Selective, on-resin N-methylation of peptide N-trifluoroacetamides

Article abstract of DOI:10.1021/ol402341u

Mild and efficient methods for site-specific methylation of peptide backbone amides are important tools for chemists seeking to modulate the pharmacokinetic properties of peptide drugs. The Mitsunobu reaction was used to selectively methylate N-trifluoroa

Full text of DOI:10.1021/ol402341u

ORGANIC
LETTERS
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Vol. XX, No. XX
000–000
Selective, On-Resin N‑Methylation
of Peptide N‑Trifluoroacetamides
Rushia A. Turner, Niels E. Hauksson, Jordan H. Gipe, and R. Scott Lokey*
Department of Chemistry and Biochemistry, University of California at Santa Cruz,
1156 High Street, Santa Cruz, California 95064, United States
slokey@ucsc.edu
Received August 14, 2013
ABSTRACT
Mild and efficient methods for site-specific methylation of peptide backbone amides are important tools for chemists seeking to modulate the
pharmacokinetic properties of peptide drugs. The Mitsunobu reaction was used to selectively methylate N-trifluoroacetamide (Tfa) protected
peptides on-resin. The Tfa group was removed quickly and completely by reduction with excess NaBH4, and it was shown to be orthogonal to
many of the protecting groups used in solid-phase peptide synthesis.
Backbone amide methylation is a hallmark feature
of cyclic peptide natural products and influences both
the physical properties and the conformational states
of these molecules. For example, the orally bioavailable
cyclic peptide immunosuppresant drug cyclosporin A
is N-methylated at seven of its 11 backbone amides, at
least three of which are essential for its bioactivity and
bioavailability.1 Several researchers have shown that
N-methylation of backbone amides can enhance the po-
tency, selectivity, and bioavailability of cyclic peptides
relativeto their unmethylated precursors.2,3 Consequently,
the growing demand for selectively N-methylated peptides
has spawned an interest in the development of mild and
efficient methods for site-specific methylation of peptide
backbone amides on the solid phase.
Even though the demand for N-methylated amino acids
has increased significantly in recent years, only a few of
these building blocks are commercially available. Methods
exist for the solution-phase conversion of unprotected,4
Boc-protected,5 or Fmoc-protected6 R amino acids into
their N-methyl cognates; however, coupling N-protected
N-methyl amino acids onto resin-bound peptides can be
especially challenging when there are multiple, contiguous
N-methylated residues in the sequence.7 As a result, meth-
ods that enable on-resin backbone amide N-methylation
are highly desirable, even though they add additional steps
to a synthesis.
Virtually all of the methods reported for on-resin, site-
specific backbone amide methylation have utilized the
N-arylsulfonamide moiety togenerate a nucleophilic anion
(1) Wenger, R. M. Angew. Chem., Int. Ed. Engl. 1985, 24, 77–85.
(4) White, K. N.; Konopelski, J. P. Org. Lett. 2005, 7, 4111–4112.
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(2) (a) Dechantsreiter, M. A.; Planker, E.; Matha, B.; Lohof, E.;
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1999, 42, 3033–3040. (b) Chatterjee, J.; Ovadia, O.; Azhn, G.; Marinelli,
L.; Hoffman, A.; Gilon, C.; Kessler, H. J. Med. Chem. 2007, 50, 5878–
5881. (c) Weltrowska, G.; Berezowska, I.; Lemieux, C.; Chung, N. N.;
Wilkes, B. C.; Schiller, P. W. Chem. Biol. Drug. Des. 2010, 75, 182–188.
(d) Doedens, L.; Opperer, F.; Cai, M.; Beck, J. G.; Dedek, M.; Palmer,
E.; Hruby, V. J.; Kessler, H. J. Am. Chem. Soc. 2010, 132, 8115–8128.
(3) (a) Biron, E.;Chatterjee, J.;Ovadia, O.;Langenegger, D.; Brueggen,
J.; Hoyer, D.; Schmid, H. A.; Jelinek, R.; Gilon, C.; Hoffman, A.;
Kessler, H. Angew. Chem., Int. Ed. 2008, 47, 2595–2599. (b) White, T. R.;
Renzelman, C. M.; Rand, A. C.; Rezai, T.; McEwen, C. M.; Gelev, V.;
Turner, R. A.; Linington, R. G.; Leung, S. F.; Kalgutkar, A. S.; Bauman,
J. N.; Zhang, Y.; Liras, S.; Price, D. A.; Mathiowetz, A. M.; Jacobson,
M. P.; Lokey, R. S. Nature Chem. Biol. 2011, 7, 810–817. (c) Ovadia, O.;
Greenberg, S.; Chatterjee, J.; Laufer, B.; Opperer, F.; Kessler, H.; Gilon,
C.; Hoffman, A. Mol. Pharm. 2011, 8, 479–487.
(6) (a) Freidinger, R. M.; Hinkle, J. S.; Perlow, D. S. J. Org. Chem.
1983, 48 (1), 77–88. (b) Reddy, G. V.; Rao, G. V.; Iyengar, D. S.
Tetrahedron Lett. 1998, 39, 1985–1986. (c) Aurelio, L.; Brownlee,
R. T. C.; Hughes, A. B.; Sleebs, B. E. Aust. J. Chem. 2000, 53, 425–
433. (d) Aurelio, L.; Brownlee, R. T. C.; Hughes, A. B. Org. Lett. 2002, 4,
3767–3769. (e) Aurelio, L.; Box, J. S.; Browlee, R. T. C.; Hughes, A. B.;
Sleebs, M. J. Org. Chem. 2003, 68, 2652–2667. (f) Zhang, S.; Govender,
T.; Norstrom, T.; Arvidsson, P. I. J. Org. Chem. 2005, 70, 6918–6920.
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M. Int. J. of Pept. Protein Res. 1992, 40, 282–293. (b) Angell, Y. M.;
Garcia-Echeverria, C.; Rich, D. H. Tetrahedron Lett. 1994, 35, 5981–
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2005, 70, 3892–3897.
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10.1021/ol402341u
XXXX American Chemical Society

that can be alkylated using methyl p-nitrobenzenesulfonate,8
dimethyl sulfate,9 alkyl halides,10 or Mitsunobu condi-
tions.9b,10a,10b,11 Following on the initial reports of Miller
and Scanlan,8 the Kessler group has published widely on
the use of the o-nitrobenzenesulfonamide (o-NBS) protec-
tion group to facilitate selective methylation of peptide
backbone amides using the Mitsunobu reaction (see ref 12
for a review). We have found that selective methylation
of the o-NBS group using Mitsunobu conditions is in-
deed efficient; however, removal of the o-NBS from
N-methylated residues is often unpredictable and se-
quence dependent. Herein, we report the uprecedented
use of N-trifluoroacetamide (Tfa) as an N-terminal pro-
tecting group to facilitate rapid and efficient on-resin
N-methylation via the Mitsunobu reaction. Importantly,
the Tfa group is readily removed using conditions that are
orthogonal to most standard SPPS protecting groups,
making this a significant advance in the area of site-
selective peptide N-methylation.
treatment with triethylamine (TEA, 10 equiv) and ethyl
trifluoroacetate (ETFA, 12 equiv; Scheme 1c). At room
temperature, on-resin trifluoroacetylation using TEA was
slower than desired and we found that the use of a micro-
wave reactor hastened the process significantly (20W,
75 °C, 10 min). Substituting DBU for TEA (12 equiv
DBU, 10 equiv ETFA, 60 min) also enhanced the rate of
the reaction at room temperature.
Scheme 1. Synthesis of N-Tfa Amino Acids and Peptides:
(a) Synthesis of Tfa-L-leucine;16 (b) Coupling of Tfa-L-leucine
to a Resin-Bound Peptide; (c) Installation of Tfa Group via
Protection Group Exchange
We investigated other amine protection groups that are
reactive toward Mitsunobu methylation while still afford-
ing mild and reliable cleavage conditions. The N-trifluor-
oacetamide is known to form a stabilized anion,13 and its
use has been demonstrated in a solution-phase, intramo-
lecular Mitsunobu reaction.14 It is also reputed to be one of
the most labile amides used as a protection group.15 These
reports led us to test the reactivity and selectivity of the Tfa
group in the site-specific N-methylation of resin-bound
peptides, which has not been reported in the literature.
The dipeptide Tfa-L-Leu-L-Leu was synthesized on the
2-chlorotrityl resin using two different methods. In the first
method, Tfa-protected L-leucine was synthesized using the
procedure reported by Curphey (Scheme 1a).16 The pro-
duct was isolated via extraction and subsequently coupled
onto a resin-bound leucine using base-free conditions in
order to prevent epimerization (Scheme 1b). The second
method entailed on-resin installation of the Tfa group
via protection group exchange. A Leu-Leu dipeptide was
synthesized on the 2-chlorotrityl chloride resin using
standard Fmoc chemistry. The Tfa group was installed
by first removing the N-terminal Fmoc group, followed by
The Tfa-protected dipeptide was subject to the same
Mitsunobu reaction conditions reported for the selective
N-methylation o-NBS protected peptides.9b,11b,12 Triphe-
nylphosphine (5 equiv) and methanol (10 equiv) were
dissolved in minimal anhydrous THF and added to a
reaction vial containing the resin-bound, Tfa-protected
dipeptide. Diisopropyl azodicarboxylate (DIAD, 5 equiv)
was added slowly with vigorous agitation, and the reaction
vessel was capped and shaken for an additional 15 min.
The N-methylated peptide was obtained in good to ex-
cellent yields of 80À99% (compound 2; see the Supporting
Information, Figure S3), with the best results obtained
when precautions were taken to exclude water from the
reactants, solvent, and the resin. When necessary, a second
treatment pushed the reaction to >99% methylation
without undesirable side reactions.
(8) (a) Miller, S. C.; Scanlan, T. S. J. Am. Chem. Soc. 1997, 119, 2301–
2302. (b) Miller, S. C.; Scanlan, T. S. J. Am. Chem. Soc. 1998, 120, 2690–
2691.
(9) (a) Biron, E.; Kessler, H. J. Org. Chem. 2005, 70, 5183–5189. (b)
Biron, E.; Chatterjee, J.; Kessler, H. J. Pept. Sci. 2006, 12, 213–219.
(10) (a) Dankwardt, S. M.; Smith, D. B.; Porco, J. A.; Nguyen, C. H.
Synlett 1997, 854–856. (b) Reichwein, J. F.; Liskamp, R. M. J. Tetra-
hedron Lett. 1998, 39, 1243–1246. (c) Andersen, T. F.; Stromgaard, K.
Tetrahedron Lett. 2004, 45, 7929–7933.
(11) (a) Mitsunobu, O. Synthesis 1981, 1–28. (b) Demmer, O.;
Dijkgraaf, I.; Schottelius, M.; Wester, H. J.; Kessler, H. Org. Lett.
2008, 10, 2015–2018.
(12) Chatterjee, J.; Gilon, C.; Hoffman, A.; Kessler, H. Acc. Chem.
Res. 2008, 41, 1331–1342.
We examined the conditions previously reported to
cleave the Tfa group on the solid phase. Contrary to
literature reports, we found that the Tfa group was quite
robust. Of the many cleavage conditions that have been
reported (K2CO3 in methanol,17 aqueous piperidine,18
hydrazine,19 reduction13,14,20), the only method that removed
(17) (a) Bergeron, R. J.; McManis, J. S. J. Org. Chem. 1988, 53, 3108–
3111. (b) Boger, D. L.; Yohannes, D. J. Org. Chem. 1989, 54, 2498–2502.
(c) Pak, J. K.; Guggisberg, A.; Hesse, M. Tetrahedron 1998, 54, 8035–
8046.
(18) (a) Goldberger, R. F.; Anfinsen, C. B. Biochemistry 1962, 1, 401–
405. (b) West, M. H. P.; Bonner, W. M. Biochemistry 1980, 19, 3238–
3245. (c) Leamon, C. P.; DePrince, R. B.; Hendren, R. W. J. Drug
Targeting 1999, 7, 157–169.
(19) Vagner, J.; Handl, H. L.; Monguchi, Y.; Jana, U.; Begay, L.; Mash,
E. A.; Hruby, V. J.; Gillies, R. J. Bioconjug. Chem. 2006, 17, 1545–1550.
(20) Weygand, F.; Frauendorfer, E. Chem. Ber. 1970, 103, 2437–
2449.
(13) Kudzin, Z. H.; Lyzwa, P.; Luczak, J.; Andrijewski, G. Synthesis
1997, 44–46.
(14) Callaghan, O.; Lampard, C.; Kennedy, A. R.; Murphy, J. A.
Tetrahedron Lett. 1999, 40, 161–164.
(15) Greene, T. W.; Wuts, P. G. M. Protective Groups in Organic
Synthesis, 3rd ed.; John Wiley & Sons, Inc.: New York, 1999.
(16) (a) Curphey, T. J. J. Org. Chem. 1979, 44, 2805–2807. (b)
Deblander, J.; Van Aeken, S.; Jacobs, J.; DeKimpe, N.; Tehrani,
K. A. Eur. J. Org. Chem. 2009, 28, 4882–4892.
B
Org. Lett., Vol. XX, No. XX, XXXX

the Tfa group completely within 30 min was reduction with
sodium borohydride in THF/ethanol (1:1 v/v; Table 1).
These results indicate that the Tfa group is sufficiently
stable for use in SPPS and that rapid and reliable removal
of the Tfa group can be accomplished using mild condi-
tions that are compatible with SPPS.
Scheme 2. Synthesis of Dipeptides Used To Evaluate the
Regioselectivity of the Tfa-Mitsunobu N-Methylation
Table 1. Reaction Conditions Investigated To Remove the Tfa
Group from a Resin-Bound Tfa-Leu-Leu Dipeptide
% Tfa
time group
reaction conditions
(h) removeda
15% N2H4, 15% MeOH, 70% DMF (v/v)
15% N2H4, 15% MeOH, 70% THF (v/v)
5% H2O, 25% MeOH, 70% THF (v/v), excess K2CO3
10% H2O, 10% piperidine, 80% DMF (v/v)
10% piperidine, 90% DMF (v/v)
1
0
0
1
noteworthy because the use of NaBH4 for the cleavage of
Dde has not been reported in the literature.
1
0
1
0
The discovery of a rapid and mild Dde cleavage protocol
is fortuitous because Dde is only quasi-orthogonal to
Fmoc. Dde is stable to the bases used to remove Fmoc,
but Fmoc is quickly cleaved by hydrazine. The discovery
that Dde can be removed quickly with NaBH4 led us
to verify the stability of Fmoc to treatment with NaBH4.
We were pleased to find that Fmoc was completely un-
reactive to NaBH4 (31, see the Supporting Information,
Figure S42). These findings reveal that Fmoc and Dde are
fully orthogonal when NaBH4 is used to deprotect Dde.
To date, there has been only one other report of Dde
cleavage conditions that enable orthogonality with Fmoc
1
0
5% H2O, 95% THF, 1 equiv of LiOH
5 equiv of DBU, 10 equiv of BME in NMP
10 equiv of NaOEt in THF
1
0
1
0
1
100b
100
10 equiv of NaBH4 in EtOH/THF (1:1 v/v)
0.5
a Percent removal of Tfa group was determined by integrating the
peaks in the HPLC trace (λ = 220 nm). b Deprotection with NaOEt
resulted in ∼10% epimerization of the N-terminal residue.
In order to assess the scope of the Tfa-Mitsunobu
method for broader application in SPPS, we examined
both the regioselectivity of the methylation reaction and
the stability of various side-chain protection groups to-
ward treatment with NaBH4. Fourteen dipeptides were
synthesized in parallel by adding different Fmoc-protected
amino acids to the 2-chlorotrityl resin preloaded with
leucine (Scheme 2). The amino acids Arg(Mtr), Arg(Pbf),
Cys(Trt), Glu(O-t-Bu), Glu(OBnz), Gln(Trt), His(Trt),
Lys(Boc), Lys(Dde), Lys(Mtt), Orn(Alloc), Ser(OBnz),
Trp(Boc), and Tyr(O-t-Bu) were deprotected, and each
dipeptide was trifluoroacetylated using ETFA and
DBU. The Tfa-dipeptides were subjected to two sequential
Mitsunobu reactions. An aliquot of each dipeptide was set
aside for LCMS and NMR analysis, and the remainder of
each resin was treated with excess NaBH4 for 30 min. In all
cases, the LCMS and NMR spectra showed complete
methylation that was regioselective for the backbone
amide. Moreover, there was no evidence of side chain
modifications nor loss of side chain protection groups
during the methylation step (see the Supporting Informa-
tion, Figures S3ÀS27).
(NH2OH HCl, imidazole);21 however, this method re-
3
quires a 3 h reaction time, and it is unknown whether these
conditions are combatible with the various protection
groups used in SPPS.
We also evaluated the efficacy of the Tfa-Mitsunobu
N-methylation method relative to that of the o-NBS
method by challenging both with a tri-N-methylated tetra-
peptide that contained sterically demanding residues such
as the bulky, β-branched Thr(O-t-Bu). H-N-MeTyr(O-t-
Bu)-N-MeTrp(Boc)-N-MeThr(OtBu)-Leu-OH was syn-
thesized using both methods (Scheme 3), and upon com-
pletion of both syntheses the crude products were analyzed
by LCMS (Figure 1; see the Supporting Information,
Figures S43 and S44).
The HPLC trace for the Tfa-Mitsunobu synthesis
showed the desired tri-N-methylated tetrapeptide as the
major product (Figure 1a, tR 8.44). The two most signifi-
cant byproducts represent truncations (-Trp and -Tyr,
respectively), and the peak at 7.49 min represents the full
tetrapeptide with the Tyr tert-butyl ether removed. The
LCMSdata for the intermediate peptides showedthateach
Tfa deprotection was complete, indicating that these trun-
cations resulted from incomplete amino acid couplings.
The o-NBS-Mitsunobusynthesis, however, providedthe
desired tetrapeptide as only one among four major com-
ponents (Figure 1b, tR 8.42), with the major byproducts
arising from incomplete removal of the o-NBS group at
different steps (see the Supporting Information for a more
Virtually all of the side chain protection groups exam-
ined in this study were found to be stable to treatment with
excess NaBH4 (see the Supporting Information, Figures
S28ÀS41). EventhoughNaBH4 isknown toreducemethyl
and allyl esters to the corresponding alcohols, we found
that tert-butyl and benzyl esters were stable to these con-
ditions. Likewise, the trityl ester linking the peptide to the
solid phase was stable to these conditions. Of the 14 amino
acids investigated, the only side chain protection group
affected by treatment with NaBH4 was the Lys-Dde
protection group, which was removed completely. This is
(21) Diaz-Mochon, J. J.; Bialy, L.; Bradley, M. Org. Lett. 2004, 6,
1127–1129.
Org. Lett., Vol. XX, No. XX, XXXX
C

Scheme 3. Synthesis of Tri-N-methylated Tetrapeptides Using
Tfa- and o-NBS-Mitsunobu Methods
Figure 1. HPLC traces of tri-N-methylated tetrapeptids synthe-
sized via the (a) Tfa-Mitsunobu method and (b) the o-NBS-
Mitsunobu method.
strategy. The efficiency and consistency with which the
Tfa group is both N-methylated and removed thus repre-
sents a potential improvement over the conventional
methodology.
We have demonstrated that the Tfa protection group
enables site-specific N-methylations under Mitsunobu con-
ditions, with no side-chain reactivity or protection group
losses. The Tfa group was shown to be stable to a variety of
conditions, but was removed quickly and reliably using
excess NaBH4 in THF/EtOH. The Tfa cleavage conditions
were determined to be orthogonal to virtually all of the
protection groups commonly used in SPPS, indicating that
the Tfa group may have more utility as a general protection
group in SPPS than has previously been reported.
complete analysis of these data). We used the standard
conditionsreportedfor efficientremoval ofo-NBS (5 equiv
of DBU, 10 equiv of BME, 2 Â 10 min in NMP); however,
these results suggest that repeated rounds of deprotection
and LCMS monitoring may be warranted for some se-
quences to ensure complete deprotection and avoid un-
wanted truncations. In contrast, the Tfa method was
consistently efficient with respect to both the N-methyla-
tion chemistry and the deprotection steps.
The o-NBS-Mitsunobu method is regarded as the stan-
dard protocol for on-resin, site-specific backbone amide
methylations. One advantage that has been attributed to
o-NBS is the removal of incompletely N-methylated by-
products, presumablydue tothe inability toremoveo-NBS
from non-N-methylated residues.2d,8,9b In principle, this
enables purification of the resulting byproducts as pro-
tected truncations. In this model system, we found that
N-methylation was equally efficient using both methods,
however, the removal of the protectiongroup from N-methy-
lated residues was the problematic step for the o-NBS
Acknowledgment. Thisworkwas supportedbythe NIH
(award 5R01GM084530). We are grateful to James Gibson
at Thermo Fisher Scientific for his invaluable help with the
laboratory’s LCQ Classic, on which all LCMS data were
acquired.
Supporting Information Available. Experimental pro-
1
cedures and H NMR and LCMS (ESI) spectra. This
material is available free of charge via the Internet at
http://pubs.acs.org.
The authors declare no competing financial interest.
D
Org. Lett., Vol. XX, No. XX, XXXX