Solid-phase synthesis of 2-amino-3-chloro-5- and 8-nitro-1,4- naphthoquinones: A new and general colorimetric test for resin-bound amines

Article abstract of DOI:10.1016/j.tetlet.2005.01.049

Treatment of resin-bound primary or secondary alkyl and arylamines with 2,3-dichloro-5-nitro-1,4-naphthoquinone leads to the rapid formation of intensely colored (red) beads. The resulting 2-amino-3-chloro-5- and 8-nitro-1,4-naphthoquinones can be cleaved rapidly from acid-labile supports in high yields and purities. The reaction is of value as a sensitive and general qualitative test for amino groups on-resin that can be followed by cleavage for characterization and quantification of the chromogen(s) responsible for the color.

Full text of DOI:10.1016/j.tetlet.2005.01.049

Tetrahedron
Letters
Tetrahedron Letters 46 (2005) 1405–1409
Solid-phase synthesis of 2-amino-3-chloro-5- and
8-nitro-1,4-naphthoquinones: a new and general
colorimetric test for resin-bound amines
Christopher Blackburn*
Millennium Pharmaceuticals, 35 Landsdowne Street, Cambridge, MA 02139, USA
Received 17 December 2004; revised 10 January 2005; accepted 11 January 2005
Abstract—Treatment of resin-bound primary or secondary alkyl and arylamines with 2,3-dichloro-5-nitro-1,4-naphthoquinone
leads to the rapid formation of intensely colored (red) beads. The resulting 2-amino-3-chloro-5- and 8-nitro-1,4-naphthoquinones
can be cleaved rapidly from acid-labile supports in high yields and purities. The reaction is of value as a sensitive and general qual-
itative test for amino groups on-resin that can be followed by cleavage for characterization and quantification of the chromogen(s)
responsible for the color.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Over the past decade solid phase synthesis1 has been ap-
plied widely but difficulties in monitoring the progress of
reactions remain a barrier toward more widespread use.
While colorimetric tests for resin-bound functional
groups2–6 are of value, those for amino groups have
their limitations. The Kaiser ninhydrin test7 is limited
to primary aliphatic amines and the harsh reaction con-
ditions can lead to false positives. Ion pairing with a col-
ored anion8 is sensitive but this test will give positive
results for any resin-bound base. Likewise, NPIT9 reacts
with resin-bound alkyl- and arylamines; however, amine
quantification relies on the subsequent release of the
dimethoxytrityl cation and thus two or more amines
on the resin cannot be differentiated. Other tests use col-
ored precursors10 making the color of the resin difficult
to distinguish from that of the solution. The colorimetric
test for secondary amines involving reaction with
chloranil and acetaldehyde11,12 is assumed, on the basis
of solution phase precedent,13,14 to give enamine quin-
ones. Recently, an additional test,15 in which resin-
bound arylamines form red products on treatment with
chloranil (in the absence of acetaldehyde) at 100 ꢁC, has
been reported. Both these tests are qualitative since the
chromogens responsible for the color have not been
cleaved for characterization or quantification. This
paper describes the reaction of weakly colored 2,3-di-
halo-1,4-naphthoquinones with resin-bound amines to
give highly colored beads that release the chromogenic
2-amino-3-chloro-1,4-naphthoquinone derivatives on
treatment with TFA.
Reaction of 2,3-dihalo-1,4-naphthoquinones, 1–bf 3
with primary amines results in the displacement of one
chlorine to give 2-amino-3-chloro-1,4-naphthoquin-
ones.16 While quinones 1–3 are pale yellow in solution
due to tailing of a n–p* absorption into the visible re-
gion, the introduction of the amino group creates p–p*
donor–acceptor chromogens17 which absorb more in-
tensely in the 450–500 nM region of the visible spectrum
and are thus orange to red in color.16
Treatment of Rink amide (RAM)18 resin with saturated
(ca. 200 mM) solutions of 1 or 2 in either DMF or
CH2Cl2 in the presence of 2,6-di-tert-butylpyridine as
base, afforded deep red resins 4-RAM and 5-RAM
(Scheme 1). These resin-bound quinones are, in essence,
vinylogous amides, and it was thus of interest to subject
them to typical amide cleavage conditions. In fact, ami-
no quinones 719 and 820 were isolated in high purities
after treatment with TFA/CH2Cl2 and good yields of
analytically pure products were obtained after filtration
through silica gel (Table 1, entries 1 and 2). In order to
Keywords: 1,4-Naphthoquinone; Solid-phase synthesis; Colorimetric
test.
*
Tel.: +1 617 761 6811; fax: +1 617 444 1483; e-mail: blackburn@
mpi.com
0040-4039/$ - see front matter ꢀ 2005Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2005.01.049

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C. Blackburn / Tetrahedron Letters 46 (2005) 1405–1409
Scheme 1. Reagents and conditions: (i) Resin–NH2, 2,6-di-tert-butylpyridine, 25 ꢁC, 2 h; (ii) TFA–CH2Cl2 (1:1), 25 ꢁC.
Table 1.
Entry Resin
temperatures although intensely colored resins were ob-
served within seconds.25 Subsequent treatment with
TFA/CH2Cl2 (1:1) afforded 9 in good yields and puri-
ties. These cleavage conditions are noteworthy in the
case of MBHA resin for which the standard conditions
for release of amides are usually TFMSA or HF.24
Product Puritya (%) Yieldb (%)
1
RAM
RAM
7
8575
9570
9590
9595
7575
955
90
2
8
3
RAM
RAM plug
9
4
5PAL
9
9
95
The reaction of quinone 3 with resin-bound secondary
amines was next investigated. Several primary amines
of variable steric and electronic properties were an-
chored to FDMP resin (Fig. 1)26 under reducing condi-
tions affording resins 11 (Scheme 2). The resultant alkyl
derivatives were found to react rapidly with quinone 3 in
the presence of 2,6-di-tert-butylpyridine to give the red
resin-bound quinones 12. Treatment with TFA:CH2Cl2
(1:4) led to very rapid cleavage of quinones 13–16 as reg-
ioisomeric mixtures in high purities.27 The isolated
yields listed in Table 1 are based on two steps and thus
also reflect the relative efficiencies of the reductive ami-
nation steps. The regioisomers were formed in approxi-
mately equal amounts and were readily separated by
chromatography on neutral alumina. In the case of mix-
ture 14, the higher Rf component was shown to be the 5-
nitro isomer 14a and the lower Rf component the 8-nitro
6
MBHA
11R = Me
11R = n-Pr
7
13
14
50
8
9570
94
9
11R = phenethyl 15
50
35
10
11
12
11R = i-Pr
11R = Ph
Wang-NH-n-Pr
16
17
14
90
5
9540
0
30
a LC–MS analysis (see Supplementary data).
b Isolated yield after chromatography.
develop an improved substrate, further reactions were
conducted using the 5-nitro compound 3 which is more
reactive toward amines in solution phase reactions
affording regioisomeric mixtures of mono-substitution
products that have higher extinction coefficients than
the parent compounds16 and is approximately 2-fold
more soluble than 1 and 2 in DMF and CH2Cl2. After
removal of the Fmoc group from commercially available
RAM resin by treatment with piperidine–DMF (1:4),21
reaction with 3 led to the instantaneous formation of
deep red beads of 6-RAM clearly visible in the yellow
solution of 3. Cleavage was also rapid, two 15min treat-
ments with TFA–CH2Cl2 (1:1) affording the regioiso-
meric mixture of amino-quinones 9 in high yield and
purity (entry 3). Similar results were obtained with
RAM resin in the plugTM format.22 The amine function-
alized resins PAL23 and MBHA24 (Fig. 1) were also al-
lowed to react with quinone 3. On a preparative scale,
reactions were allowed to proceed for 2 h at ambient
1
isomer 14b by H–13C (HMBC) analysis. For compari-
son, n-propylamine was anchored to a resin with a less
electron-rich linker by reaction with Wang chloride
(Fig. 1). Subsequent reaction with 3, followed by cleav-
age also gave 14a/b in high purity and yield (entry 12).
While reactions were allowed to proceed for longer peri-
ods on a preparative scale it was possible to conduct a
5min qualitative test in which alkyl derivatives of resin
11 were allowed to react with 3 for 2 min followed by
washing, cleavage for 2 min, and TLC analysis on neu-
tral alumina. Deep red beads that afford a red cleavage
solution on treatment with TFA which, on TLC analy-
Figure 1. Polystyrene-based resins described in the text.

C. Blackburn / Tetrahedron Letters 46 (2005) 1405–1409
1407
Scheme 2. Reagents and conditions: (i) RNH2, HOAc, NaBH(OAc)3, DMF, 25 ꢁC, 16 h; (ii) ArNH2, HOAc, NaBH3CN, DMF, 25 ꢁC, 16 h; (iii) 3,
2,6-di-tert-butylpyridine, CH2Cl2, 25 ꢁC, 2 h; (iv) TFA–CH2Cl2 (1:1), 25 ꢁC, 0.25h.
sis, affords two red bands of approximately equal inten-
sities constitutes a positive test for secondary amines. It
should be noted that prior to reaction with 3, the amines
are not readily cleaved from resins 11. In the case of re-
sin 11 (R = Ph),6 reaction with 3 was noticeably slower.
However, heating under microwave irradiation28 for
10 min in dichloroethane at 80 ꢁC afforded intensely
red beads of 12 (R = Ph), cleavage of which afforded
17 in moderate purity.
tection, allowed to react with 3 in the presence of 2,6-di-
tert-butylpyridine. A coupling reaction in which the
respective mole ratios were 1:99 afforded pink beads that
were cleaved and analyzed by TLC showing the presence
of 18; thus, the sensitivity of the test is <10 lmol/g
resin.29
Reaction of 3 with H-Ala-RAM resin proved to be
slower than that with the analogous b-Ala-RAM pre-
sumably due to both inductive and steric effects.
Although orange beads were observed within a few min-
utes at ambient temperature, overnight reactions were
necessary in order to obtain moderate yields of 19 fol-
lowing cleavage. However, microwave irradiation of
the reaction mixture for 10 min at 150 ꢁC in dichloroeth-
ane led to the formation of intensely red beads from
which 19 was isolated following cleavage in 90% yield
and 85% purity (Scheme 4).
To assess the value of quinone 3 for monitoring a typical
solid-phase synthesis, several coupling reactions between
Na-Fmoc-b-Ala-OH and RAM resin were conducted in
which the mole ratio of DIC was varied from 0.1 to
4 equiv (Scheme 3). The (partially) coupled resins were
deprotected with piperidine then treated with 3 and
2,6-di-tert-butylpyridine for 1 h at ambient temperature
and cleaved. After cleavage, the coupling involving the
use of 4 equiv of DIC afforded 18 in 80% yield.
In conclusion, this paper describes the reaction of 2,3-di-
chloro-5-nitro-1,4-naphthoquinone (3) with resin-bound
amines to give highly colored products rapidly with high
sensitivity. The resulting 2-amino-1,4-naphthoquinones
can be cleaved from acid-labile resins in high yields
and purities. The chromogenic products derived from
secondary amines are activated toward cleavage and af-
ford intensely colored solutions within seconds that can
be analyzed rapidly by TLC, LC–MS or visible spectro-
scopy. The test provided by Scheme 2 improves on the
TLC analysis of the remaining cleavage products
showed the presence of isomeric pair 9 (two closely elut-
ing orange spots) and the b-Ala derivatives 18 (two clo-
sely eluting red bands) the relative amounts of which
were in accord with the amount of DIC used in the cou-
pling step. To assess the sensitivity of this colorimetric
test, mixtures of aN-Fmoc–Ala-OH and 3-(3,4-dime-
thoxy-phenyl)-propionic acid in various mole ratios
were coupled to RAM resin, and following Fmoc depro-
Scheme 3. Reagents and conditions: (i) Fmoc-b-Ala-OH, DIEA, DIC, HOBt, DMAP, DMF, 25 ꢁC; (ii) piperidine–DMF (1:4), 0.25h (iii) 3, 2,6-di-t-
butylpyridine, CH2Cl2, 25 ꢁC, 2 h; (iv) TFA–CH2Cl2, (1:1) 25 ꢁC, 0.25h.

1408
C. Blackburn / Tetrahedron Letters 46 (2005) 1405–1409
Scheme 4. Reagents and conditions: (i) 3, 2,6-di-tert-butylpyridine, C2H2Cl2, 15 0ꢁC, lWI; (ii) TFA–CH2Cl2 (1:1).
N.; Hosten, N. G. C.; De Muynck, H.; De Clerq, P. J.;
chloranil test (the products from which are hydrolyti-
cally unstable and cannot be cleaved) and is complemen-
tary to the 2,4-DNP5,6 test for resin-bound aldehyde
groups. Further work on the quantitative aspects of
the test is in progress and will be reported in due course.
Barry, J.; Davis, A. P. Eur. J. Org. Chem. 1999, 7, 2787.
11. Christensen, T. Acta Chem. Scand. B 1979, 33, 763–766.
12. Vojkovsky, I. Pept. Res. 1995, 8, 236–237.
13. Buckley, D.; Henbest, H. B.; Slade, P. J. Chem. Soc. 1957,
4891–4900.
14. Blackburn, C.; Griffiths, J. J. Chem. Res. S 1983, 7, 168–
169, M, 1556.
15. Marik, J.; Song, A.; Lam, K. S. Tetrahedron Lett. 2003,
44, 4319–4320.
Acknowledgements
The author thanks Ian Parsons for assistance in the ori-
entation of regioisomers by NMR spectroscopy, Claire
Santos for HRMS, and Kevin Sprott for helpful
comments.
16. Chu, K. Y.; Griffiths, J. J. Chem. Soc., Perkin Trans. 1
1978, 1083–1087.
17. Griffiths, J. The Colour and Constitution of Organic
Molecules; Academic, 1976, 173–180.
18. Purchased from Polymer Labs., Amherst, MA. This resin
is also known as aminomethyl-PS with the Knorr linker,
see: Bernatowicz, M. S.; Daniels, S. B.; Koster, H.
Tetrahedron Lett. 1989, 30, 4645–4648.
19. Hoover, J. R. E.; Day, A. R. J. Am. Chem. Soc. 1954, 76,
4148–4152.
20. Gaultier, J.; Hauw, C. Acta Crystallogr. 1966, 20, 620–
625.
21. Failure to wash the resin thoroughly results in formation
of the purple piperidine substitution product of 3 which
obscures the color of the beads.
22. (a) Atrash, B.; Bradley, M.; Kobylecki, R.; Cowell, D.;
Reader, J. Angew. Chem., Int. Ed. 2001, 40(5), 938–941;
(b) Atrash, B.; Reader, J.; Bradley, M. Tetrahedron Lett.
2003, 44, 4779–4782.
Supplementary data
Synthetic details for the solid-phase synthesis of com-
pounds 7, 8, and 9. Characterization data for com-
pounds 15, 16, and 17. HMBC correlations for 14a
and 14b. TLC image of quinones prepared according
to Scheme 2. The supplementary data are available on-
line with the paper in ScienceDirect. Supplementary
data associated with this article can be found, in the on-
line version, at doi:10.1016/j.tetlet.2005.01.049.
23. Albericio, F.; Knieb-Cordonire, S.; Biancalana, L.; Gera,
L.; Masada, R. I.; Hudson, D.; Barany, G. J. Org. Chem.
1990, 55, 3730–3734.
References and notes
24. Matsueda, G. R.; Stewart, J. M. Peptides 1981, 2, 45–50.
25. No reaction was observed when the Fmoc protected
versions of the RAM or PAL resins were subjected to the
same reaction conditions.
26. Boojamra, C. G.; Burow, K. M.; Ellman, J. A. J. Org.
Chem. 1995, 60, 5742–5743.
1. (a) See for example, Solid-Phase Synthesis. A Practical
Guide; Kates, S. A., Albericio, F., Eds.; Marcel Decker:
New York, 2000; (b) Guillier, F.; Orain, D.; Bradley, M.
Chem. Rev. 2000, 100, 3859.
2. Kay, C.; Lorthioir, O. E.; Parr, N. J.; Congreve, M.;
McKeown, S. C.; Scicinski, J. J.; Ley, S. V. Biotechnol.
Bioeng. 2002, 71(2), 110–118.
3. Bunin, B. A. The Combinatorial Index; Academic, 1998,
pp. 214–220.
4. Gaggini, F.; Porcheddu, A.; Reginato, G.; Rodriquez, M.;
Taddei, M. J. Comb. Chem. 2004, 6, 805–810.
5. Shannon, S. K.; Barany, G. J. Comb. Chem. 2004, 6, 165–
170.
6. Shannon, S. K.; Barany, G. J. Org. Chem. 2004, 69, 4586–
4594.
7. (a) Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P.
I. Anal. Biochem. 1970, 34, 595–598; (b) Sarin, V. K.;
Kent, S. B. H.; Tam, J. P.; Merrifield, R. B. Anal.
Biochem. 1981, 117, 145–157.
8. (a) Krchnak, V.; Vagner, J.; Lebl, M. J. Pept. Protein Res.
1988, 32, 416–425; (b) Krchnak, V.; Vagner, J.; Safar, P.;
Lebl, M. Collect. Czech. Chem. Commun. 1998, 53, 2542–
2548; (c) Oded, A.; Houghten, R. A. Pept. Res. 1990, 3, 42.
9. Chu, S. S.; Reich, S. H. Bioorg. Med. Chem. Lett. 1995, 5,
1053–1058.
27. Resin 11 (Supplementary information) R = nPr, (250 mg,
0.35mmol) was treated with a solution of 3 (2.5equiv) in
CH2Cl2 followed by addition of 2,6-di-tert-butylpyridine
(2 equiv). The reaction mixture was protected from light
and allowed to stand for 2 h then washed with CH2Cl2,
(3·), MeOH (3·), CH2Cl2 (5·). Cleavage was effected by
treatment with TFA–CH2Cl2 (1:4) (2 · 5min). The cleav-
age solution was removed, combined with CH2Cl2 wash-
ings of the resin, evaporated under reduced pressure and
analyzed by LC–MS. Chromatography on a column of
neutral alumina eluting with hexane–CH2Cl2 gradient
afforded 14a (34 mg, 33% yield) from the more mobile
band and 14b (38 mg, 37%) from the slower eluting band.
14a 1H NMR (CDCl3, 300 MHz, d) 8.36 (1H, d, J = 9 Hz),
7.86 (1H, dd, J = 9 Hz), 7.63 (1H, d, J = 9 Hz), 3.80 (2H,
m), 1.70 (2H, m), 1.01 (3H, t, J = 7 Hz). kmax 486 nm, emax
3300 MÀ1 cmÀ1 (CH2Cl2). 14b 1H NMR (CDCl3,
300 MHz, 8.22 (1H, d, J = 9 Hz), 7.76 (1H, dd,
J = 9 Hz), 7.66 (1H, d, J = 9 Hz), 3.80 (2H, m), 1.72
(2H, m), 1.02 (3H, t, J = 9 Hz). kmax 483 nm, emax
3400 MÀ1 cmÀ1 (CH2Cl2). Compounds 13, 15, and 16
were prepared similarly and the crude products purified by
10. (a) Shah, A.; Rahman, S. S.; de Biasi, V.; Camilleri, P.
Anal. Commun. 1997, 34, 325–328; (b) Madder, A.; Farcy,

C. Blackburn / Tetrahedron Letters 46 (2005) 1405–1409
1409
filtration through a plug of silica gel and characterized as
regioisomeric mixtures (Supplementary information). All
new compounds were also characterized by HRMS.
28. More prolonged heating at higher temperatures resulted in
release of 17 into solution evidently due to thermally
induced cleavage. Quinone 17 has been reported previ-
ously: Kasai, T.; Kurabayaschi, Y.; Suzuki, Y.; Tsuruoka,
S. J. Synth. Org. Chem. Jpn. 1969, 27, 162.
(3 equiv) in DMF (Ref. 5). When Fmoc-b-Ala-OH and 3-
(3,4-dimethoxy-phenyl)-propionic acid were coupled in
the ratio 3:1; 1:1; 1:3; and 1:99 the intensity of the bead
color decreased from deep red to pink. Cleavage followed
by quantification of 18 using visible spectroscopy (the
extinction coefficient of pure 18 was found to be
3400 MÀ1 cmÀ1) gave respective loadings that were
consistent with the relative amounts of the carboxylic
acids used in the coupling step, the last of which was
ꢀ10 lmol/g. Thus, the sensitivity of the test is <10 lmol/g.
29. Mixture couplings were effected using 3 combined equiv-
alent of carboxylic acid, HATU (3 equiv) and DIEA