A Dde-based carboxy linker for solid-phase synthesis

Article abstract of DOI:10.1016/S0040-4039(01)00101-0

The Dde-derived carboxy protecting group, 4-{N-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl]amino}benzyl ester (ODmab) has been developed into a carboxy functional linker. The stability of the linker to standard acid and base conditions employ

Full text of DOI:10.1016/S0040-4039(01)00101-0

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 2189–2192
A Dde-based carboxy linker for solid-phase synthesis
Siri Ram Chhabra, Harendra Parekh, Azra N. Khan, Barrie W. Bycroft and Barrie Kellam*
School of Pharmaceutical Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK
Received 6 October 2000; accepted 17 January 2001
Abstract—The
Dde-derived
carboxy
protecting
group,
4-{N-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methyl-
butyl]amino}benzyl ester (ODmab) has been developed into a carboxy functional linker. The stability of the linker to standard
acid and base conditions employed in Fmoc/tBu SPPS has been demonstrated and its utility illustrated by the construction of
model peptides, Leu-enkephalin and Human Angiotensin II. © 2001 Elsevier Science Ltd. All rights reserved.
Over the past decade the initial stages of the drug
discovery process have been revolutionised by the
advances in high-throughput screening and combinato-
rial chemistry.1,2 The rapid progression of the latter
from small peptides to complex compound libraries has
hinged largely on the development, in solid-phase syn-
thesis (SPS), of new chemistry, more robust and sophis-
ticated solid supports, versatile linkers and new
protecting groups.3 In general, linker strategies have
evolved from protecting groups,4 and we now report
such a development for a carboxy linker based on the
protecting group ODmab 1.5 This linker, which is
stable to acid and base conditions but readily cleaved
using 2% v/v hydrazine monohydrate in DMF, has
been specifically developed to be compatible with other
protecting groups in relation to some of our interests
with atypical and glyco-peptides.
only, yet cleaved by fluoride ion, have been described
by Chao12 and Ramage,13 but they limit the use of silyl
protecting groups in SPS, particularly for carbohydrate
manipulation. A phenanthridine derived linker with
similar acid/base stability has been reported recently,14
but the oxidative cleavage conditions were unsuitable
for a number of our purposes. Other carboxy linkers,
requiring mild photochemical,15 enzymatic16 or Pd(0)17
cleavage conditions to release the product are also
available and have specific stabilities and applications.
Further methodology, which has additional advantages,
now offers the opportunity to extend the range of
compound types amenable to SPS.
ODmab 1, synthesised by the condensation of Ddiv18
and 4-aminobenzyl alcohol serves as a carboxy-protect-
ing group. This group, like Dde19 and Nde,20 displays
inherent stability towards the base-deprotection condi-
tions required for Fmoc/tBu SPPS and the acid condi-
tions required for side-chain deprotection. Cleavage is
achieved via hydrazinolysis, followed by elimination of
the resulting p-aminobenzyl group via a 1,6-electron
shift (Scheme 1).
Although a variety of carboxy linkers are available, the
majority are either acid (Wang6) or hyper acid (HAL,7
SASRIN,8 trityl9) labile. Linkers such as HMB10 and
glycolamido11 are stable to acid, but release carboxylic
acids on treatment with NaOH(aq) or Bu4NOH(aq)
.
Effective silicon based linkers which are stable to base
Scheme 1. Hydrazinolysis followed by 1“6 elimination of an ODmab protected carboxylic acid.
Keywords: carboxylic acids; linkers; solid-phase synthesis; supported reagents/reactions.
* Corresponding author. E-mail: barrie.kellam@nottingham.ac.uk
0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(01)00101-0

2190
S. R. Chhabra et al. / Tetrahedron Letters 42 (2001) 2189–2192
via DIPCDI/HOAt mediated coupling to furnish 8.
Complete acylation was confirmed by a negative TNBS
test for primary amines.24
We envisaged that by modification and functionalisa-
tion of the alkyl side chain of 1, in a manner akin to
our development of a Dde-based amine linker,21 the
product could be successfully anchored to a solid-sup-
port. The synthetic approach followed two distinct
routes and is outlined in Scheme 2.
In route B the free acid 9 was first immobilised onto the
solid support to afford 10, again complete acylation
was confirmed by a negative TNBS test. Condensation
of 10 with 4-aminobenzylalcohol using the optimised
conditions described in route A yielded the desired
linker 11. Coupling of the first residue was achieved via
either the corresponding amino acid fluoride or the
pre-formed symmetrical anhydride, the latter resulting
in higher loading (>80%) as confirmed by Fmoc loading
tests.25
Route A was undertaken primarily in solution to high-
light any potential problems in synthesis; since the
reactions could be monitored using standard chromato-
graphic techniques. This route however generated a
linker with the first amino acid (e.g. Fmoc-Leu-OH)
pre-attached. Therefore, to construct a ‘generic linker’
route B was developed in parallel to yield 11 with a free
benzylic-OH functionality available for the attachment
of any carboxylic acid.
The acid stability of the acylated linker was demon-
strated by exposing 8 to 90% TFA in DMF over a
period of 4 h. Fmoc loading remained unchanged. The
base stability of 5 was assessed following initial acyla-
tion with Boc-Leu-OH. Exposing the resultant product
to 20% v/v piperidine in DMF or 2% v/v DBU in DMF
over a 3 h period resulted in no detectable decomposi-
tion or side reactions as determined by TLC, RP-HPLC
DCCI/DMAP activated coupling of dimedone with
mono tert-butyl glutarate 3 afforded the desired C-acyl-
ated product 4 in high yield. Amidation of 4 with
4-aminobenzyl alcohol (Route A) proved problematic;
presumably due to the reduced basicity of the anilino
nitrogen and the propensity of the 4-aminobenzyl alco-
hol to undergo polymerisation via 1,6-dehydration.
These problems were circumvented by the addition of
4-aminobenzyl alcohol in aliquots over 72 hours to a
refluxing solution of 4 in THF, yielding the amidated
product 522 as a crystalline solid in 87% yield. Attach-
ment of a carboxylic acid was achieved via the corre-
sponding acid fluoride to afford 6.23 Subsequent
acidolysis afforded the free carboxylic acid 7, which
was immobilised onto aminomethyl polystyrene resin
1
and H NMR analyses.
The utility of the linker was evaluated via the synthesis
of model peptides Leucine-Enkephalin (H-Tyr-Gly-Gly-
Phe-Leu-OH) and Human Angiotensin II (H-Asp-Arg-
Val-Tyr-Ile-His-Pro-Phe-OH) using standard Fmoc
chemistry. The first amino acid was attached to 11 via
its preformed symmetrical anhydride and subsequent
couplings were performed using standard TBTU/
Scheme 2. Reagents and conditions: (i) Dimedone, DCC/DMAP, DCM, rt, 4 h, 81%; (ii) 4-aminobenzyl alcohol, THF, reflux, 72
h, 87%; (iii) Fmoc-Leu-F, rt, 1.5 h, 88%; (iv) TFA:TIPS:H2O (95:2.5:2.5), 3 h, 81%; (v) aminomethylpolystyrene resin (200–400
mesh), DIPCDI, HOAt, DMF.

S. R. Chhabra et al. / Tetrahedron Letters 42 (2001) 2189–2192
2191
Figure 1. RP-HPLC profiles of the crude native peptides (a) Leu-enkephalin and (b) Angiotensin II.
HOBt/DIEA activation. Fmoc deprotection was carried
out either with 20 % v/v piperidine or 2% v/v DBU in
DMF. However, the crude peptides in the latter case
displayed the highest purity as demonstrated by RPH-
PLC. Peptide-resin cleavage was achieved using 2% v/v
hydrazine·H2O in DMF (2×20 min), releasing the pro-
tected fragments, which on treatment with
TFA:TIPS:H2O (95:2.5:2.5) afforded the native pep-
tides (Fig. 1).26 Upon cleavage, the Dde portion of the
linker remains resin bound and is converted to the
6,6-dimethyl-4,5,6,7-tetrahydro-4(1H)-indazolone.19
7. Albericio, F.; Barany, G. Tetrahedron Lett. 1991, 32,
1015–1018.
8. Mergler, M.; Tanner, R.; Gosteli, J.; Grogg, P. Tetra-
hedron Lett. 1988, 29, 4005–4008.
9. (a) Barlos, K.; Gatos, D.; Kallitsis, I.; Papaioannou, P.;
Sotiriou, P. Leibigs Ann. Chem. 1988, 1079; (b) Barlos,
K.; Gatos, D.; Kallitsis, I.; Papaphotiu, G.; Sotiriou, P.;
Wenqing, Y.; Schafer, W. Tetrahedron Lett. 1989, 30,
3943–3946; (c) Eleftheriou, S.; Gatos, D.; Panagopoulos,
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In conclusion, we have successfully developed a new
carboxy linker stable to acidic and basic environments,
yet easily cleaved in 2% v/v hydrazine monohydrate.
The application of the linker for the synthesis of model
side-chain protected or unprotected peptide fragments
by Fmoc/tBu SPPS has been demonstrated. The
exploitation of the described linker for modified gly-
copeptide synthesis is currently under investigation in
our laboratories.
14. Li, W.-R.; Hsu, N.-M.; Chou, H.-H.; Lin, S.-T.; Lin,
Y.-S. J. Chem. Commun. 2000, 401–402.
Acknowledgements
15. (a) Ajayaghosh, A.; Pillai, V. N. R. J. Org. Chem. 1987,
52, 5714–5717; (b) Rodebaugh, R.; Frasier-Reid, B.; Gey-
sen, H. M. Tetrahedron Lett. 1997, 38, 7653–7656; (c)
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Tetrahedron Lett. 1997, 38, 1227–1230; (e) Peukert, S.;
Giese, B. J. Org. Chem. 1998, 63, 9045–9051.
16. Sauerbrei, B.; Jungmann, V.; Waldmann, H. Angew.
Chem., Int. Ed. Engl. 1998, 37, 1143.
We would like to thank the BBSRC for a studentship
(to H.P.), and the Commonwealth Association UK for
a studentship (to A.N.K)
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22. tert-Butyl 5-(4-hydroxymethylanilino)-5-(4,4-dimethylcy-
clohexylidene-2,6-dioxo)pentanoate 5: Oil, Rf 0.64 (80%
EtOAc/hexane); selected spectral data: m/z (ES-MS) 416
(M+H); lH (250 MHz, CDCl3) 1.08 (6H, s, (CH3)2), 1.34
(9H, s, (CH3)3), 1.55–1.85 (2H, m, 3-H2), 2.20 (2H, t,
2-CH2), 2.39 and 2.48 (4H, s, Dde 2-CH2), 2.85–2.95 (2H,
m, 4-H2), 3.48 (1H, br s, OH), 4.73 (2H, s, CH2OH), 7.12
and 7.45 (4H, 2×d, ArH), 14.97 (1H, s, NH); lC (62.9
MHz, CDCl3) 24.59 (3-CH2), 28.27 ((CH3)2), 28.59
((CH3)3), 30.30 (Dde 4-C), 30.41 (4-CH2), 35.86 (Dde
3,5-CH2), 53.98 (2-CH2), 64.40 (CH2OH), 80.14
(C(CH3)3), 107.49 (Dde 1-C), 126.47 (Ar 3,5-C), 128.12
(Ar 2,6-C), 135.52 (Ar 4-C), 141.84 (Ar 1-C), 172.61
(COOBut), 176.25 (5-C), 196.79 (Dde CꢀO (H bonded)),
200.92 (Dde CꢀO).
g-Me2), 24.28 (3-CH2), 24.75 (Leu a-CH), 28.00 ((CH3)3),
28.27 (Dde Me2), 29.95 (Dde 4-C), 35.47 (Dde 3,5-CH2),
41.53 (Leu b-CH2), 47.17 (Leu a-CH), 52.34 (Fmoc CH),
53.79 (2-CH2), 66.08 (benzyl alcohol CH2), 66.98 (Fmoc
CH), 80.14 (C(CH3)3), 107.25 (Dde 1-C), 119.99, 125.04,
127.70 and 129.16 (Fmoc ArCH), 126.39 and 127.04
(ArCH), 135.31 (aromatic 1-C), 136.71 (aromatic 4-C),
141.29 and 143.70 (Fmoc-C), 156.00 (NH·CꢀO), 172.16
(COOBut), 172.96 (Leu CꢀO), 175.62 (5-C), 196.28 (Dde
CꢀO (H bonded)), 200.57 (Dde CꢀO).
24. Hancock, W. S.; Battersby, J. E. Anal. Biochem. 1976, 38,
2799–2802.
25. Atherton, E.; Logan, C. J.; Sheppard, R. C. J. Chem.
Soc., Perkin Trans. 1 1981, 538–546.
26. HPLC conditions: Vydac C8 column:208TP5415; 4.6 mm
(id)×150 mm. Eluents: A: 0.06% TFA(aq). B: 90% MeCN/
H2O v/v (0.06% TFA). Gradient: 20–100% B in 30 min
linearly. Leu-enkephalin protected fragment: Rt 9.22 min;
m/z (ES-MS) 612.4 [MH+]. Leu-enkephalin: Rt 5.29 min;
m/z (ES-MS) 556.3 [MH+]. Angiotensin II protected frag-
ment Rt 19.46 min; m/z (ES-MS) 1652.6 [M+],
Angiotensin II: Rt 5.36 min; m/z (ES-MS) 1047.0 [MH+].
From the HPLC profiles obtained (cf. Fig. 1), it is
apparent that the p-iminomethine quinoid species, or its
hydrazine adduct, is either removed during peptide
workup or elutes with the solvent front and therefore
does not hinder the isolation or purification of the crude
cleavage products.
23. tert-Butyl
5-(4-(N-9-fluorenylmethoxycarbonylleucinyl-
oxymethyl)anilino) - 5 - (4,4 - dimethylcyclohexylidene - 2,6-
dioxo)pentanoate 6: Oil Rf 0.52 (40% EtOAc/hexane);
selected spectral data: m/z (ES-MS) 751 (M+H); lH (250
MHz, CDCl3) 0.95 (6H, d, J 4.15, Leu g-Me2), 1.08 (6H,
s, Dde-Me2), 1.34 (9H, s, (CH3)3), 1.44–1.78 (5H, br m,
Leu g-H, b-H2 and 3-H2), 2.22 (2H, t, J 7.55, 2-H2), 2.40
and 2.49 (4H, s, Dde 2×CH2), 2.85–2.95 (2H, m, 4-H2),
4.21 (1H, t, J 8, Fmoc-CH), 4.39–4.51 (3H, br m, Fmoc-
CH2, Leu a-H), 5.13 (2H, s, PABA CH2), 5.30 (1H, d, J
8.6, Leu-NH), 7.15 (2H, d, Fmoc-ArH), 7.26–7.41 (6H,
br m, Fmoc-ArH), 7.12 and 7.45 (4H, 2×d, ArH), 15.08
(1H, s, NH); lC (62.9 MHz, CDCl3) 21.77 and 22.85 (Leu
.