2-Iminothiolane as a useful coupling reagent for polyamine solid-phase synthesis

Article abstract of DOI:10.1055/s-0028-1083566

Here, the versatility of 2-iminothiolane to act as coupling reagent for solid-phase synthesis of polyamine conjugates is shown. This method, which supersedes the use of elaborate protection group strategies, allows the mild coupling of biomacromolecules and other functional moieties. Spermine conjugates with diverse maleimido building blocks were synthesized by a quick and selective reaction. Georg Thieme Verlag Stuttgart.

Full text of DOI:10.1055/s-0028-1083566

LETTER
2785
2-Iminothiolane as a Useful Coupling Reagent for Polyamine Solid-Phase
Synthesis
Coupling
R
eag
r
ent for
P
a
olyamine
S
n
olid-Phase
Sk
ynthesis Hahn,a Klaus Müllen,b Ute Schepers*a
a
LIMES-Institute Program Unit Membrane Biology and Lipid Biochemistry and Kekulé-Institut für Organische Chemie und Biochemie,
University of Bonn, Gerhard-Domagk-Str. 1, 53121 Bonn, Germany
Fax +49(228)737778; E-mail: schepers@uni-bonn.de
Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany
b
Received 9 May 2008
2-iminothiolane hydrochloride (1, Traut’s reagent), which
is a popular crosslinking agent for peptides.6 It has fre-
quently been used for the conversion of the side-chain
amino group of lysine into a thiol (Scheme 1). Eventually,
Abstract: Here, the versatility of 2-iminothiolane to act as coupling
reagent for solid-phase synthesis of polyamine conjugates is shown.
This method, which supersedes the use of elaborate protection
group strategies, allows the mild coupling of biomacromolecules
and other functional moieties. Spermine conjugates with diverse the thiol-modified polyamines can be covalently coupled
maleimido building blocks were synthesized by a quick and selec-
tive reaction.
to other functionalities either to maleimido groups in a
Michael addition or to other thiols in a disulfide bond as
Key words: solid-phase synthesis, chemoselectivity, conjugation,
polyamines, 2-iminothiolane
well as to a-bromocarbonyls in an SN2 reaction.
+
Cl–
+
NH2
NH2
– HCl
••
NH2
S–
R
S
R
N
In order to increase their water solubility and bioavailabil-
ity many therapeutically interesting drugs and nanoparti-
cles have been covalently conjugated to polycationic
H
1
moieties.1 Besides the well-characterized polyarginines2 Scheme 1 Reaction of 2-iminothiolane with primary amines
also polyamines bearing primary and secondary amino
groups have been shown to enhance the cellular uptake of
Some features make 2-iminothiolane suitable for the use
in solid-phase synthesis of polyamine conjugates. Name-
ly, its selectivity for primary amines and the preservation
of charge by the quick conversion of the amine into an
amidine/ amidinium salt are attractive features. The reac-
tion takes place under mild conditions and allows efficient
synthesis on acid- and base-labile resins permitting mild
cleavage conditions. Recently, this reaction was estab-
lished for the solution-phase conjugation of spermine and
cholesterol.7 So far, it was not reported for solid-phase
synthesis. To efficiently convert the reaction to solid
phase, we established a model system with diaminopro-
pane as a short diamine (Scheme 2). 2-Chlorotrityl resin
(1.3 mmol/g) was loaded with N-monoprotected diamino-
propane in order to avoid crosslinking of the symmetric
diamine. The o-nosyl group was removed with 2-mercap-
toethanol and DBU in DMF and the free primary amine
was reacted with excess of 2-iminothiolane hydrochlo-
ride.
different cargo.1,3 They are protonated at physiological pH
thereby tightly interacting with negatively charged cell-
membrane components such as proteoglycans and the
phosphate groups of phospholipids. Depending on the
length of the polyamine backbone and the covalently cou-
pled cargo, the cellular uptake is suggested to occur either
via a polyamine transport system (PAT) or via endocyto-
sis of proteoglycan-enriched membranes.1
The conjugation of polyamines with biomacromolecules
is often a laborious task due to the elaborate protection-
group strategy, which is required to differentiate between
the amines and the mild chemistry, which should not harm
the biomacromolecule. Furthermore, polyamine moieties
are highly hydrophilic, making the conjugates often water
soluble and difficult to handle in organic solvents. These
problems can be greatly reduced by solid-phase chemis-
try.4 Recently, we have established a novel protection-
group strategy for polyamines on solid supports that per-
mitted a mild cleavage procedure.5 Now, we were looking
for methods facilitating the on-bead coupling of the bio-
macromolecule or the nanomaterials. So far, one option is
the coupling of a thiol to a maleimido group in a Michael
addition yielding a thioether linkage. A very gentle meth-
od to introduce a thiol functionality in a biomacromole-
cule is the modification of a primary amino group with
N-o-nosyl-aminopropane,
DIPEA, CH2Cl2
H
N
H
Cl
N
o-nosyl
2
2-mercaptoethanol,
DBU, DMF
H
N
NH2
3
H
N
H
N
1
S–
SYNLETT 2008, No. 18, pp 2785–2790
Advanced online publication: 15.10.2008
1
4
.1
1
.2
0
0
8
4
+
NH2
DOI: 10.1055/s-0028-1083566; Art ID: G14908ST
© Georg Thieme Verlag Stuttgart · New York
Scheme 2 Loading of the resin and subsequent deprotection

2786
F. Hahn et al.
LETTER
H
H
N
H
N
H
S
N
N
S–
4
+
NH2
NH2
cleavage from
resin
H
N
N
S
– NH3
5
+
+H3N
N
NH
NH
N
S
S
SH
6
+
NH2
7
Scheme 3 Side reaction of 4 leading to restitution of a N-substituted iminothiolane
The thiol modification with 2-iminothiolane is usually (pH 8.0) and DMSO and the reaction between the 2-imi-
carried out in DMSO or in aqueous buffers at pH values nothiolane and the primary amine was prevented. Like-
above 7.0.7,8 However, both solvents are less suitable for wise, the reaction in THF proceeded very poorly,
reactions on polystyrene resins. Therefore, we investigat- probably due to the low solubility of 2-iminothiolane. Al-
ed other solvent systems as described below. 2-Imino- though, the solubility in DMF was improved, not all pri-
thiolane was added to the pre-swollen resin in the mary amines reacted even after 15 hours. The addition of
respective solvent. After 15 hours the resin was washed 2 equivalents DIPEA in order to accelerate the reaction
and the cleaved crude product was analyzed by 1H NMR did not markedly improve the conversion.
spectroscopy and mass spectrometry. As expected, the
resins showed only minimal swelling in phosphate buffer
thiolane was dissolved in water and was added to a sus-
However, complete reaction was observed, if 2-imino-
Table 1 Optimization of Reaction Conditions
O
H
H
N
N
N
O
Et
8
1
3
ethylmaleimide
O
N
H
H
Et
N
N
S
NH2+Cl–
O
9
Entry
Reaction time (h)
2-Iminothiolanea
(equiv/min)
N-Ethylmaleimidea Ratiob,c
(equiv/min)
5
8
9
1
1
1
1
2
3
15
15
15
2/0
2/0
5/780
5/2
1.69
0.13
0.10
0
0.47
0.10
1/0
1/5
1/0
1/5
4
5
6
15
15
15
1/0
1.5/1
1/1
0.3/2.5
0.08
0.10
0.06
0.10
0.31
0.04
1
1
1
0.5/0
1.5/2
1.3/2
2/1
3/0
a Amount (equiv), addition time (min), 1/5: addition of 1 equiv after 5 min
b The ratio between 5 and 9 was determined by peak integration of the characteristic triplets in the 1H NMR spectra at d = 3.7 and 3.4.
c The ratio of 8 and 5 was determined by integration of the characteristic 1H NMR dd signals at d = 4.5 and 3.9 in MeOD.
Synlett 2008, No. 18, 2785–2790 © Thieme Stuttgart · New York

LETTER
Coupling Reagent for Polyamine Solid-Phase Synthesis
2787
1
pension of the resin in THF, giving a solvent mixture of molecular, as indicated by H NMR spectroscopy and
water–THF (1:9). The clear liquid phase indicated that the ESI-MS.
2-iminothiolane had entirely dissolved, while the resin
was sufficiently swollen.
To avoid the formation of this byproduct and to optimize
the protocol for the maleimido–thiol coupling, the respec-
Analysis of the crude mixture indeed showed the absence tive maleimide and 2-iminothiolane were simultaneously
of diaminopropane but also the occurrence of a byproduct. added to resin 3. Although this mainly prevented the for-
The mass spectra showed an extra [M – 16]+ peak at 159.1 u mation of the cyclization product, the simultaneaous cou-
due to the loss of ammonia.8 The formation of this byprod- pling reaction of the amine to the maleimide resulted a
uct arose from the nucleophilic attack of the liberated thi- second byproduct 8.10 Reaction time, excess of both com-
olate onto the amidite resulting in the N-substituted ponents, and order of addition were varied in order to pre-
iminothiolane 5 (Scheme 3). If the reaction was carried vent both side reactions (Table 1). Finally, the desired
out overnight in the presence of a weak base, only this product 9 could be obtained in 95% purity after a short re-
1
byproduct can be detected by H NMR spectroscopy action with 3 equivalents of 1 and 2 equivalents of ethyl-
(Scheme 2).9 Addition of excess (5 equiv) of ethylmale- maleimide.11
imide 2 hours prior cleavage from the resin did not result
The formation of both byproducts could be suppressed by
in any coupling indicating the absence of free thiols and
adding the maleimide two minutes after the 2-iminothi-
also the irreversibility of the ring closure. After some time
olane, since the recyclization occured slower than the cou-
in a protic solvent and purification by preparative HPLC,
pling reaction. These conditions were used to perform the
reaction with longer immobilized polyamine backbones
5 further showed severe decomposition arising from the
attack of primary amines to the N-substituted imino-
such as spermine.
thiolane. This attack seems to occur intra- as well as inter-
Table 2 Regioselective Synthesis of Spermine Conjugates
Aloc
H
N
N
Aloc
N
N
H
Aloc
11
N,N'-dimethylbarbituric acid,
Pd(PPh3)4, CH2Cl2
H
H
N
N
N
NH2
H
12
1, maleimide, THF–H2O (9:1)
NH2+Cl–
O
H
N
H
N
S
N
N
R
N
H
H
13
O
Entry
1
Maleimide (Mal-R)
Isolated yield (%) Purity (%)a
O
75
82
72
93
94
98
N
Et
O
O
2
3
N
O
O
N
HOOC
O
Synlett 2008, No. 18, 2785–2790 © Thieme Stuttgart · New York

2788
F. Hahn et al.
LETTER
Table 2 Regioselective Synthesis of Spermine Conjugates (continued)
Aloc
H
N
N
Aloc
N
N
H
Aloc
11
N,N'-dimethylbarbituric acid,
Pd(PPh3)4, CH2Cl2
H
H
N
N
N
NH2
H
12
1, maleimide, THF–H2O (9:1)
NH2+Cl–
O
H
N
H
N
S
N
N
R
N
H
H
13
O
Entry
4
Maleimide (Mal-R)
Isolated yield (%) Purity (%)a
O
NO2
H
N
H
N
76
99
N
S
O2
O
O
O
H
N
N
5
6
n.d.
62
O
N
DMTr
O
N
O
O
O
HO
O
O
N
NH
n.d.
94
O
HO
O
N
NH
O
O
O
N
O
O
O
O
O
O
NH
O
O
N
O
7b
63
55c
N
O
a The purities were calculated by integration of the relative peak area of the HPLC with UV detection at 280 and 215 nm.
b Only 1.7 equiv of the maleimide were applied to avoid aggregation on the polystyrene matrix.
c The high amphipilicity of the conjugate prevents a purification on RP-C18 as well as on silica gel columns. Therefore, the conversion was
calculated from 1H NMR spectra of the crude product; n.d. = not determined.
An alkoxytrityl resin4b was loaded with tris-Aloc-sper- using the established protocol (Table 2).12,13 The alkoxy-
mine (11). Protection of the primary amine prevented any trityl resin was chosen due to its extremely mild cleavage
coupling of 11 with 4 equivalents of 1 even after 4 hours. conditions (0.1–0.5% TFA), which allow the solid-phase
Deprotection resulted in nonsymmetrically immobilized synthesis of acid- and base-labile polyamine conjugates.
spermine,5 and a variety of maleimides were crosslinked After one hour the resin was washed and the products
Synlett 2008, No. 18, 2785–2790 © Thieme Stuttgart · New York

LETTER
Coupling Reagent for Polyamine Solid-Phase Synthesis
2789
(10) Spectroscopic Data for Side Product 8
were cleaved from the resin and purified by preparative
HPLC.13 As shown in Table 2, the crosslink occurred ef-
ficiently with a variety of structurally diverse maleimides.
The small maleimides from entries 1–3 reacted smoothly
and the products were obtained in high purities. Entries 4–
7 showed that also bulkier maleimido building blocks
with a broad variation in polarity were tolerated.
1H NMR (MeOD, 400 MHz): d = 4.45 (dd, J1 = 9.1 Hz,
J2 = 5.7 Hz, 1 H), 3.57 (q, J = 7.3 Hz), 3.16 (dd, J1 = 17.9
Hz, J2 = 9.2 Hz, 1 H), 3.08 (t, J = 7.6 Hz, 2 H), 3.05 (t,
J = 7.7 Hz, 2H), 2.91 (dd, J1 = 17.9 Hz, J2 = 5.6 Hz, 1 H),
2.13 (m, 2 H), 1.17 (t, J = 7.2 Hz).
(11) Spectroscopic Data for Compound 9
1H NMR (MeOD, 400 MHz): d = 3.89 (dd, J1 = 9.1 Hz,
J2 = 2.9 Hz, 1 H), 3.52 (q, J = 7.2 Hz, 2 H), 3.38 (t, J = 7.1
Hz, 2 H), 3.19 (dd, J1 = 18.5 Hz, J2 = 9.1 Hz, 1 H), 3.03 (t,
J = 7.7 Hz, 2 H), 2.97 (dd, J1 = 13.4 Hz, J2 = 6.8 Hz, 1 H),
2.79 (dd, J1 = 13.5 Hz, J2 = 7.1 Hz, 1 H), 2.61 (t, J = 7.7 Hz,
2 H), 2.46 (dd, J1 = 18.6 Hz, J2 = 3.7 Hz, 1 H), 2.08 (tt,
J1 = 7.1 Hz, J2 = 7.1 Hz, 1 H), 2.01 (tt, J1 = 7.2 Hz, J2 = 7.2
Hz, 1 H), 1.13 (t, J = 7.2 Hz, 2 H). ESI-HRMS: m/z calcd for
C13H25N4O2S+: 301.1693; found: 301.1686.
The lower conversion rate in entry 7 (Table 2) is due to the
lower excess of the strongly fluorescent maleimido build-
ing block (1.7 equiv) to avoid the aggregation of the com-
pound on the resin.14 Nonreacted substrate can be
recovered.
In conclusion, we developed a protocol that allows the ap-
plication of 2-iminothiolane for the chemoselective cou-
pling of polyamines to maleimides for solid-phase
chemistry. This method displays a quick and mild method
that avoids both, laborious protection-group transforma-
tions and alkylation procedures, and allows the synthesis
of base- and acid-labile conjugates if carried out on highly
acid-labile alkoxytrityl resins.15
(12) Ethylmaleimide, phenylmaleimide, and 3-maleimido-
propionic acid were purchased from Sigma-Aldrich. The
maleimides from entries 4–6 were prepared by the following
procedure: 3-maleimidopropionic acid N-
hydroxysuccinimide ester (3 equiv) and DIPEA (1 equiv)
were dissolved in DMF, and amine (1 equiv) was added in
the same volume of THF. The suspension was stirred for 8 h
and THF was removed in vacuo. The remaining suspension
was partitioned between CH3Cl and H2O, and the organic
layer was washed with H2O (3 ×). The organic layer was
dried over Na2SO4, and the product was purified by flash
chromatography. The products were isolated in 47%, 49%,
and 35% yield and analyzed by HRMS and 1H NMR
spectroscopy. In entry 5, CH2Cl2 was used as solvent instead
of THF–DMF and DMAP (1 equiv) was added for additional
activation.
Acknowledgment
We would like to thank the DFG-Center for Functional Nanostruc-
tures (CFN), Karlsruhe, Prof. Bräse, Karlsruhe, and Prof. Sandhoff,
Bonn for financial support.
References and Notes
(13) Experimental Procedure
Alkoxytrityl resin (100 mg) loaded with tris-Aloc-spermine
(0.027 mmol, 1 equiv) were swollen in CH2Cl2 and a
solution of Pd(PPh3)4 (6 mg, 0.005 mmol, 0.2 equiv) and of
N,N¢-dimethylbarbituric acid (33 mg, 0.214 mmol, 8 equiv)
in CH2Cl2 (2 mL) were added. The suspension was agitated
for 16 h at 40 °C. The resin was alternately washed with a
solution of sodium N,N-dimethylaminodithiocarbamate in
CH2Cl2–MeOH (4:1) and MeOH (3 ×), THF and MeOH (3 ×),
and CH2Cl2 (3 ×). The resin was swollen in THF (1 mL) for
10 min and 2-iminothiolane (11 mg, 0.081 mmol, 3 equiv.)
in H2O (200 mL) was added. The suspension was agitated
for 2 min, then the respective maleimide (2 equiv) in THF
(800 mL) was added, and the suspension was agitated for
1 h. The resin was washed with H2O, THF, MeOH (3 ×), and
CH2Cl2 (3 ×). The crude product was cleaved from the resin
with 1% TFA in CH2Cl2. The filtrate was combined with the
filtrate of the CH2Cl2 and MeOH wash. In entry 5 the 5¢-
DMTr group was removed by quick treatment of the resin
with 1% TFA in CH2Cl2 and subsequent washing of the
polymer with a minimal amount of CH2Cl2–Et2O (2:1) until
the filtrate is clear. The product was washed from the resin
with CH2Cl2 and MeOH. The solvents were removed and the
remaining compound was purified by preparative HPLC on
C18 column with gradients of eluent A [95% TEAA (0.01 M,
pH 7.0)–5% MeCN] and eluant B [5% TEAA (0.01 M, pH
7.0)–95% MeCN), or with eluant A (95% H2O–5% MeCN–
0.1% AcOH) and eluant B (5% H2O–95% MeCN–0.1%
AcOH).
(1) For a review, see: Schepers U., Schmitz K., Hahn F., Bräse
S.; Angew. Chem. Int. Ed.; submitted
(2) Foerg, C.; Merkle, H.-P. J. Pharm. Sci. 2007, 97, 144.
(3) Hahn, F.; Schepers, U. In Combinatorial Chemistry on Solid
Supports, Vol. 278; Bräse, S., Ed.; Springer: Berlin /
Heidelberg, 2007, 135–208.
(4) (a) Manku, S.; Laplante, C.; Kopac, D.; Chan, T.; Hall, D. G.
J. Org. Chem. 2001, 66, 874. (b) Kan, T.; Kobayashi, H.;
Fukuyama, T. Synlett 2002, 1338. (c) Jönsson, D.
Tetrahedron Lett. 2002, 43, 4793. (d) Nash, I. A.; Bycroft,
B. W.; Chan, W. C. Tetrahedron Lett. 1996, 37, 2625.
(5) Hahn, F.; Schepers, U. J. Comb. Chem. 2008, 10, 267.
(6) (a) Traut, R. R.; Bollen, A.; Sun, T. T.; Hershey, J. W. B.;
Sundberg, J.; Pierce, L. R. Biochemistry 1973, 12, 3266.
(b) Jue, R.; Lambert, J. M.; Pierce, L. R.; Traut, R. R.
Biochemistry 1978, 17, 5399. (c) Miyata, K.; Kakizawa, Y.;
Nishiyama, N.; Harada, A.; Yamasaki, Y.; Koyama, H.;
Kataoka, K. J. Am. Chem. Soc. 2004, 126, 2355. (d) Tada,
T.; Mano, K.; Yoshida, E.; Tanala, N.; Kunugi, S. Bull.
Chem. Soc. Jpn. 2002, 75, 2247. (e) King, T. P.; Li, Y.;
Kochoumian, L. Biochemistry 1978, 17, 1499.
(7) Gruneich, J. A.; Diamond, S. L. J. Gene Med. 2007, 9, 381.
(8) (a) Bartlett, M. G.; Buscht, K. L. Biol. Mass Spec. 1994, 23,
353. (b) Singh, R.; Kats, L.; Blättler, W. A.; Lambert, J. M.
Anal. Biochem. 1996, 236, 114. (c) Mokotoff, M.;
Mocarski, Y. M.; Gentsch, B. L.; Miller, M. R.; Zhou, J. H.;
Chen, J.; Ball, E. D. J. Peptide Res. 2001, 57, 383.
(9) Spectroscopic Data for Side Product 6
(14) Characterization of the Products of Entries 1–7 (Table 2)
Entry 1: 1H NMR (400 MHz, MeOD): d = 1.14 (t, J = 7.2
Hz, 3 H), 1.79 (m, 4 H), 2.06 (tt, J1 = 7.7 Hz, J2 = 7.3 Hz,
4 H), 2.13 (m, 2 H), 2.47 (dd, J1 = 18.5 Hz, J2 = 3.9 Hz, 1 H),
2.62 (t, J = 7.7 Hz, 2 H), 2.80 (dt, J1 = 13.5 Hz, J2 = 7.0 Hz,
1 H), 2.98 (dt, J1 = 13.6 Hz, J2 = 6.8 Hz, 1 H), 3.00–3.15 (m,
1H NMR (MeOD, 400 MHz): d = 3.69 (t, J = 7.2 Hz, 2 H),
3.65 (t, J = 6.5 Hz, 2 H), 3.26 (t, J = 7.2 Hz, 2 H), 3.09 (t,
J = 7.5 Hz, 2 H), 2.44 (tt, J1 = 6.9 Hz, J2 = 6.8 Hz, 2 H), 2.18
(tt, J1 = 7.4 Hz, J2 = 7.4 Hz, 2 H). ESI-MS: m/z = 159.1
[M + H]+.
Synlett 2008, No. 18, 2785–2790 © Thieme Stuttgart · New York

2790
F. Hahn et al.
LETTER
10 H), 3.19 (dd, J1 = 18.5 Hz, J2 = 9.1 Hz, 1 H), 3.40 (t,
J = 7.1 Hz, 2 H), 3.53 (q, J = 7.3 Hz, 2 H), 3.90 (dd, J1 = 9.1
Hz, J2 = 3.8 Hz, 1 H). ESI-HRMS: m/z calcd [M + H]+:
429.3006; found: 429.3010.
2.58–2.68 (m, 3 H), 2.81 (m, 2 H), 2.95 (dt, J1 = 13.1 Hz,
J2 = 6.5 Hz, 1 H), 3.00–3.30 (m, 12 H), 3.42 (t, J = 6.9 Hz,
2 H), 3.70–3.90 (m, 4 H), 3.93 (dd, J1 = 9.0 Hz, J2 = 3.9 Hz,
1 H), 4.03 (dt, J1 = 3.4 Hz, J2 = 3.5 Hz, 1 H), 4.39 (dt,
J1 = 6.8 Hz, J2 = 6.5 Hz, 1 H), 6.09 (d, J = 7.8 Hz, 1 H), 6.22
(m, 1 H), 8.39 (br, J = 7.8 Hz, 1 H). ESI-MS: m/z = 682.36
[M + H]+, MS–FAB+: 682.4 [M + H]+.
Entry 2: 1H NMR (400 MHz, MeOD): d = 1.79 (br m, 4 H),
1.96–2.14 (m, 6 H), 2.60–2.70 (m, 3 H), 2.86 (dt, J1 = 13.6
Hz, J2 = 7.1 Hz, 1 H), 2.93–3.10 (m, 11 H), 3.39 (t, J = 6.5
Hz, 2 H), 3.32–3.40 (m, 1 H), 4.10 (dd, J1 = 9.1 Hz, J2 = 3.8
Hz, 1 H), 7.26–7.54 (m, 5 H). ESI-HRMS: m/z calcd [M +
H]+: 477.3006; found: 477.3002.
Entry 6: 1H NMR (400 MHz, MeOD): d = 1.70–1.85 (m,
4 H), 1.89 (s, 3 H), 2.00–2.18 (m, 6 H), 2.25–2.40 (m, 2 H),
2.45–2.55 (m, 3 H), 2.63 (t, J = 7.6 Hz, 2 H), 2.81 (dt,
J1 = 13.3 Hz, J2 = 7.2 Hz, 1 H), 2.91–3.28 (m, 12 H), 3.37–
3.45 (m, 2 H), 3.70–3.97 (m, 7 H), 4.42 (dt, J1 = 7.1 Hz,
J2 = 6.4, 1 H), 6.21 (m, 1 H), 7.87 (s, 1 H). ESI-HRMS: m/z
calcd [M + H]+: 696.3861; found: 696.3859.
Entry 3: ESI-HRMS: m/z calcd [M + H]+: 473.2905; found:
473.2907.
Entry 4: 1H NMR (400 MHz, MeOD): d = 1.67 (tt, J1 = 6.7
Hz, J2 = 6.7 Hz, 2 H), 1.73–1.88 (br m, 4 H), 2.00–2.14 (br
m, 6 H), 2.50 (dd, J1 = 17.9 Hz, J2 = 3.6 Hz, 1 H), 2.44–2.70
(m, 2 H), 2.60–2.70 (m, 2 H), 2.82 (dt, J1 = 13.9 Hz, J2 = 6.5
Hz, 1 H), 2.93 (dt, J1 = 13.8 Hz, J2 = 6.9 Hz, 1 H), 3.04–3.24
(m, 15 H), 3.42 (t, J = 7.1 Hz, 2 H), 3.68–3.83 (m, 1 H), 3.90
(dd, J1 = 9.2 Hz, J2 = 3.9 Hz, 1 H), 7.80–8.11 (m, 4 H). ESI-
HRMS: m/z calcd [M + 2H]2+: 357.6749; found: 357.6736.
Entry 5: 1H NMR (400 MHz, MeOD): d = 1.80–1.90 (m,
4 H), 2.00–2.18 (br m, 6 H), 2.21 (m, 1 H), 2.50 (m, 1 H),
Entry 7: MS (MALDI+): m/z = 1583.16 [M + H]+. ESI-
HRMS: m/z calcd [M + 2H]2+: 791.8509; found: 791.8456.
(15) A proposed sequence would be: (1) Selective introduction of
a protection group at the primary amine; (2) protection of the
secondary amines; (3) removal of the protection group on the
primary amine; (4) elongation with an S-protected building
block; (5) deprotection of the thiol; (6) coupling of the thiol
to a maleimide; (7) deprotection of the secondary amine.
Synlett 2008, No. 18, 2785–2790 © Thieme Stuttgart · New York

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