Dual linker with a reference cleavage site for information rich analysis of polymer-supported transformations

Article abstract of DOI:10.1016/j.tetlet.2004.05.029

Two dual linker systems with specific reference cleavage sites were designed and synthesized to accelerate and simplify development and optimization of reaction conditions for solid-phase synthesis. The dual linker allows simple evaluation of cleavage rate of polymer-supported compounds from the linker and, at the same time, ensures that all resin-bound components are cleaved from the solid support. The dual linker 4 was assembled from two Wang linkers connected by a three carbon spacer. The linker 9 was synthesized using the PAL and HMPB linkers.

Full text of DOI:10.1016/j.tetlet.2004.05.029

Tetrahedron
Letters
Tetrahedron Letters45 (2004) 5237–5241
Dual linker with a reference cleavage site for information
rich analysis of polymer-supported transformations
a,
b
*
ꢀꢁ
Viktor Krchnak and Greg A. Slough
aDepartment of Chemistry and Biochemistry, 251 Nieuwland Science Center, University of Notre Dame, Notre Dame,
IN 46556, USA
bChemistry Department, Kalamazoo College, 1200 Academy St., Kalamazoo, MI 49006, USA
Received 3 March 2004; revised 30 April 2004; accepted 7 May 2004
Abstract—Two dual linker systems with specific reference cleavage sites were designed and synthesized to accelerate and simplify
development and optimization of reaction conditions for solid-phase synthesis. The dual linker allows simple evaluation of cleavage
rate of polymer-supported compounds from the linker and, at the same time, ensures that all resin-bound components are cleaved
from the solid support. The dual linker 4 was assembled from two Wang linkers connected by a three carbon spacer. The linker 9
was synthesized using the PAL and HMPB linkers.
Ó 2004 Elsevier Ltd. All rights reserved.
Two cleavage sites
Optimization of reaction conditionsisa prerequisite for
successful solid-phase synthesis. Whereas the critical
task of solution phase combinatorial synthesis is the
Solid
support
isolation of the product, the most time-consuming step
of solid-phase combinatorial synthesis is optimization of
reaction conditions; the product isolation cannot be
simpler. The final step of a chemical transformation on
solid phase includes cleavage of the resin-bound reaction
componentsfollowed by analysisof the cleaved sample.
To obtain meaningful information regarding the com-
position of components on solid phase, all components
need to be cleaved from the support (a textbook exam-
ple of a reaction, where the starting synthon is not
cleaved isacylation of polymer-uspported N-alkylated
benzylamines). Information that cannot be obtained
from thissimple experiment isthe rate of cleavage from
the solid support. An independent quantification analy-
sis is required in order to assess the cleavage yield.
Linker 1
Spacer
Linker 2
Compound
Cleavage
Spacer
LLiinnkkeerr22
CCoommppoouunndd
Spacer
Linker 2
CCoommppoouunndd
+
+
Figure 1. The concept of two dual linkerswith a reference cleavage
site.
spacer (Fig. 1). The spacer is designed to allow its
cleavage from the Linker 1 under standard conditions.
Linkers1 and 2 may be, but do not need to be, the same
linkers. However, both linkers need to be cleavable
under the same reaction conditions. Simultaneous
cleavage of compoundsfrom both linkersby the asme
reagent givescleavage of all polymer-uspported com-
ponentsand allowsone to fine tune the cleavage con-
ditionsfor complete removal of the ysnthetic product.
Thus, the presence of the desired compound still
attached to Linker 2 and the spacer indicates incomplete
cleavage from Linker 2. Alternatively, observation of
the desired compound free from Linker 2 indicates
complete cleavage.
We report a general, yet very simple, concept that yields
cleavage data for all polymer-supported components
and, at the same time, assays the cleavage rate. This is
possible by carrying out the synthesis on a polymer-
supported dual linker with a reference cleavage site
consisting of two sequential linkers separated by a
Keywords: Solid-phase synthesis; Dual linker; Cleavage rate.
* Corresponding author. Tel.: +1-574-6315113; fax: +1-574-6316652;
e-mail: vkrchnak@nd.edu
The concept of dual linkersisnot a new one and
1
wasrecently reviewed. In 1979 Merrifield described a
0040-4039/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2004.05.029

5238
V. Krchꢀnꢁak, G. A. Slough / Tetrahedron Letters 45 (2004) 5237–5241
multi-detachable resin consisting of two linkers with
different cleavage characteristics, which allows cleavage
of either free or protected peptides.2;3 Geysen used the
dual linker concept to incorporate a coding moiety
The synthesis of the dual Wang linker is straightfor-
ward. Trichloroacetimidate Wang polystyrene––1% di-
vinylbenzene (PS/DVB) resin
1
(NovaBiochem,
0.66 meq/g) reactswith 1,3-propanediol according to the
reported procedure.12 A sample of the resulting resin-
bound alcohol 2 wasacylated with Fmoc-Ser(Bzl)-OH
and the product treated with TFA/DCM. The expected
serine derivative Fmoc-Ser-O-(CH2)3-OH wascleaved in
74% yield with respect to the declared resin substitution
level (quantification from HPLC tracesat 300 nm). The
resin 2 wasthen coupled to TBS protected 4-hydroxy-
benzyl alcohol21 under Mitsunobu conditions22 to yield
the TBS protected dual linker resin 3.23 A sample of the
resin 3 wasacylated with Fmoc-Ser(Bzl)-OH in order to
detect any unreacted alcohol and the product was
cleaved with 50% TFA in DCM for 30 min. Only traces
(<1%) of Fmoc-Ser-(CH2)3-OH were detected.
4
between two linkersthat facilitated library encoding.
Two different linkerswith orthogonal cleavage charac-
teristics have been connected via an analytical enhancer,
that facilitated UV and MS detection of the cleaved
product by incorporating a strong UV chromophore5;6
and MS sensitizer (with an isotope label),5;7–9 respec-
tively. Current limitationsof dual linkersare two-
fold: (i) the need to synthesize the analytical construct,
and (ii) incorporation of functionalitiesnot compati-
ble with all reagentsand chemtirsiesuesd in the
synthesis.1
In order to prove the concept of the dual linker with a
reference cleavage site, we built a tandem from two
10
Wang linkers(Scheme 1). The Wang linker
isfre-
Finally, the TBS group wascleaved by TBAF and a
sample of the polymer-supported dual linker 4 wasex-
posed to 10% TFA in the presence of 5% Et3SiH. The
expected 3-(4-methylphenoxy)propan-1-ol wasdetected.
Without the use of the scavenger silane, polymerization
of the benzyl alcohol derivative in TFA takesplace and
no simple product can be detected on HPLC traces.
quently used in solid-phase synthesis and it allows
preparation of a range of compound classes including
acids,10;11 alcohols,12 and phenols.12–14 For the prepara-
tion of amides15–19 and amines,20 one or two additional
methoxy groupshave been introduced onto the aro-
matic ring in order to increase the acid mediated
cleavage. We connected two Wang linkersvia a three
methylene carbon spacer in order to install the reference
cleavage site; thus quantitative cleavage between the
Wang Linker 1 and the spacer is achieved under stan-
dard conditions(10% trifluoroacetic acid (TFA) in
dichloromethane (DCM) for 30 min).12
Several model compoundswere prepared (Scheme 2)
and cleaved with TFA in order to prove the concept of
the dual linker with a reference cleavage site. Esterifi-
cation by Fmoc-Lys(Boc)-OH was carried out by in situ
prepared HOBt esters with DMAP catalysis. Fmoc-
NH
CCl3
i
O
OH
ii
O
Pol
O
Pol
O
2
1
OTBS
OH
iii
O
O
Pol
O
O
DL OH
Pol
O
Pol
O
3
4
Scheme 1. Synthesis of the dual Wang linker. Reagents: (i) 1,3-propanediol, BF3 (etherate), anhydrousTHF, 30 min; (ii) p-HO-Ph-CH 2-OTBS,
DEAD, PPh3, anhydrousTHF, 20 °C, overnight; (iii) 0.5 M TBAF, THF, 20 °C, 30 min.
O
O
Pol
ii
iii
DL
N
Pol
DL OH
Pol
N
DL
O
O
4
O
iv, v
iv, v
i
O
O
H
N
O
H
N
H
N
Pol
DL
O
DL
N
Fmoc
Pol
N
Fmoc
Pol
H
H
DL
O
Fmoc
H
H
N
H
N
Boc
N
Boc
Boc
Scheme 2. Synthesis of model compounds. Reagents: (i) Fmoc-Lys(Boc)-OH, DIC, HOBt, DMAP, DMF/DCM, 20 °C, 16 h; (ii) phthalimide, PPh3,
DEAD, anhydrousTHF, 20 °C, 16 h; (iii) N-hydroxyphthalimide, PPh3, DEAD, anhydrousTHF, 20 °C, 16 h; (iv) hydrazine hydrate, THF/MeOH
(1:1), 20 °C, 16 h; (v) Fmoc-Lys(Boc)-OH, DIC, DMF/DCM, 20 °C, 16 h.

V. Krchꢀnꢁak, G. A. Slough / Tetrahedron Letters 45 (2004) 5237–5241
5239
Table 1. Relative amount of products 7 and 8 asa function of TFA concentration and time
Entry
R
Bond cleaved
TFA/DCM (%)
Time (min)
Product 7 (%)
Product 8 (%)
1
A
A
A
B
C–O
C–O
C–O
C–O
C–O
C–O
C–O
C–O
C–O
C–O
C–N
C–N
10a
10a;b
50
30
30
30
30
60
30
60
30
30
30
60
60
11
11
89
89
2
3
<1
82
>99
18
4
10
5
C
C
C
C
D
D
E
1c
10
74
63
26
37
6
7
10
50
<1
<1
71
>99
>99
29
8
9
10
10
11
12
50
20
93
80
95b;d
95b;d
7
<1
F
>99
a Partial cleavage of the Boc group.
b Contains5% Et 3SiH.
c Incomplete cleavage from the Wang Linker 1 (the ‘Linker 1––spacer’ bond).
d TFA ester of 7E wasformed during cleavage, which hydrolyzed in MeOH/water solution.
Lys(Boc)-OH was selected as the acylation species since
it provides easy quantification using the Fmoc group
and the free amino group (after TFA cleavage from the
resin) enhances identification by mass spectroscopy.
Phthalimide wasuesd to convert the Linker 2 into the
amino-functionalized resin.24 Analogously, N-hydroxy-
phthalimide was used to install the hydroxylamine for
the synthesis of hydroxamic acids.25;26 In both cases, the
phthalimide wascleaved with hydrazine and the amino
groupswere acylated with Fmoc-Lys(Boc)-OH.
13% of 8B cleaved), due to the protonation of the Na-
amino group (entry 4). Acylation with Boc-Lys(Fmoc)-
OH and subsequent cleavage with TFA showed 84%
cleavage, comparable with sample A, which wasacyl-
ated by Fmoc-Lys(Boc)-OH, suggesting that the Boc
cleavage occurred only after the product wascleaved
from the resin.
Immobilized N-hydroxyphthalimide, an intermediate
for the synthesis of hydroxamic acids, was more stable
when compared to esters (entries 5–8). Quantitative
cleavage of N-hydroxyphthalimide 8C from the Wang
Linker 2 required 30 min treatment with 50% TFA.
Cleavage of hydroxamic acids was substantially slower,
71% of compound 7D remained intact after 30 min in
10% TFA. Cleavage with 50% TFA showed 20% of the
product 7D (entries9 and 10). Quantitative cleavage of
hydroxamic acid required 95% TFA for one hour.
However, under these conditions the Wang linker was
partially cleaved from PS/DVB support. Thus, the more
acid labile 4-(4-hydroxymethyl-3-methoxyphenoxy)-
butyric acid (HMPB) linker27 ismore usitable for the
synthesis of hydroxamic acids.
The results of cleavage experiments are summarized in
Table 1. Trifluoroacetic acid cleavage leadsto the for-
mation of two products, 7 and 8 (Scheme 3). The pres-
ence of compound 7 indicatesincomplete cleavage from
the Linker 2. Ester cleavage of Na-Fmoc protected
amino acid (entries1–3) isfast and after 30 min in 10%
TFA/DCM only 11% of compound 7A remainsto be
cleaved. The presence of the scavenger Et3SiH doesnot
affect the cleavage rate, however, 3-(4-methylphen-
oxy)propan-1-ol wasdetected. In contrats, cleavage of
N-unprotected amino acid (after Fmoc group cleavage
with piperidine in DMF) is substantially slower (only
R
i
R
O
O
H
R
+
HO
O
Pol
O
7
8
H-R:
O
O
O
O
H
H
N
H
NH2
O
N
H
H
O
H
N
Fmoc
O
Fmoc
H
O
N
H
H
N
NH2
N
Boc
Boc
O
D
B
A
C
O
O
H
N
H
N
Fmoc
H
N
H
NH2
O
F
E
Scheme 3. Cleavage of model compounds. Reagents: (i) TFA in DCM, for concentration and reaction time see Table 1.

5240
V. Krchꢀnꢁak, G. A. Slough / Tetrahedron Letters 45 (2004) 5237–5241
O
O
O
O
O
Fmoc
i, ii
O
N
O
N
H
H
OH
Pol
N
O
O
Pol
N
O
O
H
H
9
Scheme 4. Synthesis of the dual amide linker. Reagents: (i) 40% piperidine, DMF, 20 °C, 20 min; (ii) HMPB linker, DIC, HOBt, DMF/DCM, 20 °C,
16 h.
Expectedly, almost no cleavage of the C–N amide bond
wasobserved in any experiment (entries11 and 12). One
hour in 95% TFA (5% water or Et3SiH) released 7% of
the product 8E from the Linker 2. After acylation with
Fmoc-Lys(Boc), compound 8F wasnot detected (no
corresponding ion current during LC/MS analysis).
Thus, cleavage of amides requires a more acid labile
linker.15–19
all components present on the solid-phase support. (ii) It
allows simple estimation of the cleavage rate of polymer-
supported compounds from LC/MS traces. (iii) It is
suitable for straightforward optimization of cleavage
protocol to arrive at the mildest conditions required for
quantitative cleavage of particular productsfrom a re-
sin-bound linker, including evaluation of new cleavage
cocktails. (iv) The second linker can be replaced by a
linker with modified cleavage characteristics (more sta-
ble, more labile) in order to arrive at the most suitable
linker for given compound classes. (v) The dual linker
has a potential to be used for design of new linkers with
altered sensitivity towards cleavage conditions. (vi)
Finally, the dual linker can assist in determining the
reason of low yield. Such poor yields can be caused not
only by incomplete cleavage but also by premature loss
during synthesis or incomplete extraction of cleaved
compound from the solid support. Elimination of
incomplete cleavage narrowsthe determination of the
cause.
The next dual linker wasbuilt from linkersdesigned for
the synthesis of nitrogen-containing compounds (cleav-
age of the Ca (benzyl)-N(amide) bond). The linker 9 was
assembled from two different linkers, which are both
cleavable with reagentssuch asTFA (Scheme 4). Amino-
methylated polystyrene––1% divinylbenzene resin
(Advanced ChemTech, 1.2 meq/g) wasacylated with the
5-(4-(9-fluorenylmethyloxycarbonyl)aminomethyl-3,5-di-
methoxyphenoxy)valeric acid (PAL) linker.28 The sub-
stitution level was evaluated by quantification of Fmoc-
NH2, cleaved from the resin by 10% TFA in 60 min and
was found to be 89% with respect to the declared sub-
stitution of aminomethylated resin (repeated cleavage
did not provide additional product). The cleavage time
wasetsimated by treatment of the PAL linker (not
attached to the resin) with TFA. After exposure to 10%
TFA for 30 min, 5.8% of intact PAL linker wasdetected
by HPLC. The cleavage of Fmoc-NH2 wascomplete
after 60 min.
The synthesis of the dual linker with a reference cleavage
site is very simple and straightforward. The dual linker
doesnot incorporate any additional functional group,
hence it does not restrict the choice of chemical trans-
formations. This letter described the concept of the dual
linker with a reference cleavage site and its application
to selection of the optimal linker for the synthesis of
hydroxamic acids.
After cleavage of the Fmoc group, the resin-bound
27
amino group wasacylated with the HMPB linker.
A
sample of the resin was reacted with Fmoc-ONSu and
treated with TFA. No Fmoc-NH2 wasdetected indi-
cating complete coupling of the HMPB linker. The dual
linker 9 wasreacted with phthalimide and N-hydroxy-
phthalimide under Mitsunobu conditions and the
productscleaved when treated with 10% TFA for 30 min
(in addition to expected products, a side-product having
a strong MS signal at 484 Da was detected). In the case
of phthalimide, cleavage from the Linker 2 wasincom-
plete with approximately 46% of phthalimide remaining
attached to the HMPB linker. Complete cleavage of
phthalimide from the Linker 2 wasobesrved at higher
concentration of TFA (50% in DCM) in 30 min. N-
hydroxyphthalimide wascleaved completely with 10%
TFA in 30 min. Complete cleavage wasalos obesrved
after deprotection of the phthalyl group and acylation
by carboxylic acids, indicating that this is the linker of
choice for the synthesis of hydroxamic acids.
Acknowledgements
The work wassupported by the Department of Chem-
istry and Biochemistry University of Notre Dame.
References and notes
1. Congreve, M. S.; Ley, S. V.; Scicinski, J. Chem. Eur. J.
2002, 8, 1769–1776.
2. Tam, J. P.; Tjoeng, F. S.; Merrifield, R. B. Tetrahedron
Lett. 1979, 20, 4935–4938.
3. Tam, J. P.; Tjoeng, F. S.; Merrifield, R. B. J. Am. Chem.
Soc. 1980, 102, 6117–6127.
4. Geysen, H. M.; Wagner, C. D.; Bodnar, W. M.; Mark-
worth, C. J.; Parke, G. J.; Schoenen, F. J.; Wagner, D. S.;
Kinder, D. S. Chem. Biol. 1996, 3, 679–688.
5. Williams, G. M.; Carr, R. A. E.; Congreve, M. S.; Kay, C.;
McKeown, S. C.; Murray, P. J.; Scicinski, J. J.; Watson, S.
P. Angew. Chem., Int. Ed. 2000, 39, 3293–3296.
6. Scicinski, J. J.; Congreve, M. S.; Jamieson, C.; Ley, S. V.;
Newman, E. S.; Vinader, V. M.; Carr, A. E. J. Comb.
Chem. 2001, 3, 387–396.
In summary, the new dual linker with a reference
cleavage site can serve in development of solid-phase
synthesis. (i) The dual linker ensures cleavage of all
resin-bound components, thus allowing identification of

V. Krchꢀnꢁak, G. A. Slough / Tetrahedron Letters 45 (2004) 5237–5241
5241
7. Carrasco, M. R.; Fitzgerald, M. C.; Oda, Y.; Kent,
S. B. H. Tetrahedron Lett. 1997, 38, 6331–6334.
8. Murray, P. J.; Kay, C.; Scicinski, J. J.; McKeown, S. C.;
Watson, S. P.; Carr, R. A. E. Tetrahedron Lett. 1999, 40,
5609–5612.
9. McKeown, S. C.; Watson, S. P.; Carr, R. A. E.; Marshall,
P. Tetrahedron Lett. 1999, 40, 2407–2410.
10. Wang, S. S. J. Am. Chem. Soc. 1973, 95, 1328–1333.
11. Phoon, C. W.; Oliver, S. F.; Abell, C. Tetrahedron Lett.
1998, 39, 7959–7962.
23. When a sample of the polymer-supported linker 3 was
treated with 10% TFA in DCM, LC/MS data indicated the
presence of an ion 279 Da, corresponding to a chroman
derivative 6, rather than expected product 5. Thus, the
TBS mono-protected diol 5 wasindependently prepared
from propane diol and TBS protected 4-hydroxybenzyl
alcohol. Treatment of the alcohol 5 with 10% TFA in
DCM led to the formation of the product, indistinguish-
able from the compound obtained by cleavage from the
resin 3. Experimental detailswill be publihsed in a full
report.
12. Hanessian, S.; Xie, F. Tetrahedron Lett. 1998, 39, 733–736.
13. Richter, L. S.; Gadek, T. R. Tetrahedron Lett. 1994, 35,
4705–4706.
OTBS
OTBS
14. Wang, Y.; Wilson, S. R. Tetrahedron Lett. 1997, 38, 4021–
4024.
O
HO
O
15. Boojamra, C. G.; Burow, K. M.; Ellman, J. A. J. Org.
Chem. 1995, 60, 5742–5743.
6
5
.
16. Fivush, A. M.; Willson, T. M. Tetrahedron Lett. 1997, 38,
7151–7154.
24. Ryoo, S. J.; Kim, J.; Kim, J. S.; Lee, Y. S. J. Comb. Chem.
2002, 4, 187–190.
25. Floyd, C. D.; Lewis, C. N.; Patel, S. R.; Whittaker, M.
Tetrahedron Lett. 1996, 37, 8045–8048.
17. Jensen, K. J.; Alsina, J.; Songster, M. F.; Vagner, J.;
Albericio, F.; Barany, G. J. Am. Chem. Soc. 1998, 120,
5441–5452.
26. Gordeev, M. F.; Hui, H. C.; Gordon, E. M.; Patel, D. V.
Tetrahedron Lett. 1997, 38, 1729–1732.
18. Sarantakis, D.; Bicksler, J. J. Tetrahedron Lett. 1997, 38,
7325–7328.
€
27. Florsheimer, P.; Riniker, B. Solid-Phase Synthesis of
19. Swayze, E. E. Tetrahedron Lett. 1997, 38, 8465–8468.
20. Forns, P.; Sevilla, S.; Erra, M.; Ortega, A.; Fernandez, J.
C.; de la Figuera, N.; Fernandez-Forner, D.; Albericio, F.
Tetrahedron Lett. 2003, 44, 6907–6910.
21. Ankala, S. V.; Fenteany, G. Tetrahedron Lett. 2002, 43,
4729–4732.
Peptideswith the Highly Acid-Senistive HMPB Linker.
In Pept. 1990, Proc. Eur. Pept. Symp., 21st; Giralt,
E.; Andreu, D., Eds.; ESCOM: Leiden, 1991; pp 131–
133.
28. Albericio, F.; Kneibcordonier, N.; Biancalana, S.; Gera,
L.; Masada, R. I.; Hudson, D.; Barany, G. J. Org. Chem.
1990, 55, 3730–3743.
22. Mitsunobu, O.; Yamada, M.; Mukayima, T. Bull. Chem.
Soc. Jpn. 1967, 40, 935–939.