Introduction of functional groups into peptides via N-alkylation

Article abstract of DOI:10.1021/ol800654n

An optimized protocol for the mild and selective Fukuyama-Mitsunobu reaction was used for mono- and di-N-alkylation on solid support. Thereby, nonfunctionalized aliphatic and aromatic residues are quickly introduced into transiently protected, primary ami

Full text of DOI:10.1021/ol800654n

ORGANIC
LETTERS
2008
Vol. 10, No. 10
2015-2018
Introduction of Functional Groups into
Peptides via N-Alkylation
O. Demmer,† I. Dijkgraaf,‡ M. Schottelius,‡ H.-J. Wester,‡ and H. Kessler*,†
Department Chemie, Lehrstuhl II fu¨r organische Chemie, Technische UniVersita¨t
Mu¨nchen, Lichtenbergstraꢀe 4, D-85747 Garching, Germany, and Nuklearmedizinische
Klinik and Poliklinik, Klinikum rechts der Isar, Technische UniVersita¨t Mu¨nchen,
Ismaninger Straꢀe 22, D-81675 Mu¨nchen, Germany
kessler@ch.tum.de
Received March 21, 2008
ABSTRACT
An optimized protocol for the mild and selective Fukuyama-Mitsunobu reaction was used for mono- and di-N-alkylation on solid support.
Thereby, nonfunctionalized aliphatic and aromatic residues are quickly introduced into transiently protected, primary amines of a linear peptide.
N-Alkylation can also be used to implement alkyl chains carrying (protected) functionalities suited for subsequent modification. Applicability
of this method is demonstrated by various N-alkylated analogues of a cyclic CXCR4 receptor antagonist originally developed by Fujii et. al.
Many different ways to alkylate amines have been devel-
oped,1 including reductive methods for imines,2 amides,3
direct alkylation by halide,4 sulfonate5 or tosylate6 displace-
ment, or epoxide opening.7 However, in most cases when
used on solid support, small organic molecules are modified
that contain only little to no functionalities other than the
reaction center.
Our method of choice to modify more complex molecules
such as peptides is the Fukuyama sulfonamide alkylation,8
as it is especially mild and efficient and fits well with Fmoc
solid phase chemistry.9 Protecting the amine as a sulfonamide
† Department Chemie, Lehrstuhl II fu¨r organische Chemie.
‡ Nuklearmedizinische Klinik and Poliklinik, Klinikum rechts der Isar.
(1) Olsen, C. A.; Franzyk, H.; Jaroszewski, J. W. Synthesis 2005, 263,
1–2653, and references cited therein.
(2) (a) Sasaki, Y.; Murphy, W. A.; Heiman, M. L.; Lance, V. A.; Coy,
D. H. J. Med. Chem. 1987, 30, 1162–1166. (b) Sasaki, Y.; Coy, D. H.
Peptides 1987, 8, 119–121. (c) Meyer, J. P.; Davis, P.; Lee, K. B.; Porreca,
F.; Yamamura, H. I.; Hruby, V. J. J. Med. Chem. 1995, 38, 3462–3468. (d)
Gazal, S.; Gellerman, G.; Gilon, C. Peptides 2003, 24, 1847–1852.
(3) (a) Dankwardt, S. M.; Smith, D. B.; Porco, J. A.; Nguyen, C. H.
Synlett 1997, 854–856. (b) Kan, T.; Kobayashi, H.; Fukuyama, T. Synlett
2002, 1338–1340.
(7) (a) Rosse´, G.; Ouertani, F.; Schro¨der, H. J. Comb. Chem. 1999, 1,
397–401. (b) Bryan, W. M.; Huffman, W. F.; Bhatnagar, P. K. Tetrahedron
Lett. 2000, 41, 6997–7000. (c) Wong, J. C.; Sternson, S. M.; Louca, J. B.;
Hong, R.; Schreiber, S. L. Chem. Biol. 2004, 11, 1279–1291. (d) Tremblay,
M. R.; Wentworth, P.; Lee, G. E.; Janda, K. D. J. Comb. Chem. 2000, 2,
698–709.
(4) (a) Zuckermann, R. N.; Kerr, J. M.; Kent, S. B. H.; Moos, W. H.
J. Am. Chem. Soc. 1992, 114, 10646–10647. (b) Barn, D. R.; Morphy, J. R.;
Rees, D. C. Tetrahedron Lett. 1996, 37, 3213–3216. (c) Byk, G.; Frederic,
M.; Scherman, D. Tetrahedron Lett. 1997, 38, 3219–3222.
(5) (a) Virgilio, A. A.; Schu¨rer, S. C.; Ellman, J. A. Tetrahedron Lett.
1996, 37, 6961–6964. (b) Souers, A. J.; Virgilio, A. A.; Schu¨rer, S. S.;
Ellman, J. A.; Kogan, T. P.; West, H. E.; Ankener, W.; Vanderslice, P.
Bioorg. Med. Chem. Lett. 1998, 8, 2297–2302. (c) Souers, A. J.; Virgilio,
A. A.; Rosenquist, A.; Fenuik, W.; Ellman, J. A J. Am. Chem. Soc. 1999,
121, 1817–1825.
(8) (a) Rew, Y.; Goodman, M. J. Org. Chem. 2002, 67, 8820–8826. (b)
Andersen, T. F.; Strømgaard, K. Tetrahedron Lett. 2004, 45, 7929–7933.
(c) Beaver, K. A.; Siegmund, A. C.; Spear, K. L. Tetrahedron Lett. 1996,
37, 1145–1148. (d) Boeijen, A.; Kruijtzer, J. A. W.; Liskamp, R. M. J.
Bioorg. Med. Chem. Lett. 1998, 8, 2375–2380. (e) Fukuyama, T.; Jow, C. K.;
Cheung, M. Tetrahedron Lett. 1995, 36, 6373–6374. (f) Fukuyama, T.;
Cheung, M.; Jow, C. K.; Hidai, Y.; Kan, T. Tetrahedron Lett. 1997, 38,
5831–5834.
(9) Biron, E.; Chatterjee, J.; Kessler, H. J. Pept. Sci. 2006, 12, 213–
219.
(6) Sajjadi, Z.; Lubell, W. D. J. Pept. Res. 2005, 65, 298–310.
10.1021/ol800654n CCC: $40.75
Published on Web 04/12/2008
2008 American Chemical Society

allows alkylation either by using alcohols under Mitsunobu
conditions or by using halides, giving access to a vast amount
of alkylating agents. The Fukuyama procedure was slightly
modified, leading to an efficient and fast procedure for
N-methylation.9
Scheme 1. Synthesis of the Ornithine Derivative
In the present work, we explored this optimized procedure
toward a wider range of alkylating agents and toward the
synthesis of not only secondary but also tertiary amines. Our
aim was to use N-alkylation to introduce additional functional
groups to peptides. These functionalities need orthogonal
protection or protecting groups, which are cleaved simulta-
neously during final acidic deprotection of the peptide. They
were chosen such as to allow easy subsequent on-resin
modifications like amide and esther bond synthesis. Further-
more, functionalities needed for the so-called “click-
chemistry” (oxime ligations with aldehydes or 1,3-dipolar
addition of azides to alkynes) were also included.
To demonstrate the practical applicability of this N-
alkylation procedure, the CXCR4 chemokine receptor an-
tagonist cyclo(-D-Tyr1-Orn2-Arg3-Nal4-Gly5) (Orn ) L-or-
nithine, Nal ) L-3-(2-naphthyl)alanine) was chosen as a
scaffold for the synthesis of various N-alkylated CXCR4
ligands.10
The CXCR4 receptor and its ligands are interesting for
different medical applications as they are involved in a
variety of diseases, such as HIV-1, rheumatoid arthritis, and
at least 23 different types of cancer.11 Peptidic CXCR4
antagonists have already shown activity against these diseases
and therefore represent a novel approach for therapeutic
intervention.12
To probe which substituents are tolerated at the Orn side
chain of the modified Fujii peptide, we investigated several
mono- and dialkylated analogues of cyclo(-D-Tyr1-Orn2-Arg3-
Nal4-Gly5).
The linear peptide NR-Alloc-Orn-Nδ-Fmoc-Arg(Pbf)-Nal-
Gly-D-Tyr(tBu), which was subjected to the N-alkylation
procedure, was assembled on trityl chloride resin by the
Fmoc strategy using TBTU/HOBt as coupling reagents.
The terminal amino acid NR-Alloc-Orn-Nδ-Fmoc-OH 2
was synthesized starting from H-L-Orn-Nδ-Boc-OH 1 via
protection of NR with allylchloroformate (AllocCl), followed
by Boc deprotection with 30% TFA in DCM and Nδ
reprotection using 9-fluorenylmethyloxycarbonyl-N-hydroxy-
succinimide (FmocOSu). 2 was obtained in 87% overall yield
and was sufficiently pure for direct use on solid support
(Scheme 1). Alternatively, the amine can be protected as
o-nitrobenzenesulfonamide (Ns).13 However, as the Ns group
can only be cleaved after alkylation of the sulfonamide, it is
not as versatile as the aforementioned approach.9
On-resin alkylation of the ornithines Nδ was achieved by
transitional Ns protection14 of the primary amines, followed
by N-alkylation under Mitsunobu conditions15 or direct
alkylation with halides.16 Final deprotection of the Ns
group17 yielded the secondary amine. Tertiary amines were
synthesized by an additional alkylation step. They were built
up by first alkylating with the smaller alcohol and then using
the sterically more demanding alcohol in a second step.
Cleavage of peptides from the resin was performed after
Alloc deprotection with retention of side chain protecting
groups using hexafluoroisopropanol (HFIP) in DCM. Diphe-
nylphosphoryl azide (DPPA) was used as a cyclization
reagent with NaHCO3 as a solid base in DMF.18 Ns
deprotection of secondary amines was carried out in DMF
using ꢀ-mercaptoethanol and DBU prior to the acidic
deprotection step. TMS and TBDMS groups were cleaved
with TBAF in DMF and N-[1-(4,4-dimethyl-2,6-dioxocy-
clohex-1-ylidene)ethyl] (Dde) groups with hydrazine. The
final peptides were obtained after deprotection in TFA/H2O/
triisopropyl silane (TIPS) and subsequent RP-HPLC purifi-
cation.
One goal of this work was to explore the CXCR4 receptor
binding pocket at the Orn side chain of its ligands. Emphasis
was put on modifications with various aromatic compounds
as these have been shown to contribute beneficially to the
CXCR4 binding affinity.19
The alkylation reactions were carried out using Mitsunobu
conditions because they are milder than alkylation with
halides.20 Starting from the Ns-protected amine, the course
of the alkylation reaction was monitored via RP-HPLC. After
10 min, almost full conversion could be observed for most
small and large mono-N-alkylated compounds.
After removal of the Ns group of the N-methylated
compound, a second alkylation step led to the tertiary amines
(10) (a) Fujii, N.; Oishi, S.; Hiramatsu, K.; Araki, T.; Ueda, S.;
Tamamura, H.; Otaka, A.; Kusano, S.; Terakubo, S.; Nakashima, H.; Broach,
J. A.; Trent, J. O.; Wang, Z. X.; Peiper, S. C. Angew. Chem., Int. Ed. Engl.
2003, 42, 3251–3253. (b) Tamamura, H.; Araki, T.; Ueda, S.; Wang, Z. X.;
Oishi, S.; Esaka, A.; Trent, J. O.; Nakashima, H.; Yamamoto, N.; Peiper,
S. C.; Otaka, A.; Fujii, N. J. Med. Chem. 2005, 48, 3280–3289.
(11) (a) Feng, Y.; Broder, C. C.; Kennedy, P. E.; Berger, E. A. Science
1996, 272, 872–877. (b) Nanki, T.; Hayashida, K.; El-Gabalawy, H. S.;
Suson, S.; Shi, K. R.; Girschick, H. J.; Yavuz, S.; Lipsky, P. E. J. Immunol.
2000, 165, 6590–6598. (c) Balkwill, F. Nat. ReV. Cancer 2004, 4, 540–
550.
(14) 5 equiv of NsCl, 10 equiv of collidine, NMP, 15 min, RT.
(15) 10 equiv of ROH, 5 equiv of diisopropylazodicarboxylate (DIAD),
5 equiv of PPh3, THF, RT.
(16) 4 equiv of halide, 6 equiv of DBU, NMP, RT.
(17) 16 equiv of DBU, 20 equiv of ꢀ-mercaptoethanol, NMP, 5 min,
RT, two times.
(18) Brady, S. F. P. W. J.; Arison, B. H.; Freidinger, R. M.; Nutt, R. F.;
Veber, D. F. In 8th Annual Peptide Symposium; 1983; pp 127-130.
(19) Demmer, O.; Koglin, N.; Kessler, H.; Wester, H. J. J. Pept. Sci.
2006, 12, 172.
(12) Tamamura, H.; Fujii, N. Expert Opin. Ther. Targets 2005, 9, 1267–
1282, and references cited therein.
(20) The side chain of histidine is methylated under loss of the trityl
protecting group by alkylation with halides, but not under Mitsunobu
conditions. See ref 9.
(13) Biron, E.; Kessler, H. J. Org. Chem. 2005, 70, 5183–5189.
2016
Org. Lett., Vol. 10, No. 10, 2008

Scheme 2
.
Orthogonally Protected Functionalities Introduced by
Table 2. Comparison of Mitsunobu Reactions and Direct
Alkylation by Halide Displacement
N-Alkylation
compound reaction time purity (%)
Mitsunobu conditions
halide displacement
3
6
4
5
2 * 30 min
2 * 15 min
3 * 1 day
3 * 1 day
74
96
72
98
groups in the final deprotection step. Silyl protecting groups
were chosen for the alcohol and the alkyne groups, as they
can be cleaved orthogonally by fluorides on resin and in
solution. The hydrazine labile Dde amine protection group
is ideally suited for N-alkylation, as it is orthogonal,22 easy
to synthesize and to cleave,23 and stable under Mitsunobu
reaction conditions.23c,24 The protecting group DdeOH itself
was synthesized after optimizing an existing protocol23b by
using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI)
as the coupling reagent and by modifying the workup
conditions. Subsequent stirring with 6-aminohexanol in DCM
at RT gave 3 in 93% yield (Scheme 3).
To compare different N-alkylation procedures, two pro-
tected functionalities were introduced under Mitsunobu
conditions and two by halide displacement. While 2-(2-
bromoethyl)-1,3-dioxolane 5 and 3-(trimethylsilyl)prop-2-
yn-1-ol 6 were commercially available, 4 was obtained by
acidic ether cleavage of THF25 (Scheme 3).
within 10 min. However, in addition to the tertiary amine,
the quaternary amine was also observed as a side product
along with traces of unreacted primary amine. For the
dibenzylated amine, the reaction time was extended to 20
min to raise yields. Compared to the other tertiary amines,
a higher amount of unreacted reactant and quaternary
ammonium salt was observed in the latter case. Dibenzylation
of an amine has already been shown to be feasible using a
simpler system with virtually equal purity.21
Scheme 3. Synthesis of Starting Compounds for N-Alkylation
Table 3. Affinities of Cyclopeptides Towards the CXCR4
Receptor
To elucidate the influence of additional functionalities on
CXCR4 affinity, different protecting groups were chosen that
are stable under alkylation conditions. Emphasis was put on
orthogonal protection compatible with Fmoc solid phase
peptide synthesis (Scheme 2).
An aldehyde functionality was introduced, protected as
dioxolan, which is cleaved together with the other acid labile
R1
R2
IC50 [nM]
Me
Et
Bz
H
H
H
H
105 ( 7
38 ( 2
155 ( 63
40 ( 3
49 ( 1
38 ( 8
Table 1. Purity of (Ns Protected) Amines in Relation to the
Starting Compound after Alkylation via Mitsunobu Reaction
purity (%)
1-naphthyl(CH2)-
2-naphthyl(CH2)-
Bz
1-naphthyl(CH2)-
2-naphthyl(CH2)-
Bz
H
ROH
NδR(Ns)
NδR(Me)
NδR(Bz)
Me
Me
Me
Bz
H
H
H
H
a
MeOH
EtOH
BzOH
1-naphthyl(CH2)OH
2-naphthyl(CH2)OH
>99
84
>99
92
n.t.b
n.t.b
60
39 ( 2
38 ( 5
n.t.b
84
82
131 ( 5
57 ( 16
15 ( 3
29 ( 21
109 ( 47
n.t.b
n.t.b
HO(CH2)4-
>99
73
H2N(CH2)6-
CHO(CH2)2-
HCt CCH2-
a An inseparable mixture of mono-, di-, and trialkylated amines was
obtained. b n.t.: not tested.
Org. Lett., Vol. 10, No. 10, 2008
2017

In comparison to direct alkylation by halide displacement,
faster quantitative conversion is observed using Mitsunobu
conditions. Although both reaction types showed little to no
formation of byproducts when analyzed by ESI-MS, control
by RP-HPLC exhibited reduced purities in relation to
nonfunctionalized residues (Table 2).
Additional applications include the synthesis of peptido-
mimetics like so-called “reduced amides” or peptoids.
Acknowledgment. This work was supported by the Center
of Integrated Protein Science Munich (CIPS).
Evaluation of the N-alkylated peptide affinities toward
CXCR4 gave mixed results (Table 3). In comparison to the
starting compound cyclo(-D-Tyr1-Orn2-Arg3-Nal4-Gly5), the
IC50 values could not be improved by alkylating the Orn
side chain. Interestingly, large aromatic substituents like
naphthylmethyl are in most cases better tolerated than smaller
ones. This aromatic moiety is found in many peptidic and
nonpeptidic CXCR4 antagonists.12,19,26
The mild and selective N-alkylation method presented in
this study offers an efficient way to functionalize resin bound
peptides with a variety of functional moieties which allow
subsequent introduction of fluorescence labels, lipids, biopoly-
mers, or others.
Supporting Information Available: Experimental pro-
cedures and compound characterization data. This material
is available free of charge via the Internet at http://pubs.acs.org.
OL800654N
(23) (a) Nash, I. A.; Bycroft, B. W.; Chan, W. C. Tetrahedron Lett.
1996, 37, 2625–2628. (b) Portal, C.; Launay, D.; Merritt, A.; Bradley, M.
J. Comb. Chem. 2005, 7, 554–560. (c) Murray, P. J.; Kranz, M.; Ladlow,
M.; Taylor, S.; Berst, F.; Holmes, A. B.; Keavey, K. N.; Jaxa-Chamiec,
A.; Seale, P. W.; Stead, P.; Upton, R. J.; Croft, S. L.; Clegg, W.; Elsegood,
M. R. J. Bioorg. Med. Chem. Lett. 2001, 11, 773–776.
(24) Chhabra, S. R.; Khan, A. N.; Bycroft, B. W. Tetrahedron Lett.
2000, 41, 1099–1102.
(25) Nystro¨m, J. E.; McCanna, T. D.; Helquist, P.; Amouroux, R.
Synthesis 1988, 56–58.
(21) Zaragoza, F.; Stephensen, H. Tetrahedron Lett. 2000, 41, 1841–
1844.
(26) (a) Zhan, W. Q.; Liang, Z. X.; Zhu, A. Z.; Kurtkaya, S.; Shim, H.;
Snyder, J. P.; Liotta, D. C. J. Med. Chem. 2007, 50, 5655–5664. (b)
Tamamura, H.; Ojida, A.; Ogawa, T.; Tsutsumi, H.; Masuno, H.; Nakashima,
H.; Yamamoto, N.; Hamachi, I.; Fujii, N. J. Med. Chem. 2006, 49, 3412–
3415.
(22) (a) Augustyns, K.; Kraas, W.; Jung, G. J. Pept. Res. 1998, 51, 127–
133. (b) Rohwedder, B.; Mutti, Y.; Dumy, P.; Mutter, M. Tetrahedron Lett.
1998, 39, 1175–1178.
2018
Org. Lett., Vol. 10, No. 10, 2008