A new bio-compatible pH cleavable linker for solid-phase synthesis of a squalamine analogue

Article abstract of DOI:10.1016/S0040-4039(01)01217-5

Linkers that can be cleaved directly within the biological assay offer some advantages over traditional linkers in the range of direct screening applications that the associated libraries can be utilised for. The 1,6-elimination process is an efficient method of cleaving compounds from substituted 4-hydroxymethyl phenols, although giving rise to quinone methide by-products. Here, we report on a linker that uses an in-built amine 'activator' to cleave a phenoxy ester and hence to activate the linker to 1,6-elimination. An analogue of the antibacterial agent squalamine was synthesised and released using this linker strategy.

Full text of DOI:10.1016/S0040-4039(01)01217-5

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 6211–6214
A new bio-compatible pH cleavable linker for solid-phase
synthesis of a squalamine analogue
Bordin Chitkul, Butrus Atrash and Mark Bradley*
Department of Chemistry, University of Southampton, Southampton SO17 1BJ, UK
Received 22 May 2001; accepted 5 July 2001
Abstract—Linkers that can be cleaved directly within the biological assay offer some advantages over traditional linkers in the
range of direct screening applications that the associated libraries can be utilised for. The 1,6-elimination process is an efficient
method of cleaving compounds from substituted 4-hydroxymethyl phenols, although giving rise to quinone methide by-products.
Here, we report on a linker that uses an in-built amine ‘activator’ to cleave a phenoxy ester and hence to activate the linker to
1,6-elimination. An analogue of the antibacterial agent squalamine was synthesised and released using this linker strategy. © 2001
Elsevier Science Ltd. All rights reserved.
Linkers play a dominating role in solid-phase organic
synthesis, determining not only the method of com-
pound cleavage and attachment, but also placing
restraints on the necessary nature of library synthesis.1
Huge numbers of linkers are now known,2 falling pre-
dominantly into electrophilically cleaved systems such
as the Wang, Rink and trityl linkers,3 and nucleophili-
cally cleaved linkers such as the Kenner, Marshall and
ester based linkers,4 but also covering more exotic
linkers such as light and thermally based cleavage
processes.5
One linker type that has been used in the area of SPOS
is the so-called ‘safety catch linker’. These linkers
require some form of preactivation prior to compound
cleavage, often requiring two orthogonal steps to neces-
sitate cleavage. Some of these linkers potentially allow
compound cleavage to take place under mild and bio-
compatible conditions, either within assay wells or
within agarose gels for zone diffusion-based screens.
Such linkers have been described for example, by
Frank4 as well as by ourselves,6 but problems with
these linkers have limited their application. Here, we
Scheme 1. 1,6-Elimination process of pH cleavable linkers.
Keywords: solid-phase synthesis; linker; squalamine.
* Corresponding author. Tel.: +44 2380 593598; fax: +44 2380 596766; e-mail: mb14@soton.ac.uk
0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(01)01217-5

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B. Chitkul et al. / Tetrahedron Letters 42 (2001) 6211–6214
describe a new pH cleavable linker system, based on the
1,6-elimination process of our previously described
linker 1, but without liberation of the reactive quinone
methide by-product 2 (Scheme 1).
TFA/DCM followed by removal of volatiles and then
treatment with K2HPO4 buffer (50 mM, pH 8.0). Sam-
ples were removed and quenched with 0.1% TFA
before injection and quantification using internal stan-
dards. The production of Fmoc-Ala-OH and disappear-
ance of starting material are shown in Fig. 1.
The new linker model was initially prepared in solution
(Scheme 2), followed by solid-phase attachment. The
synthesis started with 5-formylsalicylic acid 3, which
was coupled to pentylamine to give 4a (88%). This was
then reduced with borane using the procedure described
by Hall et al.8 The amine was then protected without
isolation with Boc2O to give 5a, followed by selective
acetylation9 of the phenol using the reagent 1-acetyl-
1H-1,2,3-triazolo[4,5-b]pyridine to give 6 (90%). Ester-
The half-life of cleavage was 12 minutes and Fmoc-Ala-
OH was recovered in 96% yield. The linker was then
synthesised directly on the solid-phase using both
polystyrene and TentaGel resins. The resin was coupled
directly with the building block 3 under standard reac-
tion conditions to give 4 (b–e) (Scheme 2). The solution
chemistry was repeated on the solid-phase to afford
products 7 (b–e).
ification
of
the
hydroxymethyl
entity
with
Fmoc-Ala-OH using DIC/DMAP gave 7a7 (86%). This
was then used as a solution model for subsequent resin
based chemistries.
The kinetics of the release of Fmoc-Ala-OH from com-
pound 7b was then carried out in the same manner as
described above. As expected, product release was
slower than that obtained in solution and a half-life of
40 minutes was observed (Fig. 1). The kinetics of
Fmoc-Ala-OH release from compound 7c was identical
The kinetics of cleavage of this linker in solution were
monitored by RP-HPLC. The Boc protecting group
was first removed by treating with a 50% solution of
Scheme 2. Synthesis/cleavage of pH cleavable linker 7 (i) R1NH2, DIC, HOBt, DCM; (ii) a. BH3·THF, 65°C; b. Boc2O, DCM;
(iii) 1 M NaOH (aq.), 1-acetyl-1H-1,2,3-triazolo[4,5-b]pyridine, THF, rt; (iv) Fmoc-Ala-OH, DIC, DMAP, DCM; (v) 50%
TFA/DCM, rt, 1 h; (vi) K2HPO4 buffer (50 mM), pH 8.0.
Figure 1. Kinetics of compound release from 7a and 7b: (x) disappearance of 7a, (ꢀ) production of Fmoc-Ala-OH from 7a, (ꢁ)
production of Fmoc-Ala-OH from 7b.

B. Chitkul et al. / Tetrahedron Letters 42 (2001) 6211–6214
6213
Scheme 3. Reagents and conditions: (i) 3b-acetoxybisnor-5-chlolenic acid/DIC/HOBt; (ii) Py·SO3, CHCl3; (iii) 50% TFA in DCM
1 h then phosphate buffer (pH 8.0) 24 h.
Acknowledgements
to that of 7b. Hence the small hydrophobic spacer
attached to the resin had no effect on the kinetics of
cleavage. Other compounds released from the linker
immobilised on PS included Fmoc-Phe-Ala-OH, Fmoc-
Gly-Phe-Ala-OH and 4-hydroxy-7-trifluoromethyl-3-
quinoline carboxylic acid. In all cases a quantitative
recovery of the compounds released was achieved.
We thank the BBSRC and the Thai government for a
scholarship to B.C.
References
The linkers attached to TentaGel (compounds 7d and
7e) were found to cleave substantially upon treatment
with acid to remove the amino protecting group. Use of
various percentages of TFA (5–50%) and reaction times
(1–30 min) to remove the Boc group still resulted in
substantial cleavage of Fmoc-Ala-OH. An analogous
structure to that of 7d was synthesised, but using Bpoc
instead of Boc as the amino protecting group. Here,
after deprotection with acetic acid, most of the Fmoc-
Ala-OH was released.
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antibacterial squalamine 10 (Scheme 3). Briefly, linker
6c was transformed into an active carbonate using
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was achieved on the solid-phase. It was used for the
first solid-phase synthesis of an analogue of the shark-
derived antibacterial agent squalamine, and these link-
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7. Selected data for 7a: lH (300 MHz, CDCl3) 0.9 (3H, t, J
7, CH3), 1.14–1.36 [6H, m, -(CH2)3CH3], 1.36–1.6 (14H,
m, Boc, -NCH2 (CH2)3CH3, and Ala-CH3), 4.2 (1H, t, J

6214
B. Chitkul et al. / Tetrahedron Letters 42 (2001) 6211–6214
7, FmocCH), 4.3–4.5 [5H, m, FmocCH2, ArCH2-N, and
EtOAc:hexane); wmax (CH2Cl2)/cm−1 3541, 2963, 2952,
2923, 1760, 1726, 1691; ES-MS (+ve): m/z 676.7 (M+
NH4)+, 681 (M+Na)+, HRMS: expected for C38H46N2O8,
658.3332, found: 658.3358.
AlaCH(a)], 5.17 (2H, Abq, J 7, ArCH2), 5.45 (1H, d, J 7,
NH), 7.06 (1H, d, J 8, Ar), 7.21–7.28 (2H, m, Ar), 7.32
(2H, t, J 7, Fmoc), 7.41 (2H, t, J 7, Fmoc), 7.65 (2H, d,
J 7, Fmoc), 7.78 (2H, d, J 7, Fmoc); lC (75 MHz, CDCl3)
14.41 (CH3), 19.11 (CH3-Ala), 21.30 (CH3, Ac), 22.77
(CH2CH3), 27.98 (CH2CH2CH3), 28.83 (Me3), 29.38
[CH2(CH2)2CH3], 46.58 [N-CH2(CH2)3CH3], 47.58
(Fmoc-CH), 50.10 [Ala-CH(a)], 66.99 (Fmoc-CH2,
ArCH2-N), 67.46 (ArCH2), 80.10 (CMe3), 120.39, 123.27,
125.51, 127.48, 128.12, 128.66, 130.05 (ArCH), 131.18,
133.63, 141.72, 144.20, 144.34, (ArC), 156.01 (urethanes),
169.56, 173.20 (esters); TLC Rf 0.32 (50:50
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