Preparation of NG-substituted L-arginine analogues suitable for solid phase peptide synthesis

Article abstract of DOI:10.1021/jo801517f

(Chemical Equation Presented) A high-yielding and concise preparation of N^G-substituted L-arginine analogues, suitably protected for use in solid phase peptide synthesis, is reported. The synthesis of each analogue employed an activated thiourea intermediate that was converted under mild conditions to the desired L-arginine analogue (10 examples, each in near quantitative yield). Subsequent allyl group removal provided each analogue in a form ideally suited for use in solid phase peptide synthesis.

Full text of DOI:10.1021/jo801517f

Preparation of NG-Substituted L-Arginine
Analogues Suitable for Solid Phase Peptide
Synthesis
molecular cargoes with an efficiency and apparent mechanism
of entry similar to the Tat peptide.9,10 Aside from its numerous
biological roles in peptides and proteins, L-arginine itself is also
required for mammalian nitric oxide (NO) biosynthesis. The
nitric oxide synthases are a class of heme proteins that
oxidatively convert L-arginine to NO and L-citrulline via the
intermediate NG-hydroxy-L-arginine.11,12 In addition, the post-
translational methylation of L-arginine has recently emerged as
an important regulatory mechanism in a variety of cellular
processes including signal transduction, gene transcription, RNA
processing, chromatin remodeling, DNA repair, viral replication,
and cancer.13-18 Enzymes implicated in both the methylation
and demethylation of L-arginine have also been recently
characterized.19,20 Given the numerous roles of both L-arginine
and its NG-substituted variants in biological systems, NG-
modified L-arginine building blocks, compatible with solid phase
peptide synthesis (SPPS), are desirable. We here report a high-
yielding, concise, and general approach toward the preparation
of a wide array of NG-substituted L-arginine analogues suitable
for use in SPPS.
Nathaniel I. Martin* and Rob M. J. Liskamp
Department of Medicinal Chemistry & Chemical Biology,
UniVersity of Utrecht, Sorbonnelaan 16 3584 CA Utrecht,
The Netherlands
n.i.martin@uu.nl
ReceiVed July 10, 2008
While a variety of methods for the preparation of NG-
substituted guanidines have been described,1,21-30 few can be
considered of general use in providing ready access to NG-
modified L-arginine analogues suitably protected for use as
building blocks in SPPS.31 Given our recent experience in the
A high-yielding and concise preparation of NG-substituted
L-arginine analogues, suitably protected for use in solid phase
peptide synthesis, is reported. The synthesis of each analogue
employed an activated thiourea intermediate that was con-
verted under mild conditions to the desired L-arginine
analogue (10 examples, each in near quantitative yield).
Subsequent allyl group removal provided each analogue in
a form ideally suited for use in solid phase peptide synthesis.
¨
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L-Arginine is the most basic (pKa 12.48) of the naturally
occurring common amino acids and plays a unique role in a
number of important physiological and pathophysiological
processes.1 Positively charged at neutral pH, the guanidinium
group of the L-arginine side chain serves as an ion-pairing
moiety in several biologically relevant molecular interactions.
Proteases such as trypsin, thrombin, and Factor Xa preferentially
cleave substrates containing an L-arginine residue at the P1
position.2,3 L-Arginine is also a component of the conserved
integrin receptor-binding RGD peptide motif,4 analogues of
which have been used to modulate cell adhesion.5,6 Cell-
penetrating agents have also been developed based largely upon
a 9 amino acid sequence containing 6 L-arginine residues derived
from the HIV-1 Tat protein.7,8 Furthermore, homopolymers of
L-arginine have been shown to mediate cellular uptake of various
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10.1021/jo801517f CCC: $40.75
Published on Web 08/28/2008
2008 American Chemical Society
J. Org. Chem. 2008, 73, 7849–7851 7849

SCHEME 2. Conversion of Thiourea 1 into NG-Substituted
L-Arginine Analogue 2
preparation of NG-hydroxy and NG-amino guanidines via a Cbz
carbamate-activated thiourea precursor,24,25 we sought to employ
a similar approach in the current investigation (the group of
Clement has also recently reported using a similar Cbz-activated
thiourea approach in the racemization-free preparation of NG-
substituted L-arginine analogues26). Primary amines react rapidly
with carbamate-protected isothiocyanates (i.e., FmocNCS32,33
and CbzNCS23-26) to yield thioureas which can then be
converted to carbamate-protected, NG-substituted guanidines
under mild conditions.23 Both Fmoc and Cbz carbamates,
however, are not suitable as protecting groups for the guanidine
moiety of arginine in SPPS.34 An alternative protection scheme
was therefore sought that would still employ the thiourea
intermediate strategy desired. In this regard the Pbf (2,2,4,6,7-
pentamethyldihydrobenzofuran-5-sulfonyl)- and Pmc (2,2,5,7,8-
pentamethylchroman-6-sulfonyl)-protected isothiocyanates, re-
cently developed by the groups of Fan35 and Madalengoitia,36
respectively, presented ideal options. While both the PbfNCS35
and PmcNCS36 reagents were considered suitable for the desired
application, we chose to use the former given that Pbf protection
is preferred for the guanidine group of arginine in SPPS.37 To
this end, the appropriately protected L-ornithine precursor was
converted to Pbf-protected thiourea 1 by treatment with PbfNCS
(Scheme 1).
TABLE 1. NG-Substitued L-Arginine Analogues Prepared from
Thiourea 1
SCHEME 1. Preparation of Pbf-Protected Thiourea 1
Of note is the particular care taken with respect to the order
of reagent addition in the preparation of 1. Previous reports
involving manipulations of similarly protected L-ornithine
species have described the problematic generation of 6-mem-
bered lactam side products, formed rapidly upon neutralization
of the intermediate L-ornithine acid salt.38,39 To address this
concern, PbfNCS was first added to the intermediate L-ornithine
TFA salt prior to the addition of NEt3. This approach led to
exclusive formation of the desired thiourea in excellent yield.
Thiourea 1 was then employed as a common precursor in
the preparation of a number of NG-substituted L-arginine
analogues. Treatment of 1 with EDCI (2 equiv) followed by
addition of the amine of interest (2 equiv) yielded the expected
NG-substitued L-arginine analogue (Scheme 2). In all cases
investigated the conversion was complete in <5 min and in near
quantitative yields (Table 1).
As shown in Table 1, a number of amines with diverse steric
and electronic properties were successfully incorporated into
the guanidine moiety of the corresponding protected L-arginine
products (entries 1-5). Azido-substituted amines (entries 6 and
7) were incorporated to provide analogues bearing a “handle”
suitable for ligation to alkynes via the popular copper(I)-
a One equivalent of NEt3 added to the reaction mixture.
(32) Kearney, P. C.; Fernandez, M.; Flygare, J. A. J. Org. Chem. 1998, 63,
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mediated Huisgens 3 + 2 cycloaddition. NG-Heteroatom-
substituted L-arginine analogues (entries 8 and 9) were also
readily prepared from the appropriately protected hydrazine or
hydroxylamine. In addition, the successful incorporation of
pyrrolidine (entry 10) indicates that (as for carbamate-activated
thioureas23,26) the process is amenable to the use of secondary
amines.
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7850 J. Org. Chem. Vol. 73, No. 19, 2008

was dissolved in CH2Cl2 (5 mL) and the solution was treated with
EDCI (136 mg, 0.70 mmol) followed by benzylamine (78 µL, 0.70
mmol). Immediate TLC analysis indicated complete consumption
of the starting material along with formation of a slightly more
polar species (Rf 0.54, 1:1 EtOAc/hexane). The mixture was
concentrated under vacuum and applied directly to a silica column
eluting with a gradient of 1:1 to 2:1 EtOAc/hexane. Following
solvent removal the title compound was obtained as a white
nanocrystalline foam (265 mg, 97%). Analytical data: mp 63-65
°C; 1H NMR (300 MHz, CDCl3) δ 7.76-7.74 (m, 2H), 7.56-7.52
(m, 2H), 7.41-7.36 (m, 2H), 7.31-7.26 (m, 2H), 7.23-7.08 (m,
5H), 5.93-5.81 (m, 1H), 5.48 (br m, 1H), 5.34-5.24 (m, 2H), 4.61
(d, J ) 5.5 Hz, 2H), 4.42-4.20 (m, 5H), 4.16 (t, J ) 6.6 Hz, 1H),
3.38-3.01 (br m, 2H), 2.92 (s, 2H), 2.54 (s, 3H), 2.48 (s, 3H),
2.07 (s, 3H), 1.98-1.49 (m, 4H), 1.45 (s, 6H); 13C NMR (75 MHz,
CDCl3) δ 171.9, 158.8, 156.5, 155.0, 144.0, 143.8, 141.5, 138.6,
133.6, 132.5, 131.6, 128.9, 128.0, 127.4, 127.3, 125.3, 124.7, 120.3,
119.5, 117.6, 86.5, 67.3, 66.5, 53.3, 47.3, 45.5, 43.5, 41.1, 30.5,
28.8, 25.4, 19.5, 18.2, 12.7; HRMS (MALDI) calcd for
C44H51N4O7S [M + H]+ 779.3479, found 779.3473.
L-2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-5-(3-benzyl-
2-(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-ylsulfo-
nyl)guanidino)pentanoic Acid (12). Allyl ester 2 (180 mg, 0.23
mmol) was dissolved in THF (5 mL) and treated with N-
methylaniline (77 µL, 0.69 mmol) followed by addition of Pd(PPh3)4
(5 mol %, 0.012 mmol, 14 mg). The mixture was protected from
light and stirred under N2 (g) at room temperature for 45 min. TLC
analysis at this time indicated complete consumption of the allyl
ester with formation of a more polar product. The mixture was
then diluted with EtOAc (50 mL) and washed with saturated NH4Cl
(25 mL). The NH4Cl layer was further extracted with EtOAc (2 ×
25 mL) and the combined EtOAc layers were dried over Na2SO4,
filtered, and evaporated. The residue was applied to a silica column
eluting first with CH2Cl2/MeOH (50:1) to remove the aniline
impurities followed by CH2Cl2/MeOH/AcOH (25:1:0.1) to elute
the free acid (Rf 0.21). After evaporation of product-containing
fractions, the title compound was obtained as a white nanocrystalline
foam (169 mg, 99%). Analytical data: mp 115-117 °C; 1H NMR
(300 MHz, CDCl3) δ 9.54 (br s, 1H), 7.72-7.70 (m, 2H), 7.56-7.50
(m, 2H), 7.37-7.32 (m, 2H), 7.30-7.20 (m, 2H), 7.20-7.02 (m,
5H), 5.94 (br m, 1H), 4.47-4.04 (m, 6H), 3.34-3.00 (br m, 2H),
2.87 (s, 2H), 2.48 (s, 3H), 2.41 (s, 3H), 2.03 (s, 3H), 1.98-1.47
(m, 4H), 1.43 (s, 6H); 13C NMR (75 MHz, CDCl3) δ 175.1, 158.9,
156.8, 155.1, 144.0, 143.8, 141.5, 138.6, 133.1, 132.5, 128.8, 128.0,
127.5, 127.3, 125.4, 124.8, 120.2, 117.7, 86.6, 67.4, 53.4, 47.3,
45.5, 43.4, 41.2, 29.8, 28.8, 25.2, 19.5, 18.2, 12.7; [R]D -3.9 (c
1.2, DMF); HRMS (MALDI) calcd for C41H47N4O7S [M + H]+
739.3166, found 739.3170.
Each of the allyl-protected compounds listed in Table 1 were
cleanly deprotected by using Pd(PPh3)4 and N-methylaniline40
in THF to provide the corresponding NG-modified L-arginine
analogues in a form suitable for SPPS in good to excellent yields
(Scheme 3).
SCHEME 3. Allyl Group Deprotection of 2-11
In summary, a concise and high-yielding methodology for
the preparation of NG-substituted L-arginine analogues suitable
for use in SPPS has been developed. The generality of this
methodology has been illustrated by the successful preparation
of a number of new NG-substituted L-arginine analogues, each
bearing the requisite guanidine and R-amine protecting groups
(Pbf and Fmoc). Access to such analogues serves to expand
the repertoire of available tools for future explorations into the
numerous and unique biological roles played by L-arginine and
its NG-substituted variants. In this regard, our group is currently
examining a number of synthetic peptides containing NG-
modified L-arginine(s) residues for use in various biochemical
studies, the results of which will be reported in due course.
Experimental Section
Details pertaining to the preparation of compounds 1, 2, and 12
are provided here as representative procedures.
L-Allyl 2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-5-(3-
(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-ylsulfonyl)th-
ioureido)pentanoate (1). Fmoc-Orn(Boc)-O-All (2.23 g, 4.5 mmol)
was treated with 40 mL of TFA/CH2Cl2 (1:1) for 1 h. After removal
of TFA and CH2Cl2 under vacuum, the residue was redissolved in
CH2Cl2 (20 mL) and treated with PbfNCS35 (1.40 g, 4.5 mmol)
followed by addition of NEt3 (1.25 mL, 9.0 mmol). After stirring
1 h at ambient temperature TLC analysis revealed complete
consumption of the PbfNCS with formation of the desired product
(Rf 0.62, 1:1 EtOAc/hexane). The mixture was concentrated and
applied directly to a silica column, eluting with 1:1 EtOAc/hexane.
Following solvent removal the title compound was obtained as a
white nanocrystalline foam (3.02 g, 95%). Analytical data: mp
80-82 °C; 1H NMR (300 MHz, CDCl3) δ 8.30 (br s, 1H), 8.01 (br
t, J ) 5.0 Hz, 1H), 7.77-7.75 (m, 2H), 7.62-7.59 (m, 2H),
7.42-7.37 (m, 2H), 7.32-7.29 (m, 2H), 5.94-5.83 (m, 1H),
5.43-5.25 (m, 3H), 4.64 (d, J ) 5.5 Hz, 2H), 4.42-4.33 (m, 3H),
4.22 (t, J ) 6.9 Hz, 1H), 3.56 (br d, J ) 5.5 Hz, 2H), 2.95 (s, 2H),
2.56 (s, 3H), 2.48 (s, 3H), 2.10 (s, 3H), 1.98-1.52 (m, 4H), 1.46
(s, 6H); 13C NMR (75 MHz, CDCl3) δ 178.8, 171.9, 161.2, 156.2,
144.1, 143.9, 141.6, 140.4, 134.5, 131.6, 128.0, 127.3, 127.1, 126.1,
125.3, 120.2, 119.5, 119.0, 87.8, 67.3, 66.5, 53.7, 47.4, 45.4, 43.1,
30.0, 28.7, 24.5, 19.5, 17.9, 12.7; HRMS (MALDI) calcd for
C37H44N3O7S2 [M + H]+ 706.2621, found 706.2599.
Acknowledgment. This work was supported by The Neth-
erlands Organization for Scientific Research (NWO-VENI
fellowship to N.I.M.). Kees Versluis and Mirjam Damen are
kindly acknowledged for providing HRMS analysis.
Supporting Information Available: General experimental
procedures and analytical data for compounds 3-11 and 13-21
1
(including copies of H and 13C NMR spectra for all com-
pounds). This material is available free of charge via the Internet
at http://pubs.acs.org.
JO801517F
L-Allyl 2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-5-(3-
benzyl-2-(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-ylsul-
fonyl)guanidino)pentanoate (2). Thiourea 1 (250 mg, 0.35 mmol)
(40) Waldmann, H.; Kunz, H. Liebigs Ann. Chem. 1983, 1712–1725.
J. Org. Chem. Vol. 73, No. 19, 2008 7851