Alcohols immobilization onto 2-chlorotritylchloride resin under microwave irradiation

Article abstract of DOI:10.1016/j.tetlet.2011.03.113

The immobilization of alcohols onto 2-chlorotritylchloride resin using microwave irradiation was studied. Three different Fmoc-aminoalcohols were tested: the phenol-like Fmoc-tyramine, the primary alcohol Fmoc-ethanolamine, and the secondary alcohol Fmoc-

Full text of DOI:10.1016/j.tetlet.2011.03.113

Tetrahedron Letters 52 (2011) 2808–2811
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Alcohols immobilization onto 2-chlorotritylchloride resin under
microwave irradiation
⇑
Luca Rizzi , Katarina Cendic, Nadia Vaiana, Sergio Romeo
Dipartimento di Scienze Farmaceutiche ‘‘Pietro Pratesi’’, Università degli Studi di Milano, Via Mangiagalli 25, 20133 Milan, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The immobilization of alcohols onto 2-chlorotritylchloride resin using microwave irradiation was stud-
ied. Three different Fmoc-aminoalcohols were tested: the phenol-like Fmoc-tyramine, the primary alco-
hol Fmoc-ethanolamine, and the secondary alcohol Fmoc-4-hydroxypiperidine. Several reaction
conditions were evaluated: different bases, reaction times, temperatures, and concentrations. Microwave
immobilization resulted is effective in binding to the resin all three types of alcohols with loadings which
were superior or comparable to the ‘classical’ methods in shorter time and without employing toxic and
racemizing reagents. This method resulted also useful for the immobilization, through the hydroxyl
group, of FmocTyrOAll, FmocSerOAll, and FmocThrOAll, important building blocks for the synthesis of
cyclic peptides.
Received 12 January 2011
Revised 16 March 2011
Accepted 23 March 2011
Available online 1 April 2011
Keywords:
Solid phase synthesis
2-Chlorotritylchloride resin
Alcohol
Ó 2011 Elsevier Ltd. All rights reserved.
Microwave
Cyclic peptide
2-Chlorotrityl chloride resin (2CTC) is widely employed in solid
phase organic and peptide synthesis, because it overcomes several
problems related to the use of other types of resins, such as Merri-
field and Wang resins.1 For example, unlike Wang resin, attach-
and phenol-like alcohols to the 2CTC. As model compounds for this
study we chose tyramine, ethanolamine, and 4-hydroxypiperidine,
phenol-like, primary and secondary alcohols, respectively: the
amine moiety of these molecules was protected as Fmoc deriva-
tive,24 (Scheme 1) being one of the most frequently used amine-
protecting group in the solid phase synthesis. The cleavage of the
Fmoc group after the reaction between 2CTC and the chosen alco-
hols allowed us to verify the resin loading by UV analysis.25 Using
a polystyrene 2CTC loading of 1.55 mmol/g, a series of experiments
were carried out in order to test the effects of different bases, reac-
tion times, concentrations, and temperatures in MW assisted load-
ing of Fmoc-amino alcohols onto 2CTC.26
With the aim of finding less toxic bases than pyridine and
DMAP, often used in the immobilization of alcohols onto 2CTC,
we tested the MW reaction, maintaining constant temperature
(50 °C), time (30 min), and concentration (80 mM), with the pres-
ence of different tertiary bases: diisopropylethylamine (DIPEA), tri-
ethylamine (TEA), N-methylmorpholine (NMM), and 1,5-
diazabicyclo[4.3.0]non-5-ene (DBU); using these type of bases
the formation of activated species was avoided, so the reaction
ment of
a-amino acids to 2CTC is free from enantiomerization
and other side reactions, such as diketopiperazine formation.
Moreover, extremely mild acidolysis conditions make possible
the cleavage of protected peptide segments from the resin.2,3 How-
ever, the greater advantage of 2CTC is its vast versatility since al-
most any nucleophilic functional group is able to link to this
resin: in fact, besides carboxylic acids, trityl resins are utilized, un-
der mild conditions, to bond amines, thiols, guanidines, imidazoles,
and other basic heterocycles.4–8
Trityl chloride resins are also employed for the immobilization
of alcohols9,10 but in this case the attachment to the solid support
is not as easy as for other functional groups: often the use of strong
nucleophiles, such as DMAP, or toxic bases, such as pyridine,
dry solvents, and long reaction times (from 2 to 96 h) are re-
quired.10–19 In addition, the reaction yields are usually very low,20
reaching, in the best cases, a resin loading of 0.2–0.3 mmol/g. Since
microwave (MW) irradiation has been successfully employed for
more than 20 years in organic synthesis, in order to obtain faster
reaction times and/or higher yields respect to conventional heating
conditions,21–23 we decided to apply this technique to the immobi-
lization of alcohols onto 2CTC. Thus, in this Letter we study the
effects of MW irradiation on the attachment of primary, secondary,
OH
OH
H2N
H2N
HN
FmocHN
FmocHN
FmocN
Fmoc-OSu,
DIPEA
OH
OH
OH
OH
CH2Cl2
overnight
⇑
Corresponding author. Tel.: +39 02 50319354; fax: +39 02 50319365.
E-mail address: luca.rizzi@unimi.it (L. Rizzi).
Scheme 1. Synthesis of Fmoc-aminoalcohols.
0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2011.03.113

L. Rizzi et al. / Tetrahedron Letters 52 (2011) 2808–2811
2809
Table 1
Table 2
Effect of the bases in microwave-assisted loading of Fmoc-amino alcohols onto
2-chlorotrityl resin
Effect of the temperature in microwave-assisted loading of Fmoc-amino alcohols onto
the 2-chlorotrytyl resin
basec
base
Cl
Cl
Cl
Cl
Cl
Cl
CH2Cl2/DMFb
2
2
CH Cl /DMF
X OHa
X OH
+
O
+
O
X
X
MW, 50°C
30 min
MW, T°C
30 min
Base
Loading (mmol/g)
Temperature
Loading (mmol/g)
(°C)
OH
OH
OH
OH
OH
OH
FmocHN
FmocHN
FmocN
FmocN
FmocHN
FmocHN
0.380a
0.304b 0.560c
0.315b 0.565c
0.200c
0.208c
DIPEA
TEA
0.380
0.388
0.246
0.320
n.r.d.g
0.120
0.132
0.204
0.304
0.560
0.236
0.052
n.r.d.g
n.r.d.g
n.r.d.g
0.200
n.r.d.g
n.r.d.g
50
100
0.386a
NMM
a
b
c
DIPEA.
NMM.
DBUd
Pyridine
DIPEA,
DMAPe
DIPEA,
DMAPf
DBU (loading calculated after coupling with FmocGlyOH).
0.064
0.070
n.r.d.g
when we employed DMAP, both in catalytic and in equimolar
amounts, the loadings were very low (0.120 and 0.064 mmol/g,
respectively), while with the use of pyridine no reaction was de-
tected. In the case of Fmoc-ethanolamine, model compound for
testing the reactivity of the primary alcohols, the results showed
a different behavior than Fmoc-tyramine: in fact the best base
seemed to be DBU, which gave, in only 30 min, an excellent loading
of 0.560 mmol/g, greater than that obtained with the ‘classical’
methods.10,19,11 On the other hand, TEA, DIPEA, and NMM did not
appear as good as DBU yielding a loading of 0.204, 0.132, and
0.304 mmol/g, respectively. Contrary to what happened in the case
of tyramine, pyridine was effective in MW immobilization of
Fmoc-ethanolamine affording a loading of 0.236 mmol/g inferior
only to that obtained with DBU and NMM. Conversely, DMAP re-
mained ineffective also with primary alcohols. On the contrary,
an interesting result emerged by the immobilization of secondary
alcohols, which are extremely problematical to immobilize onto
2CTC: in fact, as it can be noticed from Table 1 MW irradiation only
DBU was able to link Fmoc-4-hydroxypiperidine affording a load-
ing of 0.200 mmol/g. Any other conditions tested resulted
ineffective.
a2 equiv, b1.8 ml CH2Cl2 and 0.1 ml DMF for 50 mg of resin, c8 equiv.
d
Loading calculated after coupling with FmocGlyOH.
0.2 equiv.
1 equiv.
No reaction detected.
e
f
g
would be free from epimerization.2 Since DBU is one of the most
efficient base for Fmoc-removal,27 we verified the resin loading
only after the coupling with Fmoc-glycine.28 Furthermore, in order
to evaluate the MW assisted synthesis under the ‘classical’ condi-
tions, we undertook MW reaction also in the presence of pyridine
and DMAP both in catalytic and in equimolar amounts (Table 1). As
it can be noticed in Table 1, in the case of phenol-like alcohols the
use of bases, such as DIPEA and TEA allowed to achieve, in a very
short time (30 min) and without the use of toxic reagents, a very
high loading (0.380 mmol/g for DIPEA and 0.388 mmol/g for TEA)
greater than the loading obtained in the ‘classical’ way. NMM
seemed to be less efficient than the other two tertiary bases: in
fact, NMM gave a loading comparable with the literature value
(0.246 mmol/g).11 On the contrary DBU, after the coupling with
FmocGlyOH, showed a good loading (0.320 mmol/g), although
smaller than that obtained with DIPEA and TEA. Moreover, it is
interesting to notice that the use of DMAP and pyridine was detri-
mental for the immobilization of phenol-like alcohols: in fact,
The effect of the reaction time during MW 2CTC immobilization
was investigated planning a series of experiments with different
irradiation times (5, 15, 30, 60, and 120 min), Fmoc-tyramine
was reacted at constant temperature (50 °C) and concentration
(80 mM), using DIPEA and TEA as bases, Fmoc-ethanolamine was
Figure 1. Effect of time in microwave-assisted loading of Fmoc-amino alcohols onto the 2-chlorotrityl resin.

2810
L. Rizzi et al. / Tetrahedron Letters 52 (2011) 2808–2811
Figure 2. Effect of concentration in microwave-assisted loading of Fmoc-amino alcohols onto the 2-chlorotrityl resin.
concentration of 80 mM. So, we tested the MW irradiation of
Twice
DIPEA,
Fmoc-tyramine (with TEA as base), Fmoc-ethanolamine (with
NMM and DBU), and Fmoc-4-hydroxypiperidine (with DBU) halv-
ing (40 mM) and doubling (160 mM) the concentration, both of
the alcohol and of the base. In Figure 2 it can be noticed that the
best concentration seemed to be 80 mM: halving the concentration
yields a three times lower loading in the cases of tyramine, etha-
nolamine and 4-hydroxypiperidine, while doubling the concentra-
tion did not affect the value. This data can be explained considering
the necessity to have a sufficient amount of CH2Cl2 in order to
maintain the resin swelled.
OH
+
Cl
Cl
Cl
CH2Cl2/DMF
O
MW,50°C
45 min
NHFmoc
NHFmoc
Loading: 0.600 mmol/g
Scheme 2. ‘Double coupling’ approach.
transformed in the presence of NMM and DBU, while in the case of
Fmoc-4-hydroxypiperidine only DBU was used. As shown in Fig-
ure 1, when DIPEA, TEA, or NMM were employed, the conversion,
both in the case of tyramine and in the case of ethanolamine,
Finally, in order to further increase the loading of phenol-like
alcohols, we carried out a ‘double coupling’ approach on the resin
(Scheme 2): after the first 45 min of reaction of Fmoc-tyramine and
DIPEA, the reaction mixture was filtered off and another amount of
Fmoc-tyramine and DIPEA was added and stirred again for extra
45 min, reaching a final loading of 0.600 mmol/g, comparable to
the loading of secondary alcohols obtained with the use of DBU.
Since 2CTC is widely employed in solid phase peptide synthesis
for the immobilization of Fmoc-amino acids,2,3 we tested the best
conditions, found with the model compounds, to bond, through
the hydroxyl group, FmocTyrOAll, FmocSerOAll, and FmocThrOAll.
This type of immobilization can be useful because it permits to
have two growth positions that can be used, for example, for the
synthesis of cyclic peptides. The three protected amino acids, syn-
thesized starting from commercially available FmocTyr(tBu)OH,
reached
a maximum value in 60 min, giving loadings of
0.504 mmol/g for Fmoc-tyramine and 0.402 mmol/g for Fmoc-eth-
anolamine. On the other hand, when DBU was utilized, the maxi-
mum loading was obtained at 30 min, both in the case of
ethanolamine and 4-hydroxypiperidine, after that time there was
a decrease in the loading, a signal of instability of the system.
An increase of temperature from 50 to 100 °C (Table 2) did not
affect the resin loading, which remained constant both for Fmoc-
tyramine, Fmoc-ethanolamine, and Fmoc-4-hydroxypiperidine.
Another parameter evaluated was the reaction concentration: as
mentioned earlier, the data shown so far refer to reactions with a
OH
OH
O
OH
O
FmocHN
FmocHN
O
O
O
FmocHN
DBU
DBU
O
Cl
DMF/CH2Cl2
MW50°C
60 min
Cl
1)
Cl
DMF/CH2Cl2
MW50°C
60 min
1)
Cl
TEA
Cl
Cl
DMF/CH2Cl2
MW50°C
60 min
3)FmocGlyOH
TBTU/DIEA
2)MeOH
3)FmocGlyOH
TBTU/DIEA
2)MeOH
Fmoc
Fmoc
Cl
Cl
NH
O
Cl
NH
O
Fmoc
O
O
H
N
O
H
N
O
NH
O
O
O
O
O
Loading: 0.432 mmol/g
Loading: 0.296 mmol/g
Loading: 0.128 mmol/g
Scheme 3. MW immobilization of FmocTyrOAll, FmocSerOAll, and FmocThrOAll onto 2CTC.

L. Rizzi et al. / Tetrahedron Letters 52 (2011) 2808–2811
2811
References and notes
O
O
O
N
H
O
1. García-Martín, F.; Bayó-Puxan, N.; Cruz, L. J.; Bohling, J. C.; Albericio, F. QSAR
Comb. Sci. 2007, 26, 1027–1035.
2. Peptide Synthesis and Application; Howl, J., Ed.; Humana Press: Totowa, New
Jersey, 2005; Vol. 298,.
3. Chemistry of Peptide Synthesis; Benoiton, N. L., Ed.; Taylor & Francis Group: Boca
Raton, 2006.
4. Matthews, D. P.; Green, J. E.; Shuker, A. J. J. Comb. Chem. 1999, 2, 19–23.
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Bioorg. Med. Chem. Lett. 2000, 10, 1935–1938.
O
O
Fmoc
N
SPPS
cycles
N
O
O
O
FmocHN
HN
O
N
O
O
N
N
H
6. Guan, Y.; Green, M. A.; Bergstrom, D. E. J. Comb. Chem. 2000, 2, 297–300.
7. Barco, A.; Benetti, S.; De Risi, C.; Marchetti, P.; Pollini, G. P.; Zanirato, V. J. Comb.
Chem. 2000, 2, 337–340.
OH
O
8. Manku, S.; Laplante, C.; Kopac, D.; Chan, T.; Hall, D. G. J. Org. Chem. 2001, 66,
874–885.
1)Pd(PPh
)
2)Piperidin3e4
N
H
O
N
O
9. Frechet, J. M. J.; Nuyens, L. J. Can. J. Chem. 1976, 54, 926–934.
10. Wenschuh, H.; Beyermann, M.; Haber, H.; Seydel, J. K.; Krause, E.; Bienert, M.;
Carpino, L. A.; El-Faham, A.; Albericio, F. J. Org. Chem. 1995, 60, 405–410.
11. Bernhardt, A.; Drewello, M.; Schutkowski, M. J. Pept. Res. 1997, 50, 143–152.
12. Seo, J.-s.; Yoon, C. M.; Gong, Y.-D. J. Comb. Chem. 2007, 9, 366–369.
13. De Luca, L.; Giacomelli, G.; Riu, A. J. Org. Chem. 2001, 66, 6823–6825.
14. Garner, A. L.; Koide, K. Org. Lett. 2007, 9, 5235–5238.
N
N
O
O
NH
3)HATU,DIPEA
4)TFA
O
O
N
N
H
15. Silva, S. G.; Rodríguez-Borges, J. E.; Marques, E. F.; do Vale, M. L. C. Tetrahedron
2009, 65, 4156–4164.
Stilysin 2
16. Nguyen, H.-H.; Imhof, D.; Kronen, M.; Schlegel, B.; Haertl, A.; Graefe, U.; Gera,
L.; Reissmann, S. J. Med. Chem. 2002, 45, 2781–2787.
17. Vidal-Ferran, A.; Bampos, N.; Moyano, A.; Pericas, M. A.; Riera, A.; Sanders, J. K.
M. J. Org. Chem. 1998, 63, 6309–6318.
Scheme 4. Solid phase synthesis of stylisin 2.
18. Anders, R.; Wenschuh, H.; Soskic, V.; Fischer-Frühholz, S.; Ohlenschläger, O.;
Dornberger, K.; Brown, L. R. J. Pept. Res. 1998, 52, 34–44.
19. Arano, Y.; Akizawa, H.; Uezono, T.; Akaji, K.; Ono, M.; Funakoshi, S.; Koizumi,
M.; Yokoyama, A.; Kiso, Y.; Saji, H. Bioconjugate Chem. 1997, 8, 442–446.
20. Tailhades, J.; Gidel, M.-A.; Grossi, B.; Lecaillon, J.; Brunel, L.; Subra, G.; Martinez,
J.; Amblard, M. Angew. Chem., Int. Ed. 2010, 49, 117–120.
21. Kappe, C.; Dallinger, D. Mol. Diversity 2009, 13, 71–193.
22. Alcazar, J.; Oehlrich, D. Future Med. Chem. 2010, 2, 169–176.
23. Kranjc, K.; Kocevar, M. Curr. Org. Chem. 2010, 14, 1050–1074.
24. General procedure for the Fmoc protection: DIPEA (4 equiv) and then Fmoc-OSu
(1.1 equiv) were added to a solution of aminoalcohol (4 mM in CH2Cl2). The
reaction mixture was stirred overnight at rt. At the end of reaction (TLC
monitoring), EtOAc was added and the organic phase was washed with HCl 1 M
(2 times), saturated NaHCO3 (2 times), water and brine. The organic phase was
dried with anhydrous Na2SO4, concentrated in vacuo and the crude product
was crystallized from CH2Cl2.
FmocSer(tBu)OH, and FmocThr(tBu)OH (see Supplementary data),
were subjected to MW irradiation for 60 min at 50 °C in the pres-
ence of TEA for tyrosine and DBU for serine and threonine
(Scheme 3). Also in the case of the two Fmoc/OAll-protected amino
acids the MW irradiation yielded good results, with a loading of
0.432 mmol/g for tyrosine, 0.220 mmol/g for serine, and
0.128 mmol/g for threonine. If we compare these data with those
obtained by Bernhardt et al.11 it can be noticed that we reached
the best loading in the case of tyrosine and an equivalent loading
in the case of serine and threonine, in a shorter time and without
the use of DMAP, a strong nucleophile which can lead to racemiza-
tion. As proof of concept the synthesis of a cycloheptapeptide char-
acterized by the presence of a tyrosine residue, stylisin 2,29 was
25. Deprotection with 20% piperidine in DMF gives the fulvene–piperidine adduct
which can be determined by quantitative spectrophotometric analysis at
301 nm (E301 = 7800). UV measurements were carried out with a Unicam
carried out (Scheme 4) immobilizing 60 lmol of FmocTyrOAll onto
Hekios
a spectrophotometer.
26. General procedure for microwave-assisted loading of Fmoc-amino alcohols onto
the 2-chlorotrityl resin: Fmoc-amino alcohol (2 equiv) was dissolved in 0.1 ml of
DMF and 1.8 ml of CH2Cl2 and 2CTC (50 mg of a 1.55 mmol/g; IRIS Biotech),
previously swelled with CH2Cl2, was added to the reactor. Then the appropriate
base (10 equiv) was added and the reaction was stirred in sealed reactor under
MW irradiation. At the end of the reaction the excess of reagents was filtered
off and the resin was washed with DMF (6 times). To cleave the Fmoc-group, a
solution of piperidine (20% in DMF) was added to the resin and the mixture
was swirled for 5 min at rt and filtered. Then, another amount of piperidine
(20% in DMF) was added and the mixture swirled for other 15 min. The excess
of reagents was initially filtered off and successively the resin was washed with
DMF (6 times). MW reactions were carried out by Biotage Initiator oven.
27. Wade, J. D.; Bedford, J.; Sheppard, R. C.; Tregear, G. W. Pept. Res. 1991, 4, 194–
199.
28. At the end of the MW reaction, methanol was added to the mixture, in order to
cap the unreacted chloride groups, and the reaction mixture was stirred for
5 min. Then, the excess of reagents was filtered off and the resin was washed
with DMF (6 times). A solution of FmocGlyOH (4 equiv), TBTU (4 equiv, 0.45 M
in DMF) and DIPEA (10 equiv) was added to the resin and the mixture was
stirred for 50 min. At the end of the reaction the excess of reagents was filtered
off and the resin was washed with DMF (6 times) and piperidine (20% in DMF)
was added to the resin to cleave the Fmoc group.
2CTC resin and generating the peptide in solid phase following the
Fmoc/tBu protocols. After the cyclization step, cleavage and purifi-
cation of stylisin 2 was obtained in a 13.3% overall yield. In conclu-
sion, we presented a study about the immobilization of primary,
secondary, and phenol-like alcohols, onto 2CTC resin by means of
MW irradiation: this technique allowed us to achieve the best or
comparable loadings than those obtained with the ‘classical’ meth-
ods in a shorter time (30–60 min maximum) and without employ-
ing toxic bases, such as pyridine, or racemizing agents, such as
DMAP. MW alcohol immobilization was found viable to link to
2CTC, through the hydroxyl group, tyrosine, serine, and threonine,
from which it was possible to obtain cyclic peptides.
Acknowledgment
This work was supported by MIUR (PRIN2008: Leads ad Attività
Antimalarica di Origine Naturale: Isolamento, Ottimizzazione e
Valutazione Biologica).
29. Dahiya, R.; Gautam, H. Mar. Drugs 2010, 8, 2384–2394.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2011.03.113.