New isocysteine building blocks and chemoselective peptide ligation

Article abstract of DOI:10.1039/b309235f

Boc-, Fmoc- and Cbz-protected isocysteine building blocks were prepared by a concise three-step procedure starting from thiomalic acid. The use of Boc/Trt-protected isocysteine provided convenient access to isocysteinyl peptides that allow the chemoselective ligation of unprotected peptide fragments in water. The pH-dependency of the isocysteine-mediated ligation was compared with that of cysteine-mediated native chemical ligation.

Full text of DOI:10.1039/b309235f

View Article Online / Journal Homepage / Table of Contents for this issue
New isocysteine building blocks and chemoselective peptide
ligation
Christian Dose and Oliver Seitz*
Institute of Chemistry, Humboldt-University of Berlin, Brook-Taylor-Str. 2, D-12489 Berlin,
Germany. E-mail: oliver.seitz@chemie.hu-berlin.de; Fax: ϩ49-30-20937266;
Tel: ϩ49-30-20937446
Received 5th August 2003, Accepted 13th October 2003
First published as an Advance Article on the web 5th November 2003
Boc-, Fmoc- and Cbz-protected isocysteine building blocks were prepared by a concise three-step procedure starting
from thiomalic acid. The use of Boc/Trt-protected isocysteine provided convenient access to isocysteinyl peptides that
allow the chemoselective ligation of unprotected peptide fragments in water. The pH-dependency of the isocysteine-
mediated ligation was compared with that of cysteine-mediated native chemical ligation.
Introduction
The replacement of naturally occurring amino acids by
isosteric or artificial amino acids is commonly employed for
probing the interaction of a protein with its ligand. Thiol-
containing building blocks are of interest due to their particular
reactivity which mediates inhibitory interactions with protein
metallo centers, conformational restriction through disulfide
bond formation and site-selective tagging reactions among
other effects. The β-mercapto amino acid cysteine is by far the
most frequently used thiol-containing building block. Its 1,2-
aminothiol structure also makes it an invaluable tool for the
efficient and convenient joining of two unprotected peptide
fragments.1–5
The introduction of α-mercapto acids provides the opportun-
ity to alter thiol acidity and/or thiolate basicity which can allow
the fine-tuning of metal–ligand interactions. For example, some
inhibitors of metalloproteases such as angiotensin-converting
enzymes contain α-mercapto acyl peptides.6,7 The β-amino-α-
mercapto amino acid isocysteine is a useful cysteine analogue
and has been shown to increase the inhibitory activity of pep-
tidic stromelysin binders when incorporated as a replacement
of cysteine.8 Isocysteine also might serve as a replacement of
β3-H cysteine in β-peptides which opens the possibility of alter-
ing thiol reactivity without changing the backbone length.9
There is, however, only one report that describes the prepar-
ation of an isocysteine derivative that suits the needs of solid-
phase peptide synthesis.8 We here present convenient syntheses
of Boc-, Fmoc- and Cbz-protected isocysteine building blocks.
Furthermore we show the utility of isocysteine to support the
ligation of unprotected peptide segments by a native chemical
Scheme 1 a) cyclopentanone, p-TsOH, PhCH3, reflux, 68%; b) 1.
diphenylphoshoryl azide, TEA, PhCH3; 2. 85 ЊC; c) PhCH2OH, 85 ЊC,
86% (3 4); 9-fluorenylmethanol, 85 ЊC, 87% (3 5); tBuOH, 75 ЊC,
77% (3 6); d) 1. 0.2 M LiOH, THF, 0 ЊC; 2. Trt-Br, 50% (5 7); 1.
0.2 M LiOH, THF, 0 ЊC; 2. PMB-Cl, 80% (5 8); e) 1. 1 M LiOH,
THF, RT; 2. PMB-Cl, 96%; f ) 1. 1 M LiOH, THF; 2. Trt-Br, 78%
(6 10); 1. 1 M LiOH, THF, RT. 2. PMB-Cl, 77% (6 11).
ligation-like reaction. To illustrate the effects of alterations of
the thiol acidity the pH-dependency of the isocysteine ligation
was compared with that of cysteine-mediated native chemical
ligation.
Results and discussion
Hanglows’ synthesis of isocysteine 7 and also our route to N,S-
protected building blocks 8–11 was based on commercially
available -thiomalic acid 1 as described by Gustavson.8,10,11
According to this, simultaneous protection of both the
α-mercapto and the α-carboxylic group was achieved upon
reaction with cyclopentanone in the presence of p-toluene-
sulfonic acid (Scheme 1). The β-carboxyl group in the formed
oxathiolanone 2 remained unaffected and was subsequently
converted to the acyl azide. In contrast to Hanglows’ Fmoc/
Trt-Isocys synthesis, in which the isocyanate 3 formed after the
Curtius rearrangement was hydrolysed to obtain isocysteine in
unprotected form, it was preferred to fashion a more direct
establishing of Fmoc-, Cbz- and Boc-protecting groups. The
isocyanate 3 was allowed to react with 9-fluorenylmethanol,
benzyl alcohol or tert-butanol, which completed a three-step
sequence and furnished the fully protected isocysteine deriv-
atives 4, 5 and 6 in 77–87% yield. We next sought a method that
would liberate both the carboxyl group and the α-mercapto
group without detriment to the acid- and base-sensitive protect-
ing groups in 4–6. The opening of the cyclic ketal was initiated
by ester hydrolysis with aqueous LiOH in THF, which proved
straightforward with Cbz- and Boc-protected isocysteines 4 and
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 5 9 – 6 5
59

View Article Online
6. The presence of the base-sensitive Fmoc-group in 5 rendered
this step challenging. It was found that careful addition of
exactly one equivalent of lithium hydroxide in THF, however,
enabled a clean and quantitative removal of the ketal group.
The liberated α-mercapto groups were subsequently masked by
omitting any attempts to isolate the N-protected isocysteine
derivatives. Instead, it was preferred to directly add p-methoxy-
benzyl chloride or trityl bromide to the basic THF solution
obtained after ester hydrolysis. This convenient one-pot pro-
cedure afforded the known Fmoc/Trt-protected isocysteine 7
and the novel Fmoc/PMB-, Cbz/PMB-, Boc/Trt- and Boc/
PMB-protected building blocks 8–11 in 50–96% yield. It is
noted that the same strategy would allow for the synthesis of
enantiomerically pure isocysteine if - or -thiomalic acid were
used.12
Having established expedient access to various protected
isocysteine derivatives we next set out to explore their utility in
the solid-phase synthesis of isocysteine containing peptides.
Previous work had demonstrated the usefulness of the Fmoc/
Trt-protected building block 7.8 We envisaged investigating
the use of isocysteine as a ligation handle (vide infra). Our
particular interest, therefore, concerned the synthesis of peptides
such as 16 bearing isocysteine as N-terminal amino acid. We
reckoned that Cbz/PMB- and Boc/PMB-protected isocysteine
building blocks 9 and 11 would enable the isocysteinyl-peptide
16 to be assembled by applying the Boc-strategy. For the final
cleavage trifluoromethanesulfonic acid was employed as a non-
toxic alternative to HF. The crude peptide obtained after cleav-
age, however, failed to precipitate upon addition of ether which
hampered its purification. After repeated unsuccessful attempts
with various scavengers, we chose to couple Boc/Trt-protected
isocysteine 10 to resin-bound peptide 14 which was assembled
by using Fmoc/tBu-protected amino acids (Scheme 2). It was
expected that the application of the Fmoc-strategy and TFA as
cleaving agent would be beneficial. TFA in contrast to TFMSA
(trifluoromethanesulfonic acid) is volatile and allows vacuum
concentration thereby facilitating ether precipitation. Indeed, in
this case purification was straightforward providing peptide 16
after HPLC purification in 69% overall yield.
selective ligation of two unprotected peptide fragments to be
performed in aqueous solution. The 1,2-aminothiol structure
of N-terminal cysteine is one of the most suitable entities for
achieving such segment couplings demonstrated best by the
numerous protein syntheses that have been enabled by the
native chemical ligation technology.2,19 This reaction (Scheme 3)
is initiated by a reversible thiol-exchange reaction of cysteine
conjugate 17 with thioester 18.20 The formed thioester inter-
mediate 19 is subject to a spontaneous S–N-acyl shift, which
establishes the α-peptide bond in product 20. In isocysteine, the
1,2-aminothiol structure is retained. Accordingly, isocysteine is
expected to support fragment ligations by a native chemical
ligation-like reaction mechanism.21 The S–N-acyl shift in
thioester-intermediate 22 would lead to the formation of a
β-peptide bond in 23. The isocysteine-mediated native chemical
ligation would hence offer prospects for a convergent assembly
of peptides with β-amino acid architecture.9,22,23
Scheme 3
To assess the feasibility of isocysteine-mediated native
chemical ligation we explored model peptides and ligation
conditions as described by Kent and co-workers.3,24 First,
peptide mercaptopropionamide thioester 24, which was
obtained through solid-phase synthesis on a mercaptopropionic
acid loaded Rink-resin, was converted to the more reactive
mercaptobenzylester 25. Peptide thioester 25 was allowed to
react with the isocysteine-peptide 16. Reactions were carried
out in argon saturated 0.1 M phosphate buffer and in the pres-
ence of 4% benzylmercaptan which helped to prevent thioester
hydrolysis and to maintain a reducing environment. At pH 7
and 1 mM concentration of reactants the isocysteine-mediated
native chemical ligation proceeded smoothly. HPLC/ESI-MS
analysis showed a new peak that appeared at 16 min with an
m/z that corresponded to ligation product 26 (Fig. 1). The
ligation product formed lacked susceptibility to attempted
hydrolysis by NaOH-treatment which provided support for the
formation of a non-hydrolysable peptide bond. It can be con-
cluded that isocysteine supports a native chemical ligation-like
reaction. It is interesting to note that the S N acyl transfer
likely to be involved in the isocysteine ligation proceeds via
main chain expansion as opposed to S N acyl transfer
involved in cysteine and auxiliary mediated ligations25–29 that
are characterised by main chain contraction. This feature might
be of less importance in convergent peptide assembly, but we
presume that it could affect ligation reactions that proceed
under geometrical constraints such as peptide-30,31 and DNA-
template controlled reactions.32,33
Scheme 2 a) 10 eq. Fmoc-Ser(tBu)-OH, 5 eq. DIC, 0.1 eq. DMAP,
DMF; b) 1. DMF/piperidine (4 : 1), 2. 4 eq. Fmoc-Aa-OH, 4 eq. HOBt,
3.6 eq. HBTU, 8 eq. DIPEA; 3. pyridine/Ac2O (5 : 1); c) 1. DMF/
piperidine (4 : 1), 2. 4 eq. Boc-Isocys(Trt)-OH, 4 eq. HOBt, 3.6 eq.
HBTU, 8 eq. DIPEA; 3. pyridine/Ac2O (5 : 1); d) CF3COOH/PhSMe/
Et3SiH/H2O (92 : 3 : 3 : 2), 69%.
Terminally modified peptides such as bromoacetamido-,13
maleinimido-,14 hydroxylamino-15 and azidopeptides16,17
among others18 are invaluable tools that can allow chemo-
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 5 9 – 6 5
60

View Article Online
we compared the initial rate of formation of isocysteine-
peptide 26 and cysteine-containing peptide 28 as a function of
pH at 1 mM concentrations of reactants and at identical reac-
tion conditions (Fig. 3). RP-HPLC-analysis revealed that the
initial rate of cysteine native chemical ligation was reduced by a
factor of 0.25 when lowering the pH from pH 7 to pH 6. This
pH decrease reduced the initial rate of isocysteine native chem-
ical ligation only by a factor of 0.56. Further pH decreases to
pH 5 led to a diminution of initial rates such that the formation
of ligation products almost ceased. At pH 7, the ligation of
cysteine-peptide 28 was 3-times faster than ligation of the iso-
cysteine-peptide 26 while at pH < 6, the isocysteine native
chemical ligation was as efficient as the cysteine native chemical
ligation.
Fig. 1 Analytical HPLC trace of the ligation reaction at λ = 280 nm
(16 ϩ 25) and pH 7.
It is worthwhile noting that both the isocysteine containing
starting material 16 and the ligation product 26 exist as a mix-
ture of diastereomers due to the use of racemic isocysteine.
Nevertheless, under the selected conditions the HPLC-traces
showed single albeit broadened peaks and it was therefore not
possible to explore whether one of the diastereomers conferred
higher ligation rates than the other.
Fig. 2 shows the time-dependent formation of ligation prod-
uct 26 at pH 7 and pH 6. It became evident that the ligation at
pH 6 is slower than the ligation at pH 7. For example, product
26 was formed in 63% yield after 240 min as opposed to 44%
yield obtained at pH 6.
Fig. 3 pH-Profile of initial ligation rates of the cysteine ligation (25 ϩ
Cys-Arg-Ala-Glu-Tyr-Ser, 27
Leu-Tyr-Lys-Ala-Gly-Cys-Arg-Ala-
Glu-Tyr-Ser, 28) and the isocysteine ligation (16 ϩ 25
conditions as specified in Fig. 1.
26). Ligation
It became apparent that the isocysteine-mediated ligation is
less pH-dependent than the cysteine-mediated native chemical
ligation. We assume that the increased acidity of the thiol
group is the decisive factor that leads to this attenuation of the
pH-dependence. This assumption is based on the notion that
native chemical ligation proceeds through the thiolate form of
the cysteine-peptide and that the thiol-exchange reaction is
rate-limiting.3 In isocysteine, the α-thiol group should be more
acidic than the β-thiol group of cysteine due to the inductive
effect of the carboxyl group. This influence becomes manifest
in the pKa-value of thioglycolic amide (pKa = 8.2) which is
considerably lower than that of β-mercaptopropionic amide
(pKa = 9.4).35 The calculated pKa-values of isocysteine amide
(pKa = 8.2) and cysteine amide (pKa = 9.1) suggest a similar
difference of thiol acidity.36 The more acidic the thiol group, the
more thiolate is provided even under acidic conditions and
hence the faster the ligation reaction. On the other hand,
inductive and steric effects are expected to decrease the nucleo-
philicity of the α-thiolate in isocysteine when compared with
the β-thiolate of cysteine which might explain why at pH > 6
the cysteine native chemical ligation proved faster than the
isocysteine native chemical ligation.
Conclusion
Fig. 2 Isocysteine-mediated chemical ligation at pH 7 and pH 6.
In conclusion, we have provided convenient synthetic access to
various isocysteine building blocks for solution and solid-phase
peptide synthesis. The replacement of cysteine by isocysteine
allows for an alteration of thiol reactivity as evidenced by
the attenuated pH-dependence of isocysteine-mediated native
It has been shown that the cysteine-mediated native chemical
ligation proceeds rapidly around pH 7 but is rendered less effi-
cient at pH < 5.5.34 To explore whether the isocysteine-mediated
native chemical ligation would exhibit a similar pH-dependence
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 5 9 – 6 5
61

View Article Online
chemical ligation of unprotected peptide fragments. Isocysteine
being a β-amino acid, also might be incorporated in β-peptides,
structurally well-defined peptide analogues that show interest-
ing biological activities. It is, for example, conceivable that the
replacement of β3-H-cysteine by isocysteine allows the thiol
reactivity to be altered while maintaining the backbone length
and the ability to confer chemoselective fragment conden-
sations. In this regard, the ability of isocysteine to support
native chemical ligation reactions might be of advantage for the
convergent assembly of modified β-peptides.
with CH2Cl2 (5×), DMF (5×) and CH2Cl2 (5×) to avoid any
side reactions.
(2-Oxo-1-oxa-4-thia-spiro[4.4]non-3-yl)-acetic acid (2)
To a solution of 2-mercaptosuccinic acid 1 (5.0 g, 33.3 mmol)
in toluene (70 ml) were added cyclopentanone (3.54 ml, 40.2
mmol) and p-toluenesulfonic acid (76 mg, 0.40 mmol). The
reaction vessel was equipped with a Dean–Stark trap and was
refluxed for 8 h. The solution was concentrated under reduced
pressure, and the residue was dissolved in sat. NaHCO3 (30 ml).
The aqueous solution was washed with CH2Cl2 (2 × 20 ml),
adjusted to pH 2 with concentrated HCl and extracted with
CH2Cl2 (3 × 30 ml). The organic phase was dried (MgSO4) and
evaporated under reduced pressure. The residue was purified by
silica gel flash chromatography to furnish 4.90 g (68%) of 2 as a
white solid. Mp = 93 ЊC. Rf = 0.21 (EtOAc : cyclohexane) = 2 : 5
ϩ 1% AcOH). 1H-NMR (CDCl3): δ 1.75 (m, 4H); 1.94 (m, 2H);
2.24 (m, 2H); 2.84 (dd, 1H, J = 9.16, J = 17.69); 3.21 (dd, 1H,
J = 4.02, J = 17.57); 4.38 (dd, 1H, J = 4.14, J = 9.16); 11.43 (s,
1H). 13C-NMR (CDCl3): 24.89 (CH2); 25.17 (CH2); 39.15
(CH2); 42.63 (CH2); 43.94 (CH2); 44.93 (CH); 97.20 (Cq);
174.83 (Cq); 177.59 (Cq). HRMS [FAB, m-NBA, pos.]: calcd.
216.0456; found 216.0468.
Experimental
General materials and methods
Reagents and chemicals were obtained from Acros, Aldrich,
Biosolve, Fluka, Novabiochem or Senn Chemicals and were
used without further purification. All solvents were freshly dis-
tilled. Proton and carbon NMR spectra were recorded on a
Varian Mercury 400 or a Bruker DRX-500 spectrometer at
room temperature. The signals of the residual protonated
solvent (CDCl3 or [D6]DMSO) were used as reference signals.
Coupling constants J are reported in Hz. Flash chromato-
graphy was performed using Merck silica gel 60. TLC was
performed with aluminium-backed silica gel 60 F254 plates
(Merck). Melting points were measured using a Büchi B-540
apparatus in open capillary tubes and are not corrected.
(2-Oxo-1-oxa-4-thia-spiro[4.4]non-3-ylmethyl)-carbamic acid
benzyl ester (4)
A solution of 2 (1.02 g, 4.73 mmol) and triethylamine (728 µl,
5.20 mmol) in toluene (20 ml) was stirred 30 min at room
temperature. The reaction mixture was cooled to 0 ЊC and di-
phenylphosphoryl azide (1.12 ml, 5.10 mmol) was added drop-
wise. After stirring for 2 h at room temperature, the solution
was heated to 85 ЊC until the evolution of N2 ceased. The
reaction mixture was cooled to room temperature and EtOAc
(30 ml) was added. The organic phase was washed with sat.
NaHCO3 (20 ml), dist. H2O (20 ml), dried (MgSO4) and con-
centrated under reduced pressure. The residue was dissolved in
toluene (20 ml) and benzyl alcohol (460 µl, 4.45 mmol) was
added. The solution was kept at 80 ЊC for 14 h and then allowed
to cool to room temperature. The solvent was evaporated under
reduced pressure and the residue was purified by silica gel
chromatography to afford 1.32 g (87%) of 4 as an yellow oil. Rf
= 0.24 (EtOAc : cyclohexane = 4 : 1). 1H-NMR (CDCl3): δ 1.75
(m, 4H); 1.94 (m, 2H); 2.24 (m, 2H); 3.65 (m, 2H); 4.19 (dd, 1H,
J = 5.65); 5.07 (s, 2H); 7.31 (m, 5H). 13C-NMR (CDCl3): 23.58
(CH2); 23.84 (CH2); 41.34 (CH2); 42.53 (2 × CH2); 48.29 (CH2);
67.12 (CH2); 95.85 (Cq); 128.24–128.73 (4 × CH); 136.33 (Cq);
156.42 (Cq); 173.43 (Cq). HRMS [FAB, m-NBA, pos.]: calcd.
321.1035; found 321.1020.
Reversed phase HPLC
Analytical reversed phase HPLC was performed with an
Agilent 1100 series system and a DAD-detector recording at
280 nm. A Macherey & Nagel Nucleosil (100-5) type CC 250/4
Nautilus RP-18 column was used at room temperature. Semi-
preparative reversed phase HPLC was performed with a Gilson
Nebula Series with automated fraction collection and Gilson
156 UV-detector. A Macherey & Nagel SP 125/10 Nucleodur
Gravity RP-18 column was used at 55 ЊC. Gradients of solvent
A (98.9% water/1% acetonitrile/0.1% TFA) and B (98.9%
acetonitrile/1% water/0.1% TFA) were adjusted according to
the separation needs.
Mass spectrometry
Matrix assisted laser desorption ionisation analysis was carried
out with a Voyager-DE Pro BioSpectrometer from PerSeptive
Biosystems using a 2,5-dihydroxybenzoic acid (DHB) matrix.
ESI-MS was carried out using a Agilent 1100 series system with
a Macherey & Nagel Nucleosil (100-5) type CC 250/4 Nautilus
RP-18 column and a Finnigan Thermoquest LCQ. FAB-MS was
recorded on a JEOL JMS-SX102A machine, applied matrices
were given for each compound.
(2-Oxo-1-oxa-4-thia-spiro[4.4]non-3-ylmethyl)-carbamic acid
9H-fluoren-9-ylmethyl ester (5)
Solid-phase peptide synthesis
A solution of 2 (200 mg, 0.92 mmol) and triethylamine (142 µl,
1.01 mmol) in toluene (10 ml) was stirred 30 min at room tem-
perature. The reaction was cooled to 0 ЊC and diphenyl-
phosphoryl azide (198 µl, 0.92 mmol) was added dropwise.
After stirring over 2 h at room temperature, the solution was
heated up to 85 ЊC until the evolution of N2 ceased. The reac-
tion was cooled to room temperature and EtOAc (20 ml) was
added. The organic phase was washed with sat. NaHCO3
(15 ml), dist. H2O (15 ml), dried (MgSO4) and concentrated
under reduced pressure. The dried residue was dissolved in
toluene (10 ml) and 9-fluorenylmethanol (157 mg, 0.8 mmol)
was added. The solution was kept at 80 ЊC for 24 h and then
allowed to cool to room temperature. The solvent was evapor-
ated under reduced pressure and the residue was purified by
silica gel chromatography to afford 320 mg (85%) of 5 as an
Reactions were carried out at room temperature and performed
manually using 5 ml polyethylene syringe reactors which were
equipped with a fritted disc. All peptides were synthesised using
HBTU/HOBt chemistry. Typically 4 eq. of the corresponding
amino acid were allowed to react for 2–3 min with 3.6 eq. of
HBTU, 4 eq. of HOBt and 8 eq. of DIPEA in DMF/CH2Cl2
(1 : 1). The solution was added to the resin and agitated for 2 h
at room temperature. Cysteine was coupled using DIC/HOBt
chemistry in order to avoid extensive racemisation. Typically
4 eq. of the amino acid were reacted with 4 eq. of HOBt and
4 eq. of DIC in DMF for 20 min and then added to the resin.
Unreacted amino groups were capped by a 15 min treatment of
pyridine/Ac2O (5 : 1) after every coupling step. Fmoc cleavage
was achieved using two times a solution of 20% piperidine in
DMF for 10 min. The Boc group was cleaved using two times
pure TFA for 5 min. The resins were washed after every step
1
yellow oil. Rf = 0.22 (EtOAc : cyclohexane = 4 : 1). H-NMR
(CDCl3): δ 1.75 (m, 4H); 1.94 (m, 2H); 2.24 (m, 2H); 3.66 (m,
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 5 9 – 6 5
62

View Article Online
2H); 4.20 (m, 1H); 4.37 (m, 2H); 7.28 (t, 2H, J = 7.40); 7.37
(t, 2H, J = 7.53); 7.56 (d, 2H, J = 7.53); 7.73 (d, 2H, J = 7.53).
13C-NMR (CDCl3): 23.67 (CH2); 23.95 (CH2); 41.42 (CH2);
42.53 (CH2); 42.66 (CH); 47.32 (CH); 48.36 (CH2); 67.28 (CH2);
95.70 (Cq); 120.22–127.93 (8 × CH); 130.01 (2 × Cq); 141.50
(Cq); 143.95 (Cq); 156.51 (Cq); 173.62 (Cq). HRMS [FAB,
m-NBA, pos.]: calcd. 409.1348; found 409.1335.
34.31 (CH2); 41.98 (CH); 46.25 (CH2); 46.98 (CH); 55.30 (CH3);
65.92 (CH2); 114.12 (2 × CH); 120.46 (2 × CH); 125.58–129.73
(8 × CH); 130.46 (Cq); 141.05 (2 × Cq); 144.17 (2 × Cq); 156.48
(Cq); 158.59 (Cq); 172.73 (Cq). HRMS [FAB, m-NBA, pos.]:
calcd. 463.1453; found 463.1432.
3-Benzyloxycarbonylamino-2-(4-methoxy-benzylsulfanyl)-
propionic acid (9)
(2-Oxo-1-oxa-4-thia-spiro[4.4]non-3-ylmethyl)-carbamic acid
tert-butyl ester (6)
To a solution of 4 (1.29 g, 4.01 mmol) in 20 ml THF was added
at 0 ЊC dropwise 1 M LiOH (15 ml) and the reaction was stirred
for 1 h at room temperature. The mixture was treated with
p-methoxybenzyl chloride (544 µl, 4.01 mmol) and stirred at
room temperature for further 3 h. The reaction was adjusted to
pH ∼ 3 with AcOH and H2O (10 ml) was added. The aqueous
solution was extracted with EtOAc (3 × 20 ml). The organic
phase was dried (MgSO4) and evaporated under reduced pres-
sure. The residue was purified by silica gel flash chromato-
graphy to obtain 1.45 g (96%) of 9 as a yellow oil. Rf = 0.34
(DCM : MeOH = 95 : 5 ϩ 1% AcOH). 1H-NMR ([D6]-DMSO):
δ 3.38 (m, 2H); 3.47 (m, 2H); 3.75 (s, 3H); 3.83 (m, 2H); 5.05 (s,
2H); 6.81 (d, 2H, J = 8.53); 7.23 (d, 2H, J = 8.53); 7.32 (m, 5H).
13C-NMR ([D6]-DMSO): 35.40 (CH2); 41.04 (CH2); 44.99
(CH); 55.15 (CH3); 66.94 (CH2); 113.95 (2 × CH); 128.04–
128.45 (4 × CH); 130.18 (2 × CH); 136.07 (Cq); 156.24 (Cq);
158.82 (Cq); 176.28 (Cq). HRMS [FAB, m-NBA, pos.]: calcd.
375.1140; found 375.1160.
A solution of 2 (200 mg, 0.70 mmol) and triethylamine (108 µl,
0.77 mmol) in toluene (10 ml) was stirred 30 min at room tem-
perature. The reaction was cooled to 0 ЊC and diphenyl-
phosphoryl azide (160 µl, 0.77 mmol) was added dropwise.
After stirring over 2 h at room temperature, the solution was
heated to 85 ЊC until the evolution of N2 ceased. The reaction
was cooled to room temperature and EtOAc (20 ml) was added.
The organic phase was washed with sat. NaHCO3 (15 ml), dist.
H2O (15 ml), dried (MgSO4) and concentrated under reduced
pressure. The residue was dissolved in toluene (10 ml) and
tert-butanol (881 µl, 7.7 mmol) was added. The solution was
kept at 75 ЊC for 40 h and then allowed to cool to room temper-
ature. The solvent was evaporated under reduced pressure and
the residue was purified by silica gel chromatography to afford
183 mg (73%) of 6 as an yellow oil. Rf = 0.26 (EtOAc : cyclo-
hexane = 4 : 1). 1H-NMR (CDCl3): δ 1.42 (s 9H); 1.75 (m 2H);
1.94 (m, 2H); 2.24 (m, 2H); 3.59 (m, 2H); 4.18 (m, 1H).
13C-NMR (CDCl3): 23.57 (CH2); 23.83 (CH2); 28.42 (CH3);
41.44 (CH2); 42.19 (CH2); 42.53 (2 × CH2); 48.35 (CH); 80.02
(Cq); 95.79 (Cq); 173.54 (Cq). HRMS [FAB, m-NBA, pos.]:
calcd. 287.1191; found 287.1186.
3-tert-Butoxycarbonylamino-2-tritylsulfanyl-propionic acid (10)
To a solution of 6 (344 mg, 1.17 mmol) in 10 ml THF was
added dropwise 1 M LiOH (5 ml) at 0 ЊC. The mixture
was heated to room temperature, stirred for 1 h before trityl
bromide (388 mg, 1.2 mmol) was added. The solution was
stirred for further 4 h and adjusted to pH ∼ 3 by AcOH. H2O
(10 ml) was added and the aqueous solution was extracted with
EtOAc (3 × 10 ml). The organic phase was dried (MgSO4),
concentrated under reduced pressure and the residue was puri-
fied by silica gel flash chromatography to furnish 419 mg (78%)
of 10 as a white solid. Mp = 58–60 ЊC. Rf = 0.32 (DCM : MeOH
= 95 : 5 ϩ 1% AcOH). 1H-NMR ([D6]-DMSO): δ 1.46 (s, 9H);
2.99 (m, 2H); 3.07 (m, 1H); 7.25–7.36 (m, 15H). 13C-NMR
([D6]-DMSO): 28.17 (3 × CH3); 36.43 (CH2); 47.07 (CH); 67.35
(Cq); 77.69 (Cq); 126.93 (3 × CH); 128.08 (3 × CH); 129.21
(3 × CH); 144.15 (Cq); 155.01 (Cq); 172.28 (Cq). HRMS [FAB,
m-NBA, pos.]: calcd. 463.1817; found 463.1835.
3-(9H-Fluoren-9-ylmethoxycarbonylamino)-2-tritylsulfanyl-
propionic acid (7)
To a solution of 5 (80 mg, 0.20 mmol) in THF (5 ml), cooled to
0 ЊC was added dropwise 0.2 M LiOH (1 ml). After 30 min the
starting material was completely consumed and trityl bromide
(110 mg, 0.40 mmol) was added. The solution was stirred 3 h at
room temperature and adjusted to pH ∼ 3 with AcOH. The
aqueous solution was extracted with EtOAc (3 × 10 ml). The
organic extract was dried (MgSO4), filtered and concentrated
under reduced pressure. The residue was purified by silica gel
flash chromatography to furnish 57 mg (50%) of 7 as a white
solid. Mp = 128–130 ЊC. Rf = 0.36 (DCM : MeOH = 95 : 5 ϩ 1%
AcOH). 1H-NMR ([D6]-DMSO): δ 2.99 (m, 1H); 3.05 (m, 2H);
4.11–4.18 (m, 3H); 7.30–7.37 (m, 19H); 7.64 (t, 2H, J = 8.48);
7.87 (d, 2H, J = 7.48). 13C-NMR ([D6]-DMSO): 36.54 (CH);
46.55 (CH); 67.40 (Cq); 79.80 (CH2); 126.89–129.24 (6 × CH);
140.68 (Cq); 143.79 (Cq); 144.17 (Cq); 155.60 (Cq); 176.94
(Cq). HRMS [FAB, m-NBA, pos.]: calcd. 585.1974; found
585.1978.
3-tert-Butoxycarbonylamino-2-(4-methoxy-benzylsulfanyl)-
propionic acid (11)
To a solution of 6 (90 mg, 0.31 mmol) in 5 ml THF was added
dropwise 1 M LiOH (3 ml) at 0 ЊC. The mixture was heated
to room temperature, stirred for 1 h before p-methoxybenzyl
chloride (43 µl, 0.31 mmol) was added. The solution was stirred
for further 3 h and adjusted to pH ∼ 3 by AcOH. H2O (10 ml)
was added and the aqueous solution was extracted with EtOAc
(3 × 10 ml). The organic phase was dried (MgSO4), concen-
trated under reduced pressure and the residue was purified by
silica gel flash chromatography to furnish 81 mg (77%) of 11 as
a yellow oil. Rf = 0.34 (DCM : MeOH = 95 : 5 ϩ 1% AcOH).
1H-NMR ([D6]-DMSO): δ 1.36 (s, 9H); 3.18 (m, 2H); 3.28 (m,
2H); 3.72 (s, 3H); 3.75 (m, 2H); 6.86 (d, 2H); 7.23 (d, 2H).
13C-NMR ([D6]-DMSO): 28.24 (3 × CH3); 33.92 (CH2); 46.13
(CH2); 55.05 (CH3); 62.61 (CH); 77.69 (Cq); 77.95 (Cq); 113.46
(CH); 113.83 (CH); 127.97 (Cq); 129.46 (CH); 130.18 (CH);
155.59 (Cq); 158.34 (Cq); 172.44 (Cq). HRMS [FAB, m-NBA,
pos.]: calcd. 341.1297; found 341.1282.
3-(9H-Fluoren-9-ylmethoxycarbonylamino)-2-(4-methoxy-ben-
zylsulfanyl)-propionic acid (8)
To a solution of 5 (80 mg, 0.20 mmol) in THF (5 ml), cooled to
0 ЊC was added dropwise 0.2 M LiOH (1 ml). After 30 min the
starting material was completely consumed and p-methoxyben-
zyl chloride (28 µl, 0.20 mmol) was added. The solution was
stirred 2 h at 0 ЊC and adjusted to pH ∼ 3 with AcOH. The
aqueous solution was extracted with EtOAc (3 × 10 ml). The
organic extract was dried (MgSO4), filtered and concentrated
under reduced pressure. The residue was purified by silica gel
flash chromatography to furnish 73 mg (80%) of 8 as a yellow
oil. Rf = 0.35 (DCM : MeOH = 95 : 5 ϩ 1% AcOH). 1H-NMR
([D6]-DMSO): δ 3.24 (m, 1H); 3.34 (m, 2H); 3.69 (m, 3H);
3.77 (m, 2H); 4.24 (m, 3H); 6.82 (d, 2H, J = 8.53); 7.23 (d, 2H,
J = 8.53); 7.31 (t, 2H, J = 7.15); 7.40 (t, 2H, J = 7.40); 7.69 (t,
2H, J = 6.40); 7.88 (d, 2H, J = 7.53). 13C-NMR ([D6]-DMSO):
Isocys-Arg-Ala-Glu-Tyr-Ser (16)
52 mg of Wang resin (0.98 mmol gϪ1, 0.051 mmol) was used and
the peptide assembly proceeded as described above (Fmoc-
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 5 9 – 6 5
63

View Article Online
Ser(t-Bu)-OH, Fmoc-Tyr(t-Bu)-OH, Fmoc-Glu(t-Bu)-OH,
Fmoc-Ala-OH, Fmoc-Arg(Pmc)-OH, Boc-Isocys(Trt)-OH).
The peptide was released and deprotected by treating the resin
with a solution of CF3COOH : PhSMe : Et3SiH : H2O (92 : 3 : 3
: 2, 2 ml) for 2 h. After filtration the resin was washed two times
with pure TFA (1 ml). The combined filtrates were concen-
trated under reduced pressure and cold ether was added (5 ml).
The precipitate was collected by centrifugation and washed two
times with ether. Purification by semi-preparative reversed
phase HPLC and lyophilization furnished 33 mg (69%) of 16 as
a white powder. MS (MALDI-TOF, DHB, pos.): m/z: calcd. for
C29H46N9O11S [M ϩ H]: 728.8, found: 729.1.
Ala-Glu-Tyr-Ser (26), C55H86N15O17S [M ϩ H]: 1260.6, found:
1260.4.
Determination of the pH–rate profile
Aliquots (50 µl) of the ligation mixture were diluted with 20%
TFA (30 µl) and analysed by RP-HPLC. The extinction co-
efficients at 280 nm of 16, 25 and 27 were equal as judged by
the peak areas obtained upon HPLC-analysis of equimolar
mixtures. The extinction coefficient of the ligation products 26
and 28 was calculated as the sum of the extinction coefficient of
16 and 25 or 25 and 27, respectively. These estimations were
reasonable since a) extinction coefficients at 280 nm are deter-
mined by the Tyr-absorbance with one tyrosine in 16, 25, 27
and two tyrosine residues in the ligation products 26 and 28, b)
hydrolysis of the benzylmercaptan thioester 25 furnished a
product which showed an unchanged peak area, thereby con-
firming that the benzylmercaptan is a weak chromophore at 280
nm. The peak areas corresponding to 16, 25, 26, 27, and 28 were
therefore calibrated by weighing factors of 1, 1, 0.5, 1, 0.5,
respectively. The relative content of the ligation products was
determined and plotted against the reaction time. The initial
rates were calculated by determining the slope of the linear
range of the resulting curve. A plot of the initial rates of Cys-
and Isocys-mediated ligations against the pH-value furnished
the pH–rate profile depicted in Fig. 3.
Leu-Tyr-Lys-Ala-Gly-COS(CH2)2CONH2 (24)
To 104 mg of MBHA resin (0.8 mmol gϪ1, 0.083 mmol) was
added a solution of 3-tritylsulfanylpropionic acid (1.47 mg,
0.42 mmol), HBTU (159.3 mg, 0.42 mmol) and DIPEA (145 µl,
0.83 mmol) in DMF/DCM (1 : 1, 2 ml). After 14 h the trityl
group was removed by means of a one hour treatment with
CF3COOH : Et3SiH (95 : 5, 1 ml). The peptide assembly pro-
ceeded as described above (Boc-Gly-OH, Boc-Ala-OH, Boc-
Lys(2-Cl-Z)-OH, Boc-Tyr(Bz)-OH, Boc-Leu-OH). The peptide
was released and deprotected by treating the resin with a solu-
tion of CF3COOH : CF3SO3H : PhSMe : EDT (77 : 10 : 10 : 3, 2
ml) for 2 h. After filtration the resin was washed two times with
pure TFA (1 ml). The combined filtrates were concentrated
under reduced pressure and cold ether was added (5 ml). The
precipitate was collected by centrifugation and washed two
times with ether. Purification by semi-preparative reversed
phase HPLC and lyophilization furnished 35 mg (49%) of 24 as
a white powder. MS (MALDI-TOF, DHB, pos.): calcd. for
C29H48N7O7S [M ϩ H]: 638.8 found: 639.6.
References
1 D. M. Coltart, Tetrahedron, 2000, 56, 3449–3491.
2 P. E. Dawson and S. B. H. Kent, Annu. Rev. Biochem., 2000, 69, 923–
960.
3 P. E. Dawson, T. W. Muir, I. Clark-Lewis and S. B. H. Kent, Science,
1994, 266, 776–779.
4 C. F. Liu and J. P. Tam, J. Am. Chem. Soc., 1994, 116, 4149–4153.
5 C. F. Liu, C. Rao and J. P. Tam, J. Am. Chem. Soc., 1996, 118, 307–
312.
6 J. A. Robl, C. Q. Sun, J. Stevenson, D. E. Ryono, L. M. Simpkins,
M. P. Cimarusti, T. Dejneka, W. A. Slusarchyk, S. Chao, L. Stratton,
R. N. Misra, M. S. Bednarz, M. M. Asaad, H. S. Cheung, B. E.
Abboa-Offei, P. L. Smith, P. D. Mathers, M. Fox, T. R. Schaeffer,
A. A. Seymour and N. C. Trippodo, J. Med. Chem., 1997, 40, 1570–
1577.
7 W. T. Ashton, C. L. Cantone, R. L. Tolman, W. J. Greenlee, R. J.
Lynch, T. W. Schorn, J. F. Strouse and P. K. S. Siegel, J. Med. Chem.,
1992, 35, 2772–2781.
8 N. Fotouhi, A. Lugo, M. Visnick, L. Lusch, R. Walsky, J. W. Coffey
and A. C. Hanglow, J. Biol. Chem., 1994, 269, 30227–30231.
9 T. Kimmerlin, D. Seebach and D. Hilvert, Helv. Chim. Acta, 2002,
85, 1812–1826.
Cys-Arg-Ala-Glu-Tyr-Ser (27)
52 mg of Wang resin (0.98 mmol gϪ1, 0.051 mmol) were used
and the peptide assembly proceeded as described above (Fmoc-
Ser(t-Bu)-OH, Fmoc-Tyr(t-Bu)-OH, Fmoc-Glu(t-Bu)-OH,
Fmoc-Ala-OH, Fmoc-Arg(Pmc)-OH, Fmoc-Cys(Trt)-OH).
The peptide was released and deprotected by treating the resin
with a solution of CF3COOH : PhSMe : Et3SiH : H2O (92 : 3 : 3
: 2, 2 ml) for 2 h. After filtration the resin was washed two times
with pure TFA (1 ml). The combined filtrates were concen-
trated under reduced pressure and cold ether was added (5 ml).
The precipitate was collected by centrifugation and washed two
times with ether. Purification by semi-preparative reversed
phase HPLC and lyophilization furnished 38 mg (78%) of 27 as
a white powder. MS (MALDI-TOF, DHB, pos.): m/z: calcd. for
C29H46N9O11S [M ϩ H]: 728.8, found: 729.2.
10 L. M. Gustavson and A. Srinivasan, Synth. Commun., 1991, 21,
221–225.
11 L. M. Gustavson, D. S. Jones, J. S. Nelson and A. Srinivasan, Synth.
Commun., 1991, 21, 249–263.
12 Enantioselective synthesis of - and -thiomalic acid: B. C. Chen,
M. S. Bednarz, O. R. Kocy and J. E. Sundeen, Tetrahedron:
Asymmetry, 1998, 9, 1641–1644.
13 M. Schnölzer and S. B. H. Kent, Science, 1992, 256, 221–225.
14 B. Bader, K. Kuhn, D. J. Owen, H. Waldmann, A. Wittinghofer and
J. Kuhlmann, Nature, 2000, 403, 223–226.
15 H. F. Gaertner, K. Rose, R. Cotton, D. Timms, R. Camble and R. E.
Offord, Bioconjugate Chem., 1992, 3, 262–268.
16 B. L. Nilsson, L. L. Kiessling and R. T. Raines, Org. Lett., 2000, 2,
1939–1941.
Chemical ligations
Leu-Tyr-Lys-Ala-Gly-COS(CH2)2CONH2 (24) (100 µl of
10 mM solution in H2O) was added to an argon saturated
0.1 M sodium phosphate buffer (800 µl) adjusted to the desired
pH-value by addition of 2 M NaOH. Benzylmercaptan (40 µl)
was added and the mixture was stirred until the starting
material was completely consumed. The corresponding
C-terminal peptide (100 µl of 10 mM solution) was added and
the ligation was monitored by analytical reversed phase HPLC.
17 E. Saxon, J. I. Armstrong and C. R. Bertozzi, Org. Lett., 2000, 2,
2141–2143.
Gradient: 0–5 min, 5% B; 5–15 min, 5
20% B; 15–20 min,
18 G. A. Lemieux and C. R. Bertozzi, Trends Biotechnol., 1998, 16,
506–513.
20
50% B; flow 1 ml minϪ1. The eluted peptides were identi-
fied by coupled electrospray mass spectrometry. ESI-MS m/z:
calcd. for Leu-Tyr-Lys-Ala-Gly-COSBn (25), C33H48N6O6S [M
ϩ H]: 657.3, found: 657.3; calcd. for Cys-Arg-Ala-Glu-Tyr-Ser
(27), C29H46N9O11S [M ϩ H]: 728.3, found 728.4; calcd. for
Isocys-Arg-Ala-Glu-Tyr-Ser (16), C29H46N9O11S [M ϩ H]:
728.3, found: 728.4; calcd. for Leu-Tyr-Lys-Ala-Gly-Cys-
Arg-Ala-Glu-Tyr-Ser (28), C55H86N15O17S [M ϩ H]: 1260.6,
found: 1260.5; calcd.; for Leu-Tyr-Lys-Ala-Gly-Isocys-Arg-
19 G. J. Cotton and T. W. Muir, Chem. Biol., 1999, 6, 247–256.
20 T. Wieland, E. Bokelmann, L. Bauer, H. U. Lang and H. Lau,
Liebigs Ann. Chem., 1953, 583, 129–149.
21 Isocysteine-peptides were observed as by-products in the reaction of
a thiocarboxylic acid with β-bromoalanine, which presumably
proceeded through an aziridine that was opened to form
intermediate 22. J. P. Tam, Y. A. Lu, C. F. Liu and J. Shao,
Proc. Natl. Acad. Sci. USA, 1995, 92, 12485–12489.
22 D. Seebach and J. L. Matthews, Chem. Commun., 1997, 2015–2022.
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 5 9 – 6 5
64

View Article Online
23 R. P. Cheng, S. H. Gellman and W. F. DeGrado, Chem. Rev., 2001,
101, 3219–3232.
24 P. E. Dawson, M. J. Churchill, M. R. Ghadiri and S. B. H. Kent,
J. Am. Chem. Soc., 1997, 119, 4325–4329.
30 A. J. Kennan, V. Haridas, K. Severin, D. H. Lee and M. R. Ghadiri,
J. Am. Chem. Soc., 2001, 123, 1797–1803.
31 S. Yao, I. Ghosh, R. Zutshi and J. Chmielewski, Angew. Chem.,
Int. Ed., 1998, 37, 478–481.
25 L. E. Canne, S. J. Bark and S. B. H. Kent, J. Am. Chem. Soc., 1996,
118, 5891–5896.
26 J. Offer and P. E. Dawson, Org. Lett., 2000, 2, 23–26.
27 T. Kawakami, K. Akaji and S. Aimoto, Org. Lett., 2001, 3, 1403–
1405.
32 A. Mattes and O. Seitz, Angew. Chem., Int. Ed., 2001, 40, 3178–
3181.
33 A. Mattes and O. Seitz, Chem. Commun., 2001, 2050–2051.
34 R. J. Hondal, B. L. Nilsson and R. T. Raines, J. Am. Chem. Soc.,
2001, 123, 5140–5141.
28 P. Botti, M. R. Carrasco and S. B. H. Kent, Tetrahedron Lett., 2001,
42, 1831–1833.
29 C. Marinzi, S. J. Bark, J. Offer and P. E. Dawson, Bioorg. Med.
Chem., 2001, 9, 2323–2328.
35 J. W. Haefele and R. W. Broge, Kosmet. Parfüm Drogen Rundsch.,
1961, 8, 1–4.
36 The pKa-values were calculated on http://ibmlc2.chem.uga.edu/
sparc/.
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 5 9 – 6 5
65