4-(9-Fluorenylmethyloxycarbonyl)phenylhydrazine (FmPH): A new chromophoric reagent for quantitative monitoring of solid-phase aldehydes

Article abstract of DOI:10.1021/jo049830b

A direct method for quantifying solid-phase aldehydes has been developed, using a new reagent, 4-(9-fluorenylmethyloxycarbonyl)phenylhydrazine (FmPH). The FmPH reagent was synthesized in three steps (24% overall yield) from commercially available p-hydraz

Full text of DOI:10.1021/jo049830b

4-(9-F lu or en ylm eth yloxyca r bon yl)p h en ylh yd r a zin e (F m P H): A
New Ch r om op h or ic Rea gen t for Qu a n tita tive Mon itor in g of
Solid -P h a se Ald eh yd es1-3
Simon K. Shannon and George Barany*
Department of Chemistry, University of Minnesota, 207 Pleasant Street S.E.,
Minneapolis, Minnesota 55455
barany@umn.edu
Received J anuary 29, 2004
A direct method for quantifying solid-phase aldehydes has been developed, using a new reagent,
4-(9-fluorenylmethyloxycarbonyl)phenylhydrazine (FmPH). The FmPH reagent was synthesized
in three steps (24% overall yield) from commercially available p-hydrazinobenzoic acid. Resin-
bound aldehydes reacted quantitatively with FmPH, in the presence of trimethylorthoformate
(TMOF) as a dehydrating agent, to form a highly conjugated, immobilized FmPH-hydrazone. Next,
mild treatment of the hydrazone with an excess of piperidine-N,N-dimethylformamide (1:1) released
the piperidine-dibenzofulvene adduct chromophore (ꢀ301nm ) 7800 M-1 cm-1) from the support.
FmPH quantitation of aldehydes proved to be a straightforward, sensitive, and reproducible
technique for monitoring resin-bound aldehydes [albeit insufficiently reactive to allow reliable
quantification of ketones]. The FmPH aldehyde assay is applicable to a range of solid supports, as
demonstrated specifically for poly(ethylene glycol)-polystyrene (PEG-PS), aminomethylpolystyrene
(AMP), and cross-linked ethoxylate acrylate resin (CLEAR).
In tr od u ction
lenges, because classical techniques for following the
course of reactions, such as TLC, do not apply to polymer-
supported intermediates. Additionally, on-bead NMR,23-31
IR,32-36 MS,22,37,38 and more recently electrochemical
impedance spectroscopy39 (ESI) experiments are not
straightforward. A viable way to probe the progress of a
solid-phase reaction is to cleave the intermediate from
the linker/support40,41 and then use classical techniques
for characterization and/or quantitation. However, such
approaches may be less than advantageous to the solid-
Methods for solid-phase synthesis4-12 and combinato-
rial chemistry12-18 have been established as essential
tools for drug discovery in the pharmaceutical and
biotechnology industries. As more and more solid-phase
reactions are worked out, reliable and robust methods
for solid-phase reaction monitoring5,9,19-22 must also be
developed. The solid-phase mode presents several chal-
(1) Taken in part from Ph.D. Thesis of Simon Kelly Shannon,
University of Minnesota, Minneapolis, MN, November 2003.
(2) Portions of this work were reported in preliminary form:
Shannon, S. K.; Barany, G. In Innovation and Perspectives in Solid-
Phase Synthesis & Combinatorial Libraries, Drug Discovery, Develop-
ment & Delivery, Antibody & Vaccine Strategies, Collected Papers,
Eighth International Symposium; Epton, R., Ed.; Mayflower Worldwide
Ltd.: London, U.K., in press.
(4) Merrifield, R. B. J . Am. Chem. Soc. 1963, 85, 2149-2154.
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Chemistry; Czarnik, A. W., DeWitt, S. H., Eds.; American Chemical
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(6) Nefzi, A.; Ostresh, J . M.; Houghten, R. A. Chem. Rev. 1997, 97,
449-472.
(3) Abbreviations used are as follows: AMP, aminomethylpolysty-
rene; BAL, backbone amide linker; Boc, tert-butyloxycarbonyl; CLEAR,
cross-linked ethoxylate acrylate resin or poly(trimethylpropane ethoxy-
late (14/3 EO/OH) triacrylate-co-allylamine); DCC, N,N′-dicyclohexyl-
carbodiimide; DIEA, N,N-diisopropylethylamine; DIPCDI, N,N′-diiso-
propylcarbodiimide; DMAP, 4-(N,N-dimethylamino)pyridine; DMF,
N,N-dimethylformamide; DNPH, 2,4-dinitrophenylhydrazine; ESI,
electrochemical impedance spectroscopy; Fm, 9-fluorenylmethyl; Fmoc,
9-fluorenylmethoxycarbonyl; FmPH, 4-(9-fluorenylmethyloxycarbonyl)-
phenylhydrazine; HRESI-MS, high-resolution electrospray ionization
mass spectrometry; HATU, N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-
b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophos-
phate N-oxide; HOAc, acetic acid; HOBt, 1-hydroxybenzotriazole;
p-PALdehyde, 4-formyl-(3,5-dimethoxyphenoxy)valeric acid; PEGA,
poly(N,N-dimethacrylamide-co-bisacrylamido-poly(ethylene glycol)-co-
monoacrylamido-poly(ethylene glycol)); PEG-PS, poly(ethylene glycol)-
polystyrene (graft resin support); Purpald, 4-amino-3-hydrazino-5-
mercapto-1,2,4-triazole; TEA, triethylamine; TFA, trifluoroacetic acid;
THF, tetrahydrofuran; TMOF, trimethyl orthoformate. All amino acids
denote the L-configuration unless indicated otherwise. Abbreviations
used for amino acids follow the IUPAC-IUB Commission of Biochemical
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10.1021/jo049830b CCC: $27.50 © 2004 American Chemical Society
Published on Web 06/08/2004
4586
J . Org. Chem. 2004, 69, 4586-4594

Quantitative Monitoring of Solid-Phase Aldehydes
phase chemist because (i) not all intermediates are stable
to cleavage conditions, (ii) low-load resins may not
provide enough product for isolation and characterization,
(iii) sophisticated equipment might be needed, and (iv)
the process may entail an unacceptable time delay in
developing the information needed to make informed
decisions on how to proceed with the synthesis. Fortu-
nately, a variety of rapid on- and off-bead analytical
techniques are available for monitoring some solid-phase
functional groups.5,9,19-22
phase aldehyde transformations involving redox chem-
istry, condensations, and C-C bond formation, among
others (see various reviews6-12,49-55), companion methods
to monitor these common conversions are needed. We
recently reported on a qualitative colorimetric test to
monitor solid-phase aldehydes using 2,4-dinitrophenyl-
hydrazine (DNPH),45,56 to go with literature methods that
use p-anisaldehyde43 or 4-amino-3-hydrazino-5-mercapto-
1,2,4-triazole (Purpald).44 Nevertheless, a simple and
robust quantitative protocol for resin-bound aldehydes
was still required.
Quantitative measurements of resin-bound functional
groups can be valuable when calculating loadings, opti-
mizing reactions, and establishing yields (extent of
conversion) of solid-phase transformations. To the best
of our knowledge, the only previously described method
to quantitate solid-phase aldehydes uses fluorescence
spectroscopy to measure the uptake of dansyl hydrazine
from a supernatant solution by resin-bound aldehydes.42
While this method is reported to be quite sensitive, it is
also tedious and only serves indirectly for quantification.
In search of a simpler and more direct route to quantify
solid-phase aldehydes, we were interested in designing
a stable chromophore-based “reagent” (1, Scheme 1) that
would react quantitatively with the aldehyde to provide
an immobilized intermediate (2). Subsequent mild and
selective release of chromophore 3, followed by its sensi-
tive measurement by ultraviolet-visible (UV-vis) spec-
troscopy, would provide an accurate result that could be
related directly to the absolute amount of aldehyde.
Literature protocols for quantifying amines,57-65
thiols,66,67 and alcohols68,69 during solid-phase peptide/
organic synthesis follow this straightforward model.
The present article describes the synthesis of a new
chromophoric hydrazine-based reagent, 4-(9-fluorenyl-
We are currently interested in the qualitative/quanti-
tative monitoring of solid-phase aldehydes using on-bead
chemical derivatization methods.42-45 Given the practical
importance of on-resin reductive amination46-48 in back-
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Shannon and Barany
SCHEME 1. P r in cip le of On -Bea d Im m obiliza tion ,
Relea se of Ch r om op h or e, a n d Off-Bea d
Sp ectr oscop ic An a lysis
FIGURE 1. 4-(9-Fluorenylmethyloxycarbonyl)phenylhydrazine
(FmPH) (4). This new reagent contains a hydrazine group that
can be made to react quantitatively with solid-phase alde-
hydes. It also contains a chromophoric 9-fluorenylmethyl group
that can be released and quantified after FmPH immobiliza-
tion on solid supports.
SCHEME 2. P r op osed Rea ction of H-Gly-OF m ·HCl
w ith BAL Su p p or ts
methyloxycarbonyl)phenylhydrazine (FmPH) (4, Figure
1), and how it can be applied according to the aforemen-
tioned concept for the sensitive and direct quantification
of resin-bound aldehydes. Both the reagent attachment
and chromophore release step are readily made to go to
completion. Note that in the FmPH assay, quantitative
removal of the 9-fluorenylmethyl (Fm) group gives the
same piperidine-dibenzofulvene adduct chromophore
that is released in 9-fluorenylmethyloxycarbonyl (Fmoc)
quantitation of amines in peptide synthesis.59,65
Consequently, efforts shifted toward design and syn-
thesis of a suitable hydrazine-based reagent that would
potentially form a hydrazone at much faster rates and
with higher yields. The reactions of aldehydes with
substituted hydrazines to form hydrazones are introduc-
tory textbook organic chemistry,71,72 and have been
applied in several solid-phase syntheses. For example,
immobilized hydrazine linkers have been condensed with
exogenous aldehydes to access R-branched amines.73-75
Alternatively, hydrazines (e.g., dansyl hydrazine, or 2,4-
dinitrophenylhydrazine) have been condensed with resin-
bound aldehydes for other specialty applications.42,45,56,76-78
P r ep a r a tion of F m P H (Sch em e 3). Commercially
available 4-hydrazinobenzoic acid (8) was treated with
di-tert-butyl dicarbonate in the presence of catalytic
4-(N,N-dimethylamino)pyridine (DMAP) at 60 °C79 to
Resu lts a n d Discu ssion
P ilot Stu d ies a n d Ra tion a le. Initial attempts to
quantify resin-bound aldehydes involved the use of the
chromophoric reagent, H-Gly-OFm‚HCl (5), which was
expected to serve for aldehyde quantification by way of
imine formation with the BAL system49,50,70 (6). Despite
extensive efforts, coupling to form 7 occurred at best to
48% under near-forcing conditions [5 (10 equiv) plus N,N-
diisopropylethylamine (DIEA, 3 equiv), in N,N-dimeth-
ylformamide (DMF)-trimethyl orthoformate (TMOF)
(1:1, 1 mL), for 2 h at 80 °C] (Scheme 2).
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4588 J . Org. Chem., Vol. 69, No. 14, 2004

Quantitative Monitoring of Solid-Phase Aldehydes
SCHEME 3. P r ep a r a tion of F m P H·TF A
SCHEME 4. Rea ction of F m P H·TF A w ith BAL
Su p p or ts To F or m Resin -Bou n d Hyd r a zon e
Rea ction of F m P H‚TF A w ith Resin -Bou n d Ald e-
h yd es. With the goal of achieving quantitative condensa-
tion, several solvent/catalyst systems were evaluated to
facilitate the reaction of hydrazine 4 with BAL-PEG-PS
(6a )49,50 (Scheme 4 and Table 1). To evaluate the level of
FmPH-hydrazone (11) formation, 6a was exposed to the
conditions listed, and at appropriate time periods, 11 was
treated with an excess of piperidine-DMF (1:1) for 30
min at 25 °C to quantitatively remove the Fm ester81-84
(Scheme 5). The resultant piperidine-dibenzofulvene
adduct (13) released into solution was monitored by UV-
vis spectroscopy59,65 at 301 nm. Conditions that gave the
value closest to the known loading of 6a were considered
optimal.
Optimal formation of 11 occurred when resin 6a was
condensed with a solution of 4 (3 equiv) plus DIEA, 3
equiv in DMF-TMOF (1:1) for 30 min at 80 °C. These
conditions gave reproducible, near-quantitative loadings
(Table 1, entry K). Other catalyst/solvent systems gave
comparable results at 80 °C [e.g., DIEA (6 equiv) in
MeOH-EtOH-TMOF (2:1:1), entry E; DIEA, 3 equiv in
MeOH-EtOH-TMOF (2:1:1), entry H; or DIEA (6 equiv)
in DMF-TMOF (1:1), entry L]. The use of TMOF as a
dehydrating agent85 was necessary to achieve quantita-
tive results (see entries E, H, K, and L and compare to
G and N). None of the acid-catalyzed protocols showed
the full expected loading, presumably due to premature
cleavage of the hydrazone over the extended reaction
periods used (entries A, B, and C). However, in the
presence of excess base, the full expected loading was
observed (compare entry E with entries H, K, and L). At
ambient temperatures, with reaction times between 2
and 24 h, the level of hydrazone formation was still
incomplete (entries M, N, and O).
provide the N,N,N′-tris[tert-butyloxycarbonyl (Boc)]-
protected hydrazino acid 9,80 which without purification
was esterified with 9-fluorenylmethanol with use of N,N′-
dicyclohexylcarbodiimide (DCC) in the presence of DMAP
as a catalyst. Silica gel chromatography gave exclusively
the N,N,N′-tris intermediate 10, which was treated with
trifluoroacetic acid (TFA) to remove all Boc groups and
provided FmPH (4, isolated as its trifluoroacetate salt)
in 24% overall yield.
Kin etics of Rea ction s. To gain further insight into
these reactions, the formation of hydrazone 11 and the
release of dibenzofulvene adduct 13 were studied as a
function of time (Figure 2). In summary, formation of
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J . Org. Chem, Vol. 69, No. 14, 2004 4589

Shannon and Barany
loading
TABLE 1. Op tim iza tion of P r otocol to P r ep a r e Hyd r a zon e 11
condensation conditions
solvent (1 mL)c
entrya
catalyst (equiv)b
temp (°C)
time (h)
(mmol/g)d
A
B
C
D
E
F
G
H
I
HOAc (5%, v/v)
H2SO4 (5%, v/v)
HCl (5%, v/v)
TEA (6)
DIEA (6)
pyridine (6)
DIEA (3)
MeOH-EtOH-TMOF (2:1:1)
MeOH-EtOH-TMOF (2:1:1)
MeOH-EtOH-TMOF (2:1:1)
MeOH-EtOH-TMOF (2:1:1)
MeOH-EtOH-TMOF (2:1:1)
MeOH-EtOH-TMOF (2:1:1)
DMF
80
80
80
80
80
80
80
80
80
80
80
80
25
25
25
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
2
0.40
0.35
0.43
0.43
0.50
0.21
0.27
0.51
0.25
0.18
0.51
0.51
0.34
0.18
0.39
DIEA (3)
MeOH-EtOH-TMOF (2:1:1)
no additional
no additional
DIEA (3)
DIEA (6)
DIEA (6)
DMF-TMOF (1:1)
J
MeOH-EtOH-TMOF (2:1:1)
DMF -TMOF (1:1)
K
L
M
N
O
DMF -TMOF (1:1)
DMF-TMOF (1:1)
DIEA (3)
DIEA (3)
DMF
2
24
DMF-TMOF (1:1)
a
For each entry, 6a (∼10 mg, 0.53 mmol of CHO per g of resin) was condensed with 4 (3 equiv), using the listed catalyst, solvent,
b
temperature, and time. All bold entries represent optimal conditions. Abbreviations are defined in footnote 2. For all acid-catalyzed
protocols (entries A, B, and C), the designated v/v ratio was used with respect to 1 mL of solvent. For all other catalysts, parentheses
indicate the number of equivalents with respect to the amount of resin. c For each solvent system, the total volume was 1 mL. In cases
where TMOF was present, 4 was first dissolved in the other cosolvent(s) (e.g., MeOH, EtOH, and/or DMF), followed by addition of the
d
catalyst (i.e., acid or base), and then TMOF. Loadings were calculated following the procedure under “FmPH quantitation” described in
the Experimental Section.
SCHEME 5. Relea se of P ip er id in e-
Diben zofu lven e Ad d u ct fr om Resin -Bou n d
Hyd r a zon e
F IGURE 2. (A) Kinetics of formation (0) of hydrazone 11 with
support 6b (Scheme 4). (B) Kinetics of release (b) of diben-
zofulvene adduct 13 from hydrazone 11 (Scheme 5). (C)
Kinetics of formation (4) of imine 7 with support 6b (Scheme
2). All calculations are described in the Experimental Section.
Note: BAL-AMP (0.73 mmol of CHO per g of resin) (6b) was
used instead of BAL-PEG-PS (6a ) for all experiments described
in this figure (see Schemes 2, 4, and 5).
7 took nearly 2 days to go to completion, it is clear that
the title approach with FmPH is the superior way to
quantify solid-phase aldehydes.
F m P H Qu a n tita tion ver su s F m oc Qu a n tita tion .
Our previous work described the qualitative monitoring
of polymer-supported aldehydes with DNPH.45,56 The
lower limit of sensitivity for the DNPH test was estab-
lished after testing several partially functionalized al-
dehyde supports (16, Scheme 6), which were prepared
hydrazone 11 proceeded quantitatively within 10-20 min
(Figure 2A, squares), and piperidine-dibenzofulvene
adduct 13 was released quantitatively from hydrazone
by coupling mixtures of 4-formyl-3,5-dimethoxyphenoxy)-
11 within 10 min (Figure 2B, circles). Studies A and B
combined show that FmPH quantitation of aldehydes
could ideally be carried out in approximately 30 min. Also
of significance, it was shown that as low as 2 µmol of
aldehyde could be quantified by FmPH, with a reproduc-
ibility of (3%.
valeric acid (PALdehyde)86 (14) and Fmoc-Gly-OH (15)
[combined 3 equiv carboxylic acid function in prespecified
ratios] to commercially available amino-functionalized
supports [poly(ethylene glycol)polystyrene (PEG-PS), 0.53
mmol/g;87,88 aminomethylpolystyrene (AMP), 0.73 mmol/
In addition, hydrazine 4 and amine 5 were condensed
with BAL supports in side-by-side experiments, to com-
pare the rates of hydrazone versus imine formation
(Figure 2, part A vs part C). Since the formation of imine
(86) Albericio, F.; Kneib-Cordonier, N.; Biancalana, S.; Gera, L.;
Masada, R. I.; Hudson, D.; Barany, G. J . Org. Chem. 1990, 55, 3730-
3743.
(87) Zalipsky, S.; Chang, J . L.; Albericio, F.; Barany, G. React. Polym.
1994, 22, 243-258.
4590 J . Org. Chem., Vol. 69, No. 14, 2004

Quantitative Monitoring of Solid-Phase Aldehydes
TABLE 2. Com p a r ison betw een F m oc a n d F m P H
Qu a n tita tion of Ald eh yd es on P EG-P S, AMP , a n d CLEAR
Su p p or ts
SCHEME 6. P r ep a r a tion of P a r tia lly Su bstitu ted
Ald eh yd e Su p p or ts
quantity of aldehyde (%)
partial aldehyde
support (16)a
Fmocb
FmPHc
BAL-PEG-PS
BAL-AMP
BAL-CLEAR
57
50
49
54
51
50
a
See Experimental Section and Scheme 6 for the procedure to
b
prepare partially substituted aldehyde supports. Indicates aver-
age of three separate experiments. Starting with ∼10 mg of 16,
Fmoc quantitation59,65 allowed loading of Fmoc-Gly to be deter-
mined. Theoretical loadings of commercially available amino-
functionalized supports were determined by coupling Fmoc-Gly-
OH to the support, and then submitting to Fmoc quantitation.
Percent Fmoc-Gly-OH was determined by taking the calculated
loading of Fmoc-Gly-OH and dividing by the theoretical loading
of the respective support. Percent aldehyde was then calculated
indirectly by subtracting the percent of Fmoc-Gly-OH from 100%.
c Indicates average of three separate experiments. 16 (∼10 mg)
was treated by using the protocol of note footnote b to release Fmoc
and provide the free NH2. The remaining aldehydes were quanti-
fied directly by using FmPH quantitation.
SCHEME 7. Red u ctive Am in a tion of Am in es w ith
BAL-AMP
g;89 cross-linked ethoxylate acrylate resin (CLEAR, 0.46
mmol/g);90 and poly(N,N-dimethacrylamide-co-bisacryla-
mido-poly(ethylene glycol)-co-monoacrylamido-polyeth-
ylene glycol) (PEGA, 0.26 mmol/g)91,92]. This experimental
design makes it possible to determine indirectly the
absolute amount of PALdehyde present on 16, by Fmoc
quantitation.45,59,65
For the present work, a goal was to compare the
indirect Fmoc quantitation59,65 versus the direct FmPH
quantitation as methods to quantify aldehydes. Thus, a
1:1 ratio of 14 and 15 was coupled to PEG-PS, AMP, and
CLEAR supports giving rise to resins 16 with ap-
proximately 50% loading of PALdehyde. The absolute
amount of aldehyde was determined to be between 49%
and 57% (Table 2), proving that both methods give
consistent and accurate results. Nevertheless, FmPH
quantitation is probably more accurate because it in-
volves direct reaction with residual resin-bound alde-
hydes, as opposed to the indirect procedure with Fmoc-
Gly-OH.
Ap p lica tion of F m P H Qu a n tita tion to a Ra n ge of
Ald eh yd e Su p p or ts. During a recent BAL solid-phase
synthesis of lidocaine and procainamide analogues,56 the
conversion of BAL to secondary amines via reductive
amination was a critical step that significantly influenced
the overall yield and purity of the final product mixture
after cleavage. We revisited this step, using FmPH
quantitation to monitor NaBH3CN-mediated reductive
aminations of 6b with a range of primary aliphatic and
aromatic amines. The aforementioned reaction provided
secondary amines 17, and in some cases, residual unre-
acted aldehydes (Scheme 7). After 30 min at 25 °C,
percent conversions for the reductive amination were
largely in the range of 70-97%, as calculated by subse-
quent FmPH quantitation of the unreacted resin-bound
aldehyde (Table 3). As predicted, conversions improved
as the amine was changed from 2,6-dimethylaniline
(entry P) to aniline (entry Q) to benzylamine (entry R);
this could be due to either steric or electronic effects to
the amino functionality. Unhindered primary aliphatic
amines, including amino acid tert-butyl esters (entries
S, T, and U), converted relative to the same extent under
the conditions evaluated. Conversion with H-Phe-OtBu‚
HCl (entry V) was slightly lower than that with the other
aliphatics, again probably due to steric interferences from
the benzyl group.
(88) Barany, G.; Albericio, F.; Kates, S. A.; Kempe, M. In Chemistry
and Biological Application of Polyethylene Glycol; Harris, J . M.,
Zalipsky, S., Eds.; American Chemical Society Books: Washington, DC,
1997; pp 239-264.
(89) Mitchell, A. R.; Kent, S. B. H.; Erickson, B. W.; Merrifield, R.
B. Tetrahedron Lett. 1976, 3795-3798.
FmPH quantitation was extended to other aldehyde
supports related to our research (Figure 3). Supports
were quantified that contained aromatic aldehydes with
activating groups [methoxy (6a /b), hydroxy (18)] in the
(90) Kempe, M.; Barany, G. J . Am. Chem. Soc. 1996, 118, 7083-
7093.
(91) Meldal, M. Tetrahedron Lett. 1992, 33, 3077-3080.
(92) Christensen, M. K.; Meldal, M.; Bock, K. J . Chem. Soc., Perkin
Trans. 1 1993, 1453-1460.
J . Org. Chem, Vol. 69, No. 14, 2004 4591

Shannon and Barany
TABLE 3. P er cen t Con ver sion of Resin -Bou n d
Ald eh yd es a fter Red u ctive Am in a tion w ith BAL-AMP
(6b)
% aldehyde
entry
primary amine
remaininga
% conversionb
P
2,6-dimethylaniline
aniline
benzylamine
68
30
6
5
4
32
70
94
95
96
97
89
Q
R
S
T
U
V
butylamine
cyclohexylamine
H-Gly-OtBu‚HCl
H-Phe-OtBu‚HCl
3
11
F IGURE 4. Structures of various resin-bound functional
groups that were taken through the assay procedure. After
FmPH quantitation, none of the supports showed any residual
dibenzofulvene absorbance, suggesting no interference with
FmPH.
a
This value was determined by first submitting 17 (∼5 mg) to
FmPH quantitation from which the loading of unreacted aldehyde
was calculated. Percent aldehyde was then determined by taking
the calculated loading and dividing by the theoretical loading of
b
BAL-AMP (0.73 mmol of aldehyde per g of resin). Percent
conversion was calculated by subtracting the percent of aldehyde
from 100%.
F IGURE 5. Structures of various resin-bound ketones as-
sayed by FmPH quantitation.
the 30-50% range. These preliminary results suggest
that FmPH is not reactive enough to be useful for reliable
quantification of resin-bound ketones.
Con clu sion s
A simple, reproducible, and direct method for quantify-
ing solid-phase aldehydes has been developed and inves-
tigated thoroughly. A new reagent, FmPH, is synthesized
and isolated as its stable TFA salt in a 27% overall yield
for three easy steps. FmPH quantitation is a useful
research tool for monitoring the progress of a number of
solid-phase conversions, including reductive aminations
and the attachment of aldehyde linkers to solid supports.
Results are obtained by UV-vis, ideally in 30 min, with
detection of aldehydes at levels as low as 2 µmol and
reproduciblility to (3%. We envision that this test could
also be applied to other solid-phase aldehyde transforma-
tions, e.g., reduction of Weinreb amides, reduction of
aldehydes to alcohols, or oxidation of alcohols to alde-
hydes, and will be a significant addition to the practical
quantitative tools available for solid-phase synthesis.
F IGURE 3. Structures of various resin-bound aldehydes
assayed by FmPH quantitation.
ortho or para positions, unsubstituted p-alkoxybenzal-
dehydes (19), aliphatic aldehydes (20), or aromatic alde-
hydes with deactivating groups, such as the nitro func-
tionality in support 21. Again, a range of different
supports (e.g., PEG-PS, AMP, CLEAR) were tolerated.
The nucleophilicity of FmPH‚TFA under the given condi-
tions did not cause any substantial interference with
other electrophilic carbon centers such as alkyl halides,
as demonstrated on Merrifield resin (22) and supported
chloroethylamine (23) (Figure 4). Furthermore, interfer-
ence from other functional groups [e.g., methoxy (6 and
21), phenolic (18), amide (24), and nitro (21 and 25)] was
negligible (Figure 4).
Exp er im en ta l Section
Gen er a l Con sid er a tion s. Materials, solvents, instrumen-
tation, and general methods were essentially as described in
previous publications from our laboratories,49,56,93,94 as detailed
further in the Supporting Information.
Representative resin-bound ketones 26-28 (Figure 5)
were reacted with FmPH under the same conditions that
were optimized for aldehydes. Release and quantitation
of the piperidine-dibenzofulvene adduct in the usual way
revealed that FmPH incorporation had been at best in
(93) Alsina, J .; Yokum, T. S.; Albericio, F.; Barany, G. J . Org. Chem.
1999, 64, 8761-8769.
(94) Yokum, T. S.; Alsina, J .; Barany, G. J . Comb. Chem. 2000, 2,
282-292.
4592 J . Org. Chem., Vol. 69, No. 14, 2004

Quantitative Monitoring of Solid-Phase Aldehydes
Nr-(ter t-Bu toxycar bon yl)glycin e 9-flu or en ylm eth yl Es-
t er . Boc-Gly-OH (1.0 g, 5.7 mmol) was taken up in freshly
distilled CH2Cl2 (25 mL), and DCC (1.4 g, 6.9 mmol), 9-fluo-
renylmethanol (1.3 g, 6.9 mmol), and DMAP (34 mg, 0.29
mmol) were added in order with stirring. The resultant
suspension was then mixed for 48 h at 25 °C, filtered while
cold, and concentrated to a brown oil. Purification by silica
gel column chromatography (hexanes-EtOAc, 3:1) provided
the title compound, Boc-Gly-OFm, as a clear colorless film;
HRESI-MS: m/z calcd for C21H18N2O2 330.1368, found 353.1246
([M + Na]+).
Anal. Calcd for C23H19F3N2O4, MW 444.40: C, 62.16; H, 4.31;
F, 12.83; N, 6.30; O, 14.40. Found: C, 62.49; H, 4.65; N, 6.27.
The title compound demonstrated excellent stability at
ambient temperatures, but was nevertheless stored at 4 °C,
as is typical for standard Fmoc-protected amino acids.
F m P H Qu a n tita tion : Dir ect Qu a n tita tion of P olym er -
Su p p or ted Ald eh yd es. Approximately 5-10 mg of dry
aldehyde resin (0.2-0.7 mmol of CHO per g of resin) was
weighed into a glass vial with a Teflon-lined cap, and then a
solution of FmPH‚TFA (4)/DIEA (3 equiv each) in DMF (0.5
mL) was added. [For AMP aldehyde resins, DMF-CH2Cl2 (1:
1, 0.5 mL) was used as solvent.] TMOF (0.5 mL) was added
immediately and the resulting suspension was rotated on an
orbital mixer for 30 min at 80 °C. The cooled suspension was
transferred to a fritted plastic syringe and the resin was
washed thoroughly with DMF (5 × 1.5 mL), MeOH (5 × 1.5
mL), and again with DMF (5 × 1.5 mL). The FmPH-deriva-
tized support was treated with piperidine-DMF (1:1, 1 mL)
for 30 min at 25 °C to release the piperidine-dibenzofulvene
chromophore (13), and the filtrate was collected in a 25 mL
volumetric flask. The resin was washed with piperidine-DMF
(1:1, 5 × 2 mL), and on each wash, drained directly into the
same 25-mL volumetric flask, followed by dilution up to 25
mL with piperidine-DMF (1:1). The absorbance of the solution
was measured with an ultraviolet spectrometer [using piperi-
dine-DMF (1:1) as a blank] at 301 nm, and incorporated in
the following formula to calculate the loading of aldehyde:
1
yield 609 mg (30%). H NMR (200 MHz, CDCl3): δ 7.76 (d, J
) 7.0 Hz, 2H), 6.58 (d, J ) 7.0 Hz, 2H), 7.41 (t, J ) 7.4 Hz,
2H), 7.3-7.4 (m, 2H), 5.12 (br s, 1H), 4.43 (d, J ) 7.2 Hz, 2H),
4.23 (t, J ) 7.2 Hz, 1H), 4.02 (d, J ) 5.6 Hz, 2H), 1.47 (s, 9H).
ESI-MS: m/z calcd for C21H23NO4 353.41, found 376.3 ([M +
Na]+).
Glycin e 9-F lu or en ylm eth yl Ester , Hyd r och lor id e Sa lt
(5). Boc-Gly-OFm (500 mg, 1.4 mmol) was taken up in 4 M
HCl-dioxane (9 mL) and stirred for 30 min at 25 °C. The
burgundy solution was concentrated under a stream of N2 (to
remove HCl) and subsequently under reduced pressure to
provide a brown oil. After triturating with Et2O (3 × 15 mL),
compound 5 precipitated as a white solid (HCl salt), and was
filtered and dried (2 mm, overnight, desiccator); yield 274 mg
(77%) (23% overall from Boc-Gly-OH). 1H NMR (500 MHz, CD3-
OD-d6): δ 7.73 (d, J ) 12.0 Hz, 2H), 7.54 (d, J ) 12.0 Hz, 2H),
7.33 (t, J ) 12.0 Hz, 2H), 7.24 (t, J ) 12.0 Hz, 2H), 4.53 (d, J
) 11.0 Hz, 2H), 4.20 (t, J ) 10.0 Hz, 1H), 3.75 (s, 2H). LRESI-
MS: m/z calcd for C16H15NO2 253.30, found 267.3 ([M + Na]+).
4-(9-F lu or en ylm eth yloxyca r bon yl)[N,N,N-tr is(ter t-bu -
tyloxyca r bon yl)p h en ylh yd r a zin e (10). A solution of di-tert-
butyl dicarbonate (8.6 g, 39 mmol) in tetrahydrofuran (THF,
10 mL) was added to a stirred solution of 4-hydrazinobenzoic
acid (8) (1.9 g, 13 mmol) in THF-DMF (2:1, 50 mL), and the
solution was mixed for 30 min at 25 °C. DMAP (39 mg, 0.33
mmol) was then added to the solution, following which the
temperature was increased to 60 °C and stirring continued
for 4 h.79 The reaction mixture was then cooled, partially
concentrated in vacuo (to remove THF), diluted with Et2O (125
mL), and washed with 1 M aqueous KHSO4-brine (1:1) (3 ×
25 mL) and brine (3 × 15 mL), dried (MgSO4), and concen-
trated to give 9 as a tan solid; yield 4.5 g (76%). Without
further purification, 9 (2.0 g, 3.2 mmol) was taken up in freshly
distilled CH2Cl2 (50 mL) and then DCC (1.1 g, 5.3 mmol),
9-fluorenylmethanol (1.0 g, 5.3 mmol), and DMAP (27 mg, 0.22
mmol) were added in sequence with stirring. The resultant
suspension was then stirred for 48 h at 25 °C, after which it
was cooled, filtered, concentrated to a brown oil, and purified
by silica gel column chromatography (hexanes-EtOAc, 4:1)
to provide 10 as a white solid; mp 125-127 °C; yield 1.2 g
(43%) (33% overall for two steps). 1H NMR (500 MHz, CDCl3):
δ 8.06 (d, J ) 11.0 Hz, 2H), 7.81 (d, J ) 12.5 Hz, 2H), 7.66 (d,
J ) 12.5 Hz, 2H), 7.55 (d, J ) 11.5 Hz, 2H), 7.44 (t, J ) 12.5
Hz, 2H), 7.34 (t, J ) 12.5 Hz, 2H), 4.61 (d, J ) 11.5 Hz, 2H),
4.41 (t, J ) 12.0 Hz, 1H), 1.55 (s, 9H), 1.51 (s, 18H). HRESI-
MS: m/z calcd for C36H42N2O8 630.7274, found 653.2838 ([M
+ Na]+).
loading of CHO (mmol/g) ) [1000(A)(V)]/[ꢀ × M]
where A is the average absorbance after three measurements,
V is the dilution volume in mL, ꢀ ) 7800 M-1 cm-1 (the
extinction coefficient for the dibenzofulvene chromophore), and
M is the resin weight in mg.
Kin etic Stu d y A: F or m a tion of Hyd r a zon e 11. Dry
samples of BAL-AMP (5 mg, 0.73 mmol of CHO per g of resin)
(6b) were weighed into glass vials (15 × 45 mm, 1 dram) and
a solution of FmPH‚TFA (4)/DIEA (3 equiv each) in DMF-
CH2Cl2 (1:1, 0.5 mL) was added to each vial, followed im-
mediately by TMOF (0.5 mL). The suspensions were mixed
on an orbital mixer at 80 °C, and reactions were stopped
individually after 1, 2, 3, 4, 5, 7, 10, 20, 25, and 30 min. The
cooled suspensions were transferred to fritted plastic syringes,
washed, and processed by the experimental protocol under the
subsection FmPH Quantitation. The calculated loadings, as
determined by the amount of 13 quantified, were plotted as a
function of time (Figure 2A, squares).
Kin etic Stu d y B: Relea se of Diben zofu lven e Ad d u ct
fr om Hyd r a zon e 11. Dry samples of resin 11 (5 mg) [pre-
pared from AMP-BAL (6b), 0.73 mmol of CHO per g of resin]
were transferred to fritted plastic syringes (3 mL each), swollen
in CH2Cl2 (1.5 mL, 5 min), and washed thoroughly with DMF
(5 × 1.5 mL). The resins were then treated with piperidine-
DMF (1:1, 1 mL) at 25 °C, and after 1, 2, 3, 4, 5, 7, 10, 15, 20,
and 30 min, each reaction was stopped and the released
piperidine-dibenzofulvene adduct (13) was quantified by the
experimental protocol given under the subsection FmPH
Quantitation. The amounts of Fm remaining on the support
(calculated indirectly from the amount of 13 released subse-
quently) were plotted as a function of time (Figure 2B, circles).
Kin etic Stu d y C: F or m a tion of Im in e 7. This experi-
ment was conducted similar to what was described in the
subsection Kinetic Study A, except that H-Gly-OFm‚HCl (5)/
DIEA (3 equiv each) was used instead of FmPH‚TFA (4)/DIEA;
reactions were stopped after 1, 2, 3, 4, 5, 7, 10, 15, 20, and 30
min, 24 h, and 48 h, respectively. The loadings of resins 7, as
determined by the amount of 13 quantified, were plotted as a
function of time (Figure 2C, triangles).
Anal. Calcd for C36H42N2O8, MW 639.73: C, 68.55; H, 6.71;
N, 4.44; O, 20.29. Found: C, 68.47; H, 6.74; N, 4.42.
4-(9-F lu or en ylm et h yloxyca r b on yl)p h en ylh yd r a zin e,
Tr iflu or oa ceta te Sa lt (4). Compound 10 (350 mg, 0.55 mmol)
was taken up in CH2Cl2-TFA (1:1, 10 mL) and stirred for 1 h
at 25 °C. The burgundy solution was concentrated under a
stream of N2 (to remove TFA) and subsequently under reduced
pressure to provide a brown oil. The oil was triturated with
Et2O (3 × 25 mL) while title compound 4 precipitated as a
yellow solid (TFA salt), which was filtered and dried (2 mm,
overnight, desiccator); mp 169-171 °C; yield 182 mg (74%)
1
(24% overall from 8). H NMR (500 MHz, CD3OD-d6): δ 7.92
(d, J ) 14.0 Hz, 2H), 7.80 (d, J ) 9.0 Hz, 2H), 7.70 (m, 2H),
7.42 (t, J ) 8.0 Hz, 2H), 7.34 (t, J ) 8.0 Hz, 2H), 7.05 (d, J )
8.0 Hz, 2H), 4.55 (d, J ) 6.5 Hz, 2H), 4.41 (t, J ) 6.5 Hz, 1H).
F m oc a n d F m P H Qu a n tita tion Com p a r ison : P r ep a r a -
tion of P a r tia lly Su bstitu ted P ALd eh yd e Su p p or ts (16).
PEG-PS (0.53 mmol of NH2 per g), AMP (0.73 mmol of NH2
J . Org. Chem, Vol. 69, No. 14, 2004 4593

Shannon and Barany
per g), and CLEAR (0.46 mmol of NH2 per g) supports [all
obtained as the amine hydrochloride salts, approximately 50
mg each] were swollen in CH2Cl2 (1.5 mL, 5 min) and washed
thoroughly with DMF-DIEA (4:1, 5 × 1.5 mL). PALdehyde86
plus Fmoc-Gly-OH were mixed in a 1:1 ratio (3 equiv combined
carboxylic acid function) with HATU/DIEA (3 equiv each),
dissolved in DMF (1.5 mL), and the resultant solutions were
added to the respective resins which were rotated on an orbital
shaker for 24 h at 25 °C. The partially substituted aldehyde
supports (16) were washed with DMF (5 × 1.5 mL), MeOH (5
× 1.5 mL), and DMF (5 × 1.5 mL). The resins were then
processed first by Fmoc quantitation45,59,65 to indirectly deter-
mine the percent aldehyde, and then following Fmoc, the same
resins, which contained residual aldehydes, were subjected to
FmPH quantitation. The percent loading of aldehydes were
calculated as described in footnotes b and c of Table 2.
Solid -P h a se Red u ctive Am in a tion : P r ep a r a tion of 17.
BAL-AMP (6b, 0.73 mmol of CHO per g of resin) (5 mg) was
swollen in CH2Cl2 (1.5 mL, 5 min) and washed thoroughly with
DMF (5 × 1.5 mL). A solution of NaBH3CN (2 mg, 18 µmol, 5
equiv), the primary amine (18 µmol, 5 equiv), and 1% HOAc
in CH2Cl2-DMF-MeOH (2:1:1; 1 mL) was added to the
support and the sample was rotated on an orbital shaker for
30 min at 25 °C. The resultant secondary amine supports (17)
were washed with DMF (5 × 1.5 mL), MeOH (5 × 1.5 mL),
and CH2Cl2 (5 × 1.5 mL). The resins were checked by the
chloranil test95 and the DNPH test,45 and then subjected to
FmPH quantitation to determine the amount of residual
aldehyde. Percent conversions were calculated by using the
procedure described in footnotes a and b of Table 3. The
primary amines used were 2,6-dimethylaniline, aniline, benz-
ylamine, butylamine, cyclohexylamine, H-Gly-OtBu‚HCl, and
H-Phe-OtBu‚HCl.
Ack n ow led gm en t. We thank Drs. Daniel G. Mullen
and Steven A. Kates for critical readings of the manu-
script and NIH (GM 42722 and GM 08347) for financial
support.
Su p p or tin g In for m a tion Ava ila ble: General procedures,
1H NMR and HRESI-MS spectra, and elemental analysis for
compounds 4 and 10. This material is available free of charge
via the Internet at http://pubs.acs.org.
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