A convenient approach to synthesizing peptide C-terminal N-alkyl amides

Article abstract of DOI:10.1002/bip.21600

Peptide C-terminal N-alkyl amides have gained more attention over the past decade due to their biological properties, including improved pharmacokinetic and pharmacodynamic profiles. However, the synthesis of this type of peptide on solid phase by current available methods can be challenging. Here we report a convenient method to synthesize peptide C-terminal N-alkyl amides using the well-known Fukuyama N-alkylation reaction on a standard resin commonly used for the synthesis of peptide C-terminal primary amides, the peptide amide linker-polyethylene glycol-polystyrene (PAL-PEG-PS) resin. The alkylation and oNBS deprotection were conducted under basic conditions and were therefore compatible with this acid labile resin. The alkylation reaction was very efficient on this resin with a number of different alkyl iodides or bromides, and the synthesis of model enkephalin N-alkyl amide analogs using this method gave consistently high yields and purities, demonstrating the applicability of this methodology. The synthesis of N-alkyl amides was more difficult on a Rink amide resin, especially the coupling of the first amino acid to the N-alkyl amine, resulting in lower yields for loading the first amino acid onto the resin. This method can be widely applied in the synthesis of peptide N-alkyl amides.

Full text of DOI:10.1002/bip.21600

A Convenient Approach to Synthesizing Peptide C-Terminal
A Convenient Approach to Synthesizing Peptide C-Terminal
N-Alkyl Amides
N-Alkyl Amides
Wei-Jie Fang,* Tatyana Yakovleva, Jane V. Aldrich
Department of Medicinal Chemistry, The University of Kansas, Lawrence, KS 66045
Received 30 July 2010; revised 3 January 2011; accepted 10 January 2011
Published online 9 February 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bip.21600
can be widely applied in the synthesis of peptide N-alkyl
ABSTRACT:
#
amides. 2011 Wiley Periodicals, Inc. Biopolymers (Pept
Peptide C-terminal N-alkyl amides have gained more
Sci) 96: 715-722, 2011.
attention over the past decade due to their biological
Keywords: peptide C-terminal N-alkyl amides;
Fukuyama amine synthesis
properties, including improved pharmacokinetic and
pharmacodynamic profiles. However, the synthesis of this
type of peptide on solid phase by current available
methods can be challenging. Here we report a convenient
method to synthesize peptide C-terminal N-alkyl amides
using the well-known Fukuyama N-alkylation reaction
on a standard resin commonly used for the synthesis of
peptide C-terminal primary amides, the peptide amide
linker-polyethylene glycol-polystyrene (PAL-PEG-PS)
resin. The alkylation and oNBS deprotection were
conducted under basic conditions and were therefore
compatible with this acid labile resin. The alkylation
reaction was very efficient on this resin with a number of
different alkyl iodides or bromides, and the synthesis of
model enkephalin N-alkyl amide analogs using this
method gave consistently high yields and purities,
demonstrating the applicability of this methodology. The
synthesis of N-alkyl amides was more difficult on a Rink
amide resin, especially the coupling of the first amino
acid to the N-alkyl amine, resulting in lower yields for
loading the first amino acid onto the resin. This method
This article was originally published online as an accepted
preprint. The ‘‘Published Online’’date corresponds to the preprint
version. You can request a copy of the preprint by emailing the
Biopolymers editorial office at biopolymers@wiley.com
INTRODUCTION
olid-phase peptide synthesis (SPPS), first developed
by R. Bruce Merrifield,1 permits the rapid synthesis
of peptides and related biologically active com-
pounds. Because SPPS almost always starts from the
S
C-terminus of the peptide, C-terminal modifications
of peptides are usually more difficult to introduce than
modifications at the N-terminus.
C-Terminal amide alkylation of peptides can have signifi-
cant effects on their biological properties. Groups such as a
C-terminal ethyl amide can increase stability toward pepti-
dases,2,3 and can also increase affinity for specific biological
targets.4–6 In the case of lutenizing hormone-releasing
hormone (LH-RH), several N-ethyl and N-methyl amide
derivatives of LH-RH analogs are four to five times more
potent in releasing pituitary luteinizing hormone than the
corresponding primary amide peptides.4,5 In addition, C-ter-
minal alkylation can increase lipophilicity and reduce hydro-
gen bonding potential, which can facilitate the penetration
of peptides across biological membranes and/or improve
pharmacokinetic properties.7,8 Therefore, C-terminal amide
alkylation has considerable potential for application to thera-
peutically relevant peptides.
Correspondence to: Jane Aldrich; e-mail: jaldrich@ku.edu
*Present affiliation: Department of Chemical and Biological Engineering, Univer-
sity of Colorado, Boulder, CO 80309
Given the interest in these modified peptides, a number of
approaches to the synthesis of peptide C-terminal N-alkyl
amides have been described.9 However, the synthesis of this
Contract grant sponsor: National Institute on Drug Abuse
Contract grant number: R01 DA018832
C
VV
2011 Wiley Periodicals, Inc.
PeptideScience Volume 96 / Number 6
715

716
Fang, Yakovleva, and Aldrich
FIGURE 1 Structures of resins 1–6.
type of modified peptide on solid phase is not straightfor- peptides synthesized on an oxime resin,13 but again this resin
ward. One approach used is to first synthesize the corre- is only compatible with the Boc synthetic strategy.
sponding peptides with a C-terminal carboxylic acid. Follow-
Another approach involves the use of a modified resin
ing cleavage from the resin the free carboxylic acid is then linker for introduction of an alkyl amine onto the resin
activated and coupled to a primary or secondary amine in before peptide chain assembly begins. For example, reductive
solution.10 This approach has some limitations, however, as amination of the 9-aminoxanthen-3-yloxymethylpoly(styr-
other carboxylic acid groups, i.e. those on the side chains of ene) resin (2, Figure 1) with an aldehyde followed by stand-
Asp and Glu, can also react, and racemization may occur at ard peptide synthesis afforded C-terminal alkylated amides.14
the C-terminal amino acid.11 A related approach is amino- However, reductive alkylation on solid phase with an excess
lysis of a resin-bound ester. For example, a 4-bromomethyl- of aldehyde often results in dialkylation15; this essentially
3-nitrobenzamidobenzyl polystyrene (4-bromomethyl-Nbb) caps the resin, since an amino acid cannot couple to a terti-
resin (1, Figure 1), developed by Nicolas et al., has been used ary amine, decreasing the yield of the desired peptides. It was
for the solid phase synthesis of peptide C-terminal N-alkyl also reported that the PAL-PEG-PS (Peptide Amide Linker-
amides using the Boc (tert-butyloxycarbonyl) synthetic strat- polyethylene glycol-polystyrene) resin (3, Figure 1), a stand-
egy.12 This approach is not compatible with the standard ard resin for the preparation of peptide primary amides,
Fmoc (9-fluorenylmethoxycarbonyl) synthetic strategy, how- was not applicable for the synthesis of peptide C-terminal
ever, since the secondary amine piperidine used for removal alkylated amides by this methodology because the imine
of the Fmoc group following each coupling can also amino- intermediate could not be obtained.14 Alternatively, reductive
lyze the resin-bound peptide ester.12 Similarly peptide C-ter- amination of an aldehyde-containing resin such as one
minal N-alkyl amides have been prepared by aminolysis of containing the backbone amide linker16,17 (BAL, 4, Figure 1)
Biopolymers (Peptide Science)

Synthesis of Peptide C-Terminal N-Alkyl Amides
717
SCHEME 1 Introduction of the C-terminal N-alkyl groups onto the PAL-PEG-PS-resin.
followed by peptide chain assembly can be used to prepare the ninhydrin test.32 Cleavage of an aliquot of the resin
peptides with a C-terminal N-alkyl amide. This approach, afforded pure oNBS-NH2 as confirmed by HPLC (5%–50%
however, has been reported only for higher molecular weight MeCN with 0.1% trifluoroacetic acid (TFA) over 45 min at
amines,18–21 and could be difficult with smaller, highly vola- 1 mL/min, tR ¼ 3.8 min, see below).
tile amines such as ethylamine.
The newly formed sulfonamide (7-resin) was deprotonated
Another commercially available resin used for the synthe- using 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in DMF29
sis of these C-terminally modified peptide amides is the and the resulting anion then reacted with several different
‘‘safety catch’’ resin (5, Figure 1).3,6,22–25 With this resin pep- alkyl halides (Scheme 1). The yields of these reactions were
tide assembly is first completed, and then the resin is acti- monitored by HPLC analysis of aliquots for the appearance of
vated by treatment with a reagent such as iodoacetonitrile to oNBS-NHR (8) and the disappearance of oNBS-NH2 (7). The
yield the N-alkyl-N-acylsulfonamide; the peptide is then results for these reactions are shown in Table I. The conversion
cleaved from the resin with nucleophiles such as amines to was quantitative for most of the alkylating reagents (methyl
yield the N-alkyl amide.
iodide, ethyl iodide, allyl bromide, propargyl bromide, cyclo-
Efficient SPPS methodologies for the preparation of propylmethyl (CPM) bromide, and benzyl bromide) after
N-alkyl amides would facilitate the preparation of this im- reaction at room temperature for 2 days (Table I, entries A–F),
portant type of peptide. Here we report a convenient method except for phenylethyl chloride and phenylethyl bromide (Ta-
for synthesizing peptide C-terminal N-alkyl amides using the ble I, entries G–K). Even with potassium iodide (KI) as a cata-
standard PAL-PEG-PS resin (3) and comparison to the syn- lyst and/or heating the reaction to 808C (Table I, entries H
thesis on a resin containing the Rink amide linker (6). We and I), there was no apparent conversion when the alkylating
applied a well-known N-alkylation reaction, the Fukuyama reagent was the less reactive phenylethyl chloride. With phe-
amine synthesis,26,27 to the preparation of peptide C-termi- nylethyl bromide there was only 20% conversion after reaction
nal N-alkyl amides. This type of reaction has been applied to for 2 days at room temperature (Table I, entry J). Increasing
the synthesis of N-methylamino acid-containing peptides by the reaction temperature to 808C did not increase the yield
both solid and solution phase methods.28–31 However, to our (21%, Table I, entry K) under the same reaction conditions.
knowledge this type of reaction has not been reported previ-
We next examined various conditions for oNBS deprotec-
ously for the synthesis of peptide C-terminal N-alkyl amides. tion26 from the oNBS-N(Et)-resin using different bases and
thiols (Table II). The completion of this reaction was moni-
tored by the disappearance of the oNBS-NH-Et by HPLC.
RESULTS AND DISCUSSION
The use of 4-dimethylaminopyridine (DMAP) resulted in
only partial cleavage of the oNBS group (Table II, entry A).
Use of the stronger base DBU resulted in complete removal
Introduction of the C-Terminal N-Alkyl Group
The synthesis of the peptide C-terminal N-alkyl amides was of the oNBS, likely as a result of its stronger basicity com-
examined on the Fmoc-PAL-PEG-PS resin (Fmoc-3, 0.19 pared to DMAP and more efficient deprotonation of the
mmol/g, Scheme 1). The Fmoc group was first removed thiol group. Different thiols (2-mercaptoethanol, thiophenol,
using piperidine in N,N-dimethylformamide (DMF, 1/4, v/v) and 2,20-(ethylenedioxy)diethanethiol ((HSCH2CH2OCH2)2))
to give the free amine, which was then reacted with 4 equiv were also investigated; they all resulted in complete deprotec-
each of ortho-nitrobenzenesulfonyl chloride (oNBS-Cl) and tion of the N-ethylamide under these reaction conditions
N,N-diisopropylethylamine (DIEA) in dichloromethane (Table II, entries B–D). The nonvolatile 2,20-(ethylenedioxy)-
(DCM). The completion of this reaction was monitored by diethanethiol was subsequently used in combination with
Biopolymers (Peptide Science)

718
Fang, Yakovleva, and Aldrich
Table I Yields of the Alkylation Reactions With Different Alkyl Halides on the PAL-PEG-PS
Resin, and the Retention Times (tR) of the Resulting oNBS-NH-Ra
Yield (%)/tR (min)
of oNBS-NH-Rb,c Entry
Yield (%)/tR (min)
of oNBS-NH-Rb,c
Entry
R-X
Me-I
Et-I
R-X
A
B
C
D
E
>98/7.9
>98/12.7
>98/14.9
>98/12.9
>98/20.1
>98/27.0
G
H
I
PhCH2CH2Cl
<5
<5
<5
20/31.4
21/31.6
PhCH2CH2Cl/KI
PhCH2CH2Cl/KId
PhCH2CH2Br
PhCH2CH2Brd
All-Br
Propargyl-Br
CPM-Br
Bn-Br
J
K
F
a After reaction for 2 days at room temperature except where noted.
b The yields were determined from HPLC chromatograms by comparing the relative area under the peaks of
oNBS-NH-R (8) with nonalkylated oNBS-NH2 (7).
c HPLC conditions: 5–50% solvent B over 45 min (solvent A ¼ H2O, solvent B ¼ MeCN, both containing
0.1% TFA) at 1 mL/min, monitored at 214 nm.
d At 808C.
DBU to deprotect the oNBS from the other alkylated resins. resin. oNBS-Cl was reacted with the amine on a Rink-PEG-
To the best of our knowledge the use of 2,20-(ethylenedioxy)- PS resin as described above; completion of the reaction was
diethanethiol to deprotect the oNBS group has not been monitored with ninhydrin. The alkylation of the sulfonamide
previously reported.
was then examined with ethyl iodide and also the bulky alkyl
While oNBS was completely removed from most of the N- halide CPM bromide; the latter was examined to determine
alkyl resins using DBU and 2,20-(ethylenedioxy)diethanethiol whether steric hinderance from the Rink amide linker would
as the base and thiol, respectively (Table III, entries A–E), the affect more difficult alkylations. The alkylation with ethyl
deprotection of the oNBS-N(CPM)-resin turned out to be iodide proceeded almost to completion, yielding 95% of the
difficult (Table III, entry F). Only a trace amount of the oNBS-NH-R after reaction at room temperature for a total
oNBS was removed under standard conditions, and increas- of 3 days (see Experimental). The alkylation with CPM
ing the temperature to 808C only increased the deprotection bromide, however, was only 76% complete using these con-
to around 30% (entry G). This sluggish reaction is likely due ditions, as determined by HPLC of an aliquot, consistent
to steric hindrance by the CPM group. Thiophenol was with a slower reaction due to steric hinderance from the
found to be a much better reagent to remove the oNBS group Rink amide linker. The conversion to oNBS-N(CPM)-resin
from this N-alkylated resin (Table III, entry H), probably due was increased to 93% by an additional treatment with CPM
to increased nucleophilicity. Any remaining unreacted oNBS bromide at 80oC for one day. The oNBS group was then
group would act as a capping group and would not interfere removed as described above; disappearance of the oNBS-
with subsequent peptide synthesis.
NH-R peak in the HPLC of aliquots verified that the reaction
We also investigated the use of this methodology on a on both resins was complete.
Rink amide resin to prepare peptide C-terminal alkyl amides
using the reaction conditions developed for the PAL-PEG-PS
Table III Cleavage of the oNBS Group From Different oNBS-N(R)-
PAL-PEG-PS Resins (DBU/2,20-(ethylenedioxy)diethanethiol/DMF
Table II Exploration of Different Conditions for the Cleavage
of oNBS From the oNBS-N(Et)-PAL-PEG-PS Resin (Base/Thiol/
DMF ¼ 1/1/2, v/v/v, 1 Day at Room Temperature)
¼ 1/1/2, 1 Day at Room Temperature)
Entry
R
Yield (%)a Entry
R
Yield (%)a
Entry
Base
Thiol
Yield (%)a
A
Me
Et
Quantitative
Quantitative
Quantitative
E
F
Bz
CPM Trace
Quantitative
B
C
D
Allyl
G
H
CPMb 30%
A
DMAP
DBU
DBU
DBU
HSCH2CH2OH
HSCH2CH2OH
PhSH
24
Propargyl Quantitative
CPMc Quantitative
B
>99
>99
>99
C
D
a Determined by HPLC; see Table I for conditions.
(HSCH2CH2OCH2)2
b At 808C.
a Determined by HPLC; see Table I for conditions.
c Thiophenol used instead of 2,20-(ethylenedioxy)diethanethiol.
Biopolymers (Peptide Science)

Synthesis of Peptide C-Terminal N-Alkyl Amides
719
Table IV Resin Loadings of Different Fmoc-Leu-N(R)-PAL-PEG-
PS Resins, as Determined by Quantitative Fmoc Analysis
the N-ethyl resin using the procedure described above and
cleavage from the resin with TFA, two products were detected
by HPLC that appeared to be Fmoc-Leu-NH-Et (tR ¼ 36.3
min, 71%) and Fmoc-Leu-NH2 (tR ¼ 32.5 min, 29%). The
presence of Fmoc-Leu-NH2 suggests that the amine groups
on this resin may not have reacted completely with oNBS-Cl,
even though a negative ninhydrin test was obtained. An addi-
tional coupling of Fmoc-Leu-OH with the more reactive
6-chloro derivative of PyBOP PyClock (6-chlorobenzotri-
azole-1-yloxy-tris-pyrrolidinophosphonium hexafluorophos-
phate)34 increased the yield of Fmoc-Leu-NH-Et to 93%,
decreasing the second product to 7% as determined by
HPLC. Thus nonalkylated amine represented a small percent-
age of the resin, and the larger percentage of Fmoc-Leu-NH2
following the initial double coupling was due to the higher
Fmoc Quantititation Loading
(mmol/g)a
Entry
Resin
Yield (%)
A
B
C
D
E
Fmoc-Leu-N(Me)-resin
Fmoc-Leu-N(Et)-resin
Fmoc-Leu-N(All)-resin
0.171 6 0.001
0.169 6 0.004
0.171 6 0.001
92
91
92
95
95
87
Fmoc-Leu-N(Propargyl)-resin 0.176 6 0.003
Fmoc-Leu-N(CPM)-resin
Fmoc-Leu-N(Bz)-resin
0.175 6 0.001
0.160 6 0.001
F
a Mean 6 SEM of three samples.
Peptide Synthesis and Analysis
To evaluate this method of synthesizing peptide C-terminal reactivity of the primary amine in the coupling compared to
N-alkyl amides we then introduced Fmoc-Leu-OH onto the the secondary amine. In the case of the N-CPM resin after the
N-alkyl amines in preparation for the synthesis of the initial double coupling Fmoc-Leu-NH-CPM (tR ¼ 40.3 min)
C-terminal N-alkyl amide derivatives of the model pentapep- was the minor product by HPLC (42%), and the peak at 32.7
tide Leu-enkephalin, Tyr-Gly-Gly-Phe-Leu-NH-R. Coupling min that appeared to be Fmoc-Leu-NH2 was the major prod-
of Fmoc-Leu-OH (using benzotriazole-1-yloxy-tris-pyrrol- uct (58%). An additional coupling with PyClock increased
idinophosphonium
hexafluorophosphate
(PyBOP), the yield of Fmoc-Leu-NH-CPM to 86%. Following the cou-
1-hydroxybenzotriazole (HOBt), and DIEA in DMF (4/4/4/ pling with PyClock the resin substitutions for Fmoc-Leu-NH-
10) as coupling reagents) to the secondary amino groups on Et and Fmoc-Leu-NH-CPM were both determined to be 0.11
the PAL-PEG-PS resin was relatively slow and required dou- mmol/g by Fmoc quantitation, corresponding to loading
ble couplings with extended reaction times (overnight). Any yields of only 48% for the first amino acid on this resin, in
unreacted free amino groups were then blocked using an contrast to loading yields of >90% for the PAL-PEG-PS resin.
excess of acetic anhydride and DIEA in DMF, and the loading
Following the successful installation of the first amino
of the first amino acid determined by Fmoc quantitation acid, the synthesis of the Leu-enkephalin C-terminal N-alkyl
(Table IV).11,33 Coupling yields of the first amino acid Fmoc- amides was continued on the PAL-PEG-PS resin using stand-
Leu to these secondary amino groups on the PAL-PEG-PS ard SPPS methods described previously.35 These peptides
resin were generally >90% (Table IV).
were then cleaved from the resin with 95% TFA with 5%
Coupling of Fmoc-Leu-OH to the N-ethyl or N-CPM water for 2 h. Electrospray ionization mass spectrometry
amines on the Rink amide resin, however, proved to be more (ESI-MS) analysis showed the desired molecular weights for
difficult. Following the double coupling of Fmoc-Leu-OH to all of the peptides (Table V), and HPLC analysis indicated
Table V HPLC and MS Analysis of Crude Peptide C-Terminal Alkylated Amides Synthesized on
the PAL-PEG-PS Resin
ESI-MS (m/z, [M + H]+)
HPLCa tR
Entry
Peptide Sequence
(min)/% Purity
Calculated
Observed
A
B
C
D
E
Y-G-G-F-L-NH2
Y-G-G-F-L-NH-Me
Y-G-G-F-L-NH-Et
14.6/98.4
14.8/90.4
16.8/97.0
17.9/88.0
17.6/98.9
20.0/90.7
23.5/93.8
555.3
569.3
583.3
595.3
593.3
609.3
645.3
555.3
569.3
583.3
595.3
593.3
609.3
645.3
Y-G-G-F-L-NH-All
Y-G-G-F-L-NH-propargyl
Y-G-G-F-L-NH-CPM
Y-G-G-F-L-NH-Bz
F
G
a See Table I for HPLC conditions.
Biopolymers (Peptide Science)

720
Fang, Yakovleva, and Aldrich
FIGURE 2 HPLC chromatogram of the crude peptide Y-G-G-F-L-NH-Bz synthesized on the PAL-
PEG-PS resin. See Table I for HPLC conditions.
that most of these crude peptides had a purity of >90%
(Table V). There was no trace of the non-alkylated control
peptide Tyr-Gly-Gly-Phe-Leu-NH2, either in the HPLC or
MS spectra (HPLC tR ¼ 14.6 min, see Table V for condi-
tions; calculated [M+H]+ ¼ 555.3), for any of these pep-
CONCLUSIONS
In conclusion, we applied a well-known N-alkylation reac-
tion to the efficient synthesis of peptide C-terminal N-alkyl
amides. The acid labile PAL-PEG-PS and Rink amide resins
are compatible with the reaction conditions since the alkyla-
tion and oNBS deprotection reactions are conducted under
basic conditions. The alkylation reaction on the PAL-PEG-PS
resin proceeded in high yields with a variety of alkyl bro-
mides and iodides at room temperature. We have introduced
the use of the nonvolatile 2,20-(ethylenedioxy)diethanethiol
to deprotect the oNBS group, which offers a significant
advantage over the malodorous thiols typically used in this
reaction. The synthesis of the model enkephalin N-alkyl am-
ide analogs on the PAL-PEG-PS resin using this methodology
gave consistently high yields and purities, demonstrating the
applicability of this synthetic strategy. This methodology can
be widely applied in the combinatorial synthesis of peptide
C-terminal N-alkyl amides.
tides. Figure
2 shows the HPLC tracing of crude
Leu-enkephalin-NH-Bz (Tyr-Gly-Gly-Phe-Leu-NH-Bz) as
an example.
The methodology described here offers an alternative to
other methods to synthesize peptide C-terminal N-alkyl
amides. It is complementary to the use of a BAL resin, espe-
cially for the incorporation of small alkyl groups such as
methyl or ethyl into the C-terminal amide. As noted earlier,
these groups are of considerable interest in the design of
therapeutically relevant peptides. In both these methods the
C-terminal N-alkyl group is installed in the initial steps in
the synthesis. Once the C-terminal N-alkyl group is intro-
duced, the peptide sequence can be easily varied using stand-
ard SPPS methodology, making it especially useful for the
synthesis of combinatorial peptide libraries containing a
modified C-terminus. In contrast, with the ‘‘safety catch’’
resin the N-alkyl group is introduced during the last step
when the peptide is cleaved from the resin.3,6,22–25 There are
limitations with regard to the nucleophile used in this reac-
tion, and hindered amines do not always yield the desired
peptide.6
In contrast, the synthesis of N-alkyl amides on a Rink am-
ide resin was more difficult than on the PAL-PEG-PS resin.
Alkylation of the Rink amide resin with the bulky alkyl halide
CPM bromide required modified conditions (elevated tem-
perature) to drive the reaction to close to completion. Cou-
pling to the N-alkyl amine on the bulky Rink amide linker
was much more difficult than on the PAL-PEG-PS resin, and
even an additional third coupling with the more reactive
Biopolymers (Peptide Science)

Synthesis of Peptide C-Terminal N-Alkyl Amides
721
case the alkylation with CPM bromide and DBU (20 equiv) was
repeated at 80oC for an additional day; this increased the yield of
oNBS-NH-CPM to 93%.
coupling reagent PyClock resulted in low loading yields for
the first amino acid on the Rink amide resin.
EXPERIMENTAL
Coupling of Fmoc-Leu-OH to the N-Alkyl Resins
Fmoc-Leu-OH (4 equiv) was double coupled to the 9-PAL-PEG-PS
resins with PyBOP, HOBt, and DIEA (4/4/10) in DMF for 12 h. The
reactions were monitored using the chloranil test.36 Any unreacted
free amino groups were capped using an excess of acetic anhydride
and DIEA (20 equiv each) in DMF for 30 min. Loading of the first
amino acid was then determined by Fmoc quantitation (see
below).11,33
Materials
All Fmoc-protected amino acids were purchased from Bachem
(King of Prussia, PA), Novabiochem (San Diego, CA), Applied Bio-
systems (Foster City, CA), or Peptides International (Louisville,
KY). Fmoc-PAL-PEG-PS resin was purchased from Applied Biosys-
tems, and the Rink amide-PEG-PS resin (NovaSyn TGR resin) was
purchased from Novabiochem. PyBOP and PyClock were purchased
from Novabiochem. All solvents, methyl iodide, and TFA were pur-
chased from Fisher Scientific (Hampton, NH). HOBt, allyl bromide,
benzyl bromide, propagyl bromide, phenylethyl chloride, and thio-
phenol were purchased from Acros Chemical Co. (Pittsburgh, PA).
All other chemicals, including oNBS-Cl, DBU, DMAP, ethyl iodide,
cyclopropylmethyl bromide, phenylethyl bromide, potassium iodide,
2-mercaptoethanol, 2,20-(ethylenedioxy)diethanethiol, and piperi-
dine, were purchased from Aldrich Chemical Co. (Milwaukee, WI).
The N-ethyl and N-CPM Rink amide resins were initially double
coupled with PyBOP, HOBt and DIEA as described above. Aliquots
of each resin were cleaved with TFA, and the products analyzed by
HPLC using the conditions listed above. The coupling with Fmoc-
Leu-OH (4 equiv) was repeated with PyClock,34 HOAt (1-hydroxy-
azabenzotriazole) and DIEA (4/4/10) in DMF for an additional 12
h; additional aliquots of each resin were then cleaved with TFA and
analyzed by HPLC.
Fmoc Quantitation11,33
Installation of the C-Terminal N-Alkyl Group
About 5 mg of resin (three samples for each resin) was deprotected
using piperidine (0.4 mL) in DCM (0.4 mL) for 30 min. Methanol
(1.6 mL) was then added, and the mixture diluted to a total volume
of 10 mL with DCM. The UV absorbance was measured at 301 nm
compared to a blank, and the Fmoc loading calculated using an
The Fmoc group of the Fmoc-PAL-PEG-PS resin (Fmoc-3, 0.19
mmol/g) was first removed using piperidine in DMF (1/4, v/v, 2 3
10 min) to yield the free amine, which was then coupled with 4
equiv each of oNBS-Cl and DIEA in DCM for 30 min to afford the
oNBS-NH-resin (7-resin, Scheme 1). The completion of this reac-
tion was monitored by the ninhydrin test.32 Cleavage of an aliquot
of the resin afforded pure oNBS-NH2 (7, HPLC (5–50% aqueous
MeCN containing 0.1% TFA over 45 min at 1 mL/min, monitored
at 214 nm): tR ¼ 3.8 min). The oNBS-NH-resin was then treated
with an alkyl halide and DBU (10 equiv each) in DMF for 2 days at
room temperature to afford the oNBS-NR-resins (8-resins). The
yields of these reactions were monitored by HPLC analysis (see con-
ditions above) of aliquots to monitor the appearance of oNBS-NHR
and the disappearance of oNBS-NH2 following cleavage from the
resin using TFA (Table I). For phenylethyl chloride, KI (0.1 equiv)
was also added as a catalyst; the reaction was run at room tempera-
ture or at 808C to increase the conversion rate (Table I).
The oNBS-NR-resins (8-resins) with quantitative alkylation were
then treated with different bases and thiols in DMF (1/1/2) at room
temperature for 1 day to afford the RNH-resins (9-resins). A small
quantity of resin (around 5 mg) was cleaved using TFA (1 mL) for
3 h. Following filtration and washing the resin with TFA (1 mL), the
TFA was evaporated and the residue was analyzed by HPLC using
the conditions described above. The difference in the absorbance
of oNBS-NHR before and after oNBS deprotection was used to
estimate the conversion rate.
extinction coefficient of 7800 Mꢀ1 cmꢀ1
.
Synthesis of the Model Peptides
Following successful installation of the first amino acid, the synthe-
sis of the Leu-enkephalin C-terminal N-alkyl amides was continued
on the PAL-PEG-PS resin using the standard SPPS method as
described previously.35 The model peptides were cleaved from the
resins using 95% TFA and 5% water for 2 h. Following filtration the
TFA was evaporated, and the peptides analyzed as described below.
Analysis of the Peptides
The crude peptides were analyzed by HPLC using a linear gradient
of 5% to 50% solvent B (solvent A aqueous 0.1% TFA, and solvent
B MeCN containing 0.1% TFA) over 45 min, at a flow rate of 1 mL/
min, with monitoring at 214 nm (Table V). The molecular weights
of these peptides were determined by ESI-MS.
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