Postsynthetic modification of peptides via chemoselective N-alkylation of their side chains

Article abstract of DOI:10.1021/ol300437w

A chemoselective, mild, and versatile method for performing postsynthetic modifications of peptide sequences is described. It requires only activated molecular sieves in the presence of an alkyl halide in order to N-alkylate lysine side chains. This reaction is fully compatible with most of the peptide functionalities, discriminates the reactivity of differently protected lysines, and proceeds in good yield. The mild conditions employed were further proved by performing the N-alkylation of a peptide containing a disulfide bridge.

Full text of DOI:10.1021/ol300437w

ORGANIC
LETTERS
2012
Vol. 14, No. 7
1664–1667
Postsynthetic Modification of Peptides via
Chemoselective N-Alkylation of Their Side
Chains
Luca Monfregola,† Marilisa Leone,‡ Enrica Calce,‡ and Stefania De Luca*,‡
Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309,
United States, and Institute of Biostructures and Bioimages, National Research Council,
80138 Naples, Italy
stefania.deluca@cnr.it
Received January 18, 2012
ABSTRACT
A chemoselective, mild, and versatile method for performing postsynthetic modifications of peptide sequences is described. It requires only
activated molecular sieves in the presence of an alkyl halide in order to N-alkylate lysine side chains. This reaction is fully compatible with most of
the peptide functionalities, discriminates the reactivity of differently protected lysines, and proceeds in good yield. The mild conditions employed
were further proved by performing the N-alkylation of a peptide containing a disulfide bridge.
The development of new methodologies for the selective
and straightforward chemical modification of peptides is
eagerly requested and represents a scientific challenge, due
toits important implications indrug discovery, aswell asin
structureÀactivity relationship (SAR) studies in peptide
chemistry.1
Instead of a stepwise synthetic approach where unnat-
ural amino acids are incorporated by a traditional protocol
into a peptide chain, direct and selective peptide modifica-
tion represents a flexible and versatile alternative approach
to optimize lead structures.
during the coupling of modified amino acids with the
peptide chain on the resin.2 However, the key to the success
of the postsynthetic peptide modification approach lies in
the chemoselective outcome of the employed reaction.
In recent years, many synthetic protocols have been
published to introduce modifications on already pre-
formed peptide sequences.3
A possible synthetic route to modify peptide side chains
is the N-alkylation reaction. In this regard, the most cited
Kessler protocol4 reported a synthetic route to N-alkylate
The main goal of this synthetic strategy is avoiding
sterical hindrance problems, which are often encountered
(3) (a) Ooi, T.; Tayama, E.; Maruoka, K. Angew. Chem., Int. Ed.
~
2003, 42, 579–582. (b) Espuna, G.; Arsequell, G.; Valencia, G.; Barluenga,
ꢀ
J.; Alvarez-Gutierrez, J. M.; Ballesteros, A.; Gonzales, J. M. Angew.
Chem., Int. Ed. 2004, 43, 325–329. (c) Ruiz-Rodriguez, J.; Albericio, F.;
Lavilla, R. Chem.;Eur. J. 2010, 16, 1124–1127. (d) Tedaldi, L. M.; Smith,
M. E. B.; Nathani, R. I.; Baker, J. R. Chem. Commun. 2009, 43, 6583–6585.
(e) Huang, R.; Holbert, M. A.; Tarrant, M. K.; Curtet, S.; Colquhoun,
D. R.; Dancy, B. M.; Dancy, B. C.; Hwang, Y.; Tang, Y.; Meeth, K.;
Marmorstein, R.; Cole, R. N.; Khochbin, S.; Cole, P. A. J. Am. Chem. Soc.
2010, 132, 9986–9987. (f) Carrasco, M. R.; Silva, O.; Rawls, K. A.;
Sweeney, M. S.; Lombardo, A. A. Org. Lett. 2006, 8, 3529–3532. (g)
Chan, A. O.-Y.; Ho, C.-M.; Chong, H.-C.; Leung, Y.-C.; Huang, J.-S.;
Wong, M.-K.; Che, C.-M. J. Am. Chem. Soc. 2012, 134, 2589–2598.
(4) Demmer, O.; Dijkgraaf, I.; Schottelius, M.; Wester, H.-J.; Kessler,
H. Org. Lett. 2008, 10, 2015–2018.
† University of Colorado.
‡ National Research Council.
(1) Jungheim, L. N.; Shepperd, T. A.; Baxter, A. J.; Burguess, J.;
Hatch, S. D.; Lubbehusen, P.; Wiskerchen, M.; Muesing, M. A. J. Med.
Chem. 1996, 39, 96–108.
(2) (a) Dal Pozzo, A.; Bergonzi, R.; Minghong, N. Tetrahedron Lett.
2001, 42, 3925–3927. (b) Dal Pozzo, A.; Minghong, N.; Muzi, L.;
Caporale, A.; De Castiglione, R.; Kaptein, B.; Broxterman, Q. B.;
Formaggio, F. J. Org. Chem. 2002, 67, 6372–6375. (c) Minghong, N.;
Esposito, E.; Kaptein, B.; Broxterman, Q. B.; Dal Pozzo, A. Tetrahedron
Lett. 2005, 46, 6369–6371.
r
10.1021/ol300437w
Published on Web 03/12/2012
2012 American Chemical Society

nosyl-protected amino acids during the peptide assembly,
according to traditional protocols (Mitsunobu conditions
or alkyl halides in presence of DBU), which were not fully
compatible with Fmoc-chemistry.
Scheme 1. Synthetic Strategy of Mono-N-alkylated Peptides
We have recently reported the usage of molecular sieves
to N-alkylate several basic amino acid derivatives (Fmoc-
Lys(Ns)-OH, Fmoc-Orn(Ns)-OH, Fmoc-Dab (Ns)-OH,
Fmoc-Dap(Ns)-OH).5
Starting from these results, we have developed an in-
novative protocol to perform peptide postsynthetic mod-
ifications. Itallowspeptides on the amino group of a Lys to
be selectively N-alkylated, which were previously pro-
tected with the nosyl group,6 by only employing molecular
sieves in the presence of alkyl halides. In particular, it relies
on the earlier reported reaction conditions, which does not
affect the stereochemistry of the employed amino acid
chiral center.5 To the best of our knowledge, postsynthetic
N-alkylation of peptides promoted by molecular sieves is
not reported in literature. Herein we report a broad study
carried out on several peptide models through a fine-
tuning of the reaction parameters.
For each peptide model, amino acids were chosen in
order to cover the range of functionalities present on the
side chains of natural peptide molecules (Scheme 1).
In detail, the generic peptide sequence 1 was first synthe-
sized, and thenseveral amino acid derivatives were inserted
at its N-terminal side (peptides 2À12) (Scheme 1). Each
peptide, acetylated at the N-terminus and free at the
carboxylic extremity, is alkylated after cleavage from the
solid support. The peptide sequences were dissolved in
DMF under an Ar atmosphere and in the presence of
was effectively achieved on the peptide came from com-
parison of 1D and 2D proton NMR spectra of peptide 1b
(Scheme 1) with those of the parent peptide containing a
free Lys. In Figure 1A it can be clearly seen that, upon
insertion of the benzyl ring on the Lys side chain, chemical
shifts for Ala NH and Lys NHζ atoms undergo major
˚
activated 4 A molecular sieves, then alkyl bromide was
added (1.2 equiv), and stirring of the obtained mixture
occurred at rt for 12À24 h.
As we have previously discussed, the molecular sieves
are supposed to act as a base, capturing the proton of the
amino group.5,7
1
changes. NMR H/2H exchange experiments noticeably
We used benzyl bromide to alkylate the whole set of
peptides and methylnaphtyl bromide to obtain peptide 1c.
The aim was to demontrate that the efficiency of the
reaction doesnotrelyonthespecific employedalkyl group.
The course of the alkylation reaction was followed by
HPLC-ES-MS analysis; the yields were estimated by
HPLC integration of the alkylated peptide compared to
the starting peptide and other byproduct, if any (see Table 1).
The reaction mixture was centrifuged, and the super-
natant was collected to remove the Ns group from the Lys
side chain. This reaction was easily performed by adding
the appropriate amount of DBU (10 equiv) and 2-mercap-
toethanol (10 equiv) to the DMF solution and stirring for
20 min at rt. It is worth noting that the whole synthetic
protocol does not require intermediate purification steps.
The first alkylation reactions were performed on peptide
1 in good yield (Table 1). Good evidence that benzylation
indicatedthe presenceofthe benzylic group on1b, in factin
an excess of 2H2O;signals from labile NH groups disappear
whereas peaks from nonexchangeable aromatic protons
are still visible in the 1D proton spectrum (Figure 1B).
1
Similar NMR H/2H exchange experiments were carried
out to demonstrate the insertion of a naphtylic ring in the
AcGlyLys(MeNaph)ValAla peptide (Figure 1C).
The benzylation performed on peptide sequences 2À8
(Scheme 1) enabled the assessment that these molecules,
containing unprotected functional groups, were chemose-
lectively alkylated in good yield (Table 1) on the Lys nosyl-
protected ε-NH2, under the mild conditions employed.
Most of the features observed in the NMR spectra of 1b
could be also recognized in the spectra of all the mono-
alkylated peptides (see Supporting Information (SI)). In
particular the shift of the Lys NHζ group near 7.64 ppm
was considered diagnostic of the good accomplishment of
the alkylation reaction.
We also analyzed a peptide sequence containing the Lys
to be alkylated and an unprotected Arg residue; in this
case, the desired monoalkylated peptide was characterized
by a lower yield, due to the reactivity of the guanidine
(5) Monfregola, L.; De Luca, S. Amino Acids 2011, 41, 981–990.
(6) De Luca, S.; Della Moglie, R.; De Capua, A.; Morelli, G.
Tetrahedron Lett. 2005, 46, 6637–6640.
(7) Hasegawa, M.; Ono, F.; Kanemasa, S. Tetrahedron Lett. 2008, 49,
5220–5223.
Org. Lett., Vol. 14, No. 7, 2012
1665

Table 1. Efficiency of the N-Alkylation Reaction Performed on
Different Peptide Models
Figure 1. (A) Comparison of 1D NMR spectra, recorded in
DMSO-d6 at 298 K, of AcGlyLys(Bn)ValAla (1b, black) and
AcGlyLysValAla (red) peptides. Spectral regions containing
resonances from backbone and side-chain NH groups as well
as aromatic protons are shown. (B, C) 1H/2H exchange experi-
ments (see SI for details about samples preparation) for AcGlyLys-
(Bn)ValAla (B) and AcGlyLys(MeNaph)ValAla (C) peptides.
on both the Cys and Lys side chain, resulted in being the
main product (data not shown). However, it should not be
considered a real synthetic problem, since several Fmoc-
Cys derivatives protected on their side chain with no acid-
labile substituents are routinely used for the peptide synthe-
sis. For instance, we succeeded in preparing in high yield the
peptide 10a (Table 1) where the Cys protecting group Acm
can be removed, by a standard method,10 after having
performed the alkylation.
To explore the reactivity exhibited, upon alkylation, by
differently protected lysines, we synthesized peptide se-
quences 11 and 12. In our previous work, we investigated
the effects of the newly developed N-alkylation procedure
on amino groups protected with several substituents which
are routinely used in solid-phase peptide synthesis.5 For
instance, Fmoc-Lys(ivDde)-OH and Fmoc-Lys(Alloc)-
OH are widely employed. We have already tested the benzy-
lation of the Lys amino group protected with ivDde;5 thus,
we decided to study the same reaction on N-ε-Alloc-Lys (see
SI). These N-alkylation reactions were slower with respect to
the reactions performed on a nosyl-protected amino group.
Peptides 11 and 12 were alkylated as reported in
Scheme 1. After 12 h for 11a and after 24 h for 12a, the
most abundant products resulted in being monoalkylated
(see Table 1).
group (pKa = 12.5) which was alkylated together with the
Lys side chain, thus providing a dialkylated byproduct
with a yield of 70À80% (data not shown).
This result prompts us to explore the yield of the
monoalkylation reaction in a peptide sequence containing
a tosyl-protected Arg residue (peptide 9a). This protecting
group, in fact, survives in the employed cleavage conditions
(70% TFA in DCM for 30 min)8 and allows us to obtain
benzylation of the Lys side chain with an ∼75% yield. The
isolated final product contains the tosyl group, clearly identi-
fied during NMR analysis, which can be removed in a
subsequent step, by using the known reaction protocols.9
Afterwards, we explored a peptide containing the Lys to
be alkylated and a free Cys residue; in this case, the desired
monoalkylated peptide was not obtained in an appreciable
yield. In fact, due to the high nucleophilicity of the Cys
sulfhydryl group, the dialkylated peptide, with modifications
(8) De Luca, S.; Ulhaq, S.; Dixon, M. J.; Essex, J.; Bradley, M.
Tetrahedron Lett. 2003, 44, 3195–3197.
(9) Kiso, Y.; Satomi, M.; Ukawa, K.; Akita, T. J. Chem. Soc., Chem.
(10) Chan, W. C.; White, P. D. Fmoc solid phase peptide synthesis. A
practical approach; Oxford University Press: USA, 2000.
Commun. 1980, 22, 1063–1064.
1666
Org. Lett., Vol. 14, No. 7, 2012

We employed 1D H and 2D [1H, H] NMR spectro-
scopy (see SI) to further prove that the desidered mono-
alkylated peptide products were effectively obtained.
NMR studies, performed on 11b and 12b, confirmed
that the chemoselective alkylation occurred on the nosyl-
protected Lys, while the ivDde/Alloc-protected lysines
resulted free of a benzyl substituent. It is worth noting that
the ivDde and Alloc groups were kept on the Lys residues
to facilitate NMR analysis. However, the ivDde can be
removed together with the Ns group (by preparing an
appropriate mixture of hydrazine, 2-mercaptoethanol, and
DBU), while the Alloc group can be removed under
usually standard conditions.10
1
1
Scheme 2. Synthetic Strategy of N-Alkylation Reaction Per-
formed on Peptide Containing Oxidized Cysteines
1
In 2D [1H, H] NMR spectra, such as TOCSY and
ROESY, signals from Lys(Bn) and Lys(ivDde)/ Lys-
1
(Alloc) could be easily identified (SI). In 2D [1H, H]
TOCSY spectra, the characteristic NHζ spin system of
Lys(Bn) at 7.64 ppm is clearly identifiable for both 11b and
12b peptides.
on 16 (Table 1). The final peptide was obtained in quite
good yield, thus demonstrating that the mild conditions
employed to alkylate the peptide do not affect the dis-
ulfide bridge. NMR analysis of the peptide confirmed the
presence of a benzylated Lys (see SI), whereas Cys
residues appear unaffected by peptide postsynthetic che-
mical modification.
In summary, we describe an original method for per-
forming postsynthetic modifications via alkylation of a
nosyl-protected Lys side chain, inserted in already pre-
formed peptide sequence. The mild conditions employed
(only molecular sieves used to catalyze the reaction) were
proven to be compatible with most of the natural peptide
functionalities, providing high chemoselectivity and excel-
lent conversion yields. We believe that our versatile and
straightforward approach will find wide application in the
field of peptide-based drug development. In fact, to the
best of our knowledge a protocol that allows generating a
large number of analogs by performing the same reaction,
with different alkylating agents, on a single presynthesized
peptide sequence, has not been reported in literature.
To further examine the chemoselectivity shown by the
Lys Ns-NH upon alkylation reaction with respect to the
position into the peptide sequence of other competing
functional groups, peptides 13a and 14a were synthesized
in high yield (Table 1). Thus, it was demonstrated that the
close proximity of an amino acid, e.g. Trp (13a), or even of
two nucleophilic amino acids, e.g. Trp and His (14a), does
not influence the reactivity of the Lys nosyl protected
ε-NH2 during the benzylation reaction.
Peptide 15a was synthesized to prove that the proposed
methodology allowed the one pot synthesis of polyalky-
lated molecules in good yield (Table 1) and that the
reactivity of the substrate does not rely on its specific
situation within the peptide sequence. For this peptide,
we could not carry out a detailed NMR characterization
due to the appearance in the 2D TOCSY and ROESY
spectra of many duplicated spin systems indicative of the
occurrence of different conformations in solutions and/or
aggregation processes.
The very mild conditions of the proposed procedure
were further proven by performing the N-alkylation of a
peptide containing a disulfide bridge. In fact, in the case of
the Cys oxidation reaction being performed in solid
phase,10 the postsynthetic alkylation cannot be accom-
plished on nosyl-protected Lys residue, whose deprotec-
tion conditions (thiol in the presence of a base) are not
compatible with a disulfide bridge.
For instance, we synthesized peptide 16a (Scheme 2) by
using the Fmoc-Lys(Dde)-OH derivative. After the oxi-
dized peptide was cleaved from the resin under appropriate
reaction conditions, the benzylation reaction was performed
Acknowledgment. We thank Mr. Leopoldo Zona
(National Research Council, Naples) for NMR technical
assistance.
Supporting Information Available. The content in-
cludes the following: (1) experimental section, (2) LC-
MS spectra, (3) NMR chemical shift tables, (4) NMR
spectra. This information is available free of charge via
the Internet at http://pubs.acs.org.
The authors declare no competing financial interest.
Org. Lett., Vol. 14, No. 7, 2012
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