Synthesis and comparative properties of two amide-generating resin linkers for use in solid phase peptide synthesis

Article abstract of DOI:10.1002/psc.1279

Well-characterized resins of high purity are critical for effective solid phase peptide synthesis (SPPS). The quality of commercial (4-methyl)benzhydrylamine-resin (MBHA-resin), used for the synthesis of peptide amides, is not consistent and residual ketone functionalities are frequently present. Such ketone or aldehyde impurities lead to the formation of acylation-resistant deletion peptides in SPPS. To avoid these undesirable side reactions, we have optimized the preparation of two amide-generating linkers, which, in combination with aminomethyl-resin prepared directly from polystyrene resin, serve as alternatives to MBHA-resin for peptide amide synthesis. Then we have explored their comparative properties in SPPS. Use of sonication in reductive amination facilitated the synthesis of both benzhydrylamine (BHA) and MBHA linkers. We have optimized the preparation of two amide-generating linkers for use in stepwise solid phase peptide synthesis. In combination with aminomethyl-resin prepared directly from polystyrene resin, these linkers serve as alternatives to MBHA-resin for peptide amide synthesis. Copyright

Full text of DOI:10.1002/psc.1279

Research Article
Received: 1 June 2010
Revised: 30 June 2010
Accepted: 1 July 2010
Published online in Wiley Online Library:
(wileyonlinelibrary.com) DOI 10.1002/psc.1279
Synthesis and comparative properties
of two amide-generating resin linkers
for use in solid phase peptide synthesis‡
Fang-Kun Deng,∗ Kalyaneswar Mandal, Sam Luisier and Stephen B. H. Kent
Well-characterized resins of high purity are critical for effective solid phase peptide synthesis (SPPS). The quality of commercial
(4-methyl)benzhydrylamine-resin (MBHA-resin), used for the synthesis of peptide amides, is not consistent and residual ketone
functionalities are frequently present. Such ketone or aldehyde impurities lead to the formation of acylation-resistant deletion
peptides in SPPS. To avoid these undesirable side reactions, we have optimized the preparation of two amide-generating
linkers, which, in combination with aminomethyl-resin prepared directly from polystyrene resin, serve as alternatives to
MBHA-resin for peptide amide synthesis. Then we have explored their comparative properties in SPPS. Use of sonication in
c
reductive amination facilitated the synthesis of both benzhydrylamine (BHA) and MBHA linkers. Copyright ꢀ 2010 European
Peptide Society and John Wiley & Sons, Ltd.
Supporting information may be found in the online version of this article
Keywords: solid phase peptide synthesis; MBHA resin; peptide amide; peptide thioester
Introduction
resin with 4-methylbenzoyl chloride, followed by reductive amina-
tion of the resulting benzophenone [3]. Incomplete reduction of
thebenzophenoneimineoroximewouldgiverisetoresidualresin-
bound ketone moieties. Formation of deletion peptides in Boc
chemistry SPPS has been attributed to the formation of acylation-
resistant Schiff’s base compounds involving residual aldehyde or
ketone functionalities on the resin with the alpha-amino group of
a resin-bound peptide at each stage of SPPS [2].
The preparation by SPPS of high-purity peptides, peptide
amides, and peptide-thioesters of 30–50 amino acids is essential
for chemical protein synthesis by modern chemical ligation
methods [9]. Well-characterized amide-generating resins as an
alternative to MBHA-resin would be useful for the unambiguous
synthesisofpeptide-thioesters[10].Wesetouttomakesuchawell-
definedamide-generatingresinbyaroutethatwouldprecludethe
presence of residual ketone functionalities. Our starting point was
aminomethyl-resinmadedirectlyfrompolystyreneresinaccording
to published procedures that not only avoid the potential
introduction of aldehyde or ketone functionalities but which also
chemically scavenge any such functionalities that may be present
in the starting polystyrene resin [2,8,11]. This aminomethyl-resin
was used in combination with a well-characterized, high-purity
amide-generating linker prepared by solution chemistry (Figure 3,
linker 4 and linker 5). The Boc-(4-OCH2COOH)benzhydrylamine
(BHA) linker of Matsueda (Figure 3, linker 4) [12] is well designed,
with a carboxylic acid that can be linked to aminomethyl-resin;
The nature and amount of resin-bound reactive functional groups
plays a critical role in solid phase peptide synthesis [1]. High
purity peptides of defined chemical composition can only be
synthesized on stable, well-defined functionalized resins free
of extraneous reactive functionality [2]. Amide-generating (4-
methyl)benzhydrylamine (MBHA) resin [3] (Figure 1, resin 1) can
be particularly problematic: properly prepared, such resins give
good results; however, the quality of commercial MBHA-resins,
like other commercial resins for use in SPPS [4,5], is not consistent.
Like all functionalized solid phase synthesis resins, MBHA-resins
are difficult to characterize and cannot be purified.
Results and Discussion
Recently, we had occasion to synthesize the model peptide YGGFL
amide [(Leu5)enkephalin amide] on commercial MBHA-resin [6],
using Boc chemistry ‘in situ neutralization’ SPPS [7]. The purity
of the crude peptide product was unacceptable; there were
extensive deletion products including YGFL amide and YGGF
amide (Figure 2(a)).
Using an identical SPPS synthetic protocol, the pep-
tide YGGFL acid was synthesized in high purity (data not
shown) on Boc-Leucine-4-OCH2Pam-resin (Boc-Leucine acyl-
4-(oxymethyl)phenylacetamidomethyl-resin) (Figure 1, resin 3)
prepared from Boc-Leucine-(4-carboxymethyl)benzyl ester and
aminomethyl-resin (Figure 1, resin 2) made directly from
polystyrene resin according to published procedures [8]. We con-
cludedthatthepoorqualityofcommercialMBHA-resinhadcaused
the low purity of the original peptide products. IR spectroscopy
examination of the starting MBHA-resin showed the presence of
carbonyl moieties (C O stretch peaks, 1700 cm−1). Conventional
MBHA-resin is prepared by Friedel–Crafts acylation of polystyrene
∗
Correspondence to: Fang-Kun Deng, Department of Chemistry, University of
Chicago, Chicago, IL, 60615, USA. E-mail: dengprotein@uchicago.edu
Department of Chemistry, University of Chicago, Chicago, IL 60615, USA
‡
Special issue devoted to contributions presented at the Symposium ‘‘Probing
Protein Function through Chemistry’’, 20–23 September 2009, Ringberg Castle,
Germany.
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J. Pept. Sci. 2010; 16: 545–550
Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd.

DENG ET AL.
more than 27 h. However, the reaction was slow and was not clean,
giving less than 50% conversion after 24 h. Further crystallization
from ether and ethanol gave poor yields.
We found that ethanol was not necessary for dissolving
p-hydroxybenzophenone oxime. The oxime could be dissolved
just in ammonium hydroxide aqueous solution. And in order to
enhance the speed of the reduction, we employed sonication
to make a fresh zinc surface. The resulting reaction was clean
and efficient; it gave almost full conversion after 6 h. In addition,
the workup was very easy. On adjusting the solution to pH 8
in an ice-water bath, the product precipitated as a white solid.
After filtration, washing with water, and drying, the desired (4-
OH)benzhydrylamine product was obtained in good yield (>85%)
and high purity. Conversion to the Boc-(4-OH)benzhydrylamine,
followed by reaction with sodium iodoacetate gave the Matsueda
linker (Figure 3, linker 4) in good yield and high purity.
The obtained Boc-(4-OCH2COOH)benzhydrylamine linker was
coupled to aminomethyl-resin [13], and the resulting BHA-
aminomethyl-resin 6 was used for the synthesis of the model
peptide YGGFL amide using the Boc chemistry ‘in situ neutraliza-
tion’ SPPS protocols described above. Very high quality crude
peptide was obtained (Figure 2(b)). However, HF cleavage of
117 mg of peptide-polystyrene resin gave only 3.3 mg of the
YGGFL amide peptide after lyophilization. It seemed to us that
the BHA-aminomethyl-resin was too stable to HF cleavage, and
that it might be necessary to tune the acid lability of the linker by
addition of a methyl group to the benzene ring (Figure 3, linker 5),
in order to increase its acid lability and improve HF cleavage yields.
The stability to repetitive TFA treatment of the modified MBHA
linker as peptidyl-MBHA aminomethyl-resin would also need to
be measured.
Figure 1. Chemical structures of (a) MBHA-resin 1; (b) aminomethyl-resin
2; and (c) Boc-Leucine-4-OCH2-Pam-resin 3.
removal of the Boc group gives a substituted benzhydrylamine-
resin which can be reacted with any desired C-terminal amino acid
as the first step in a solid phase synthesis.
Initial attempts to make Matsueda’s BHA linker following the
protocol described in the original paper were unsuccessful [12]. In
our hands, the synthesis was awkward and failed to reproducibly
give good yields. The key step for the synthesis is reductive
amination of the p-hydroxybenzophenone oxime. The original
reaction conditions used a large excess of zinc dust at 50 ◦C in a
mixture of concentrated ammonium hydroxide and ethanol for
Table 1. Comparative peptidyl-resin weights for syntheses on BHA-aminomethyl-resin and MBHA-aminomethyl-resin
Weight of
Weight of
Calculated weight
of peptide-resin (mg)
peptide resin for
BHA-aminomethyl-resin (mg)
peptide resin for
MBHA- aminomethyl-resin (mg)
Peptide
Scale (mmol)a
YGGFL amide
0.2
0.2
0.2
0.2
0.5
422
407
328.3
305.1
351.8
339.8
2288
300.8
286.8
293.3
321.2
1684
LFPGFL amide
CFRANK amide
501
Thioester LYRAGXA amideb
hGHRH(1–29) amidec
489
3217
a Aminomethyl-resin loading 0.98 mmol g−1
.
b X = SCH2CH2CO.
c hGHRH, human growth hormone-releasing hormone.
Table 2. Comparative yields of peptides synthesized on BHA-aminomethyl-resin and MBHA- aminomethyl-resin
Weight of crude
peptide cleaved from
Weight of crude
peptide cleaved from
Resin cleaved
HF (mg)a
Peptide
BHA-aminomethyl-resin (mg)
MBHA-aminomethyl-resin (mg)
YGGFL amide
200
200
200
200
500
5.6
17.8
18.3
31.4
LFPGFL amide
CFRANK amide
11.7
34.8
thioester LYRAGXA amideb
hGHRH(1–29) amidec
17.5
29.4
184.8
228.1
a Peptide-resin cleavage by HF was carried out at 0 ◦C for 1 h using 10 ml HF and 500 ul p-cresol.
b X = SCH2CH2CO.
c hGHRH, human growth hormone-releasing hormone.
c
wileyonlinelibrary.com/journal/psc Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2010; 16: 545–550

TWO AMIDE-GENERATING RESIN LINKERS
Figure 2. (a) Synthesis of YGGFL amide on commercial MBHA-resin. (b) Synthesis of YGGFL amide on homemade BHA-aminomethyl-resin. (c) Synthesis
of YGGFL amide on homemade MBHA-aminomethyl-resin. Note that in (b) and (c) slightly different, ore highly resolving gradients were used.
Synthesis of MBHA aminomethyl-resin is shown in Figure 4.
4-Methoxy-benzoyl chloride 8 reacted with toluene to give
4-methoxy-4ꢁ-methyl-benzophenone 9. Then BBr3 was used
to deprotect methoxy group to give 4-hydroxy-4ꢁ-methyl-
benzophenone 10 [14]. Sonication-assisted reductive amination
of oxime 11, followed by Boc protection of the resulting amino
group of 12, and reaction with sodium iodoacetate gave MBHA
linker 5. MBHA linker was coupled to aminomethyl-resin by di-
isopropylcarbodiimide activation, to afford MBHA-aminomethyl-
resin 7.
peptides and hGHRH(1–29)(human growth hormone-releasing
hormone) amide. Weights of peptide-resins after SPPS assembly
of the protected peptide chains are shown in Table 1. The yields of
protected peptide-resins were greater for BHA-aminomethyl-resin
than for the MBHA-aminomethyl-resin, suggesting that the MBHA
linker was labile to the conditions of Boc chemistry SPPS chain
assembly (presumably the repetitive TFA treatments). The weights
of crude peptides obtained after HF cleavage of the peptide-
resins are shown in Table 2. Peptides synthesized on MBHA-
aminomethyl-resin gave higher crude yields than peptides made
on BHA-aminomethyl-resin, for an equal weight of peptide-resin
cleaved.
We used both BHA-aminomethyl-resin and MBHA-amino-
methyl-resin for comparative syntheses of a series of model
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J. Pept. Sci. 2010; 16: 545–550 Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/psc

DENG ET AL.
thioester and peptide amide products from both the BHA-
aminomethyl-resin and MBHA-aminomethyl-resin (Figure 5). Both
MBHA-aminomethyl-resinandBHA-aminomethyl-resingavehigh-
quality crude peptide products. Full experimental details of the
synthesis of the resin linkers and peptide syntheses can be found
in the Supporting Information.
Conclusion
We have developed robust syntheses of two resin linkers for
making peptide amides on aminomethyl-{polystyrene resin} free
of extraneous functional groups. Both MBHA-aminomethyl-resin
and BHA-aminomethyl-resin give crude peptide products of
high purity, reflecting the unambiguous protocols used for the
syntheses of the two resins. MBHA-aminomethyl-resin gave higher
yields of crude peptide product for short peptides. For the 29-
residue hGHRH amide peptide, the two resins gave comparable
overall yields of high-quality crude peptide products. It would
seem that the BHA-aminomethyl-resin of Matsueda is somewhat
too stable to HF, while the MBHA- aminomethyl- resin is too labile
to TFA.
Either of these amide-generating resins can be used to make
peptidethioesters,cleanlyandingoodyield,foruseinthechemical
synthesis of proteins by modern ligation methods. Further fine
tuning of acid stability of amide-generating resins will be done in
our future research.
Figure 3. Structures of (a) Matsueda’s linker 4; (b) the modified MBHA
linker 5; (c) BHA- aminomethyl-resin 6; and (d) MBHA-aminomethyl-resin
7.
The stability to TFA of the peptidyl-resins was examined for
both the BHA-aminomethyl-resin and MBHA-aminomethyl-resin.
This was done by putting peptide resin in neat TFA and using
HPLC to monitor amounts of peptide cleaved from resin in
TFA. The results for LFPGFL amide- were that peptidyl-MBHA-
aminomethyl-resin lost 2% peptide per hour in neat TFA, and
peptidyl-BHA-aminomethyl-resin lost 0.9% peptide per hour in
neat TFA.
Acknowledgements
Analytic HPLC-electrospray mass spectrometry (LCMS) was
used to evaluate the comparative purity of the crude peptide
This research was supported by the Office of Science (BER), US
Department of Energy, Grant No. DE-FG02-07ER64501.
Figure 4. Synthesis of MBHA-aminomethyl-resin 7, by a variation of the route described by Gaehde and Matsueda [12].
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TWO AMIDE-GENERATING RESIN LINKERS
Figure 5. LC-MS data of (a) crude thioester LYRAGXA amide (X = SCH2CH2CO) synthesized on MBHA-aminomethyl-resin 7; (b) crude thioester LYRAGXA
amide (X = SCH2CH2CO) syntesized on BHA-aminomethyl-resin 6; (c) crude hGHRH (human growth hormone releasing hormone)(1–29) amide
synthesized on MBHA-aminomethyl-resin 7 and (d) crude hGHRH (1–29) amide synthesized on BHA-aminomethyl-resin 6. Different columns were used
in (a) and (b).
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J. Pept. Sci. 2010; 16: 545–550 Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/psc

DENG ET AL.
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