Investigation of unanticipated alkylation at the N(π) position of a histidyl residue under Mitsunobu conditions and synthesis of orthogonally protected histidine analogues

Article abstract of DOI:10.1021/jo201599c

We had previously reported that Mitsunobu-based introduction of alkyl substituents onto the imidazole N(π)-position of a key histidine residue in phosphothreonine-containing peptides can impart high binding affinity against the polo-box domain of polo-like kinase 1. Our current paper investigates the mechanism leading to this N(π)-alkylation and provides synthetic methodologies that permit the facile synthesis of histidine N(π)-modified peptides. These agents represent new and potentially important tools for biological studies.

Full text of DOI:10.1021/jo201599c

Article
pubs.acs.org/joc
Investigation of Unanticipated Alkylation at the N(π) Position of a
Histidyl Residue Under Mitsunobu Conditions and Synthesis of
Orthogonally Protected Histidine Analogues
Wenjian Qian, Fa Liu,† and Terrence R. Burke, Jr.*
Chemical Biology Laboratory, Molecular Discovery Program, Center for Cancer Research, National Cancer Institute, National
Institutes of Health, Frederick, Maryland 21702, United States
S
* Supporting Information
ABSTRACT: We had previously reported that Mitsunobu-based introduction of alkyl
substituents onto the imidazole N(π)-position of a key histidine residue in phosphothreonine-
containing peptides can impart high binding affinity against the polo-box domain of polo-like
kinase 1. Our current paper investigates the mechanism leading to this N(π)-alkylation and
provides synthetic methodologies that permit the facile synthesis of histidine N(π)-modified
peptides. These agents represent new and potentially important tools for biological studies.
intended products. These byproducts exhibited exceptionally
high affinities in Plk1 PBD-binding assays, and in some, these
affinities were approximately 3 orders of magnitude greater
than the parent peptide (1). A Plk1 PBD cocrystal structure of
the highest affinity peptide byproduct, which was obtained in a
small amount along with the expected major product (2)
following Mitsunobu reaction with C6H5(CH2)8-OH, showed it
to be the modified peptide 3, in which alkylation had occurred
on the histidine imidazole N(π) group (Figure 2).16 The high
affinity and unexpected nature of this histidine adduct
prompted us to investigate potential mechanisms leading to
its formation and to develop synthetic methodologies to
prepare related peptide analogues.
INTRODUCTION
■
The serine/threonine specific polo-like kinase 1 (Plk1) is one
of four Plks involved in cell cycle regulation. Plk1 is of
particular interest as a therapeutic target, due to its ability to
promote oncogenic transformation.1−3 Pharmacological inhib-
ition of Plk1 is being approached through kinase catalytic site-
directed agents.4−11 Inhibitors of Plk1 function that interfere
with the actions of its C-terminal polo-box domains (PBDs) are
also being investigated. These latter homologous protein
modules facilitate subcellular localization of Plk1 by recognizing
and binding to phosphoserine (pSer)/phosphothreonine
(pThr)-containing sequences.12,13
The polo-box interacting protein 1 (PBIP1) is a Plk1
substrate, which undergoes phosphorylation at its T78 residue
to form a Plk1 PBD-binding site that is critical for recruiting
Plk1 to the interphase and mitotic kinetochores.14 We had
previously shown that the wild-type PBIP1-derived five-mer
sequence, 74-PLHSpT-78, represents a minimal peptide (1)
that specifically interacts with the Plk1 PBD with high affinity
(Kd = 0.45 μM).15 More recently, in an effort to investigate
whether reduction in the phosphoryl anionic charge could also
be accomplished with retention of binding affinity, we
performed synthetic experiments to esterify one of the two
pThr phosphoryl hydroxyls of the parent peptide 1.16 Our
approach was to conduct solid-phase Mitsunobu coupling17 of a
series of alcohols with the protected resin-bound peptide, Ac-
Pro-Leu-His[N(τ)Trt]-Ser(OBut)-Thr[OPO(OBn)(OH)]-
amide,18 which contained a single free phosphoryl hydroxy
group (Figure 1). Applications of Mitsunbu chemistries to
resin-supported substrates and to the synthesis of phosphoryl
esters are known.19,20
RESULTS AND DISCUSSION
■
We rationalized that alkylation at the His N(π) of peptide 3
could potentially have occurred either indirectly by intra-
molecular alkyl transfer of the phosphoryl ester group
(mechanism 1, Figure 3) or directly through alkylation by the
alcohol reactants under Mitsunobu conditions (mechanism 2,
Figure 3). There are literature precedents for both mechanisms.
For example, in 1-substituted imidazoles, alkylation of the 3-
nitrogen [corresponding to the N(π)-position of histitine] can
be effected by phosphoryl esters. However, these reactions
require elevated temperatures.21,22 Similarly, alkylation of
imidazole by alcohols can be achieved using Mitsunobu
chemistries.23−25
To examine whether phosphoryl ester-mediated transfer was
involved in the formation of 3 (mechanism 1), Mistunobu
esterification of the free phosphoryl hydroxyl of resin-bound
N(α)-Fmoc-O-(benzyl phosphoryl)-threonine using
C6H5(CH2)8-OH was conducted prior to coupling of the
Following coupling reactions, peptides were cleaved from the
resin under mild acid conditions. In addition to the expected
phosphoryl esters, unanticipated synthetic peptide byproducts
were isolated having the same molecular weights as the
Received: August 5, 2011
Published: September 27, 2011
© 2011 American Chemical Society
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Figure 1. Solid-phase Mistunobu reaction.
alcohol used in the Mitsunobu reacton or from preformed
phosphoryl ester under Mitsunobu catalysis, the above reaction
was repeated using a homologous alcohol having a shorter
chain length [C6H5(CH2)7-OH)] as compared with the
C6H5(CH2)8-OH used in the second Mitsunobu reaction.
This allowed differentiation between sources of the alkylating
species. In this case, it was found that the mass of the bis-adduct
was consistent with the addition of C6H5(CH2)7− (peptide 5,
Figure 2), indicating that histidine alkylation occurred directly
from the alcohol used in the second Mitsunobu reaction
(mechanism 2). We concluded from these studies that the high
affinity byproduct 3 was formed by direct alkylation with
C6H5(CH2)8−OH during the Mitsunobu phosphoryl esertifi-
cation reaction.
As exemplified by 3, N(π)-alkyl-histidine residues can impart
exceptional Plk1 PBD-binding affinity.16 However, the adducts
in the referenced study were obtained as minor reaction
byproducts. To facilitate a broader application of N(π)-alkyl-
histidine analogues, we undertook the preparation of N(α)-
Fmoc-[N(π)-R]-His [8 for R = C6H5(CH2)8−], which is
suitably protected for incorporation of histidine derivatives into
peptides using standard Fmoc protocols. We employed
Hodges' regiospecific synthesis of N(π)-substituted histidines,
which relies on the use of N(τ)-Boc-protected histidines and
alkyl triflates as the alkyting species.26 Our approach started
with N(α),N(τ)-bis(tert-butoxycarbonyl)-L-histidine methyl
ester (6).26 A solution of 8-phenyloctan-1-ol in CH2Cl2 at
−75 °C was treated with triflic anhydride and diisopropylethyl-
amine (DIEPA, 1 equiv) to form the triflate ester in situ. This
was then reacted with the N(τ)-Boc histidine derivative 6 to
form the corresponding N(π)-adduct 7 in 60% yield (Scheme
Figure 2. Structures of peptides discussed in the text.
N(α)-Fmoc-[N(τ)-Trt]-His residue. Following Mitsunobu
phosphoryl ester formation, the resin was washed, and peptide
synthesis was completed in standard fashion using Fmoc
protocols. A portion of the resulting finished resin was cleaved
and examined by HPLC. The chromatogram showed the
presence of the peptide phosphoryl ester 2 with no evidence of
product 3. This indicated that N(π)-alkylation did not occur by
intramolecular transfer from the phosphoryl ester.
Direct Mitsunobu-induced N(π)-alkylation (mechanism 2)
was examined next by again subjecting a portion of the above,
fully formed resin-bound peptide to a second Mitsunobu
coupling with C6H5(CH2)8-OH prior to resin cleavage. HPLC
analysis of the resulting peptide products showed that in
addition to 2, a new product was obtained having a mass
consistent with the bis-alkylated product (4, Figure 2). This
indicated that histidine alkylation had occurred during the
second Mitsunobu reaction. To unambiguously discriminate
whether the additional alkyl adduct originated directly from the
Figure 3. Two possible mechanisms of histidine alkylation.
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a
obtained only as a minor byproduct from the synthesis of
intended product 2. The successful completion of this synthesis
confirmed the utility of 8 as providing a much improved route
to peptides containing N(π)-modified histidine residues.
The general solid-phase methodology outline above using
modified histidine analogue 8 is inherently limited because it
requires that a new histidine reagent be prepared for each
unique N(π)-substituent installed. A more useful approach that
would allow the generation of multiple peptides containing
unique N(π)-substituents could be realized through on-resin
modification of the histidyl residue in postpeptide synthesis
fashion. Scheme 3 shows the application of such a protocol to
the synthesis of peptide 13. Peptide 13 is a variant of 3 having
the labile pThr residue replaced by (2S,3R)-2-amino-3-methyl-
4-phosphonobutyric acid (Pmab), which is a phosphatase-stable
pThr mimetic32 that retains the PBD-binding affinity of parent
pThr-containing peptides.15,16 A key feature of the route
outlined in Scheme 3 is its use of commercially available N(α)-
Fmoc-[N(τ)-Trt]-His for the incorporation of an N(τ)-Trt-
protected histidine residue. Although N(α)-Fmoc-[N(τ)-Boc]-
His is also commercially available, the N(τ)-Boc group suffers
from a lack of stability against prolonged or repeated exposure
to piperidine used in Fmoc-based protocols.31
In conclusion, our current paper provides evidence that the
formation of high affinity Plk1 PBD-binding peptide by-
products previously observed as minor components following
on-resin Mitsunobu esterification result from direct alkylation
at the N(π)-position. We detail the high yield preparation of
N(α)-Fmoc-N(π)-modified histidine reagents from N(α)-Boc-
and N(α)-Fmoc-protected precursors, which permit facile
Fmoc-based solid-phase synthesis of N(π)-modified peptides.
We extend the utility of this chemistry by demonstrating on-
resin triflate-mediated derivatization of fully formed peptides
that should permit the ready preparation of libraries of N(π)-
alkyl peptides. Although only a very narrow range of alcohols is
specifically examined in our current report, it is anticipated that
the approach should provide access to a broad range of N(π)-
modified histidine analogues that may be useful in a variety of
biological contexts.
Scheme 1.
a
Reagents and conditions: (i) Ph(CH2)7CH2OH (1.1 equiv), Tf2O
(1.1 equiv), DIPEA (1.1 equiv), CH2Cl2, −75 °C−rt, 16−18 h, 60%
yield. (ii) LiOH·H2O (2.0 equiv), THF, H2O (v/v 4:1), 0 °C−rt, 1 h.
(iii) 4 M HCl in dioxane (10 equiv), rt, 1 h. (iv) Fmoc-OSu (1.5
equiv), NaHCO3 (4.0 equiv), THF − H2O (v/v 1:1), rt, 12 h (82%
over three steps).
1). Without purification, global deprotection was performed,
and the crude product was reacted with 9-fluorenylmethyl
succinimidyl carbonate (Fmoc-OSu) and aqueous NaHCO3 in
tetrahydrofuran to yield the final, protected amino acid
analogue, 8 (83% from 7, Scheme 1).
N(π)-Derivatization in the presence of N(τ)-Trt protection
has also previously been reported using alkyl halides as the
alkylating species; however, these conditions either involve
elevated temperatures, prolonged reaction times, or large
excesses of reagent.27−29 In model reactions on N(α)-Fmoc-
[N(τ)-Trt]-His methyl ester (9)30 (prepared from commer-
cially available N(τ)-trityl-L-histidine methyl ester), we found
that N(π)-alkylated product (11) could be obtained in high
yield at room temperature using in situ-generated alkyl triflate.
The crude product from this reaction contained a mixture of
the N(τ)-trityl, N(π)-alkyl bis-adduct (10) as well as the
desired product (11) (Scheme 2). Treatment of this mixture
a
Scheme 2.
a
EXPERIMENTAL SECTION
Reagents and conditions: (i) Ph(CH2)7CH2OH (1.1 equiv), Tf2O
■
(1.1 equiv), DIPEA (1.1 equiv), CH2Cl2, −75 °C−rt, 16−18 h. (ii)
TFA (10 equiv), triisopropylsilane (1.1 equiv), CH2Cl2, rt, 2 h, (90%
over two steps). (iii) LiI (6.0 equiv), EtOAc, reflux, 20 h, 80%.
Synthesis of N(α)-Fmoc-[N(π)-(8-phenyloctyl)]-histidine (8
from 6) . N(α),N(τ)-Bis[(1,1-dimethylethoxy)carbonyl]-L-histidine
Methyl Ester (6). 26 Triethylamine (11.51 mL, 83 mmmol) was added
to a stirred suspension of L-histidine methyl ester hydrochloride (10.0
g, 41.3 mmol) in MeOH (98 mL), and stirring was continued at room
temperature until dissolution was complete (30 min). A solution of di-
tert-butyl dicarbonate (18.03 g, 83 mmol) in MeOH (49 mL) was then
added dropwise over 30 min, and stirring was continued at room
temperature (48 h). Solvent was removed in vacuo, and the residue
was partitioned between CH2Cl2 and H2O. The organic layer was
washed with 10% citric acid, dried (MgSO4), concentrated, and
purified by gel column chromatography (EtOAc:hexanes; from = 4:1
to 1.5:1) to afford known 6 as a white amorphous solid (11.06 g, 72%
yield).
N(α)-[(1,1-Dimethylethoxy)carbonyl]-N(π)-(8-phenyloctyl)-L-histi-
dine Methyl Ester (7). To a stirred solution of triflic anhydride (1.51
mL, 8.93 mmol) in CH2Cl2 (18 mL) under nitrogen at −75 °C was
added a solution of 8-phenyloctan-1-ol (1.88 mL, 8.93 mmol) and
diisopropylethylamine (DIEPA) (1.56 mL, 8.93 mmol) in CH2Cl2
(12.0 mL) dropwise over 10 min. Stirring was continued at −75 °C
(20 min), then a solution of 6 (3.0 g, 8.12 mmol) in CH2Cl2 (21 mL)
was added dropwise, and the mixture was allowed to gradually warm to
room temperature over a period of 16−18 h. The mixture was poured
into a solution of aqueous NaHCO3 and stirred vigorously (30 min).
with TFA gave 11 in 90% yield from 9, and demethylation
under nucleophilic conditions gave 8 in good yield. This
approach represents a more direct synthesis of 8 than that
described above using starting material 6 (Scheme 1).
Traditionally, during peptide synthesis, protection of the
histidine imidazole ring is important to avoid side product
formation arising from ring acylation and subsequent migration
of acyl species to other regions of the peptide. Protection also
minimizes intramolecular base-catalyzed racemization of
activated histidine by the N(π)-group. Acid-labile N(τ)-Trt
derivatization is commonly employed for this purpose in
combination with N(α)-Fmoc protection.31 Analogue 8 is
somewhat unique in that its N(π)-adduct serves a dual capacity
of protecting the imidazole ring during peptide synthesis and
providing potentially important biological functionality. To
verify the synthetic utility of 8, we employed the reagent for the
solid-phase synthesis of peptide 3, which had previously been
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Scheme 3. Postpeptide Synthesis On-Resin N(π)-Alkylation
The organic layer was diluted with CH2Cl2 and washed with aqueous
NaHCO3 and brine, then dried (Na2SO4), and concentrated to a gum.
Purification by silica gel flash chromatography (CHCl3:MeOH; from
100:1 to 100:2) provided 7 as colorless gum (2.23 g, 60% yield).
mmol) in THF:H2O (v/v 1:1) at 0 °C was added Fmoc-OSu (0.9 g,
2.67 mmol), and the mixture was allowed to come to room
temperature and stirred (overnight). The organic layer was collected,
the aqueous layer was extracted (EtOAc), and the combined organic
layers were washed with H2O and brine and dried (Na2SO4).
Concentration in vacuo provided a residue, which was purified by silica
gel column chromatography (EtOAc:petroleum ether; from 1:10 to
21
1
[α]D 23.99 (c 1.045, CHCl3). H NMR (400 MHz, CDCl3): δ 7.43
(s, 1H), 7.30−7.24 (m, 2H), 7.20−7.14 (m, 3H), 6.78 (s, 1H), 5.18 (d,
J = 8 Hz, 1H), 4.54 (q, J = 4 Hz, 1H), 3.82 (t, J = 8 Hz, 2H), 3.74 (s,
3H), 3.16−2.96 (m, 2H), 2.60 (t, J = 8 Hz, 2H), 1.83 (brs, 1H), 1.77−
1.65 (m, 2H), 1.65−1.55 (m, 2H), 1.43 (s, 9H), 1.31 (brs, 8H). 13C
NMR (100 MHz, CDCl3): δ 171.7, 155.0, 142.7, 137.4, 128.3, 128.2,
128.0, 125.8, 125.5, 80.2, 53.0, 52.5, 44.7, 35.9. 31.3, 30.9, 29.3, 29.1,
29.0, 28.2, 26.9, 26.6. IR νmax 2929, 2361, 1705, 1164 cm−1. HR-ESI
MS calcd for C26H40N3O4 (M + H)+, 458.3013; found, 458.3023.
N(α)-[(9H-Fluoren-9-ylmethoxy)carbonyl]-N(π)-(8-phenyloctyl)-L-
histidine (8). To a solution of 7 (0.7 g, 1.53 mmol) in dissolved in
THF (10 mL) and H2O (2.5 mL) at 0 °C was added LiOH·H2O (128
mg, 3.06 mmol), and the mixture was stirred at 0 °C (1.5 h). The
solution was acidified to pH 1−2 (aqueous HCl), then THF was
removed in vacuo, the residue was extracted (EtOAc), and the organic
layer was dried and concentrated in vacuo. The residue was dissolved
in a solution of 4 M HCl in dioxane (4 mL) and EtOAc (4 mL),
stirred at room temperature (1 h), and then concentrated in vacuo.
The residue was dissolved in solution of THF (7 mL) and H2O (7
mL), and 9-fluorenylmethyl-succinimidyl carbonate (Fmoc-OSu) (774
mg, 2.30 mmol) and NaHCO3 (514 mg, 6.12 mmol) were added, and
the mixture was stirred at room temperature (overnight). The mixture
was neutralized by the addition of saturated NH4Cl and extracted
(EtOAc), and the organic layer was dried and concentrated in vacuo.
The resulting residue was purified by silica gel column chromatog-
raphy first by elutiom with (CHCl3:MeOH; from 100:1 to 100:3) to
remove byproduct and then (CHCl3:MeOH; from 10:1 to 3:1) to
provide the desire product 8 as a white wax (716 mg, 83% yield from
7). [α]D21 38.78 (c 0.90, CHCl3). 1H NMR (400 MHz, DMSO-d6): δ
8.39 (s, 1H), 7.89 (d, J = 8 Hz, 2H), 7.85 (d, J = 8 Hz, 1H), 7.67 (d, J
= 8 Hz, 2H), 7.41 (t, J = 8 Hz, 2H), 7.32 (t, J = 8 Hz, 2H), 7.25 (t, J =
8 Hz, 2H), 7.20−7.10 (m, 3H), 7.8 (s, 1H), 4.35−4.15 (m, 4H), 4.10−
3.90 (m, 2H), 3.20−2.90 (m, 2H), 2.60 (s, 1H), 2.55−2.47 (m, 2H),
1.75−1.60 (m, 2H), 1.60−1.45 (m, 2H), 1.23 (brs, 9H). 13C NMR
(100 MHz, DMSO-d6): δ 172.4, 155.9, 143.7, 142.2, 140.7, 135.9,
129.1, 128.20, 128.16, 127.6, 127.1, 125.5, 125.2, 121.8, 120.1, 65.7,
53.0, 46.6, 45.0, 35.1, 30.9, 29.8, 28.7, 28.5, 28.4, 25.8, 25.2. IR νmax
2930, 2361, 1713, 1452, 1255 cm−1. HR-ESI MS calcd for C35H40N3O4
(M + H)+, 566.3013; found, 566.3022.
1:4) to yield known 930 as a white foam (84% yield). [α]D 9.55 (c
23
1.0, CHCl3). 1H NMR (400 MHz, CDCl3): δ 7.77 (d, J = 8.0 Hz, 2H),
7.64 (t, J = 8.0 Hz, 2H), 7.44 (s, 1H), 7.40 (t, J = 8.0 Hz, 2H), 7.37−
7.32 (m, 9H), 7.30 (t, J = 8.0 Hz, 2H), 7.16−7.10 (m, 6H), 6.60 (s,
1H), 6.55 (d, J = 8.0 Hz, 1H), 4.68−4.63 (m, 1H), 4.40−4.24 (m,
3H), 3.65 (s, 3H), 3.14−3.17 (m, 2H). 13C NMR (125 MHz, CDCl3):
δ 172.3, 156.4, 144.3, 144.1, 142.5, 141.4, 139.0, 136.6, 129.9, 128.26,
128.25, 127.8, 127.2, 125.6, 125.5, 120.1, 119.8, 75.5, 67.4, 54.5, 52.4,
47.4, 30.3. IR νmax 2361, 1719, 1492, 1445, 1248 cm−1. HR-ESI MS
calcd for C41H35N3O4Na (M + Na)+, 656.2525; found, 656.2538.
N(α)-[(9H-Fluoren-9-ylmethoxy)carbonyl]-N(τ)-(8-phenyloctyl)-L-
histidine Methyl Ester (11). To a stirred solution of triflic anhydride
(0.101 mL, 0.60 mmol) in CH2Cl2 (3.8 mL) under nitrogen at −75 °C
was added a solution of 8-phenyloctan-1-ol (124 mg, 0.60 mmol) and
DIEA (0.104 mL, 0.60 mmol) in CH2Cl2 (2.6 mL) dropwise over 10
min at −75 °C. Stirring was continued at −75 °C (20 min), then a
solution of 9 (0.34 g, 0.54 mmol) in CH2Cl2 (4.5 mL) was added
dropwise, and the mixture was allowed to gradually warm to room
temperature and stirred (16 −18 h). The mixture was poured into
aqueous NaHCO3 and stirred vigorously (30 min) and then extracted
with CH2Cl2, and the organic extract washed with aqueous NaHCO3
and brine, dried (Na2SO4), and concentrated in vacuo. To a solution
of the resulting gum in CH2Cl2 (4.2 mL) was added trifluoroacetic
acid (0.42 mL, 5.4 mmol) and triisopropylsilane (TIS) (0.12 mL, 0.60
mmol), and the mixture was stirred at room temperature until reaction
was complete as shown by TLC (2 h). The solvent was removed in
vacuo, and the residue was purified by silica gel column
chromatography (CH2Cl2:MeOH; from 100:1 to 20:1) to provide
11 as a white foam (90% yield from 9). [α]D22.4 1.35 (c 1.01, CHCl3).
1H NMR (400 MHz, CDCl3): δ 8.62 (s, 1H), 7.75 (d, J = 8.0 Hz, 2H),
7.58 (d, J = 8.0 Hz, 2H), 7.39 (t, J = 8.0 Hz, 3H), 7.40−7.23 (m, 4H),
7.22−7.10 (m, 4H), 6.05 (d, J = 8.0 Hz, 1H), 4.56 (d, J = 4.0 Hz, 1H),
4.38 (d, J = 8.0 Hz, 2H), 4.18 (d, J = 8.0 Hz, 2H), 4.06−4.01 (m, 2H),
3.77 (s, 3H), 3.25−3.14 (m, 2H), 2.58 (t, J = 8.0 Hz, 2H), 1.81−1.73
(m 2H,), 1.63−1.53 (m, 2H), 1.28 (s, 8H). 13C NMR (100 MHz,
CDCl3): δ 170.6, 156.2, 143.79, 143.74, 142.9, 141.5, 134.9, 129.8,
128.6, 128.4, 128.0, 127.4, 125.8, 125.2, 120.2, 119.1, 67.5, 53.3, 53.1,
47.2, 36.1, 31.5, 30.2, 29.4, 29.3, 29.0, 26.5, 26.3. IR νmax 2928, 2361,
1718, 1166, 1028 cm−1. HR-ESI MS calcd for C36H42N3O4 (M + H)+,
580.3175; found, 580.3178.
Synthesis of N(α)-[(9H-Fluoren-9-ylmethoxy)carbonyl-[N(π)-
(8-phenyloctyl)]-histidine (8 from 9). N(α)-[(9H-Fluoren-9-
ylmethoxy)carbonyl]-N(τ)-(triphenylmethyl)-L-histidine Methyl
Ester (9). To a solution of N(τ)-(triphenylmethyl)-L-histidine methyl
ester hydrochloride (0.8 g, 1.78 mmol) and NaHCO3 (0.45 g, 5.3
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N(α)-[(9H-Fluoren-9-ylmethoxy)carbonyl]-N(τ)-(8-phenyloctyl)-L-
histidine (8). Lithium iodide (1.13 g, 8.45 mmol) was added to a
stirred solution of 11 (0.82 g, 1.41 mmol) in dry EtOAc (28 mL)
under nitrogen, and the mixture was heated at reflux (20 h). The
mixture was brought to room temperature, and the reaction was
quenched by the addition of 10% aqueous HCl and extracted
(EtOAc). The organic extract was dried (Na2SO4) and concentrated in
vacuo, and the residue was purified by silica gel flash chromatography
(CH2Cl2:MeOH; from 95:5 to 80:20) to provide 8 as a colorless gum
(80% yield).
Table 1. Peptide ESI-MS Spectral Data and HPLC Retention
Times
expected (M +
H)+
observed (M +
H)+
retention
(min)
a
no.
HPLC
216
316
4
863.4
863.4
863.4
863.4
25.2
24.3
29.8
29.2
23.3
II
II
I
1051.6
1037.6
861.5
1051.5
1037.4
861.4
5
1316
I
I
General Solid-Phase Peptide Synthesis. Protected amino acids
used were Fmoc-Thr(PO(OBzl)OH)-OH, Fmoc-Ser(OtBu), N(τ)-
Trt-N(α)-Fmoc-His, Fmoc-Leu, and Fmoc-Pro (purchased from
Novabiochem). Peptides were synthesized on NovaSynTGR resin
(Novabiochem, catalog no. 01-64-0060) using standard Fmoc solid-
phase protocols in N-methyl-2-pyrrolidone (NMP). 1-O-Benzotria-
zole-N,N,N′,N′-tetramethyl-uronium-hexafluoro-phosphate (HBTU)
(5.0 equiv), hydroxybenzotriazole (HOBT) (5.0 equiv), and N,N-
diisopropylethylamine (DIPEA) (10.0 equiv) were used as coupling
reagents. Amino terminal acetylation was achieved using 1-
acetylimidazole. Finished resins were washed with N,N-dimethylfor-
amide (DMF), MeOH, CH2Cl2, and Et2O, then dried under vacuum
(overnight), and then cleaved by treatment with a solution of
TFA:H2O:TIS (95:2.5:2.5) (4 h). The resin was removed by filtration,
and the filtrate was concentrated under vacuum and then precipitated
with ether, and the precipitate was washed with ether. The resulting
solid was dissolved in 50% aqueous MeCN (5 mL) and purified by
reverse phase preparative HPLC using a Phenomenex C18 column (21
mm × 250 mm, catalog no. 00G- 4436-P0) with a linear gradient from
0% aqueous MeCN (0.1% TFA) to 65% MeCN (0.1% TFA) over 30
min at a flow rate of 10.0 mL/min (detection at 220 nm).
Lyophilization provided products as white powders. For the synthesis
of Pmab-containing peptides, (2S,3R)-4-[di(tert-butyl)-oxyphosphin-
yl]-N-Fmoc-L-valine32 was used in place Fmoc-Thr(PO(OBzl)OH)-
OH.
a
HPLC method: λ = 220 nm; flow rate, 10 mL/min. Solvent A: H2O,
0.1% TFA. Solvent B: MeCN, 0.1% TFA. Gradients as indicated
below:
I
time 0 30 30.1 35
B% 0 100 100 100
II
time 0 30 30.1 35
B% 0 90 100 100
95:2.5:2.5) (4 h). Peptide 13 was obtained following work up and
HPLC purification as indicated in General Solid-Phase Synthesis.
HPLC and mass spectral data are provided in Table 1.
ASSOCIATED CONTENT
■
S
* Supporting Information
1H and 13C NMR spectra of 7−9 and 11, including 1D NOESY
assignment of N(π)-alkylation in 11 and HPLC charts of
peptide 2−5 and 13. This material is available free of charge via
the Internet at http://pubs.acs.org.
Synthesis of Peptide 3 using N(α)-[(9H-Fluoren-9-
ylmethoxy)carbonyl]-N(τ)-(8-phenyloctyl)-L-histidine (8). The
synthesis of peptide 3 was accomplished according to the procedures
listed under General Solid-Phase Synthesis, except that reagent 8 was
used in place of N(τ)-Trt-N(α)-Fmoc-His.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: +1-301-846-5906. Fax: +1-301-846-6033.E-mail:
tburke@helix.nih.gov.
On-Resin N(π)-Alkylation Using Mitsunobu Reaction Con-
ditions. Peptides 2 and 3 were prepared as previously reported.16 For
the synthesis of peptides 4 and 5, resin-bound Fmoc-Thr[(OP(OBn)-
OH]-amide, prepared as indicated above (400 mg, 0.1 mmol), was
swelled in CH2Cl2 (15 min) and then treated with triphenylphosphine
(262 mg, 1.0 mmol), diethyl azidodicarboxylate (DEAD) (0.46 mL,
40% solution in toluene, 1.0 mmol), and 8-phenyloctan-1-ol (206 mg,
1.0 mmol) in dry CH2Cl2 at room temperature (2 h). Coupling of the
remaining peptide residues was conducted as described above in
General Solid-Phase Synthesis. The resulting resin bond Ac-Pro-Leu-
[(τ)-Trt-His]-Ser(OtBu)-Thr[(OP(OBn)(O(CH2)8Ph)]-amide was
subjected to Mitsunobu coupling using triphenylphosphine (262 mg,
1.0 mmol), DEAD (0.46 mL, 40% solution in toluene, 1.0 mmol), and
the appropriate alchohol [8-phenyloctan-1-ol (206 mg, 1.0 mmol) for
peptide 4 and 7-phenylheptan-1-ol (192 mg, 1.0 mmol) for peptide 5]
in dry CH2Cl2 at room temperature (2 h). The resins were then
washed (CH2Cl2), dried under vacuum (2 h), and cleaved by
treatment with [TFA:H2O:TIS (95:2.5:2.5) (4 h)]. Peptides 4 and 5
were following procedures indictated in General Solid-Phase Synthesis.
HPLC and mass spectral data are provided in Table 1.
Synthesis of Peptide 13 by On-Resin N(π)-Alkylation. To a
stirred solution of triflic anhydride (42 μL, 0.25 mmol) in CH2Cl2 (3
mL) under nitrogen at −75 °C was added a solution of 8-phenyloctan-
1-ol (52 mg, 0.25 mmol) and DIEA (45 μL, 0.26 mmol) in CH2Cl2
(2.0 mL) dropwise over 10 min. Stirring was continued at −75 °C (20
min), and then, the mixture was warmed to room temperature and
added to peptide resin 1216 (200 mg, 0.05 mmol) that had been
swelled in CH2Cl2 for 15 min. Coupling was conducted at room
temperature (12 h), and then, the resin was washed (CH2Cl2 and
Et2O), dried under vacuum (2 h), and cleaved (TFA:H2O:TIS;
Present Address
†Lilly Research Laboratories, Indianapolis, Indiana 46285.
ACKNOWLEDGMENTS
■
This work was supported in part by the Intramural Research
Program of the NIH, Center for Cancer Research, NCI-
Frederick, and the National Cancer Institute, National
Institutes of Health.
REFERENCES
■
(1) Strebhardt, K.; Ullrich, A. Nature Rev. Cancer 2006, 6, 321−330.
(2) Eckerdt, F.; Yuan, J. Oncogene 2005, 24, 267−276.
(3) Takai, N.; Hamanaka, R.; Yoshimatsu, J.; Miyakawa, I. Oncogene
2005, 24, 287−291.
(4) Goh, K. C.; Wang, H.; Yu, N.; Zhou, Y.; Zheng, Y.; Lim, Z.;
Sangthongpitag, K.; Fang, L.; Du, M.; Wang, X.; Jefferson, A. B.; Rose,
J.; Shamoon, B.; Reinhard, C.; Carte, B.; Entzeroth, M.; Ni, B.; Taylor,
M. L.; Stuenkel, W. Drug Dev. Res. 2004, 62, 349−361.
(5) McInnes, C.; Mezna, M.; Fischer, P. M. Curr. Top. Med. Chem.
2005, 5, 181−197.
(6) Gumireddy, K.; Reddy, M. V. R.; Cosenza, S. C.; Nathan, R. B.;
Baker, S. J.; Papathi, N.; Jiang, J.; Holland, J.; Reddy, E. P. Cancer Cell
2005, 7, 275−286.
(7) Lansing, T. J.; McConnell, R. T.; Duckett, D. R.; Spehar, G. M.;
Knick, V. B.; Hassler, D. F.; Noro, N.; Furuta, M.; Emmitte, K. A.;
Gilmer, T. M.; Mook, R. A.; Cheung, M. Mol. Cancer Ther. 2007, 6,
450−459.
8889
dx.doi.org/10.1021/jo201599c|J. Org. Chem. 2011, 76, 8885−8890

The Journal of Organic Chemistry
Article
(8) Lenart, P.; Petronczki, M.; Steegmaier, M.; Di Fiore, B.; Lipp, J.
J.; Hoffmann, M.; Rettig, W. J.; Kraut, N.; Peters, J.-M. Curr. Biol.
2007, 17, 304−315.
(9) Lu, L.-Y.; Yu, X. Cell Div. 2009, 4, 4 DOI: 10.1186/1747-1028-4-
4.
(10) Reindl, W.; Yuan, J.; Kraemer, A.; Strebhardt, K.; Berg, T.
ChemBioChem 2009, 10, 1145−1148.
(11) Strebhardt, K. Nat. Rev. Drug Discovery 2010, 9, 643−659.
(12) Elia, A. E. H.; Cantley, L. C.; Yaffe, M. B. Science (Washington,
DC, U. S.) 2003, 299, 1228−1231.
(13) Cheng, K.-Y.; Lowe, E. D.; Sinclair, J.; Nigg, E. A.; Johnson, L.
N. EMBO J. 2003, 22, 5757−5768.
(14) Kang, Y. H.; Park, J.-E.; Yu, L.-R.; Soung, N.-K.; Yun, S.-M.;
Bang, J. K.; Seong, Y.-S.; Yu, H.; Garfield, S.; Veenstra, T. D.; Lee, K.
S. Mol. Cell 2006, 24, 409−422.
(15) Yun, S.-M.; Moulaei, T.; Lim, D.; Bang Jeong, K.; Park, J.-E.;
Shenoy Shilpa, R.; Liu, F.; Kang Young, H.; Liao, C.; Soung, N.-K.;
Lee, S.; Yoon, D.-Y.; Lim, Y.; Lee, D.-H.; Otaka, A.; Appella, E.;
McMahon, J. B.; Nicklaus, M. C.; Burke, T. R. Jr.; Yaffe, M. B.;
Wlodawer, A.; Lee Kyung, S. Nat. Struct. Mol. Biol. 2009, 16, 876−882.
(16) Liu, F.; Park, J.-E.; Qian, W.-J.; Lim, D.; Graber, M.; Berg, T.;
̈
Yaffe, M. B.; Lee, K. S.; Burke, T. R. Jr. Serendipitous alkylation of a
Plk1 ligand uncovers a new binding channel. Nat. Chem. Biol. 2011, 7,
81−91.
(17) Mitsunobu, O. Synthesis 1981, 1−28.
(18) The designation of imidazole nitrogens is according to IUPAC
nomenclature.
(19) Ma, X.; Shi, R.; Zhang, B.; Yan, B. J. Comb. Chem. 2009, 11,
438−445.
(20) Khaled, A.; Gravier-Pelletier, C.; Le, M. Y. Tetrahedron:
Asymmetry 2007, 18, 2121−2124.
(21) Kuhlmann, E.; Himmler, S.; Giebelhaus, H. Green Chem. 2007,
9, 233−242.
(22) Vijayaraghavan, R.; Surianarayanan, M.; Armel, V.; MacFarlane,
D. R.; Sridhar, V. P. Chem. Commun. (Cambridge, U. K.) 2009, 6297−
6299.
(23) Nicolaou, K. C.; Cao, G.-Q.; Pfefferkorn, J. A. Angew. Chem., Int.
Ed. Engl. 2000, 39, 739−743.
(24) Kim, E. J.; Ko, S. Y.; Dziadulewicz, E. K.. Tetrahedron Lett. 2005,
46, 631−633.
(25) Petit, S.; Azzouz, R.; Fruit, C.; Bischoff, L.; Marsais, F.
Tetrahedron Lett. 2008, 49, 3663−3665.
(26) Hodges, J. C. Synthesis 1987, 20−24.
(27) Jones, J. H.; Rathbone, D. L.; Wyatt, P. B. Synthesis 1987,
1110−1113.
(28) Kimbonguila, A. M.; Boucida, S.; Guibe, F.; Loffet, A.
Tetrahedron 1997, 53, 12525−12538.
(29) Kim, B. M.; Park, J. S.; Cho, J. H. Tetrahedron Lett. 2000, 41,
10031−10034.
(30) Himes, R. A.; Park, G. Y.; Barry, A. N.; Blackburn, N. J.; Karlin,
K. D. J. Am. Chem. Soc. 2007, 129, 5352−5353.
(31) Sieber, P.; Riniker, B. Tetrahedron Lett. 1987, 28, 6031−6034.
(32) Liu, F.; Park, J.-E.; Lee, K. S.; Burke, T. R. Jr. Tetrahedron 2009,
65, 9673−9679.
8890
dx.doi.org/10.1021/jo201599c|J. Org. Chem. 2011, 76, 8885−8890