Synthesis of hydrophilic and flexible linkers for peptide derivatization in solid phase

Article abstract of DOI:10.1016/j.bmcl.2003.09.067

Four N-Fmoc protected polyoxyethylene-based amino acid type linkers were designed and synthesized for peptide derivatization in solid phase. Three of them were obtained in a crystalline form. The crystallized linkers can be stored at 4°C for 2 years without significant decomposition. Protocols for biotinylation and fluorescent labeling of peptides in solid phase were developed. The linkers also provide good ionization ability for single-bead mass spectrometry analysis of peptides.

Full text of DOI:10.1016/j.bmcl.2003.09.067

Bioorganic & Medicinal Chemistry Letters 14 (2004) 161–165
Synthesis of hydrophilic and flexible linkers for peptide
derivatization in solid phase
a
a
b
a
b
Aimin Song, Xiaobing Wang, Jinhua Zhang, Jan Marık, Carlito B. Lebrilla
ˇ
´
a,
and Kit S. Lam *
aDivision of Hematology and Oncology, Department of Internal Medicine, UC Davis Cancer Center, University of California,
Davis, 4501 X Street, Sacramento, CA95817, USA
b
Department of Chemistry, University of California, Davis, CA95616, USA
Received 25 August 2003; revised 16 September 2003; accepted 27 September 2003
Abstract—Four N-Fmoc protected polyoxyethylene-based amino acid type linkers were designed and synthesized for peptide deri-
ꢀ
vatization in solid phase. Three of them were obtained in a crystalline form. The crystallized linkers can be stored at 4 C for 2 years
without significant decomposition. Protocols for biotinylation and fluorescent labeling of peptides in solid phase were developed.
The linkers also provide good ionization ability for single-bead mass spectrometry analysis of peptides.
#
2003 Elsevier Ltd. All rights reserved.
Since 1963, the introduction of solid-phase synthesis has
tremendously improved the synthesis of peptides and
proteins, and even small organic molecule compounds.
labeling of cell binding ligands is needed for flow cyto-
metry. Preferably, the labeling reagent is connected to
C-terminus of the peptide or ligand via a hydrophilic
spacer to enhance water solubility and to simulate the
presentation of ligands on TentaGel bead. For this
purpose, a PEG linker would have been an ideal choice.
However, the commercially available PEGs usually
consist of a heterogenous mixture of PEG molecules
with different molecular weights, thus causing problems
in purification and characterization. There is a need for
the development of a series of hydrophilic linkers with
various length, but with a well-defined molecular masses.
1
Based on the solid-phase peptide synthesis (SPPS) tech-
nique, Lam et al. first reported the ‘one-bead one-com-
pound’ (OBOC) combinatorial peptide library method
2
in 1991. In essence, when a split-mix synthesis method
2
,3
is used to generate a combinatorial library, each resin
2
,4
bead expresses only one chemical entity. We routinely
use TentaGel to synthesize our OBOC libraries, in
which library compounds are linked to the polystyrene
4
scaffold via a poly(ethylene glycol) (PEG) linker. The
resin-supported libraries are screened with a high-
throughput assay, and the discovered ligands are resyn-
thesized and derivatized on a releasable resin for further
biological characterization.
In the present study, we designed four short hydrophilic
linkers that can be conveniently used as building blocks
in SPPS or solid phase organic synthesis (Scheme 1).
Each linker consists of a diamine component and an
0
Using the OBOC combinatorial library method, a
number of peptide or small molecule ligands for various
anhydride component. We chose 2,2 -oxybis(ethyl-
0
amine) and 2,2 -(ethylenedioxy)-bis(ethylamine) as the
diamine components and succinic anhydride and digly-
colic anhydride as the anhydride components. These
4
,5
targets have been identified in our laboratory.
To
confirm the biological activities of the discovered
ligands, they need to be resynthesized in a soluble form
and derivatized with proper reporter groups. For
example, biotinylation of protein binding ligands is
required for Western blot analysis and fluorescent
*
Corresponding author. Tel.: +1-916-734-8012; fax: +1-916-734-
946; e-mail: kit.lam@ucdmc.ucdavis.edu
7
Scheme 1. Design of the linkers.
0
960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.09.067

162
A. Song et al. / Bioorg. Med. Chem. Lett. 14 (2004) 161–165
reagents are all commercially available. At the begin-
ning of synthesis, a lysine derivative with an orthogonal
side chain protecting group is coupled to the resin to
provide an additional amino group for attachment of
the reporter group such as fluorescent dyes or biotin.
The linker is then built on the a-amino group of lysine
and the peptide or small molecule ligand is synthesized
on its amino terminus. The reporter groups can be
coupled to the e-amino group of lysine either prior to or
after the synthesis of the ligands.
50% acetonitrile/water and chilled in an ice bath for 30
min. A solution of Fmoc-OSu (4.4 g, 13 mmol) in 25
mL acetonitrile was added dropwise under vigorous
magnetic stirring over 1 h. The pH of reaction mixture
was maintained at 8–9 by adding diisopropylethylamine
(DIEA) throughout the reaction. The reaction was
allowed to proceed overnight at room temperature. The
solvents were removed in vacuo. The residue was dis-
solved in 100 mL of 5% aqueous NaHCO solution and
3
washed with ethyl acetate. The aqueous phase was then
acidified with 1 M HCl to pH 2 and extracted with ethyl
acetate (50 mLꢁ3). The combined organic phase was
We attempted to synthesize the linkers in solid phase
directly, but failed. The attachment of succinic anhy-
dride or diglycolic anhydride was very smooth and the
reaction was usually completed in 30 min. However, the
coupling of diamines to the resin-supported carboxyl
group was problematic. First, the coupling reaction
proceeded very slowly at a low diamine concentration,
but a high diamine concentration was not compatible
with base-labile resin linkers such as 4-hydroxy-
washed with brine, dried over anhydrous MgSO and
4
then concentrated to about 15 mL. Hexanes was slowly
added to the solution until it became slightly turbid.
Seed crystals were added. The resulting mixture was
allowed to stand at room temperature for 24 h. The
crystallized product was collected, washed with ethyl
acetate/hexanes (v/v 3:2) and dried in vacuo. The yields
1
0
of four N-Fmoc protected linkers are listed in Table 1.
6
methylbenzoic acid (HMBA linker) or 3-mercaptopro-
7
pionic acid. Second, the coupling results were
inconsistent even with strong coupling reagents such as
Since all of the N-Fmoc protected linkers have a
hydrophobic Fmoc group and a long hydrophilic tail,
they are very difficult to crystallize. We have previously
reported another hydrophilic linker with a similar but
0 0
O - 7 - azabenzotriazol - 1 - yl) - N,N,N ,N - tetra-
(
methyluronium hexafluorophosphate (HATU). There-
fore, we decided to prepare the N-Fmoc protected
linkers in solution phase so that they can be used as
1
1
longer structure, which was used for preparing peptide
1
2
microarrays. We failed to crystallize that linker and it
was used in a viscous oily form, which was difficult to
handle. Furthermore, the oily linker was highly hygro-
scopic and unstable. The oil slowly changed from col-
orless to yellow and 9-fluorenylmethanol was found as
the hydrolysis product, even though it was stored in the
8
special amino acids in standard Fmoc SPPS.
The synthetic route of N-Fmoc protected linkers is illu-
strated in Scheme 2. The diamine 1 was allowed to react
with the cyclic anhydride 2 in acetonitrile to form the
mono-acylated intermediate 3, which was subsequently
protected with N-(9-fluorenylmethoxycarbonyloxy)-suc-
cinimide (Fmoc-OSu) in situ. Acetonitrile was chosen as
the solvent for the first acylation step because the pro-
duct precipitated immediately as an inner salt to avoid
dual acylation and the waxy product could be easily
separated from the unreacted starting materials. In a
typical experiment, 10 mmol of the diamine was dis-
ꢀ
dark at 4 C. In this work, we were able to crystallize
three of the four N-Fmoc protected linkers. Compounds
4a–c were obtained in crystalline forms with sharp
melting points. The crystallized N-Fmoc protected lin-
ꢀ
kers 4a–c are quite stable. They can be stored at 4 C for
two years without significant decomposition according
to HPLC and NMR analysis.
9
solved in 50 mL acetonitrile. A solution of the anhy-
Using these N-Fmoc protected linkers, protocols for
various peptide derivatization in solid phase have been
developed. For example, biotinylation of peptides can
be readily achieved in solid phase (Scheme 3). Fmoc-
Lys(Alloc)-OH was used for this purpose. The lysine
derivative was first attached to Rink resin and the allyl-
oxycarbonyl (Alloc) group on side chain was removed.
Biotin was then coupled to the e-amino group of lysine.
We found that biotin is poorly soluble in either N,N-
dimethylformamide (DMF) or dichloromethane (DCM)
at room temperature even with addition of DIEA and,
therefore, prevents efficient coupling. This problem was
dride (10 mmol) in 25 mL of acetonitrile was added
dropwise under vigorous magnetic stirring over 1 h. The
stirring was stopped after 3 h. After the waxy product
settled down, the liquid phase was decanted and dis-
carded. The waxy product was redissolved in 100 mL
ꢀ
solved by warming biotin in DMF at 60 C. Biotin dis-
solved well in warm DMF and did not precipitate after
cooling to room temperature. The linker was attached
Table 1. Synthesis of N-Fmoc protected linkers 4a–d (Scheme 2)
ꢀ
Entry
m
n
Yield (%)
Mp ( C)
a
b
c
1
1
2
2
0
1
0
1
62
49
72
76
145–146
114–115
111–113
—
d
Scheme 2. Solution-phase synthesis of N-Fmoc protected linkers.

A. Song et al. / Bioorg. Med. Chem. Lett. 14 (2004) 161–165
163
to the a-amino group of lysine after biotinylation and
Fmoc deprotection. The peptide was then constructed
on the linker using standard Fmoc chemistry, followed
by trifluoroacetic acid (TFA) cleavage. A number of
biotinylated peptides have been synthesized using this
procedure and successfully applied to peptide micro-
Fluorescent probes may be unstable under the condi-
tions used for peptide synthesis. Therefore, they should
be attached to the peptide after the synthesis to avoid
undesired side reactions. In this case, Fmoc-Lys(Dde)-
OH was used (Scheme 4). The peptide was synthesized
on the linker at the a-amino group of lysine and its N-
terminus was then protected with Boc anhydride. The 1-
(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde)
side-chain protecting group was removed by treatment
with hydrazine. The fluorescent probe was attached to
the e-amino group of lysine, followed by TFA cleavage.
As an example, a non-Hodgkin’s lymphoma cell binding
5
arrays. A B-lymphoma cell ligand peptide, wGeyidvk
1
3
(
lowercase letters represent d-amino acids), was selec-
ted as an example. The HPLC and MS analysis results
of the crude biotinylated peptide after TFA cleavage are
shown in Figure 1.
1
4
ligand peptide, pLDI, was labeled with FITC using
this procedure. The HPLC and MS spectra of the crude
product are shown in Figure 2.
The hydrophilic linkers can also facilitate MS analysis
of peptides by providing good ionization ability for
poorly ionizable short peptides or small molecule com-
1
5
pounds. In single-bead MS analysis of combinatorial
peptide libraries, an ionization linker consisting of sev-
1
6,17
eral amino acids is usually needed.
Besides increas-
ing the synthetic steps and steric hindrance, the
ionization linker itself may interfere during the biologi-
cal screening. To demonstrate the ionization ability of
our linkers, a peptide ligand for anti-b-endorphin anti-
2
body, YGGFL, was synthesized and encoded on
Scheme 3. Synthetic route for solid-phase peptide biotinylation.
Reagents and conditions: (i) 2 equiv of Fmoc-Lys(Alloc)-OH, 2 equiv
of benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluoro-
TentaGel resin with linker 4c using a laddering
1
6
method. In each coupling step, 10% of the corre-
sponding Boc protected amino acid was added to effect
partial termination. The single-bead MALDI-FTMS
analysis result is shown in Figure 3. All of the frag-
ments are clearly shown, indicating the good ionization
ability of the linker.
1
phosphate (PyBOP ) and 4 equiv of DIEA in DMF, rt, 1 h; (ii) 0.25
in DCM, Ar, rt, 30 minꢁ2;
equiv of Pd(PPh
(iii) 2 equiv of biotin, 2 equiv of PyBOP and 4 equiv of DIEA in
3
)
4
, 24 equiv of PhSiH
3
1
DMF, rt, 1 h; (iv) 20% piperidine in DMF, rt, 15 minꢁ2; (v) 2 equiv
1
of linker, 2 equiv of PyBOP and 4 equiv of DIEA in DMF, rt, 1 h;
2
(vi) peptide synthesis; (vii) TFA/H O/TIS (v/v/v 95:2.5:2.5).
2
Figure 1. MALDI-TOF MS and HPLC (insert) spectra of crude biotinylated peptide wGeyidvk-linker(4c)-K(biotin)-NH after TFA cleavage.

164
A. Song et al. / Bioorg. Med. Chem. Lett. 14 (2004) 161–165
Scheme 4. Synthetic route for FITC labeled peptides. Reagents and conditions: (i) 2 equiv of Fmoc-Lys(Dde)-OH, 2 equiv of PyBOP1 and 4 equiv
1
of DIEA in DMF, rt, 1 h; (ii) 20% piperidine in DMF, rt, 15 minꢁ2; (iii) 2 equiv of linker, 2 equiv of PyBOP and 4 equiv of DIEA in DMF, rt, 1
h; (iv) peptide synthesis; (v) 20 equiv of Boc
FITC, 3 equiv of DIEA in DMF, rt, overnight; (viii) TFA/H O/TIS (v/v/v 95:2.5:2.5).
2
O and 4 equiv of DIEA in DCM, rt, 30 min; (vi) 2% hydrazine in DMF, rt, 5 minꢁ2; (vii) 1.5 equiv of
2
2
Figure 2. MALDI-TOF MS and HPLC (insert) spectra of crude FITC labeled peptide pLDI-linker(4b)-K(FITC)-NH after TFA cleavage.
Figure 3. Single-bead MALDI-FTMS analysis result of YGGFL encoded by a laddering method.

A. Song et al. / Bioorg. Med. Chem. Lett. 14 (2004) 161–165
165
1
In conclusion, four flexible and hydrophilic linkers for
peptide derivatization in solid phase were designed and
synthesized. Three of the N-Fmoc protected linkers
were obtained in a crystalline form. They can be pre-
pared in large scale and readily used in standard Fmoc
SPPS. Protocols for biotinylation and fluorescent label-
ing of peptides using these linkers have been developed.
Various biological assays using these peptide derivatives
are currently underway in our laboratory. The linkers
also provide good ionization ability for single-bead
mass spectrometry analysis of peptides.
10. Spectral data for synthesized compounds. Linker 4a: H
NMR (DMSO-d
7
3
) d 12.06 (s, broad, 1H), 7.88 (m, 3H),
.69 (d, J=7.2 Hz, 2H), 7.41 (t, J=7.2 Hz, 2H), 7.33 (m,
H), 4.31 (d, J=6.5 Hz, 2H), 4.22 (t, J=6.5 Hz, 1H), 3.39
6
(
m, 4H), 3.20 (m, 2H), 3.16 (m, 2H), 2.43 (t, J=6.8 Hz,
1
3
2
1
1
H), 2.33 (t, J=6.8 Hz, 2H). C NMR (DMSO-d ) d
6
74.6, 171.8, 156.9, 144.6, 141.5, 128.3, 127.8, 125.9,
20.8, 69.7, 69.6, 66.1, 47.5, 40.8, 39.3, 30.7, 29.8. ESI-MS
+
1
m/z 427.2 (MH ). Linker 4b: H NMR (DMSO-d ) d
6
1
2.82 (s, broad, 1H), 7.88 (d, J=7.2 Hz, 2H), 7.83 (t,
J=5.7 Hz, 1H), 7.69 (d, J=7.2 Hz, 2H), 7.41 (t, J=7.2
Hz, 2H), 7.33 (m, 3H), 4.30 (d, J=6.5 Hz, 2H), 4.22 (t,
J=6.5 Hz, 1H), 4.11 (s, 2H), 3.97 (s, 2H), 3.42 (m, 4H),
1
3
3
1
1
6
.27 (m, 2H), 3.14 (m, 2H). C NMR (DMSO-d ) d
72.1, 169.6, 156.9, 144.6, 141.5, 128.3, 127.8, 125.9,
20.8, 70.8, 69.6, 69.4, 68.5, 66.1, 47.5, 40.8, 38.8. ESI-MS
Acknowledgements
+
1
m/z 443.2 (MH ). Linker 4c: H NMR (DMSO-d ) d
6
This work was supported by NIH R33CA-86364. The
5
1
2.69 (s, broad, 1H), 7.90 (t, J=5.7 Hz, 1H), 7.88 (d,
00 MHz NMR spectrometer was purchased in part
J=7.2 Hz, 2H), 7.69 (d, J=7.2 Hz, 2H), 7.41 (t, J=7.2
Hz, 2H), 7.33 (m, 3H), 4.29 (d, J=6.5 Hz, 2H), 4.21 (t,
J=6.5 Hz, 1H), 4.12 (s, 2H), 3.50 (m, 4H), 3.39 (m, 4H),
3.18 (m, 2H), 3.13 (m, 2H), 2.40 (t, J=6.8 Hz, 2H), 2.31
with a grant from the National Sciences Foundation,
NSF 9724412. We thank Dr. Alan Lehman for editorial
assistance.
1
3
(
t, J=6.8 Hz, 2H). C NMR (DMSO-d
56.9, 144.6, 141.5, 128.3, 127.8, 125.9, 120.8, 70.2, 69.8,
66.0, 47.4, 40.8, 39.3, 30.8, 30.1. ESI-MS m/z 471.2
6
) d 174.6, 171.9,
1
References and notes
+
1
(
6
MH ). Linker 4d: H NMR (DMSO-d ) d 12.51 (s,
1
2
. Merrifield, R. B. J. Am. Chem. Soc. 1963, 85, 2149.
. Lam, K. S.; Salmon, S. E.; Hersh, E. M.; Hruby, V. J.;
Kazmierski, W. M.; Knapp, R. J. Nature 1991, 354, 82.
. (a) Houghten, R. A.; Pinilla, C.; Blondelle, S. E.; Appel,
J. R.; Dooley, C. T.; Cuervo, J. H. Nature 1991, 354, 84.
broad, 1H), 7.88 (d, J=7.2 Hz, 2H), 7.82 (t, J=5.7 Hz,
1H), 7.70 (d, J=7.2 Hz, 2H), 7.41 (t, J=7.2 Hz, 2H), 7.33
(m, 3H), 4.30 (d, J=6.5 Hz, 2H), 4.21 (t, J=6.5 Hz, 1H),
4.12 (s, 2H), 3.98 (s, 2H), 3.51 (m, 4H), 3.42 (m, 4H), 3.28
3
1
3
(m, 2H), 3.15 (m, 2H). C NMR (DMSO-d ) d 172.1,
6
(
b) Furka, A.; Sebestye
J. Pept. Protein Res. 1991, 37, 487.
. Lam, K. S.; Lebl, M.; Krchn k, V. Chem. Rev. 1997, 97,
11.
´
n, F.; Asgedom, M.; Dibo
´
, G. Int.
169.6, 156.9, 144.6, 141.5, 128.3, 127.8, 125.9, 120.8, 70.8,
70.2, 69.8, 69.6, 68.5, 66.1, 60.5, 47.5, 40.9, 38.8. ESI-MS
m/z 487.2 (MH ).
11. Zhao, Z. G.; Im, J. S.; Lam, K. S.; Lake, D. F. Bioconju-
gate Chem. 1999, 10, 424.
+
4
5
6
7
8
9
ˇ
a
´
4
. Aina, O. H.; Sroka, T. C.; Chen, M.-L.; Lam, K. S. Bio-
polymers (Pept. Sci.) 2002, 66, 184.
. Sheppard, R. C.; Williams, B. J. Int. J. Pept. Protein Res.
12. Falsey, J. R.; Renil, M.; Park, S.; Li, S.; Lam, K. S. Bio-
conjugate Chem. 2001, 12, 346.
1
982, 20, 451.
13. Lam, K. S.; Lou, Q.; Zhao, Z. G.; Chen, M. L.; Smith, J.;
Pleshko, E.; Salmon, S. E. Biomed. Pept. Proteins Nucleic
Acids 1995, 1, 205.
14. Park, S. I.; Renil, M.; Vikstrom, B.; Amro, N.; Song,
L.-W.; Xu, B.-L.; Lam, K. S. Lett. Pept. Sci. 2002, 8,
171.
15. (a) Song, A.; Zhang, J.; Lebrilla, C. B.; Lam, K. S. J. Am.
Chem. Soc. 2003, 125, 6180. (b) Trester-Zedlitz, M.;
Kamada, K.; Burley, S. K.; Fenyo, D.; Chait, B. T.; Muir,
¨
. Camarero, J. A.; Cotton, G. J.; Adeva, A.; Muir, T. W. J.
Pept. Res. 1998, 51, 303.
. Fields, G. B.; Noble, R. L. Int. J. Pept. Protein Res. 1990,
3
5, 161.
0
. 2,2 -Oxybis(ethylamine) which came as dihydrochloride
salt, (1.77 g, 10 mmol) was dissolved in 25 mL of metha-
nol. To the solution 4.59 mL of 25% sodium methoxide
solution in methanol was added dropwise under magnetic
stirring. The resulting mixture was stirred at room tem-
perature for 30 min. The solvent was removed in vacuo.
The residue was suspended in 50 mL of acetonitrile for
subsequent acylation in situ.
T. W. J. Am. Chem. Soc. 2003, 125, 2416.
16. Youngquist, R. S.; Fuentes, G. R.; Lacey, M. P.; Keough,
T. J. Am. Chem. Soc. 1995, 117, 3900.
17. Davies, M.; Bradley, M. Tetrahedron Lett. 1997, 38, 8565.