Forced complexation of nitrogen leading to a weakening of amide bonds: Application to a new linker for solid-phase chemistry

Article abstract of DOI:10.1002/ejoc.200800642

Cu²⁺ complexation of a bispicolylamide entity leads to a weakening of the N-C-amide bond, which subsequently can be cleaved under very mild conditions by methanolysis. On the basis of this principle we developed a versatile and robust linker for solid-phase chemistry. The stability of the linker was demonstrated under acidic and basic conditions, and it was evaluated for its utility in peptide synthesis as well as for reductive amination, Pd-mediated C-C-coupling, and metathesis reactions. Wiley-VCH Verlag GmbH & Co. KGaA, 2008.

Full text of DOI:10.1002/ejoc.200800642

SHORT COMMUNICATION
DOI: 10.1002/ejoc.200800642
Forced Complexation of Nitrogen Leading to a Weakening of Amide Bonds:
Application to a New Linker for Solid-Phase Chemistry
Manuel C. Bröhmer[a] and Willi Bannwarth*[a]
Keywords: Solid-phase synthesis / Linkers / Amides / Cleavage reactions / Copper
Cu2+ complexation of a bispicolylamide entity leads to a
weakening of the N–C-amide bond, which subsequently can
be cleaved under very mild conditions by methanolysis. On
the basis of this principle we developed a versatile and ro-
bust linker for solid-phase chemistry. The stability of the
linker was demonstrated under acidic and basic conditions,
and it was evaluated for its utility in peptide synthesis as well
as for reductive amination, Pd-mediated C–C-coupling, and
metathesis reactions.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2008)
One of the most stable carbon–heteroatom bonds of car-
boxylic acid derivatives is the amide bond. Its stability origi-
nates from the high resonance energy as well as from the
poor nature of the leaving “NHR–” group.[2,3] Enzymes like
serine or cysteine proteases are able to perform such cleav-
ages under mild conditions by orienting the substrates into
their active sites and by enhancing the nucleophilicity of the
OH and SH functionalities by an adjacent Asp and His
residue forming so-called catalytic triades.
The nonenzymatic hydrolysis of amide bonds under neu-
tral conditions is a field of intensive research. The focus is
almost exclusively on the application of oxophilic metal
ions or their complexes coordinating to the carbonyl group
to allow attack by a suitable nucleophile. Nearly all rare-
earth and transition-state metals have been investigated so
far.[4] In nature, metalloproteases use the same mechanism
for hydrolytic cleavage of peptide bonds.
Introduction
In the realm of synthetic chemistry, solid-phase organic
synthesis has gained more and more interest over the last
decades. It allows the application of excess amounts of rea-
gents, which in turn leads to higher yields in comparison to
the same reaction performed in solution and with equi-
molar amounts of reacting components. The excess
amounts of reagents may be removed by simple filtration,
thus avoiding time-consuming purifications.
A major disadvantage is that two additional steps are
involved, namely, the attachment of the starting material
and the release of the product. Due to the limited range of
modifications available on polymer resins, which also limits
the range of functionalities that are directly attachable to
the solid support, there is a need for suitable linker entities
connecting the support with either the starting material, in-
termediates, or the aspired product.[1]
An alternative but only rarely discussed mechanism of
metal-assisted hydrolytic cleavage of amide bonds was pro-
posed for the peptide sequences Xaa–Ser–His and Xaa–
Thr–His. Copper(II) coordinates not to the oxygen atoms
of the carbonyl groups but rather to two nitrogen atoms of
the amide bonds and one of the imidazole nitrogen atoms
leading to a weakening of the amide N–C-bond. An intra-
molecular attack of the hydroxy group of the serine OH
leads first to an NǞO acyl rearrangement, which is fol-
lowed by hydrolysis of the ester bond (Scheme 1).[5,6] The
cleavage according to Scheme 1 is certainly facilitated by
the entropically favorable attack of the serine side chain hy-
droxy functionality.
When designing a linker molecule a number of issues
have to be considered. The envisaged chemical steps should
not lead to its modification nor should it result in the cleav-
age of intermediates from it. Yet, it should be possible to
cleave the final product with high efficiency when the syn-
thesis is complete. This final cleavage step should proceed
in high yield under mild conditions. Such conditions can be
achieved by chemical modification of the linker prior to
cleavage, as is the case for linkers dubbed as “safety-catch
linkers” or by orthogonal release procedures. A further
requirement is the straightforward synthetic accessibility of
an envisaged linker.
A similar reaction but without involvement of an intra-
molecular attack is the direct Cu2+-assisted methanolysis of
bispicolylamides (Scheme 2).[7–10] Alsfasser used them as a
model system for metal-binding peptides and proteins,
where the two pyridine rings mimic the imidazole rings of
the histidine side chains.[8–10] The participation of the amide
[a] Institut für Organische Chemie und Biochemie
Albert-Ludwigs-Universität Freiburg
Albertstraße 21, 79104 Freiburg
Fax: +49-761-203-8705
E-mail: willi.bannwarth@organik.chemie.uni-freiburg.de
Supporting information for this article is available on the
WWW under http://www.eurjoc.org or from the author.
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A New Linker for Solid-Phase Chemistry
Scheme 3. Synthesis of Boc-protected bispicolylamide 12. Reagents
and conditions: (a) I2 (1.0 equiv.), tBuI (0.4 equiv.), TFA
(3.0 equiv.), DMSO, 120 °C, 3 h, 69%; (b) 2-picolylamine
(1.0 equiv.), NaBH(OAc)3 (1.4 equiv.), 1,2-DCE, 1 h; (c) Boc2O
(1.2 equiv.), NEt3 (1.4 equiv.), CH2Cl2, 0 °C Ǟ r.t., 2 h, 69% over
2 steps; (d) NaOH (1.1 equiv.), MeOH/H2O, reflux, 2 h, Ͼ98%.
The synthesis of 12 started with the oxidation of com-
mercially available methyl-6-methylnicotinate (8) to the cor-
responding aldehyde 9 according to a literature pro-
cedure.[11] Reductive amination with 2-picolylamine and so-
dium triacetoxyborohydride in 1,2-DCE[12] gave secondary
amine 10, which was directly treated with Boc2O to yield
the N-Boc-protected methyl ester 11 in 69% yield over two
steps. Saponification with sodium hydroxide in methanol/
water resulted in carboxylic acid 12 in quantitative yield.
Boc-protected linker 12 was coupled to the amino-func-
tionalized solid support by applying a 1.5 molar excess and
by using TBTU as coupling reagent.[13] A negative Kaiser
test[14] indicated completion of the coupling after 4 h. De-
protection of the Boc group from 13 yielded 14 (Scheme 4)
ready for the attachment of substrates through amide
bonds.
Scheme 1. Proposed mechanism for Cu2+-assisted hydrolysis of a
Xaa–Ser–His sequence.
nitrogen atom in the complexation was proven by X-ray
analysis. Due to the spatial arrangement of the ligand the
carbonyl oxygen atom cannot coordinate to the metal,
whereas the amide nitrogen atom is in a preferred position
for complexation resulting in a weakening of the amide
bond.
Scheme 2. Cu2+-assisted hydrolysis of bispicolylamides; X = Cl,
OTf, ClO4.
The synthetic potential of this mild amide bond cleavage
due to involvement of the amide nitrogen atom in complex-
ation has, to the best of our knowledge, not been utilized
yet.
We reasoned that such a bispicolylamine (bpa) ligand
bound to a solid support would comply with most require-
ments for a linker to be used in a versatile way in solid-
phase chemistry. It would allow attachment of substrates
through a stable amide bond with release possible after
complexation under mild conditions leading directly to an
ester. Furthermore, the linker is chemically inert and would
survive a plethora of reaction conditions without suffering
modifications.
Scheme 4. Coupling of 12 to the solid support and cleavage of the
Boc group. Reagents and conditions: (a) HypoGel-400-NH2, DMF,
10 min; then 12 (1.5 equiv.), TBTU (1.5 equiv.), DIPEA
(6.0 equiv.), DMF, r.t., 12 h; (b) iPr3SiH (3.0 equiv.), CH2Cl2/TFA
(1:1), 0 °C Ǟ r.t., 4 h.
With the functionalized support in hand, we coupled
Boc-Ala-OH to the linker by using a standard amide coup-
ling protocol. A negative chloranil test[15] indicated total
conversion after 12 h. In order to probe the envisaged cleav-
age step, we treated the solid phase with equimolar
amounts of Cu(OTf)2 in methanol, which led directly to a
Results and Discussion
In order to test the above possibilities we synthesized 3- green color of the support indicating complex formation.
carboxy-bpa ligand 12 according to Scheme 3. After coup- From the methanol solution the expected Boc-Ala methyl
ling to the solid support and cleavage of the Boc group it ester 17 was isolated in a yield of 50% over four steps start-
should allow for the attachment of substrates through an ing from 12 and based on the initial loading of the support
amide linkage.
(Scheme 5).
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M. C. Bröhmer, W. Bannwarth
SHORT COMMUNICATION
In order to extend the scope of application of the new
linker for classic organic synthesis we next demonstrated its
utility in a reductive amination, a Suzuki–Miyaura coup-
ling, and a ring-closing metathesis reaction (Scheme 7; A,
B, and C, respectively).
Scheme 5. Coupling of Boc-Ala-OH and Cu2+-assisted cleavage to
methyl ester 17. Reagents and conditions: (a) Boc-Ala-OH
(3.0 equiv.), TBTU (3.0 equiv.), DIPEA (12 equiv.), DMF, r.t., 12 h;
(b) Cu(OTf)2 (1.0 equiv.), MeOH, r.t., 12 h, 50% over 4 steps.
Despite its easy and effective synthesis, the recycling of
used linker 16 would be desirable. This could be achieved
by decomplexation of the linker by using a better complex-
ing agent. Application of EDTA failed, but surprisingly the
use of 1.1 equiv. of free bpa ligand led to decomplexation of
the linker, indicated by loss of the green color. The solution,
however, changed its color to green. Alternatively, treat-
ment with a methanolic solution of potassium cyanide
(2 equiv.) was used for the regeneration.
The regenerated linker was then treated with Boc-Phe-
OH. Cleavage under the same conditions as before gave
pure Boc-Phe methyl ester in a yield of 41%, without any
trace of Boc-Ala methyl ester, indicating that the initial
cleavage yielding Boc-Ala-OMe had been quantitative.
Next, the stability of amide 15 was demonstrated under
acidic (50% TFA in CH2Cl2) and basic (0.2  LiOH in
H2O) conditions as well as in the presence of fluoride ions
(0.1  Bu4NF in THF). After these positive results we
tested the suitability of the linker for peptide synthesis.
For this reason, we synthesized decapeptide 18 by using
the Fmoc/tBu strategy on a 70-µmol scale (linker-modified
Scheme 7. Reactions on the bpa linker. Reagents and conditions:
(a) 4-carboxybenzaldehyde (3.0 equiv.), TBTU (3.0 equiv.), DIPEA
(12 equiv.), DMF, r.t., 12 h; (b) piperidine (10 equiv.), NaBH-
(OAc)3 (10 equiv.), 1,2-DCE, r.t., 12 h; (c) Cu(OTf)2 (1.0 equiv.),
MeOH, r.t., 12 h, 49% over 5 steps; (d) 4-iodobenzoic acid
(3.0 equiv.), TBTU (3.0 equiv.), DIPEA (12 equiv.), DMF, r.t., 12 h;
(e) PhB(OH)2 (5.0 equiv.), K3PO4 (10 equiv.), Pd(PPh3)4 (10 mol-
%), DMF, 90 °C, 12 h; (f) Cu(OTf)2 (1.0 equiv.), MeOH, r.t., 12 h,
31% over 5 steps; (g) 2-allyl-4-pentenoic acid (3.0 equiv.), TBTU
(3.0 equiv.), DIPEA (12 equiv.), DMF, r.t., 12 h; (h) Grubbs 2nd
generation catalyst (5 mol-%), CH2Cl2, r.t., 24 h; (i) Cu(OTf)2
(1.0 equiv.), MeOH, r.t., 12 h, 34% over 5 steps.
In the reductive amination, coupling of 4-carboxybenzal-
support; Scheme 6). The assembly on the support was fol- dehyde to linker 14 was followed by reductive amination
lowed by cleavage from the linker with Cu2+/MeOH and with piperidine and sodium triacetoxyborohydride in 1,2-
deprotection with TFA, which yielded the crude peptide in DCE. Methanolysis after complexation with Cu2+ gave de-
Ͼ90% purity (210 nm) as judged from HPLC and LC–MS sired methyl ester 21 in 49% yield over five steps starting
(see Supporting Information).
from the amino-modified support.
Scheme 6. Cleavage of decapeptide 19. Reagents and conditions: (a) Cu(OTf)2 (1.0 equiv.), MeOH; (b) TFA/CH2Cl2/iPr3SiH, 95:3:2.
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A New Linker for Solid-Phase Chemistry
(3 mL) was added. The mixture was agitated for 12 h at room tem-
perature. The solid support was filtered off, and the methanolic
solution was evaporated. The residue was taken up in CH2Cl2
(5 mL) and extracted with aqueous EDTA solution (0.5 ,
2ϫ10 mL). The organic layer was dried with Na2SO4 and evapo-
rated to give pure methyl ester.
Next, a Pd-mediated C–C-coupling reaction was carried
out. Here we were not sure whether a possible complexation
of Pd would take place thereby hampering the catalytic ac-
tion of the Pd. After coupling of 4-iodobenzoic acid to the
linker a Suzuki–Miyaura coupling was carried out with
phenyl boronic acid and Pd(PPh3)4 as catalyst. We were
very pleased that Cu2+-assisted cleavage gave pure corre-
sponding methyl ester 23 in 31% over five steps. The Cu
content as estimated by AAS was Ͻ8 ppm.
Supporting Information (see footnote on the first page of this arti-
cle): Experimental details for the preparation of 11–14; solid-phase
synthesis of decapeptide 18; selected HPLC traces.
Finally, a ring-closing metathesis (RCM) reaction was
performed by attaching 2-allyl-4-pentenoic acid[16] to the
linker. The RCM reaction was carried out by using 5 mol-
% of Grubbs II catalyst in CH2Cl2. Methanolysis after Cu2+
complexation gave desired methyl ester 25 in 34% yield over
five steps.
Acknowledgments
We would like to thank Dr. M. Keller, Mrs. M. Schonhardt, and
Mr. F. Reinbold for recording the NMR spectra, Mr. C. Warth and
Dr. J. Wörth for recording the mass spectra, and Mr. E. Hickl for
performing the elemental analyses.
The spectroscopic data of compounds 21, 23, and 25
were identical to those reported in the literature.[17–19]
[1] W. Bannwarth, B. Hinzen in Combinatorial Chemistry, 2nd ed.
(Eds: R. Mannhold, H. Kubinyi, G. Folkers), Wiley-VCH,
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[3] G. M. Polzin, J. N. Burstyn, Met. Ions Biol. Syst. 2001, 38, 103.
[4] K. B. Grant, M. Kassai, Curr. Org. Chem. 2006, 10, 1035–1049.
[5] G. Allen, R. O. Campbell, Int. J. Pept. Res. 1996, 48, 265.
[6] G. Allen, Met. Ions Biol. Syst. 2001, 38, 197.
[7] R. P. Houghton, R. R. Puttner, Chem. Commun. 1970, 1270–
1271.
[8] N. Niklas, F. Hampel, G. Liehr, A. Zahl, R. Alsfasser, Chem.
Eur. J. 2001, 7, 5135–5142.
[9] N. Niklas, F. W. Heinemann, F. Hampel, T. Clark, R. Alsfasser,
Inorg. Chem. 2004, 43, 4663–4673.
[10] N. Niklas, R. Alsfasser, Dalton Trans. 2006, 26, 3188–3199.
[11] P. Bigey, S. Frau, C. Loup, C. Claparols, J. Bernadou, B. Meun-
ier, Bull. Soc. Chim. Fr. 1996, 133, 679–689.
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Conclusions
We have developed a chemically robust linker for solid-
phase synthesis based on a rather uncommon involvement
of an amide nitrogen atom in a Cu2+ complexation and
from which amide-bound compounds can be cleaved under
very mild conditions. The linker entity 12 is accessible by a
straightforward synthesis over four steps starting from com-
mercially available methyl-6-methylnicotinate (8). We have
demonstrated the recyclability of the linker and its versatil-
ity by applying it to peptide synthesis, reductive amination,
Suzuki–Miyaura coupling, as well as to ring-closing me-
tathesis. We think with these demonstrated examples the
potential of the linker is by no means exhausted and further
applications are currently in progress.
Experimental Section
General Procedure for Coupling of Carboxylic Acids to the Linker:
The solid phase carrying linker 14 (0.123 mmol) was suspended in
DMF (2 mL). After 10 min, a solution of the acid (0.369 mmol,
3.0 equiv.), TBTU (0.369 mmol, 3.0 equiv.), and DIPEA
(1.48 mmol, 12 equiv.) in DMF (3 mL) was added. The mixture was
agitated for 12 h at room temperature. The solid support was fil-
tered and washed alternately with DMF/iPrOH (5ϫ3 mL), and
subsequently with CH2Cl2 (3 mL) and MeOH (3 mL).
General Procedure for Cu2+-Assisted Cleavage of the Linker: The
solid support (0.123 mmol) was suspended in MeOH (2 mL). After
10 min, a solution of Cu(OTf)2 (0.123 mmol, 1.0 equiv.) in MeOH
[16] B. Xu, A. Stephens, G. Kirschenheuter, A. F. Greslin, X.
Cheng, J. Sennelo, M. Cattaneo, M. L. Zighetti, A. Chen, S.-A.
Kim, H. S. Kim, N. Bischofberger, G. Cook, K. A. Jacobson, J.
Med. Chem. 2002, 45, 5694–5709.
[17] G. Molander, D. Sandrock, Org. Lett. 2007, 9, 1597–1600.
[18] A. Nuñez, A. Sánchez, C. Burgos, J. Álvarez-Builla, Tetrahe-
dron 2004, 60, 6217–6224.
[19] T. Ho, C. Chen, Helv. Chim. Acta 2005, 88, 2764–2770.
Received: June 30, 2008
Published Online: July 30, 2008
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