Synthesis of an amino acid with protected cyclen side chain functionality

Article abstract of DOI:10.1081/SCC-120037922

Starting from glutamic acid an artificial amino acid with 1,4,7,10-tetraazacyclododecane (cyclen) side chain functionality is prepared. The orthogonal protected building block allows the facile introduction of a metal ion binding site into peptide structu

Full text of DOI:10.1081/SCC-120037922

SYNTHETIC COMMUNICATIONSw
Vol. 34, No. 11, pp. 2077–2084, 2004
Synthesis of an Amino Acid with
Protected Cyclen Side Chain
Functionality
*
¨
Stefan Miltschitzky and Burkhard Konig
¨
Institut fu¨r Organische Chemie, Universitat Regensburg,
Regensburg, Germany
ABSTRACT
Starting from glutamic acid an artificial amino acid with 1,4,7,10-tetra-
azacyclododecane (cyclen) side chain functionality is prepared. The
orthogonal protected building block allows the facile introduction of a
metal ion binding site into peptide structures.
Key Words: 1,4,7,10-Tetraazacyclododecane; Molecular recognition;
Peptide synthesis; Transition metal binding.
*
¨ ¨
¨
Correspondence: Burkhard Konig, Institut fur Organische Chemie, Universitat
Regensburg, D-93040 Regensburg, Germany; E-mail: burkhard.koenig@chemie.
uni-regensburg.de.
2077
DOI: 10.1081/SCC-120037922
0039-7911 (Print); 1532-2432 (Online)
www.dekker.com
Copyright # 2004 by Marcel Dekker, Inc.

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INTRODUCTION
Metal ions are an important feature of protein structure and function.
In many cases they are crucial for the activity of allosteric and catalytic
centers.[1] During the last years, many applications and studies based on
functionalized metallopeptides appeared in the literature. Burger and Still have
accomplished the split/pool synthesis of encoded libraries bearing peptidic
appendages attached to a 1,4,7,10-tetraazacyclododecane (cyclen) core bind-
ing to Cu(II) ions in aqueous solution. The structural data indicate that specific
amino acid combinations produce more effective metal binders.[2] Such
ligands that display high affinity and selectivity for metal ions may find use
as sensors. With the goal of developing artificial nucleases for DNA hydroly-
sis, metal-coordinating peptides have been tethered to a DNA intercalating
Rh(III) complex to position metal ions close to the sugar–phosphate back-
bone. The intercalator provides DNA binding affinity, and a metal binding
peptide contributes reactivity.[3] In a combinatorial approach Long reported
a method to optimize the desoxyribose-based cleavage of B-DNA by
.
Ni(II) Xaa–Xaa–His metallopeptides. This metallotripeptide domain is
based on the amino-terminal, square-planar Ni(II) chelating domain of
serum albumin.[4] Scrimin et al. reported a dinuclear Zn(II) complex of a hepta-
peptide active in catalyzing the intramolecular transphosphorylation of the
RNA model substrate 2-hydroxypropyl-p-nitrophenyl phosphate.[5] In further
studies, they showed that these Zn(II) complexes in addition have a remark-
able activity in the hydrolytic cleavage of plasmid DNA.[6] Although structure
and application of the mentioned metallopeptides are different, a common
feature are metal ion coordination sites created by artificial and natural
amino acids. We report here the efficient synthesis of an artificial amino
acid containing a protected cyclen unit as metal ion chelate in the side
chain of glutamine. The compound may serve as a versatile building block
to introduce a metal ion binding site with high affinity in peptide sequences,
and in the construction of synthetic receptors.
Cyclen 1 was converted into the three fold Boc protected derivative 2
following reported procedures.[7] The protected cyclen 2 was used in a
peptide coupling reaction with Cbz–Glu(OH)–OBn. Best results in amide
bond formation were obtained with 1-hydroxy-7-azabenzotriazole (HOAt),
O-(7-azabenzotriazole-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate
(HATU), and collidin. The simultaneous deprotection of the benzyl ester
and the Cbz nitrogen protecting group of 3 is achieved by hydrogenation on
Pd/C. The clean reaction yields amino acid 4 after separation from the catalyst
by simple filtration in 78%. To prepare 4 for use in peptide coupling reactions,
its amino group was fluorenyl-9-methoxycarbonyl (Fmoc) protected. Treat-
ment with Fmoc-Cl and Na2CO3 as base in aqueous dioxane solution gives

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5, which is isolated by precipitation after acidification. To demonstrate the
ability of 5 to undergo peptide coupling a simple dipeptide with glycine
was prepared. The benzyl ester of glycine hydrotosylate is coupled to 5
using HOAt, HATU, and Hu¨nig’s base in DMF solution. The resulting dipep-
tide 6 is precipitated with water (Sch. 1).
In summary, we have reported the synthesis of a protected Glu(cyclen)–
Gly dipeptide. The protected cyclen side chain moiety serves after deprotec-
tion as a high affinity ligand for many transition metal ions. The orthogonal
Scheme 1. Synthesis of cyclen amino acid 5 and its coupling to dipeptide 6.

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protection allows a versatile use of the artificial cyclen amino acid in peptide
synthesis, including automated solid phase synthesis and combinatorial tech-
niques. In combination with natural or non-natural amino acids peptides with
one or more metal ion coordination sites can be prepared for applications in
molecular recognition and catalysis.
EXPERIMENTAL
General
Compound 2 was prepared by a known method. (The major side product
of the reaction, a 4-fold protected cyclen, can be recycled by deprotection with
TFA and eluation from basic ion exchanger giving 1 quantitative).[7] Melting
points were measured on a hot-plated microscope apparatus and are not
corrected. UV/VIS spectra: Varian Cary 50 Bio. IR spectra: Bio-Rad FTS
1
3000 FT-IR. H- and 13C-NMR spectra: Bruker AC 250 or Bruker Avance
300; at 300 K; samples of approximately 30 mg of substance in 0.8 mL of deut-
erated solvents with TMS as the internal standard; s, singlet; bs, broad singlet;
m, multiplet; the multiplicity of the 13C signals was determined using the
DEPT technique and is quoted as (þ) for CH3 or CH, (2) for CH2 and
(Cquat) for quaternary carbons. Elemental analyses were carried out by the
microanalytical laboratory University of Regensburg. Commercially available
reagents were used as received. Organic extracts were dried over anhydrous
Na2SO4 and concentrated in vacuo. TLC was performed on alumina sheets
(Merck KgaA, 20 ꢀ 20 cm, Silica gel 60 F254).
10-(4-Benzyloxycarbonyl-4-benzyloxycarbonylamino-butyryl)-1,4,7,10-
tetraazacyclododecane-1,4,7-tricarboxylic acid tri-tert-butyl ester (3).1 In a
flame dried flask, 2 (142 mg, 0.3 mmol), Cbz–Glu(OH)–OBn (122 mg,
0.33 mmol), HOAt (49 mg, 0.36 mmol), HATU (137 mg, 0.36 mmol), and
collidine (360 mL, 2.74 mmol) were dissolved in 5 mL dichlormethane
(DCM) under a nitrogen atmosphere and stirred for 65 hr at room temperature
(rt). The crude product was purified by column chromatography (SiO2, ethyl
acetate/hexane, 3 : 1, Rf ¼ 0.56) to give 198 mg (88%) of compound 3, as a
white solid.
M.P.: 748C.
1H-NMR (250 MHz, CDCl3): d ¼ 1.45 (m, 27H, Boc–CH3), 2.05 (m, 1H,
CH2–CON–cyclen), 2.18 (m, 1H, CH2–CON–cyclen), 2.38 (m, 2H, CH2–
CH2–CON–cyclen), 3.39 (m, 16H, cyclen–CH2), 4.37 (m, 1H, CH–CO2Bn),
5.08 (s, 2H, CH2–Ph), 5.17 (s, 2H, CH2–Ph), 6.00 (br s, 1H, NH–CO),
7.33 (m, 10H, Ph–H).

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13C-NMR (63 MHz, CDCl3): d ¼ 27.1 (2, CH2–CON–cyclen), 28.4
(þ, CH3–Boc), 28.5 (þ, CH3–Boc), 29.2 (2, CH2–CH2–CON–cyclen),
49.7 (2, cyclen–CH2), 49.9 (2, cyclen–CH2), 50.3 (2, cyclen–CH2), 51.3
(2, cyclen–CH2), 53.9 (þ, CH–CO2BN), 66.9 (2, CH2–Ph), 67.1 (2,
CH2–Ph), 80.3 (Cquat, Boc), 80.4 (Cquat, Boc), 128.1 (þ, Ph–H), 128.2 (þ,
Ph–H), 128.4 (þ, Ph–H), 128.5 (þ, Ph–H), 128.6 (þ, Ph–H), 135.4
(Cquat, Ph), 136.4 (Cquat, Ph), 155.5 (Cquat, OCO–NH), 156.2 (Cquat, CON–
cyclen), 157.1 (Cquat, C55O Boc), 171.9 (Cquat, CO2–Bn).
MS (ESI, DCM/MeOH þ 1% AcOH): m/z (%) ¼ 826.6 [MH]þ (53),
848.6 [M þ Na]þ (100), 864.7 [M þ K]þ (5), 1674.0 [2M þ Na]þ (30).
UV (CH3CN): lmax (log e) ¼ 258 nm (3.780), 263 (3.785), 267 (3.708).
IR (KBr): v˜ ¼ 3030 cm21, 2976, 2932, 1737, 1696, 1648, 1527, 753, 698.
Anal. calcd. for C43H63N5O11: C, 62.52; H, 7.69; N, 8.48; Found: C,
62.22; H, 7.56; N, 8.37.
10-(4-Aminoxycarboxy-4-carboxy-butyryl)-1,4,7,10-tetraazacyclodo-
decane-1,4,7-tricarboxylic acid tri-tert-butyl ester (4). A mixture of 3
(1.8g, 2.2 mmol) in 10mL of dry methanol and 234 mg palladium on
activated charcoal was hydrogenated at 10bar at rt for 48hr. The catalyst was
filtered off, washed thoroughly with methanol and the combined filtrate was
concentrated under reduced pressure to give 3 (1.1g, 85%), as a colorless solid.
M.P.: 1478C.
1H-NMR (250 MHz, CDCl3): d ¼ 1.44–1.46 (m, 27H, Boc–CH3), 2.22–
2.25 (m, 2H), 2.63–2.67 (m, 2H), 3.12–3.14 (m, 1H, C–H), 3.25–3.50
(m, 16H, cyclen–CH2).
13C-NMR (DMSO-[d6], 75 MHz): d ¼ 26.8 (2, CH2–CO–cyclen),
28.0 (þ, Boc–CH3), 29.3 (2, CH2–CH), 48.6 (2, CH2–cyclen), 48.7
(2, CH2–cyclen), 48.9 (2, CH2–cyclen), 49.0 (2, CH2–cyclen), 49.2 (2,
CH2–cyclen), 49.4 (2, CH2–cyclen), 49.8 (2, CH2–cyclen), 49.9 (2,
CH2–cyclen), 79.3 (Cquat, Boc), 79.4 (Cquat, Boc), 155.1 (Cquat, C55O Boc),
155.7 (Cquat, C55O Boc), 156.0 (Cquat, C55O Boc), 169.9 (Cquat, C55O
amid), 172.4 (Cquat, C55O acid).
IR (KBr): v˜ ¼ 3448 cm21, 2976, 2932, 2363, 1699, 1647, 1473, 1411,
1366, 1163.
UV (CH3CN): lmax (log 1) ¼ 263 nm (3.691).
MS (ESI, MeOH þ 1% AcOH): m/z (%) ¼ 602.4 [MH]þ (100), 624.4
[M þ Na]þ (40), 1203.9 [2M þ H]þ (1), 1225.8 [2M þ Na]þ (1).
Anal. calcd. for C28H51N5O9: C, 55.9; H, 8.5; N, 11.6; Found: C, 55.6;
H, 8.7; N, 11.1.
10-[4-Carboxy-(9H-fluoren-9-ylmethoxycarbonylamino)-butyryl]-1,4,7,
10-tetraazacyclododecane-1,4,7-tricarboxylic acid tri-tert-butyl ester (5).
Compound 4 (206 mg, 0.34 mmol) was dissolved in 21 mL of a water/dioxane
mixture (5 : 2) and cooled to 08C. At this temperature Na2CO3 (108 mg,

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2082
1 mmol) and a solution of Fmoc-Cl (96 mg, 0.37 mmol) in 3 mL dioxane were
added. The yellowish solution was stirred 30 min at 08C, allowed to warm up
to rt and stirred additional 26 hr. The solution was acidified with 2 N HCl and
extracted three times with 25 mL of ethyl acetate. The solvent was removed to
give 5 (224mg, 80%), as a colorless solid.
M.P.: 1578C.
1H-NMR (250 MHz, CDCl3): d ¼ 1.43–1.49 (m, 27H, Boc–CH3), 2.22–
2.25 (m, 2H), 2.63–2.67 (m, 2H), 3.12–3.67 (m, 16H, cyclen–CH2), 4.23–
4.27 (m, 4H), 7.30–7.34 (m, 4H), 7.55–7.62 (m, 2H), 7.71–7.82 (m, 2H),
6.12 (br, 1H, NH).
13C-NMR (63 MHz, CDCl3): d ¼ 27.41 (þ, Boc–CH3), 27.43 (þ, Boc–
CH3), 27.47 (þ, Boc–CH3), 27.9 (2, CH2CON), 29.2 (2, CH2CH2CON),
46.1 (þ, Fmoc), 48.6 (2, cyclen–CH2), 48.9 (2, cyclen–CH2), 49.5 (2,
cyclen–CH2), 49.6 (2, cyclen–CH2), 50.5 (2, cyclen–CH2), 52.7 (þ, C–
H), 66.1 (2, CH2 Fmoc), 79.6 (Cquat, Boc), 79.7 (Cquat, Boc), 79.8 (Cquat
,
Boc), 118.9 (þ, Ar-H), 119.0 (þ, Ar-H), 123.7 (þ, Ar-H), 124.1 (þ, Ar-
H), 124.2 (þ, Ar-H), 126.1 (þ, Ar-H), 126.5 (þ, Ar-H), 126.7 (þ, Ar-H),
140.2 (Cquat, Fmoc), 140.5 (Cquat, Fmoc), 142.7(Cquat, Fmoc), 142.9 (Cquat
,
Fmoc), 154.5 (Cquat, C55O Fmoc), 155.1 (Cquat, cyclen–C55O), 156.1
(Cquat, C55O Boc), 172.0 (Cquat, C55O acid).
IR (KBr): v˜ ¼ 3433 cm21, 2975, 2932, 1695, 1470, 1412, 741.
UV (CH3CN): lmax (log 1) ¼ 248 nm (4.987), 256 (5.122).
MS (ESI, MeOH þ 1% AcOH): m/z (%) ¼ 846.7 [M þ Naþ] (11), 824.6
[M þ Hþ] (72), 724.5 [MHþ 2 Boc] (100), 624.4 [MHþ 2 2Boc] (24).
Anal. calcd. for C43H61N5O11: C, 62.7; H, 7.5; N, 8.5; Found: C, 63.0;
H, 7.3; N, 7.5.
10-[4-(Benzyloxycarbonylmethyl-carbamoyl)-4-(9H-fluoren-9-ylmethoxy-
carbonylamino)-butyryl]-1,4,7,10-tetraazacyclododecane-1,4,7-tricarboxylic
acid tri-tert-butyl ester (6). A mixture of Tos–O2 þH3N–Gly–OBn
(101 mg, 0.30 mmol), 5 (275 mg, 0.33 mmol), HOAt (45 mg, 0.33 mmol),
HOAT (125 mg, 0.33 mmol), and Hunig’s base (102 mL, 0.60 mmol) was stir-
red at rt for 26 hr. The solution was cooled in a ice bath and the crude product
was precipitated with water. The solid was filtered off and washed thoroughly
with ice water giving 6 as a colorless solid (228 mg, 78%). For analysis a
sample was purified by column chromatography (SiO2, ethyl acetate/hexane,
3 : 1, Rf ¼ 0.24).
M.P.: 1478C.
1H-NMR (600 MHz, CDCl3): d ¼ 1.43–1.45 (m, 27H, Boc–CH3), 1.96–
2.05 (m, 1H), 2.12–2.19 (m, 1H,), 2.45–2.56 (m, 1H), 2.68–2.79 (m, 1H),
3.18–3.74 (m, 16H, cyclen–CH2), 3.90–4.46 (m, 6H, CH Fmoc, CH2
Fmoc, CH Glu, CH2–Gly), 6.44 (bs, 1H, N–H Fmoc), 7.20–7.45 (m, 9H,
Ar-H), 7.54 (m, 2H, Ar-H), 7.69–7.80 (m, 3H, Ar-H, N–H amid).

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13C-NMR (63 MHz, CDCl3): d ¼ 28.4ppm (þ, Boc–CH3), 28.5 (þ, Boc–
CH3), 29.1 (2), 29.7 (2), 41.4 (2), 47.1 (þ, CH), 50.1 (2, cylclen–CH2), 50.6
(2, cyclen–CH2), 51.4 (2, cyclen–CH2), 54.3 (þ, CH), 67.0 (2, CH2 ester),
67.1 (2, CH2 Fmoc), 80.4 (Cquat, Boc), 80.5 (Cquat, Boc), 119.9 (þ, Ar-H),
125.2 (þ, Ar-H), 125.2 (þ, Ar-H), 125.3 (þ, Ar-H), 127.0 (þ, Ar-H), 127.1
(þ, Ar-H), 127.7 (þ, Ar-H), 128.0 (þ, Ar-H), 128.1 (þ, Ar-H), 128.4 (þ,
Ar-H), 128.5 (þ, Ar-H), 128.6 (þ, Ar-H), 129.1 (þ, Ar-H), 135.3 (Cquat),
141.2 (Cquat), 141.3 (Cquat), 143.8 (Cquat), 144.0 (Cquat), 155.5 (Cquat), 156.4
(Cquat), 157.1 (Cquat), 169.4 (Cquat), 172.1 (Cquat).
IR (KBr): v˜ ¼ 3433 cm21, 2975, 2932, 1695, 1470, 1412, 741.
UV (CH3CN): lmax (log 1) ¼ 266 nm (5.666).
MS (ESI, MeCN): m/z (%) ¼ 988.6 [M þ NHþ4 ] (28), 971.6 [M þ Hþ]
(100).
ABBREVIATIONS
Cyclen
DCM
1,4,7,10-Tetraaza cyclododecane;
Dichloromethane;
DIEA (Hu¨nig’s base)
Fmoc
HATU
(N,N)-Diisopropylethylamine;
Fluorenyl-9-methoxycarbonyl;
O-(7-Azabenzotriazole-1-yl)-N,N,N,N-
tetramethyluronium hexafluorophosphate;
1-Hydroxy-7-azabenzotriazole;
Room temperature.
HOAt
rt
ACKNOWLEDGMENT
We thank the Schering AG, Berlin, and DSM, Regensburg, for support
of this work.
REFERENCES
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2. Francis, M.; Jamison, T.; Jacobsen, E. Combinatorial libraries of transition
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422–428.

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Received in Poland December 11, 2003

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