To investigate if the Cam ester could be further improved for
Alcalase, a library of Fmoc-Val-Ala-OGlyc-Xxx-NH2 esters,
wherein Xxx stands for all 20 proteinogenic amino acids, with
an either protected or unprotected side chain functionality, was
synthesised using specifically designed SPPS techniques. Two
methods were developed, one approach is based on the attach-
ment of iodoacetic acid to a Sieber resin (Scheme 1A) while the
other method is based on the attachment of Fmoc-Xxx-
OCH2CO2H building blocks to the same resin (Scheme 1B).
Both methods have their appealing characteristics. The advan-
tage of method A is that no special amino acid building blocks
have to be used, however, the esterification has to be performed
at 50 °C for 24 h, which complicates automated peptide syn-
thesis. Although method B requires the use of special Fmoc
amino acid building blocks, the advantage is that all reactions on
the resin are performed using standard SPPS protocols at
ambient temperature. Furthermore, the Fmoc-Xxx-OCH2CO2H
building blocks are easily accessible. The dipeptide Glyc-Xxx-
NH2 esters were obtained in very high yield and purity and no
side reactions were observed upon cleavage from the resin with
trifluoroacetic acid.
Scheme 1 Two different solid phase methods developed for the syn-
thesis of peptide Glyc-Xxx-NH2 esters.
coupling using H-Phe-NH2 as the nucleophile, as shown in
Table 2. The product peak was analysed using HPLC and the
yields were calculated using a calibration curve of Fmoc-Val-
Ala-Phe-NH2 (18).There are large differences between the
The relative activity of the Fmoc-Val-Ala-OGlyc-Xxx-NH2
esters was tested in an Alcalase-CLEA-catalysed peptide
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Table 2 Relative activity of various Fmoc-Val-Ala-OCam-Xxx-NH2
esters in the Alcalase-CLEA-catalysed peptide coupling with H-Phe-
NH2
Table 3 Relative activity of various Cbz-D-Phe-OR esters in Alcalase-
CLEA-catalysed peptide coupling with H-Phe-NH2
Entry
1
R =
Relative activity
100
52
2
3
53
54
98
90
Entry
Xxx =
Relative activity
1
2
3
4
5
6
7
8
Phe
Tyr
Ser
Leu
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
204
198
169
165
163
143
143
133
131
124
114
108
108
106
104
100
92
90
88
88
84
84
82
63
53
Arg
Asp(OtBu)
Ser(tBu)
Gln(Trt)
Ala
4
55
54
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
Gly
Cys
Thr
Glu(OtBu)
Arg(Pbf)
Pro
reported that no peptide product was obtained at all using Alca-
lase and Cbz-D-Phe as the acyl donor.18
As is evident from Table 3, the elongated Cam ester (entry 3)
shows comparable reactivity to the substituted phenol esters
(entries 1 and 2). An equally active, but more conveniently
accessible ester was thus developed for Alcalase-CLEA-cata-
lysed peptide synthesis. Another advantage is that no racemisa-
tion occurred on the activated amino acid ester, i.e. D-Phe, using
the Cbz-D-Phe-OGlyc-Phe-NH2 ester (54, entry 3, ee of D-Phe
>99.5), this is in contrast to the Cbz-D-Phe-OTMAP (52, entry 1,
ee of D-Phe 85.8%) ester.
Although the Glyc-Xxx-NH2 esters can be conveniently syn-
thesised on the solid phase, the Sieber or Rink resins required to
obtain the C-terminal amide functionality are relatively expens-
ive. The resins which are commonly used to obtain a C-terminal
carboxylic acid functionality (Wang or 2-chlorotritylchloride
resin) are generally much cheaper and thus better suited for
large-scale applications. Therefore, the relative activity of a
C-terminal carboxamide functionality (Fmoc-Val-Ala-OGlyc-
Leu-NH2, 22) was compared to a C-terminal carboxylic acid
functionality (Fmoc-Val-Ala-OGlyc-Leu-OH, 56) in the Alca-
lase-CLEA-catalysed peptide coupling with H-Phe-NH2 as the
nucleophile. Gratifyingly, as demonstrated in Table 4, compar-
able reaction rates were obtained.
--- (= Cam)
Cys(Trt)
Trp(Boc)
Tyr(tBu)
Ile
Gln
Thr(tBu)
Lys
Trp
Met
Lys(Boc)
Asn(Trt)
Glu
Val
Asn
His
His(Trt)
Asp
45
41
37
33
31
16
16
12
Glyc-Xxx-NH2 esters and almost an equal part of them is better
(entries 1–15) or worse (entries 17–33) than the non-elongated
Cam ester (entry 16). The best results were obtained using Phe
and Tyr (19 and 20, entries 1 and 2), resulting in a two-fold
enhancement.
The optimal Glyc-Xxx-NH2 ester (Table 2, entry 1, Glyc-Phe-
NH2, 19) was compared to the most active substituted phenol
esters from the first Cbz-Gly-OR screening (Table 1, entries 1
and 2). To clearly discern the intrinsic reactivities, the challen-
ging substrate Cbz-D-Phe was chosen as the acyl donor with
H-Phe-NH2 as the nucleophile (Table 3). D-Amino acids are
notoriously difficult substrates for Alcalase, in fact, Chen et al.
Conclusions
In conclusion, the performance of various novel and known
C-terminal esters in subtilisin A-catalysed peptide synthesis in
anhydrous organic solvents was compared. It appeared that
C-terminal phenolic esters were most active but they are difficult
6770 | Org. Biomol. Chem., 2012, 10, 6767–6775
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Table 4 Relative activity of Cam-Leu-NH2 and Cam-Leu-OH esters in
the Alcalase-CLEA-catalysed peptide coupling with H-Phe-NH2
lyophilisation. The 3 Å molecular sieves (Acros, 8–12 mesh)
were activated (200 °C under vacuum overnight), crushed and
sieved (0.5 mm pore size) to remove large particles. To deter-
mine the ee of Phe the samples were concentrated in vacuo and
the residue suspended in excess 6 N HCl and refluxed overnight.
Chiral HPLC was performed on a crown ether (+) column
(150 mm length, 4.0 mm internal diameter, 5 μm particle size) at
25 °C with 30 mM aqueous HClO4 (pH = 2.0) as the eluent.
UV detection was performed at 210 nm using a UV-VIS linear
spectrometer. The flow was 1 mL min−1. Injection volumes were
5 μL. Rt (D-Phe) = 6.90 min, Rt (L-Phe) = 8.82 min, synthesis
details and characterisation data of the Cbz-Gly-OR library have
been published previously.19 Synthesis16 and analytical data23 of
Cbz-D-Phe-OCam were identical to those reported in the
literature.
Entry
X =
Relative activity
1
2
NH2
OH
56
22
91
100
General procedure A: synthesis of peptide Glyc-Xxx-NH2 esters
The Fmoc protecting group was removed from a Sieber resin
(1 g, loading = 0.5 mmol g−1) using piperidine in N-methyl-
2-pyrrolidone (NMP, 10 mL, 1 : 4, v/v) followed by washing
steps with NMP (10 mL, 2 min, 3×), CH2Cl2 (10 mL, 2 min,
3×) and NMP (10 mL, 2 min, 3×). Fmoc-Xxx-OH was coupled
to the amine using O-benzotriazole-N,N,N′,N′-tetramethyl-
to synthesise in a chemically and stereochemically pure form.
The slightly less reactive C-terminal carboxamidomethyl (Cam)
esters, however, are easily accessible and are not prone to race-
misation. It was demonstrated that the activity of Cam esters can
be increased to the level of the most active phenolic esters by
elongating them with (apolar) amino acids and amino acid
amides. Additionally, the convenient accessibility of these substi-
tuted Cam-esters via two different solid-phase strategies was
demonstrated.
uronium-hexafluorophosphate (HBTU,
2 mmol, 758 mg),
1-hydroxy-7-azabenzotriazole (HOAt, 2 mmol, 272 mg) and di-
isopropylethylamine (DIPEA, 4 mmol, 697 μL) in NMP (10 mL,
60 min). The resin was washed with NMP (10 mL, 2 min, 3×),
CH2Cl2 (10 mL, 2 min, 3×) and NMP (10 mL, 2 min, 3×) fol-
lowed by removal of the Fmoc protecting group using piperidine
in NMP (10 mL, 1 : 4, v/v). After washing with NMP (10 mL,
2 min, 3×), CH2Cl2 (10 mL, 2 min, 3×) and N,N-dimethylforma-
mide (DMF, 10 mL, 2 min, 3×) the free amine was reacted with
iodoacetic acid (1 mmol, 186 mg), diisopropylcarbodiimide
(DIC, 1 mmol, 155 μL) and HOAt (1 mmol, 136 mg) in DMF
(10 mL, 30 min) followed by washing steps with DMF (10 mL,
2 min, 2×), CH2Cl2 (10 mL, 2 min, 2×), and DMF (10 mL,
2 min, 2×). Subsequently, Fmoc-Xxx-OH (2 mmol) and DIPEA
(2.5 mmol, 436 μL) in DMF–tetrahydrofuran (DMF–THF, 1 : 1,
v/v, 10 mL) were added and the mixture was shaken at 50 °C
with 200 rpm for 20 h. Afterwards, the resin was washed with
10 vol% H2O in DMF (10 mL, 2 min, 2×) and DMF (10 mL,
2 min, 3×). Further Fmoc deprotection and Fmoc amino acid
coupling cycles were performed using standard SPPS protocols.5
The peptide Cam ester was cleaved from the resin using 2.5 vol
% trifluoroacetic acid (TFA) in CH2Cl2 (20 mL) for 30 min.
After filtration the volatiles were partly concentrated in vacuo to
Experimental
General information
All resins and reagents used for SPPS were purchased from GL
Biochem (Changhai, China) and all peptide grade solvents from
Biosolve (Valkenswaard, The Netherlands). Before use, 3 g
Alcalase-CLEA (type OM, CLEA-Technologies, 580 U g−1
)
t
was suspended in 100 mL BuOH and crushed with a spatula.
After filtration, the enzyme was resuspended in 50 mL MTBE
followed by filtration. Large enzyme particles were removed by a
sieve (0.5 mm pore size). 1H and 13C NMR spectra were
recorded on a Bruker Avance 300 MHz NMR spectrometer and
chemical shifts are given in ppm (δ) relative to TMS (0.00 ppm),
DMSO-d6 (2.50 ppm for 1H or 39.9 ppm for 13C) or CDCl3
(77.0 ppm for 13C). Analytical HPLC chromatograms were
recorded on an HP1090 Liquid Chromatograph, using a
reversed-phase column (Phenomenex, C18, 5 μm particle size,
150 × 4.6 mm) at 40 °C. The gradient program was: a 0–25 min
linear gradient ramp from 5% to 98% eluent B and from
25.1–30 min with 5% eluent B (eluent A 0.5 mL L−1 methane
sulfonic acid (MSA) in H2O, eluent B 0.5 mL L−1 MSA in
acetonitrile). The flow was 1 mL min−1 from 0–25.1 min and
2 mL min−1 from 25.2–29.8 min, then back to 1 mL min−1 until
stopping at 30 min. Injection volumes were 20 μL. Preparative
HPLC was performed on a Varian PrepStar system using a
stationary-phase column (Pursuit XRs, C18, 10 μm particle size,
500 × 41.4 mm). Pure fractions were pooled, followed by
i
a volume of 10 mL. Subsequently, PrOH in H2O (1 : 3, v/v,
50 mL) was added followed by partial evaporation of the vola-
tiles to a volume of 30 mL. The precipitates were removed by
filtration and washed with H2O (5 mL, 2×), followed by
lyophilisation from MeCN–H2O (3 : 1, v/v).
General procedure B: synthesis of peptide Glyc-Xxx-NH2 esters
Coupling and Fmoc deprotection of the first amino acid was
identical to general procedure A. Fmoc-Xxx-OCH2COOH
(1 mmol) (Fmoc-Val-Ala-OCH2COOH for the library synthesis)
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was coupled to the free amine using HBTU (1 mmol, 379 mg),
1-hydroxybenzotriazole (HOBt, 1 mmol, 136 mg) and DIPEA
(2 mmol, 349 μL) in NMP (10 mL, 90 min). Further Fmoc
deprotection and Fmoc amino acid coupling cycles were per-
formed using standard SPPS protocols.5 After synthesis of the
desired sequence, the peptide was cleaved from the resin and
precipitated as described in general procedure A.
reaction mixture was concentrated in vacuo. The residue was
resuspended in EtOAc (100 mL) and this solution was washed
with sat. aq. NaHCO3 (100 mL, 2×), 0.1 N HCl (100 mL, 2×),
brine (100 mL) and dried (Na2SO4). The solution was concen-
trated in vacuo and the residue was resuspended in 2 N HCl in
dioxane (25 mL). After stirring for 1 h, cold diethyl ether (Et2O,
100 mL) was added and the precipitate was filtered off and
washed with cold Et2O (20 mL, 2×). HCl·H-Ala-OCH2C(vO)
O-Bn was dried at 40 °C in vacuo for 24 h and obtained in an
overall yield of 87%. Rt(HPLC) 5.19 min; purity = 98%;
1H NMR (CDCl3, 300 MHz): δ = 1.62 (d, J = 6.9 Hz, 3H),
4.19–4.28 (m, 1H), 4.51–4.77 (m, 2H), 5.03 (s, 2H), 7.21
(s, 5H), 8.64 (s, 3H); 13C NMR (CDCl3, 75 MHz): δ = 16.0,
49.2, 61.8, 67.4, 128.5, 128.6, 128.7, 134.8, 166.9, 169.7.
General procedure C: side chain deprotection of
Fmoc-Val-Ala-OGlyc-Xxx-NH2 peptides
Fmoc-Val-Ala-OGlyc-Xxx-NH2 (0.02 mmol) was dissolved in
TFA–H2O (1 mL, 95 : 5, v/v) and stirred for 1 h. Afterwards, the
volatiles were concentrated by nitrogen flow and the residue
lyophilised from CH3CN–H2O (3 : 1, v/v). The lyophilised
powders were dissolved in DMF (100 μL) and used as such for
the relative activity determination assays described below.
Additional piperidine (0.02 mmol, 2 μL) was added to the stock
solutions of Fmoc-Val-Ala-OGlyc-Asp-NH2 and Fmoc-Val-Ala-
OGlyc-Glu-NH2.
Fmoc-Val-Ala-OCH2COOH
To a solution of Fmoc-Val-OH (1 mmol, 339 mg) in CH2Cl2
(50 mL) which was cooled on ice to 0 °C, 1-ethyl-3(3-dimethyl-
aminopropyl)carbodiimide hydrochloride (EDCl, 1.1 mmol,
211 mg) and HOBt (1.1 mmol, 150 mg) were added. After stir-
ring for 30 min, HCl·H-Ala-OCH2C(vO)O-Bn (1.1 mmol,
300 mg) and DIPEA (1.1 mmol, 192 μL) were added. The
obtained reaction mixture was stirred at ambient temperature for
6 h, followed by washing with sat. aq. NaHCO3 (100 mL, 2×),
0.1 N HCl (100 mL, 2×), and brine (100 mL). The CH2Cl2 solu-
tion was dried (Na2SO4) and subsequently concentrated in vacuo
and the residue was resuspended in MeOH–toluene (1 : 1, v/v,
250 mL). Then, the mixture was hydrogenated using Pd/C
(10 mol%) with 5 bar H2 at 25 °C for 24 h. Afterwards, the reac-
tion mixture was filtrated over Celite followed by concentration
of the solvents in vacuo and purification of the residue by pre-
parative HPLC. Fmoc-Val-Ala-OCH2COOH was obtained in an
overall yield of 73%. Rt(HPLC) 15.76 min; purity = 99%;
1H NMR (DMSO-d6, 300 MHz) δ = 0.88 (2 × d, J = 6.6 and
6.9 Hz, 6H), 1.34 (d, J = 7.2 Hz, 3H), 2.92–2.03 (m, 1H),
3.88–3.94 (m, 1H), 4.18–4.39 (m, 4H), 4.51–4.65 (m, 2H),
7.29–7.43 (m, 5H), 7.75 (q, J = 3.6 and 3.3 Hz, 2H), 7.89 (d,
J = 7.5 Hz, 2H), 8.42 (d, J = 6.6 Hz, 1H), 13.05 (s, 1H);
13C NMR (DMSO-d6, 75 MHz) δ = 16.7, 18.1, 19.0, 30.3, 46.6,
47.2, 59.6, 60.7, 65.6, 120.0, 125.3, 126.9, 127.5, 140.6, 143.7,
143.8, 156.0, 168.7, 171.1, 172.0.
General procedure D: synthesis of peptide Glyc-Xxx-OH esters
2-Chlorotritylchloride resin (1 g, loading = 1.2 mmol g−1) was
reacted with Fmoc-Xxx-OH (2 mmol) and DIPEA (5 mmol) in
CH2Cl2 (10 mL, 30 min) followed by washing with DMF
(10 mL, 2×). Afterwards, the unreacted tritylchloride moieties
were capped with MeOH–CH2Cl2–DIPEA (10 mL, 15 : 85 : 5,
v/v/v) followed by washing steps with NMP (10 mL, 2 min, 3×),
CH2Cl2 (10 mL, 2 min, 3×) and NMP (10 mL, 2 min, 3×).
Fmoc-Xxx-OCH2COOH (1 mmol) was coupled to the free
amine using HBTU (1 mmol, 379 mg), HOBt (1 mmol, 136 mg)
and DIPEA (2 mmol, 349 μL) in NMP (10 mL, 90 min). Further
Fmoc deprotection and Fmoc amino acid coupling cycles were
performed using standard SPPS protocols.5 After synthesis of
the desired sequence, the peptide was cleaved from the resin and
precipitated as described in general procedure A.
General procedure E: relative activity determination for
Alcalase-CLEA catalysed peptide coupling
To a suspension of Alcalase-CLEA (4.5 mg), H-Phe-NH2
(0.54 mg) and crushed 3 Å molecular sieves (4.5 mg) in THF
(900 μL), amino acid or peptide ester stock solution (20 mM) in
DMF (100 μL) was added. The reaction mixture was shaken at
50 °C with 200 rpm for 60 min. Afterwards, the reaction mixture
was filtrated and analysed by analytical HPLC by integrating the
peptide coupling product peak. Integration areas of different
reactions were compared to each other to determine the relative
activity (ester which gave the highest peptide product integration
area = 100%).
Fmoc-Val-Ala-Phe-NH2 (18)
Rt(HPLC) 22.48 min; purity = 99%; 1H NMR (DMSO-d6,
300 MHz) δ = 0.82 (2 × d, J = 2.7 and 3.6 Hz, 6H), 1.15 (d, 3H,
J = 7.2 Hz), 1.92–1.99 (m, 1H), 2.79–3.02 (m, 2H), 3.86 (q, 1H,
J = 7.2 Hz and 1.2 Hz), 4.19–4.43 (m, 5H), 7.05 (s, 1H),
7.16–7.44 (m, 11H), 7.73 (t, 2H, J = 6.6 Hz), 7.84–8.00 (m,
4H); 13C NMR (DMSO-d6, 75 MHz) δ = 16.7, 19.0, 21.2, 22.7,
24.1, 30.3, 46.6, 47.5, 49.9, 59.6, 62.0, 65.6, 120.0, 125.3, 126.9,
127.5, 140.6, 143.7, 143.8, 155.9, 166.2, 171.3, 171.8, 173.5.
HCl·H-Ala-OCH2C(vO)O-Bn
To a solution of Boc-Ala-OH (1 mmol, 189 mg) in THF
(50 mL) were added DIPEA (2.5 mmol, 436 μL) and benzyl
iodoacetate (2.0 mmol, 552 mg). This mixture was shaken for
20 h at 50 °C with 200 rpm and after this period of stirring, the
Fmoc-Val-Ala-OGlyc-Phe-NH2 (19)
Rt(HPLC) 16.85 min; purity = 98%; 1H NMR (DMSO-d6,
300 MHz) δ = 0.87 (2 × d, J = 7.2 and 7.8 Hz, 6H), 1.32
6772 | Org. Biomol. Chem., 2012, 10, 6767–6775
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Fmoc-Val-Ala-OGlyc-Ala-NH2 (27)
(d, J = 7.2 Hz, 3H), 1.87–2.15 (m, 1H), 2.77–3.05 (m, 2H),
3.86–3.92 (m, 1H), 4.20–4.46 (m, 7H), 7.11–7.46 (m, 11H),
7.72–7.76 (m, 2H), 7.89 (d, J = 7.5 Hz, 2H), 8.09 (d, J =
8.4 Hz, 1H), 8.44 (d, J = 6.3 Hz, 1H); 13C NMR (DMSO-d6,
75 MHz) δ = 16.7, 18.1, 19.0, 30.3, 37.5, 46.6, 47.5, 53.5, 59.6,
62.1, 65.6, 120.0, 125.3, 126.1, 126.9, 127.5, 129.0, 137.7,
140.6, 143.7, 143.8, 166.0, 171.1, 171.7, 172.4.
Rt(HPLC) 14.69 min; purity = 98%; 1H NMR (DMSO-d6,
300 MHz) δ = 0.87 (2 × d, J = 7.2 and 7.8 Hz, 6H), 1.32 (d,
J = 7.2 Hz, 3H), 1.87–2.15 (m, 4H), 2.77–2.85 (m, 1H),
2.99–3.05 (m, 1H), 3.86–3.92 (m, 1H), 4.20–4.61 (m, 6H),
7.12–7.46 (m, 11H), 7.72–7.76 (m, 2H), 7.90 (d, J = 7.5 Hz,
2H), 8.09 (d, J = 8.4 Hz, 1H), 8.44 (d, J = 6.3 Hz, 1H);
13C NMR (DMSO-d6, 75 MHz) δ = 16.6, 18.2, 19.0, 30.3, 46.6,
47.5, 47.7, 59.6, 62.3, 65.6, 120.0, 125.3, 126.9, 127.5, 140.6,
143.7, 143.8, 155.9, 165.8, 171.3, 171.9, 173.7.
Fmoc-Val-Ala-OGlyc-Leu-NH2 (22)
Rt(HPLC) 16.60 min; purity = 98%; 1H NMR (DMSO-d6,
300 MHz) δ = 0.82–0.90 (m, 12H), 1.33–1.61 (m, 6H),
1.94–2.00 (m, 1H), 3.86–3.92 (m, 1H), 4.18–4.38 (m, 5H), 4.53
(m, 2H), 7.01 (s, 1H), 7.29–7.44 (m, 6H), 7.74 (q, J = 4.5 Hz
and 2.4 Hz, 2H), 7.88–7.98 (m, 3H), 8.46 (d, J = 6.0 Hz, 1H);
13C NMR (DMSO-d6, 75 MHz) δ = 16.7, 18.1, 19.0, 21.4, 22.9,
24.1, 30.3, 40.8, 46.6, 47.8, 50.5, 59.6, 62.2, 65.6, 120.0, 125.3,
126.9, 127.5, 140.6, 143.7, 143.8, 155.9, 166.0, 171.3, 171.9,
173.6.
Fmoc-Val-Ala-OGlyc-Gly-NH2 (28)
Rt(HPLC) 13.65 min; purity = 92%; 1H NMR (DMSO-d6,
300 MHz) δ = 0.88 (2 × d, J = 7.2 and 7.5 Hz, 6H), 1.34 (d,
J = 6.9 Hz, 3H), 1.93–2.00 (m, 1H), 3.66 (d, J = 5.4 Hz, 2H),
3.87–3.93 (m, 1H), 4.22–4.59 (m, 6H), 7.08 (s, 1H), 7.30–7.44
(m, 6H), 7.74–7.90 (m, 4H), 8.12–8.16 (m, 1H), 8.47 (d, J =
6.3 Hz, 1H); 13C NMR (DMSO-d6, 75 MHz) δ = 16.7,
18.1, 19.0, 30.3, 41.5, 46.6, 47.5, 59.6, 62.4, 65.6, 120.0,
125.3, 126.9, 127.5, 140.6, 143.7, 143.8, 156.0, 166.6, 170.4,
171.3, 171.9.
Fmoc-Val-Ala-OGlyc-Asp(OtBu)-NH2 (24)
Rt(HPLC) 17.00 min; purity = 98%; 1H NMR (DMSO-d6,
300 MHz) δ = 0.87 (2 × d, J = 10.2 and 7.1 Hz, 6H), 1.32–1.37
(m, 12H), 1.93–2.00 (m, 1H), 2.41–2.70 (m, 2H), 3.87–3.93 (m,
1H), 4.17–4.58 (m, 7H), 7.15 (s, 1H), 7.31–7.44 (m, 6H), 7.73
(m, 2H), 7.89 (d, J = 7.5 Hz, 2H), 8.18 (d, J = 8.1 Hz, 1H), 8.45
(d, J = 6.3 Hz, 1H); 13C NMR (DMSO-d6, 75 MHz) δ = 16.7,
18.1, 19.0, 27.5, 30.3, 31.2, 37.4, 46.6, 47.5, 49.0, 59.6, 62.2,
65.6, 80.0, 120.0, 125.3, 126.9, 127.5, 140.6, 143.7, 143.8,
156.0, 166.1, 169.3, 171.3, 171.8.
Fmoc-Val-Ala-OGlyc-Glu(OtBu)-NH2 (31)
Rt(HPLC) 17.30 min; purity = 98%; 1H NMR (DMSO-d6,
300 MHz) δ = 0.88 (2 × d, J = 6.9 and 7.2 Hz, 6H), 1.33–1.39
(m, 12H), 1.66–1.79 (m, 1H), 1.87–2.02 (m, 2H), 2.17–2.27 (m,
2H), 3.87–3.93 (m, 1H), 4.17–4.37 (m, 5H), 4.48–4.60 (m, 2H),
7.11 (s, 1H), 7.29–7.44 (m, 6H), 7.74 (dd, J = 4.5 Hz and
2.7 Hz, 2H), 7.89 (d, J = 7.5 Hz, 2H), 8.00 (d, J = 8.1 Hz, 1H),
8.46 (d, J = 6.3 Hz, 1H); 13C NMR (DMSO-d6, 75 MHz) δ =
16.7, 18.1, 19.0, 28.0, 30.3, 31.2, 32.5, 46.6, 47.5, 51.9, 59.6,
62.2, 65.6, 69.1, 120.0, 125.3, 126.2, 126.9, 127.3, 127.4,
127.5, 128.4, 140.6, 143.7, 143.8, 144.8, 166.1, 171.2, 171.3,
171.9, 172.0.
Fmoc-Val-Ala-OGlyc-Ser(tBu)-NH2 (25)
Rt(HPLC) 16.85 min; purity = 97%; 1H NMR (DMSO-d6,
300 MHz) δ = 0.87 (2 × d, J = 7.2 and 8.4 Hz, 6H), 1.10
(s, 9H), 1.35 (d, J = 7.2 Hz, 3H), 1.94–2.00 (m, 1H), 3.30–3.53
(m, 2H), 3.87–3.92 (m, 1H), 4.20–4.36 (m, 5H), 4.50–4.63
(m, 2H), 7.11 (s, 1H), 7.29–7.44 (m, 6H), 7.73–7.75 (m, 2H),
7.87–7.90 (m, 3H), 8.42 (d, J = 6.0 Hz, 1H); 13C NMR (DMSO-
d6, 75 MHz) δ = 16.7, 18.1, 19.0, 27.1, 30.3, 31.2, 46.6, 47.4,
52.9, 59.6, 61.7, 62.2, 65.6, 72.6, 120.0, 125.3, 126.9, 127.5,
140.6, 143.7, 156.0, 166.1, 171.2, 171.2, 171.2.
Fmoc-Val-Ala-OGlyc-Arg(Pbf)-NH2 (32)
Rt(HPLC) 17.94 min; purity = 94%; 1H NMR (DMSO-d6,
300 MHz) δ = 0.86 (2 × d, J = 6.9 and 7.1 Hz, 6H), 1.10 (s,
3H), 1.32–1.40 (m, 9H), 1.96–2.02 (m, 4H), 2.94–3.03 (m, 3H),
3.80–3.92 (m, 1H), 4.17–4.35 (m, 5H), 4.53 (m, 2H), 7.07
(s, 1H), 7.28–7.43 (m, 5H), 7.75 (q, J = 4.8 Hz and 2.4 Hz, 2H),
7.88 (d, J = 7.5 Hz, 2H), 7.99 (d, J = 8.1 Hz, 1H), 8.45 (d,
J = 6.3 Hz, 1H); 13C NMR (DMSO-d6, 75 MHz) δ = 12.1, 16.7,
17.5, 18.1, 18.8, 19.0, 28.2, 30.3, 31.2, 42.4, 46.6, 47.4, 51.6,
59.6, 62.2, 65.6, 86.2, 116.1, 120.0, 124.2, 125.3, 126.9, 127.5,
131.3, 137.2, 140.6, 143.7, 143.8, 156.0, 157.3, 166.1, 171.3,
171.9, 173.0.
Fmoc-Val-Ala-OGlyc-Gln(Trt)-NH2 (26)
Rt(HPLC) 19.63 min; purity = 97%; 1H NMR (DMSO-d6,
300 MHz) δ = 0.80 (2 × d, J = 6.6 Hz, 6H), 1.26 (d, J = 7.2 Hz,
3H), 1.55–1.68 (m, 1H), 1.74–1.93 (m, 2H), 2.20–2.25 (m, 2H),
3.80–3.85 (m, 1H), 4.06–4.28 (m, 5H), 4.40–4.53 (m, 2H),
7.00–7.37 (m, 22H), 7.67 (q, J = 3.6 Hz, 2H), 7.82 (d, J =
7.5 Hz, 2H), 7.96 (d, J = 8.1 Hz, 1H), 8.37 (d, J = 6.6 Hz, 1H),
8.54 (s, 1H); 13C NMR (DMSO-d6, 75 MHz) δ = 16.6, 18.1,
19.0, 27.2, 27.6, 30.3, 31.1, 46.6, 47.5, 51.3, 59.6, 62.2, 65.6,
79.6, 120.0, 125.3, 126.9, 127.5, 140.6, 143.7, 143.8, 155.9,
166.2, 171.3, 171.5, 171.9, 172.6.
Fmoc-Val-Ala-OGlyc-Pro-NH2 (33)
Rt(HPLC) 14.42 min; purity = 97%; 1H NMR (DMSO-d6,
300 MHz) δ = 0.87 (2 × d, J = 6.9 and 9.9 Hz, 6H), 1.36 (d, J =
7.2 Hz, 3H), 1.78–2.03 (m, 5H), 3.30–3.47 (m, 2H), 3.87–3.93
(m, 1H), 4.15–4.45 (m, 5H), 4.61–4.89 (m, 2H), 6.94 (s, 1H),
7.22–7.44 (m, 6H), 7.73–7.77 (m, 2H), 7.89 (d, J = 7.5 Hz, 2H),
This journal is © The Royal Society of Chemistry 2012
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8.39 (d, J = 6.3 Hz, 1H); 13C NMR (DMSO-d6, 75 MHz) δ =
16.8, 18.2, 19.0, 24.0, 29.0, 30.3, 45.1, 46.6, 47.3, 59.6, 61.6,
65.6, 120.0, 125.3, 126.9, 127.5, 140.6, 143.7, 143.8, 155.9,
164.6, 171.1, 171.9, 173.3.
Fmoc-Val-Ala-OGlyc-Ile-NH2 (38)
Rt(HPLC) 16.46 min; purity = 96%; 1H NMR (DMSO-d6,
300 MHz) δ = 0.79–0.93 (m, 12H), 1.01–1.44 (m, 4H),
1.68–1.75 (m, 2H), 1.93–2.00 (m, 1H), 3.86–3.92 (m, 1H),
4.13–4.35 (m, 5H), 4.48–4.71 (m, 2H), 7.05 (s, 1H), 7.29–7.44
(m, 6H), 7.72–7.90 (m, 5H), 8.45 (d, J = 6.6 Hz, 1H); 13C NMR
(DMSO-d6, 75 MHz) δ = 11.0, 15.3, 16.7, 18.1, 190.0, 24.0,
30.3, 36.5, 46.6, 47.4, 56.3, 59.6, 62.2, 65.6, 120.0, 125.3,
126.9, 127.5, 140.6, 143.7, 143.8, 155.9, 166.0, 171.2, 171.9,
172.5.
Fmoc-Val-Ala-OCam (34)
Rt(HPLC) 14.67 min; purity = 98%; 1H NMR (DMSO-d6,
300 MHz) δ = 0.88 (2 × d, J = 6.6 and 6.9 Hz, 6H), 1.33 (d,
J = 7.2 Hz, 3H), 1.93–2.00 (m, 1H), 3.86–3.91 (m, 1H),
4.18–4.48 (m, 6H), 7.32–7.46 (m, 7H), 7.75 (q, J = 3.9 Hz
and 2.7 Hz, 2H), 7.89 (d, J = 7.2 Hz, 2H), 8.49 (d, J = 6.6 Hz,
1H); 13C NMR (DMSO-d6, 75 MHz) δ = 16.6, 18.2, 18.9,
30.3, 46.6, 47.6, 59.7, 62.2, 65.6, 120.0, 125.3, 126.9, 127.5,
140.6, 143.7, 143.8, 156.0, 168.4, 171.4, 171.8.
Fmoc-Val-Ala-OGlyc-Thr(tBu)-NH2 (40)
Rt(HPLC) 17.32 min; purity = 98%; 1H NMR (DMSO-d6,
300 MHz) δ = 0.88 (2 × d, J = 6.9 Hz, 6H), 1.00 (d, J = 6.3 Hz,
3H), 1.13 (s, 9H), 1.35 (d, J = 7.2 Hz, 3H), 1.94–2.00 (m, 1H),
3.84–3.98 (m, 2H), 4.13–4.36 (m, 5H), 4.52–4.69 (m, 2H),
7.16–7.44 (m, 7H), 7.62–7.76 (m, 3H), 7.89 (d, J = 7.5 Hz, 2H),
8.43 (d, J = 6.6 Hz, 1H); 13C NMR (DMSO-d6, 75 MHz)
δ = 16.8, 171.1, 19.0, 19.4, 27.9, 30.3, 46.6, 47.4, 57.3, 59.6,
62.3, 65.6, 66.7, 73.4, 120.0, 120.0, 121.3, 125.3, 126.9, 127.2,
127.5, 128.8, 140.6, 143.7, 143.8, 155.9, 166.2, 171.1, 171.2,
171.4, 171.7.
Fmoc-Val-Ala-OGlyc-Cys(Trt)-NH2 (35)
Rt(HPLC) 20.91 min; purity = 91%; 1H NMR (DMSO-d6,
300 MHz) δ = 0.87 (2 × d, J = 6.0 and 6.9 Hz, 6H), 1.34 (d,
J = 7.2 Hz, 2H), 1.93–2.00 (m, 1H), 2.19–2.50 (m, 2H),
3.87–3.93 (m, 1H), 4.13–4.38 (m, 4H), 4.48–4.64 (m, 2H),
6.43–7.10 (m, 20H), 7.46–7.76 (m, 2H), 7.89 (d, J = 6.0 Hz,
2H), 8.20 (d, J = 8.4 Hz, 1H), 8.44 (d, J = 6.9 Hz, 1H);
13C NMR (DMSO-d6, 75 MHz) δ = 14.0, 14.3, 16.7, 18.1, 18.2,
19.0, 22.1, 30.3, 33.8, 46.6, 47.4, 55.7, 120.0, 125.3, 126.1,
126.6, 126.9, 127.4, 127.5, 127.7, 127.9, 128.2, 128.9, 129.0,
140.6, 143.7, 143.8, 144.1, 166.0, 171.1, 171.8.
Fmoc-Val-Ala-OGlyc-Met-NH2 (43)
Rt(HPLC) 15.93 min; purity = 91%; 1H NMR (DMSO-d6,
300 MHz) δ = 0.87 (2 × d, J = 7.2 and 7.8 Hz, 6H), 1.33 (d,
J = 6.9 Hz, 3H), 1.72–2.02 (m, 6H), 2.41–2.50 (m, 2H),
3.82–3.92 (m, 1H), 4.20–4.35 (m, 5H), 4.54 (m, 2H), 7.10
(s, 1H), 7.29–7.43 (m, 5H), 7.74 (dd, J = 3.6 Hz, 2H), 7.88 (d,
J = 7.5 Hz, 2H), 8.04 (d, J = 8.1 Hz, 1H), 8.47 (d, J = 6.3 Hz,
1H); 13C NMR (DMSO-d6, 75 MHz) δ = 14.5, 16.6, 18.1,
19.0, 29.5, 30.3, 31.7, 46.5, 47.5, 51.3, 59.6, 62.3, 65.6, 120.0,
125.3, 126.9, 127.5, 140.6, 143.7, 143.8, 155.9, 166.3, 171.3,
172.0, 172.7.
Fmoc-Val-Ala-OGlyc-Trp(Boc)-NH2 (36)
Rt(HPLC) 19.42 min; purity = 92%; 1H NMR (DMSO-d6,
300 MHz) δ = 0.84 (2 × d, J = 6.9 and 7.2 Hz, 6H), 1.31
(d, J = 7.2 Hz, 3H), 1.61 (s, 9H), 1.89–2.08 (m, 1H), 2.90–3.16
(m, 1H), 3.85–3.91 (m, 1H), 4.19–4.58 (m, 7H), 7.18–7.81
(m, 13H), 7.88 (d, J = 7.5 Hz, 2H), 8.02 (d, J = 8.1 Hz, 1H),
8.18 (d, J = 8.1 Hz, 1H), 8.44 (d, J = 6.3 Hz, 1H); 13C NMR
(DMSO-d6, 75 MHz) δ = 16.6, 18.1, 18.9, 27.2, 27.6,
30.3, 46.6, 47.5, 52.0, 59.6, 62.1, 65.6, 83.3, 114.5,
116.4, 119.2, 120.0, 122.3, 123.7, 124.1, 125.6, 126.9, 127.5,
130.2, 134.5, 140.6, 143.7, 143.8, 148.9, 155.9, 166.1, 171.2,
171.7, 172.4.
Fmoc-Val-Ala-OGlyc-Lys(Boc)-NH2 (44)
Rt(HPLC) 17.06 min; purity = 95%; 1H NMR (DMSO-d6,
300 MHz) δ = 0.86 (2 × d, J = 6.9 and 7.1 Hz, 6H), 1.21–1.63
(m, 15H), 1.92–2.00 (m, 1H), 2.83–2.89 (m, 2H), 3.80–3.91
(m, 1H), 4.12–4.34 (m, 5H), 4.50–4.60 (m, 2H), 6.70–6.75 (m,
1H), 7.02 (s, 1H), 7.28–7.43 (m, 5H), 7.71–7.95 (m, 5H),
8.45 (d, J = 6.6 Hz, 1H); 13C NMR (DMSO-d6, 75 MHz) δ =
12.1, 16.7, 17.5, 18.1, 18.8, 190.0, 22.5, 28.2, 29.1, 30.3, 31.1,
46.6, 47.5, 51.6, 59.6, 62.2, 65.6, 86.2, 120.0, 124.2, 125.3,
126.9, 127.5, 128.8, 131.3, 140.6, 143.7, 143.8, 156.0, 166.1,
171.3, 171.9, 173.0.
Fmoc-Val-Ala-OGlyc-Tyr(tBu)-NH2 (37)
Rt(HPLC) 17.86 min; purity = 94%; 1H NMR (DMSO-d6,
300 MHz) δ = 0.87 (2 × d, J = 6.9 and 8.4 Hz, 6H), 1.25
(s, 9H), 1.32 (d, J = 7.5 Hz, 3H), 1.93–2.00 (m, 1H), 2.72–3.01
(m, 2H), 3.86–3.92 (m, 1H), 4.17–4.45 (m, 7H), 6.84 (d, J =
7.5 Hz, 2H), 6.98 (d, J = 7.5 Hz, 2H), 7.29–7.44 (m, 6H),
7.72–7.76 (m, 2H), 7.89 (d, J = 7.5 Hz, 2H), 8.05 (d, J =
8.4 Hz, 1H), 8.45 (d, J = 6.3, 1H); 13C NMR (DMSO-d6,
75 MHz) δ = 16.1, 17.6, 18.4, 27.9, 29.7, 46.0, 46.9, 52.9, 59.1,
61.5, 65.0, 76.9, 114.2, 119.3, 122.6, 124.7, 126.4, 127.0, 129.0,
129.4, 131.7, 140.0, 143.1, 143.2, 152.7, 155.4, 165.4, 170.7,
171.1, 171.9.
Fmoc-Val-Ala-OGlyc-Asn(Trt)-NH2 (45)
Rt(HPLC) 19.68 min; purity = 96%; 1H NMR (DMSO-d6,
300 MHz) δ = 0.88 (2 × d, J = 6.6 Hz, 6H), 1.32 (d, J = 7.2 Hz,
2H), 1.97–2.02 (m, 1H), 2.63–2.65 (m, 2H), 3.79–3.94 (m, 1H),
4.17–4.33 (m, 4H), 4.44–4.61 (m, 3H), 7.11–7.43 (m, 21H),
7.74 (q, J = 4.5 Hz and 2.1 Hz, 2H), 7.89 (d, J = 7.5 Hz, 2H),
6774 | Org. Biomol. Chem., 2012, 10, 6767–6775
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8.23 (d, J = 8.1 Hz, 1H), 8.46 (d, J = 6.3 Hz, 1H), 8.59 (s, 1H);
13C NMR (DMSO-d6, 75 MHz) δ = 16.7, 18.1, 19.0, 30.4, 46.6,
47.5, 49.6, 62.1, 65.6, 69.3, 120.0, 125.3, 126.2, 126.9, 127.3,
127.5, 128.4, 140.6, 144.6, 166.0, 168.9, 171.3, 171.9, 172.0.
4.22–4.36 (m, 5H), 4.46–4.60 (m, 2H), 7.32–7.44 (m, 5H), 7.74
(q, J = 4.5 and 2.7 Hz, 2H), 7.89 (d, J = 3.6 Hz, 2H), 8.15
(d, J = 3.6 Hz, 1H), 8.46 (d, J = 2.7 Hz, 1H); 13C NMR
(DMSO-d6, 75 MHz) δ = 18.2, 19.0, 21.4, 22.9, 24.1, 30.3,
46.5, 50.5, 51.5, 59.7, 62.3, 65.6, 69.1, 120.0, 125.2, 126.2,
126.9, 127.3, 127.5, 128.2, 128.4, 140.6, 143.6, 143.8, 144.7,
156.0, 166.1, 170.9, 171.3, 171.6, 173.6.
Fmoc-Val-Ala-OGlyc-Val-NH2 (47)
Rt(HPLC) 15.80 min; purity = 98%; 1H NMR (DMSO-d6,
300 MHz) δ = 0.81–0.90 (m, 12H), 1.34 (d, J = 7.2 Hz, 3H),
1.91–2.04 (m, 2H), 3.86–3.92 (m, 1H), 4.11–4.38 (m, 5H), 4.56
(m, 2H), 7.06 (s, 1H), 7.29–7.44 (m, 6H), 7.72–7.90 (m, 5H),
8.45 (d, J = 6.6 Hz, 1H); 13C NMR (DMSO-d6, 75 MHz)
δ = 16.7, 17.7, 18.1, 19.0, 19.1, 30.3, 46.6, 47.4, 57.1, 59.6,
62.2, 65.6, 120.0, 125.3, 126.9, 127.5, 140.6, 143.7, 143.8,
155.9, 166.1, 171.2, 171.8, 172.5.
Notes and references
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2005, 60, 241; K. Adermann, Curr. Protein Pep. Sci., 2005, 6, 205.
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J. Torres-Llanez, B. Vallejo-Cordoba and A. F. González-Córdova, Arch.
Latinoam. Nutr., 2005, 55, 111; H. Korhonen and A. Pihlanto, Curr.
Pharm. Des., 2007, 13, 829; M. B. Roberfroid, J. Nutr., 2007, 137, 2493.
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4 F. Guzmán, S. Barberis and A. Illanes, J. Biotech., 2007, 10, 279.
5 W. C. Chan, Fmoc Solid Phase Peptide Synthesis: A Practical Approach,
Oxford University Press, 2000.
6 J. Bedford, C. Hyde, T. Johnson, W. Jun, D. Owen, M. Quibell and
R. C. Sheppard, Int. J. Pept. Protein Res., 1992, 40, 300.
7 F. Bordusa, Chem. Rev., 2002, 102, 4817.
8 V. Čeřovský and F. Bordusa, J. Pept. Res., 2000, 55, 325.
9 T. Miyazawa, E. Ensatsu, N. Yabuuchi, R. Yanagihara and T. Yamada,
J. Chem. Soc., Perkin Trans. 1, 2002, 390.
10 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.
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27, 1199; F. Bordusa, Braz. J. Med. Biol. Res., 2000, 33, 469.
12 M. Capellas, M. D. Benaiges, G. Caminal, G. Gonzalez, J. Lopez-Santín
and P. Clapés, Biotechnol. Bioeng., 2000, 50, 700.
Fmoc-Val-Ala-OGlyc-His(Trt)-NH2 (50)
Rt(HPLC) 14.08 min; purity = 90%; 1H NMR (DMSO-d6,
300 MHz) δ = 0.85 (2 × d, J = 6.0 and 6.3 Hz, 6H), 1.30 (d,
J = 7.2 Hz, 3H), 1.90–1.98 (m, 1H), 2.73–3.10 (m, 2H),
3.87–3.92 (m, 1H), 4.17–4.32 (m, 4H), 4.57–4.58 (m, 3H), 6.97
(s, 1H), 7.09–7.44 (m, 24H), 7.73 (dd, J = 4.5 and 2.4 Hz, 2H),
7.89 (d, J = 7.5 Hz, 2H), 8.14 (d, J = 8.1 Hz, 1H), 8.46 (d, J =
6.3 Hz, 1H); 13C NMR (DMSO-d6, 75 MHz) δ = 16.6, 18.1,
18.9, 30.3, 46.6, 47.5, 51.5, 55.7, 59.6, 62.1, 65.6, 120.0, 120.2,
125.3, 126.1, 126.5, 126.9, 127.4, 127.5, 127.6, 128.2, 128.4,
128.4, 128.9, 129.1, 136.7, 140.6, 140.8, 143.7, 143.8, 166.1,
171.3, 171.8, 171.8.
13 M. K. Dhaon, R. K. Olsen and K. Ramasamy, J. Org. Chem., 1982, 47,
1962.
14 F. Bordusa, C. Dahl, H.-D. Jakubke, K. Burger and B. Koksch, Tetra-
hedron: Asymmetry, 1999, 10, 307.
Cbz-D-Phe-OGlyc-Phe-NH2 (54)
1
15 N. Wehofsky, N. Koglin, S. Thust and F. Bordusa, J. Am. Chem. Soc.,
2003, 125, 6126.
ee D-Phe > 99.5; Rt(HPLC) 17.43 min; purity = 97%; H NMR
(DMSO-d6, 300 MHz) δ = 2.79–2.89 (m, 2H), 3.01–3.16 (m,
2H), 4.37–4.57 (m, 5H), 4.98 (s, 2H), 7.14–7.49 (m, 17H), 7.86
(d, J = 8.1 Hz, 1H), 8.19 (d, J = 8.4 Hz, 1H); 13C NMR
(DMSO-d6, 75 MHz) δ = 36.2, 37.3, 55.6, 55.2, 63.9, 65.3,
126.1, 126.4, 127.4, 127.6, 127.9, 128.1, 128.2, 129.0, 136.7,
137.4, 137.8, 156.0, 165.9, 171.1, 172.5.
16 T. Nuijens, C. Cusan, C. H. M. Schepers, J. A. W. Kruijtzer,
D. T. S. Rijkers, R. M. J. Liskamp and P. J. L. M. Quaedflieg, J. Mol.
Catal. B: Enzym., 2011, 71, 79–84.
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J. Chem. Soc., Perkin Trans. 1, 2002, 396.
Fmoc-Val-Ala-OGlyc-Leu-OH (56)
Rt(HPLC) 21.45 min; purity = 92%; 1H NMR (DMSO-d6,
300 MHz) δ = 0.85–0.87 (m, 12H), 1.34 (d, J = 3.5 Hz, 3H),
1.54–1.60 (m, 3H), 1.98–2.02 (m, 1H), 3.87–3.92 (m, 1H),
22 L. Abrahmsen, J. Tom, J. Burnier, K. Butcher, A. A. Kossiakoff and
J. A. Wells, Biochemistry, 1991, 30, 4151.
23 S. M. A. Salam, K. Kagawa and K. Kawashiro, Tetrahedron: Asymmetry,
2006, 17, 22.
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 6767–6775 | 6775