Syntheses and properties of silicon- and germanium-containing α-amino acids and peptides: A study on C/Si/Ge bioisosterism

Article abstract of DOI:10.1021/om000169l

The unnatural silicon-containing α-amino acids (R)- and (S)-H₂NCH(CH₂SiMe₃)COOH [(R)-2 and (S)-2], (R)-H₂NCH(CH₂SiMe₂Ph)COOH [(R)-4], and (R)-H₂NCH(CH₂SiMe₂CH=CH₂)COOH [(R)-6] as well as the unnatural germanium-containing α-amino acids (R)- and (S)-H₂NCH(CH₂GeMe₃)COOH [(R)-3 and (S)-3] and (R)-H₂NCH(CH₂GeMe₂Ph)COOH [(R)-5] were prepared in three-step syntheses, starting from (R)-3,6-diethoxy-2-isopropyl-2,5-dihydropyrazine [(R)-10]. All amino acids were isolated as enantiomerically pure (≥99percent ee) compounds. The (R)- and (S)-enantiomers of β-(trimethylsilyl)alanine [(R)-2 and (S)-2] and β-(trimethylgermyl)alanine [CR)-3 and (S)-3] are sila-analogues and germa-analogues, respectively, of the (S)- and (R)-enantiomers of the nonproteinogenic amino acid β-tert-butylalanine [(S)- and (R)-H₂NCH(CH₂CMe₃)COOH; (S)-l and (R)-1]. The C/Si/Ge-analogous (L-configurated) amino acids (S)-l, (R)-2, and (R)-3 were treated with (fluoren-9-yl)methyl chloroformate to give the corresponding N-Fmoc derivatives (S)-26, (R)-27, and (R)-28. These N-Fmoc-protected amino acids were used as building blocks for the solid-phase syntheses of the C/Si/Ge-analogous decapeptides 7-9 [Ac-D-Nal¹-4-Cl-D-Phe²-D-Pal³-Ser ⁴-Me₃El-Ala⁵-D-Cit⁶-Leu ⁷-Arg⁸-Pro⁹-D-Ala¹⁰-NH₂ (7, El = C; 8, El = Si; 9, El = Ge)]. The C/Si/Ge analogues 7-9 are derivatives of the GnRH antagonist Cetrorelix^INN, which bears an (S)-tyrosine residue [instead of the (S)-Me₃C-Ala, (R)-Me₃Si-Ala, or (R)-Me₃Ge-Ala residue] in position 5 of its decapeptide backbone. The decapeptides 7-9 were studied in vitro in receptor binding and functional assays using recombinant cell lines expressing the human GnRH receptor. All compounds behaved as potent GnRH antagonists, the binding affinities and antagonistic potencies of the three C/Si/Ge analogues being quite similar. Compounds 7-9 were also studied for their in vivo activities in the male rat after s.c. administration. They produced both a strong testosterone suppression (single-dose treatment, 1.5 mg/kg) and a strong LH suppression (castrated male rat; single-dose treatment, 0.05 mg/kg). For the silicon- and germanium-containing decapeptides 8 and 9 the testosterone and LH suppression lasted for a significantly longer period of time compared with the effects of the carbon analogue 7.

Full text of DOI:10.1021/om000169l

3486
Organometallics 2000, 19, 3486-3497
Syn th eses a n d P r op er ties of Silicon - a n d
Ger m a n iu m -Con ta in in g r-Am in o Acid s a n d P ep tid es:
A Stu d y on C/Si/Ge Bioisoster ism §
Reinhold Tacke,*,† Markus Merget,† Ru¨diger Bertermann,† Michael Bernd,‡
Thomas Beckers,‡ and Thomas Reissmann‡
Institut fu¨r Anorganische Chemie, Universita¨t Wu¨rzburg, Am Hubland,
D-97074 Wu¨rzburg, Germany, and ASTA Medica AG, Konzernforschung,
Weismu¨llerstrasse 45, D-60315 Frankfurt, Germany
Received February 23, 2000
The unnatural silicon-containing R-amino acids (R)- and (S)-H2NCH(CH2SiMe3)COOH [(R)-
2 and (S)-2], (R)-H2NCH(CH2SiMe2Ph)COOH [(R)-4], and (R)-H2NCH(CH2SiMe2CHdCH2)-
COOH [(R)-6] as well as the unnatural germanium-containing R-amino acids (R)- and (S)-
H2NCH(CH2GeMe3)COOH [(R)-3 and (S)-3] and (R)-H2NCH(CH2GeMe2Ph)COOH [(R)-5]
were prepared in three-step syntheses, starting from (R)-3,6-diethoxy-2-isopropyl-2,5-
dihydropyrazine [(R)-10]. All amino acids were isolated as enantiomerically pure (g99% ee)
compounds. The (R)- and (S)-enantiomers of â-(trimethylsilyl)alanine [(R)-2 and (S)-2] and
â-(trimethylgermyl)alanine [(R)-3 and (S)-3] are sila-analogues and germa-analogues,
respectively, of the (S)- and (R)-enantiomers of the nonproteinogenic amino acid â-tert-
butylalanine [(S)- and (R)-H2NCH(CH2CMe3)COOH; (S)-1 and (R)-1]. The C/Si/Ge-analogous
(L-configurated) amino acids (S)-1, (R)-2, and (R)-3 were treated with (fluoren-9-yl)methyl
chloroformate to give the corresponding N-Fmoc derivatives (S)-26, (R)-27, and (R)-28. These
N-Fmoc-protected amino acids were used as building blocks for the solid-phase syntheses of
the C/Si/Ge-analogous decapeptides 7-9 [Ac-D-Nal1-4-Cl-D-Phe2-D-Pal3-Ser4-Me3El-Ala5-D-
Cit6-Leu7-Arg8-Pro9-D-Ala10-NH2 (7, El ) C; 8, El ) Si; 9, El ) Ge)]. The C/Si/Ge analogues
7-9 are derivatives of the GnRH antagonist CetrorelixINN, which bears an (S)-tyrosine residue
[instead of the (S)-Me3C-Ala, (R)-Me3Si-Ala, or (R)-Me3Ge-Ala residue] in position 5 of its
decapeptide backbone. The decapeptides 7-9 were studied in vitro in receptor binding and
functional assays using recombinant cell lines expressing the human GnRH receptor. All
compounds behaved as potent GnRH antagonists, the binding affinities and antagonistic
potencies of the three C/Si/Ge analogues being quite similar. Compounds 7-9 were also
studied for their in vivo activities in the male rat after s.c. administration. They produced
both a strong testosterone suppression (single-dose treatment, 1.5 mg/kg) and a strong LH
suppression (castrated male rat; single-dose treatment, 0.05 mg/kg). For the silicon- and
germanium-containing decapeptides 8 and 9 the testosterone and LH suppression lasted
for a significantly longer period of time compared with the effects of the carbon analogue 7.
In tr od u ction
addition, unnatural amino acids are of interest as
precursors of drugs and plant-protective agents and are
used as chiral auxiliaries in asymmetric organic syn-
thesis. In the course of our systematic studies on
bioorganosilicon and bioorganogermanium chemistry,2
we became interested in enantiomerically pure silicon-
and germanium-containing amino acids of the formula
type H2NCH(CH2SiR3)COOH and H2NCH(CH2GeR3)-
COOH (R ) organyl). With their CH2SiR3 and CH2GeR3
groups as side chains, these â-(triorganylsilyl)alanine
Enantiomerically pure amino acids with unnatural
side chains are gaining increasing importance in chem-
istry and life sciences.1 Synthetic unnatural amino acids
have proven useful for probing the structural require-
ments for the biological activity of numerous peptides
and proteins and have been used as building blocks for
the synthesis of novel biologically active peptides. In
* To whom correspondence should be addressed.
§ Dedicated to Professor Horst Kessler on the occasion of his 60th
birthday.
(2) Reviews dealing with bioorganosilicon and bioorganogermanium
chemistry: (a) Tacke, R.; Linoh, H. In The Chemistry of Organic Silicon
Compounds, Part 2; Patai, S., Rappoport, Z., Eds.; Wiley: Chichester,
U.K., 1989; pp 1143-1206. (b) Lukevics, E.; Ignatovich, L. Appl.
Organomet. Chem. 1992, 6, 113-126. (c) Tacke, R.; Wagner, S. A. In
The Chemistry of Organic Silicon Compounds, Vol. 2, Part 3; Rap-
poport, Z., Apeloig, Y., Eds.; Wiley: Chichester, U.K., 1998; pp 2363-
2400. (d) Tacke, R.; Heinrich, T.; Kornek, T.; Merget, M.; Wagner, S.
A.; Gross, J .; Keim, C.; Lambrecht, G.; Mutschler, E.; Beckers, T.;
Bernd, M.; Reissmann, T. Phosphorus, Sulfur, Silicon 1999, 150/151,
69-87.
† Universita¨t Wu¨rzburg.
‡ ASTA Medica AG.
(1) (a) Giannis, A.; Kolter, T. Angew. Chem. 1993, 105, 1303-1326;
Angew. Chem., Int. Ed. Engl. 1993, 32, 1244-1267. (b) Gante, J .
Angew. Chem. 1994, 106, 1780-1802; Angew. Chem., Int. Ed. Engl.
1994, 33, 1699-1720. (c) Kleemann, A.; Leuchtenberger, W.; Hoppe,
B.; Tanner, H. In Ullmann’s Encyclopedia of Industrial Chemistry;
Gerhartz, W., Ed.; VCH: Weinheim, Germany, 1985; pp 57-97. (d)
Ishida, H.; Inoue, Y. Rev. Heteroat. Chem. 1998, 19, 79-142.
10.1021/om000169l CCC: $19.00 © 2000 American Chemical Society
Publication on Web 08/01/2000

Si- and Ge-Containing R-Amino Acids and Peptides
Organometallics, Vol. 19, No. 18, 2000 3487
Ch a r t 1
and â-(triorganylgermyl)alanine derivatives are unique
amino acids combining hydrophobicity and sterically
demanding bulkiness with the common hydrophilic
behavior of amino acids. We report here on the synthe-
ses of the enantiomerically pure R-amino acids (R)-2,
(S)-2, (R)-3, (S)-3, (R)-4, (R)-5, and (R)-6. The amino
acids â-(trimethylsilyl)alanine (2) and â-(trimethylger-
myl)alanine (3) are sila- and germa-analogues, respec-
tively, of the nonproteinogenic amino acid â-tert-
butylalanine (1) (see Chart 1).
and the ethyl esters of rac- and (R)-4,4-dimethyl-4-sila-
proline13 have been described. In addition, there are
short reports on some tripeptides bearing the silicon-
containing amino acid (R)-2.7,11 To the best of our
knowledge, germanium-containing R-amino acids and
peptides have not yet been described in the literature.
The studies presented here were carried out as part
of our systematic investigations on biologically active
organosilicon and organogermanium compounds (in this
context, see refs 2a, 2c, and 2d). Some preliminary
results of these studies have already been published
elsewhere.2d,14
Furthermore, we report on the syntheses and phar-
macological characterization of the decapeptides 7-9.
These peptides contain the C/Si/Ge-analogous L-con-
figurated amino acid residues Me3C-Ala, Me3Si-Ala, or
Me3Ge-Ala in position 5 of their backbone. The C/Si/Ge
analogues 7-9 are derivatives of the GnRH antagonist
CetrorelixINN, which bears an (S)-tyrosine residue in
position 5 [instead of the Me3El-Ala (El ) C, Si, Ge)
residues derived from (S)-1, (R)-2, or (R)-3].3 The
pharmacological studies of 7-9 presented here were
carried out with a special emphasis on C/Si/Ge bioiso-
sterism.
Resu lts a n d Discu ssion
Syn th eses of th e R-Am in o Acid s. The silicon- and
germanium-containing amino acids (R)-2, (S)-2, (R)-3,
and (S)-3 were prepared by three-step syntheses ac-
cording to the Scho¨llkopf approach,15 starting from the
dihydropyrazine (R)-10 (Scheme 1).16
Metalation of the dihydropyrazine (R)-10 with n-
butyllithium and subsequent treatment with ClCH2-
(4) (a) Birkofer, L.; Ritter, A. Angew. Chem. 1956, 68, 461-462. (b)
Birkofer, L.; Ritter, A. Liebigs Ann. Chem. 1958, 612, 22-33.
(5) Porter, T. H.; Shive, W. J . Med. Chem. 1968, 11, 402-403.
(6) Fitzi, R.; Seebach, D. Tetrahedron 1988, 44, 5277-5292.
(7) Walkup, R. D.; Cole, D. C.; Whittlesey, B. R. J . Org. Chem. 1995,
60, 2630-2634.
Silicon-containing R-amino acids [rac-2,4,5 (R)-2,6-9
(S)-2,8 rac-4,10 (R)-4,7 (R)-H2NCH(SiMePh2)COOH,7 rac-
H2NCH[Si(CH2)nMe]COOH10 (n ) 4, 5)] have already
been described in the literature; however, alternative
strategies have been used for their synthesis. Further-
more, the ethyl ester of (R)-2,11 the N-Fmoc,7,11 and
N-Boc derivatives11 of (R)-2, the N-Boc derivatives of
the R-amino acid esters (S)-H2NCH(CH2R)COOMe (R
) SiPh3, SiMe2Ph, SiMePh2, SitBuPh2),12 the N-Fmoc
derivatives of (R)-4 and (R)-H2NCH(SiMePh2)COOH,7
(8) Yamanaka, H.; Fukui, T.; Kawamoto, T.; Tanaka, A. Appl.
Microbiol. Biotechnol. 1996, 45, 51-55.
(9) Myers, A. G.; Gleason, J . L.; Yoon, T.; Kung, D. W. J . Am. Chem.
Soc. 1997, 119, 656-673.
(10) Smith, R. J .; Bratovanov, S.; Bienz, S. Tetrahedron 1997, 53,
13695-13702.
(11) Weidmann, B. Chimia 1992, 46, 312-313.
(12) Sibi, M. P.; Harris, B. J .; Shay, J . J .; Hajra, S. Tetrahedron
1998, 54, 7221-7228.
(13) Handmann, V. I.; Merget, M.; Tacke, R. Z. Naturforsch. 2000,
55b, 133-138.
(3) Reviews dealing with CetrorelixINN: (a) Kutscher, B.; Bernd, M.;
Beckers, T.; Polymeropoulos, E. E.; Engel, J . Angew. Chem. 1997, 109,
2240-2254; Angew. Chem., Int. Ed. Engl. 1997, 36, 2148-2161. (b)
Reissmann, T.; Engel, J .; Kutscher, B.; Bernd, M.; Hilgard, P.; Peukert,
M.; Szelenyi, I.; Reichert, S.; Gonzales-Barcena, D.; Nieschiag, E.;
Comaru-Schally, A. M.; Schally, A. V. Drugs Future 1994, 19, 228-
237.
(14) Merget, M.; Bartoschek, S.; Willeke, R.; Tacke, R. In Organo-
silicon Chemistry IV-From Molecules to Materials; Auner, N., Weis,
J ., Eds.; VCH: Weinheim, Germany, 2000; pp 33-36.
(15) Scho¨llkopf, U. Tetrahedron 1983, 39, 2085-2091.
(16) This method has also been used for the synthesis of the ethyl
ester of (R)-2 (see ref 11).

3488 Organometallics, Vol. 19, No. 18, 2000
Tacke et al.
Sch em e 1
dihydropyrazines (2R,5R)-20, (2R,5R)-21, and (2R,5R)-
22 [diastereomers (2S,5R)-20, (2S,5R)-21, and (2S,5R)-
22 not isolated]. Some experimental data for these
syntheses are listed in Table 1. Treatment of the
dihydropyrazines (2R,5R)-20, (2R,5R)-21, and (2R,5R)-
22 with hydrochloric acid at 0 °C and subsequent liquid-
chromatographic separation of the byproduct (R)-valine
ethyl ester (silica gel as stationary phase) yielded the
respective enantiomerically pure (g99% ee) amino acid
esters (R)-23-(R)-25 (for the yields, see Table 2).
Attemps to prepare the amino acids (R)-4-(R)-6 by
hydrolysis of the respective ethyl esters (R)-23-(R)-25
in boiling hydrochloric acid failed: Under the conditions
applied to the hydrolysis of (R)-15, (S)-15, (R)-16, and
(S)-16, Si-C (Si-Ph, Si-CHdCH2) and Ge-C (Ge-Ph)
bond cleavage was observed. However, this problem
could be solved by avoiding acidic conditions. Thus,
treatment of the amino acid esters (R)-23-(R)-25 with
lithium hydroxide (molar ratio 1:1) in a water/dioxane
mixture, followed by a special workup (including an Li+/
H+ exchange; see Experimental Section), yielded the
respective amino acids (R)-4-(R)-6 (for the yields, see
Table 3).
The amino acids (R)-2, (S)-2, (R)-3, (S)-3, and (R)-4-
(R)-6 were isolated as colorless crystalline solids, whereas
the dihydropyrazines (2R,5R)-13, (2S,5R)-13, (2R,5R)-
14, (2S,5R)-14, and (2R,5R)-20-(2R,5R)-22 as well as
the amino acid esters (R)-15, (S)-15, (R)-16, (S)-16, and
(R)-23-(R)-25 were obtained as colorless liquids. The
identity of all compounds was established by elemental
analyses (C, H, N), NMR-spectroscopic studies (1H, 13C,
29Si), and mass-spectrometric investigations (EI MS).
Deter m in a tion of th e Absolu te Con figu r a tion s.
The assignment of the absolute configurations of the
(2R,5R)- and (2S,5R)-diastereomers of 13, 14, and 20-
22 is based on the well-established stereochemistry of
the Scho¨llkopf approach15 and is supported by the
SiMe3 (11) or ClCH2GeMe3 (12) yielded mixtures of the
respective diastereomeric dihydropyrazines (2R,5R)-13/
(2S,5R)-13 and (2R,5R)-14/(2S,5R)-14 (for the diaster-
eomeric excess, see Table 1), along with the nonreacted
educt (R)-10. These mixtures were separated by medium-
pressure liquid chromatography (MPLC) on silica gel
to yield the diastereomerically pure (g99% de) dihydro-
pyrazines (2R,5R)-13, (2S,5R)-13, (2R,5R)-14, and
(2S,5R)-14 (for the yields, see Table 1). After treatment
of the dihydropyrazines (2R,5R)-13, (2S,5R)-13, (2R,5R)-
14, and (2S,5R)-14 with hydrochloric acid at 0 °C and
subsequent liquid-chromatographic separation of the
byproduct (R)-valine ethyl ester (silica gel as stationary
phase), the respective enantiomerically pure (g99% ee)
amino acid ethyl esters (R)-15, (S)-15, (R)-16, and (S)-
16 were obtained (for the yields, see Table 2). Hydrolysis
of these esters in boiling hydrochloric acid and subse-
quent treatment of the resulting amino acid hydrochlo-
rides with propylene oxide finally yielded the amino
acids (R)-2, (S)-2, (R)-3, and (S)-3 (for the yields, see
Table 3).
5
1
characteristic J GK coupling constants in the H NMR
spectra of the (2R,5R)-isomers (trans-isomers of 13, 14,
5
and 20-22: J GK ) 3.3-3.7 Hz) and (2S,5R)-isomers
5
(cis-isomers of 13 and 14: J GK ) 4.1 and 4.2 Hz) (see
Figure 1). As the hydrolytic cleavage of the dihydropy-
razines 13, 14, and 20-22 does not affect the absolute
configurations of the two centers of chirality, the
absolute configurations of the resulting amino acids can
be deduced from the absolute configurations of the
respective dihydropyrazines (see Schemes 1 and 2).
Deter m in a tion of th e Dia ster eom er ic P u r ities.
The (2R,5R)- and (2S,5R)-diastereomers of the dihydro-
pyrazines 13, 14, and 20-22 could be separated by
analytical capillary gas chromatography. This is dem-
onstrated for the diastereomers of 14 in Figure 2. The
retention times of the respective stereoisomers of the
dihydropyrazines 13, 14, and 20-22 are listed in Table
4. This gas-chromatographic method was used as an
analytical tool to determine the molar ratio of the
(2R,5R)- and (2S,5R)-diastereomers of 13, 14, and 20-
22.
The structurally related amino acids (R)-4-(R)-6 were
prepared according to Scheme 2. Thus, metalation of
the dihydropyrazine (R)-10 with n-butyllithium and
subsequent treatment with ClCH2SiMe2Ph (17), ClCH2-
GeMe2Ph (18), or ClCH2SiMe2CHdCH2 (19), followed
by medium-pressure liquid chromatography on silica
gel, yielded the diastereomerically pure (g99% de)
Deter m in a tion of th e En a n tiom er ic P u r ities.
The enantiomeric purities of the amino acid esters (R)-
15, (S)-15, (R)-16, (S)-16, and (R)-23-(R)-25 were
1
determined by H NMR experiments using the chiral
solvating agent (R)-2,2,2-trifluoro-1-(9-anthryl)ethanol
[(R)-TFAE]. As shown for 16 in Figure 3, the two

Si- and Ge-Containing R-Amino Acids and Peptides
Organometallics, Vol. 19, No. 18, 2000 3489
Ta ble 1. Exp er im en ta l Da ta for th e Syn th eses of th e Dih yd r op yr a zin es 13, 14, a n d 20-22 Sta r tin g fr om
(R)-10
ratio
ratio
yield
yield
product
(2R,5R)/(2S,5R)a
[(2R/5R),(2S,5R)]/(R)-10b
(2R/5R)c
(2S/5R)d
13
14
20
21
22
85:15 (70% de)
86:14 (72% de)
80:20 (60% de)
83:17 (66% de)
87:13 (74% de)
75:25
85:15
65:35
82:18
80:20
52%
54%
41%
41%
58%
8%
6%
n.i.e
n.i.e
n.i.e
a
Molar ratio of the (2R,5R)- and (2S,5R)-diastereomers (analysis of the crude product by capillary gas chromatography); diastereomeric
b
excess in parentheses. Molar ratio of the dihydropyrazines (mixture of diastereomers) and the educt (R)-10 (analysis of the crude product
by capillary gas chromatography). c Yield [relative to (R)-10] of the diastereomerically pure (g99% de) (2R,5R)-isomer after liquid-
d
chromatographic separation. Yield [relative to (R)-10] of the diastereomerically pure (g99% de) (2S,5R)-isomer after liquid-
chromatographic separation. e Not isolated.
Ta ble 2. Yield s of th e Am in o Acid Ester s 15, 16,
a n d 23-25 Obta in ed by Hyd r olysis of th e
Resp ective Dih yd r op yr a zin es 13, 14, a n d 20-22
Sch em e 2
product
yield [educt]
product
yield [educt]
(R)-15
(S)-15
(R)-16
(S)-16
86% [(2R,5R)-13]
84% [(2S,5R)-13]
73% [(2R,5R)-14]
80% [(2S,5R)-14]
(R)-23
(R)-24
(R)-25
83% [(2R,5R)-20]
74% [(2R,5R)-21]
70% [(2R,5R)-22]
Ta ble 3. Yield s of th e Am in o Acid s 2-6 Obta in ed
by Hyd r olysis of th e Resp ective Am in o Acid Ester s
15, 16, a n d 23-25 a s Well a s Over a ll Yield s
overall
overall
product yield [educt] yielda product yield [educt] yielda
(R)-2
(S)-2
(R)-3
(S)-3
79% [(R)-15]
60% [(S)-15]
64% [(R)-16]
39% [(S)-16]
35%
4%
25%
2%
(R)-4
(R)-5
(R)-6
53% [(R)-23]
45% [(R)-24]
42% [(R)-25]
18%
14%
15%
a
Overall yield relative to (R)-10.
enantiomers of this amino acid ester can be clearly
discriminated by NMR spectroscopy and therefore quan-
titatively determined by integration of their character-
istic resonance signals. According to this method, the
enantiomeric purities of (R)-15, (S)-15, (R)-16, (S)-16,
and (R)-23-(R)-25 were determined to be g99% ee. As
the hydrolysis of these amino acid esters does not affect
the absolute configurations of the centers of chirality,
the same enantiomeric purities (g99% ee) can be
assumed for the resulting amino acids (R)-2, (S)-2, (R)-
3, (S)-3, (R)-4, (R)-5, and (R)-6.
Syn th eses of th e Deca p ep tid es. To incorporate the
amino acids (S)-1, (R)-2, and (R)-3 into the decapeptides
7-9, the N-(fluoren-9-yl)methoxycarbonyl (N-Fmoc)
derivatives of these amino acids were prepared. The
N-Fmoc derivatives (S)-26, (R)-27, and (R)-28 were
synthesized according to Scheme 3 by treatment of the
amino acids (S)-1, (R)-2, and (R)-3 with (fluoren-9-yl)-
methyl chloroformate (Fmoc-Cl) and were isolated as
colorless solids [yields: (S)-26, 80%; (R)-27, 69%; (R)-
28, 47%].
and germanium were very helpful in these studies.
As an example of this, the ESI mass spectrum of the
germanium-containing decapeptide 9 is shown in Figure
4.
P h a r m a cologica l Ch a r a cter iza tion of th e De-
ca p ep tid es. The decapeptides 7-9 were characterized
in vitro in receptor binding and functional assays using
recombinant cell lines expressing the human GnRH
receptor. All compounds behaved as potent GnRH
antagonists. As can be seen from Table 5, the binding
affinities (KA values) of 7-9 for the human GnRH
receptor ([125I]Cetrorelix as the radio ligand) are quite
similar, but slightly lower compared with the reference
compound Cetrorelix. These findings are in accordance
with the results obtained in a reporter gene assay with
D-Trp6-GnRH (Triptorelin) as the agonist: The antago-
nistic potencies (IC50 values) of 7-9 are quite similar,
but slightly reduced compared with that of Cetrorelix.
Thus, incorporation of the (S)-Me3C-Ala, (R)-Me3Si-Ala,
The decapeptides 7-9 were prepared by solid-phase
syntheses, using standard methods17 for the sequential
synthesis of oligopeptides and starting with the C-
terminal attachment of the D-alanine residue. The
decapeptides 7-9 were purified by high-performance
liquid chromatography, and their identities were estab-
lished by mass-spectrometric studies (ESI MS and
ESI MS/MS). The characteristic isotopic ratios of silicon
(17) Stewart, J . M.; Young, J . D. Solid-Phase Peptide Synthesis;
Pierce Chemical Co: Rockford, 1984.

3490 Organometallics, Vol. 19, No. 18, 2000
Tacke et al.
Ta ble 4. Reten tion Tim esa for th e An a lytica l Ga s
Ch r om a togr a p h ic Sep a r a tion s of th e (2R,5R)- a n d
(2S,5R)-Dia ster eom er s of th e Dih yd r op yr a zin es 13,
14, a n d 20-22
retention time
(min)
retention time
(min)
compound
compound
(2R,5R)-13
(2R,5R)-14
(2R,5R)-20
(2R,5R)-21
(2R,5R)-22
18.8
20.8
28.5
30.0
21.9
(2S,5R)-13
(2S,5R)-14
(2S,5R)-20
(2S,5R)-21
(2S,5R)-22
19.6
21.5
29.1
30.4
22.5
a
For experimental conditions, see Experimental Section.
F igu r e 1. Structure of the trans-isomers [(2R,5R)-config-
uration] and cis-isomers [(2S,5R)-configuration] of the
dihydropyrazines 13, 14, and 20-22 showing the two
coupling protons HG and HK.
F igu r e 3. Quantitative NMR-spectroscopic determination
of the enantiomeric purities of the antipodes of the amino
acid ethyl ester 16. 1H NMR partial spectra (GeCH3 groups)
of the (R)- and (S)-enantiomers of 16 in the presence of
the chiral solvating agent (R)-TFAE: (a) racemic mixture;
(b) (R)-enantiomer obtained by hydrolysis of (2R,5R)-14;
(c) (S)-enantiomer obtained by hydrolysis of (2S,5R)-14. For
details, see Experimental Section.
F igu r e 2. Quantitative gas-chromatographic determina-
tion of the molar ratio of the diastereomers of the dihy-
dropyrazine 14. Gas chromatograms: (a) 86:14 mixture of
(2R,5R)-14/(2S,5R)-14 obtained by diastereoselective alky-
lation of (R)-10 (analysis of the crude product); (b) diaster-
eomerically pure (g99% de) isomer (2R,5R)-14 obtained by
liquid-chromatographic separation; (c) diastereomerically
pure (g99% de) isomer (2S,5R)-14 obtained by liquid-
chromatographic separation. For details, see Experimental
Section.
F igu r e 4. Electrospray mass spectrum of the germanium-
containing decapeptide 9.
antagonistic potencies of the C/Si/Ge analogues 7-9 are
quite similar.
The decapeptides 7-9 were also studied for their in
vivo activities in the male rat after s.c. administration.
With respect to the suppression of testosterone, all three
compounds (single-dose treatment, 1.5 mg/kg) produced
or (R)-Me3Ge-Ala residue in position 5 of Cetrorelix
causes a marginal reduction in both binding affinity and
antagonistic potency, whereas the binding affinities and

Si- and Ge-Containing R-Amino Acids and Peptides
Organometallics, Vol. 19, No. 18, 2000 3491
Sch em e 3
F igu r e 6. Mean LH serum levels in castrated male rats
after single-dose treatment (0.05 mg/kg) with 7-9. For
experimental details, see Experimental Section.
In conclusion, the silicon- and germanium-containing
decapeptides 8 and 9 showed an advantage over their
carbon analogue 7 in vivo, but no significant differences
between the Si/Ge analogues 8 and 9 could be detected
in both in vivo settings. Although the improved in vivo
activities of 8 and 9 cannot yet be explained in terms of
differences in the chemical and physicochemical proper-
ties of the C/Si/Ge analogues 7-9, the results reported
here clearly demonstrate that sila- and germa-substitu-
tion of amino acids may be a useful tool to improve the
biological properties of peptides. Further chemical and
pharmacological studies are in progress to elucidate the
potential of this approach systematically.
Ta ble 5. Bin d in g Affin ities (KA Va lu es) a n d
An ta gon istic P oten cies (IC50 Va lu es) of th e
Deca p ep tid es 7-9 a n d Cetr or elix a t th e Hu m a n
Gn RH Recep tor a
decapeptide
KA [nM-1
]
IC50 [nM]b
7
8
9
2.20 ( 0.10
0.93 ( 0.30
2.82 ( 0.33
2.01 ( 0.15
0.75 ( 0.17
0.50 ( 0.21
Exp er im en ta l Section
Gen er a l P r oced u r es. Unless otherwise indicated, the
reactions were carried out under dry nitrogen. Tetrahydrofu-
ran (THF) and diethyl ether were dried and purified according
to standard procedures and stored under nitrogen. 1H NMR
spectra were recorded at room temperature on a Bruker DMX-
600 (1H, 600.1 MHz), Bruker AMX-400 (1H, 400.1 MHz), or
Bruker DRX-300 NMR spectrometer (1H, 300.1 MHz). 13C and
29Si NMR spectra were recorded at room temperature on a
Bruker AMX-400 (13C, 100.6 MHz; 29Si, 79.5 MHz) or Bruker
DRX-300 NMR spectrometer (13C, 75.5 MHz; 29Si, 59.6 MHz).
Chemical shifts (ppm) were determined relative to internal
CHCl3 (1H, δ 7.24; solvent CDCl3), CDCl3 (13C, δ 77.05; solvent
CDCl3), and HDO (1H, δ 4.82; solvent D2O), and external TMS
(13C, δ 0; solvent D2O; 29Si, δ 0, solvents CDCl3 and D2O).
Assignment of the 1H NMR data of 13, 14, and 20-22 was
supported by 1H,1H COSY experiments, and the 1H spin
systems of 2-6, 13-16, and 20-25 were analyzed by simula-
tions using the Bruker software program WIN-DAISY 4.0.18
Assignment of the 13C NMR data was supported by DEPT 135
and 13C,1H COSY experiments (13, 14, 20-22). Mass spectra
were obtained with a Varian MAT-711 (EI MS, 70 eV),
Finnigan MAT-8430 (EI MS, 70 eV; CI MS, ammonia as
reactant gas), Finnigan MAT-8200 (EI MS, 70 eV), or Ther-
moQuest TRIO 1000 mass spectrometer (EI MS, 70 eV). Mass
spectra of the decapeptides were measured with a Finnigan
LCQ instrument [ESI MS; c(sample), 5 µg/mL; eluent, CH3-
CN/H2O/AcOH (80:20:0.01 (v/v/v)); flow rate, 3 µL/min]. The
selected m/z values given refer to the isotopes 1H, 12C, 14N,
16O, 23Na, 28Si, 35Cl, and 74Ge. IR spectra were obtained with
a Bruker Equinox 55 FT-IR spectrometer. Preparative liquid
chromatography [column: 60 mm i.d. × 350 mm] was per-
formed using silica gel as stationary phase (silica gel 60,
0.063-0.200 mm; Merck, 15111). The ion exchanger (Aldrich,
21,739-5) was activated by washing with 0.5 M hydrochloric
acid (50 mL) and water (3 × 50 mL).
Cetrorelix
5.94 ( 1.85 (n ) 27)
0.25 ( 0.06 (n ) 14)
a
b
For experimental details, see Experimental Section. Average
of three independent experiments.
F igu r e 5. Mean testosterone plasma levels in male rats
after single-dose treatment (1.5 mg/kg) with 7-9. For
experimental details, see Experimental Section.
an immediate and strong decrease in hormone levels.
For the silicon- and germanium-containing decapeptides
8 and 9 this suppression lasted for 48 h, whereas it was
24 h after injection of the carbon analogue 7 until
hormone concentrations returned to lower normal levels
(Figure 5). To further discriminate the potencies of the
C/Si/Ge analogues 7-9, their suppressive effects on the
more sensitive parameter of LH were investigated.
Again, single-dose treatment (0.05 mg/kg) with 8 and 9
resulted in the most pronounced effects with a strong
LH suppression lasting for 24 h, whereas 7 was less
effective, with a duration of suppression of only 6 h
(Figure 6). Thus, the results obtained for testosterone
were also reproduced for the parameter of LH.
(18) Weber, U.; Thiele, H.; Spiske, R.; Ho¨ffken, H.-W.; Ha¨gele, G.
WIN-DAISY 4.0; Bruker-Franzen GmbH, Bremen, Germany, 1998.

3492 Organometallics, Vol. 19, No. 18, 2000
Tacke et al.
(S)-2-Am in o-4,4-d im et h ylp en t a n oic Acid [(S)-â-ter t-
Bu tyla la n in e; (S)-1]. This compound was commercially avail-
able (Bachem AG).
solid (245 mg, 1.07 mmol). A mixture of this lithium salt (245
mg, 1.07 mmol) and an activated ion exchanger (11 g) in water
(50 mL) was shaken at room temperature for 30 min. After
the pH value was adjusted to pH 10-11 by addition of an 0.1
M aqueous diethylamine solution, the mixture was shaken for
a further 30 min. The ion exchanger was filtered off, washed
with water (20 mL), and then resuspended in 0.5 M hydro-
chloric acid (35 mL). After shaking at room temperature for 5
min, the ion exchanger was filtered off and resuspended in
0.5 M hydrochloric acid (35 mL), and the mixture was shaken
once again at room temperature for 5 min. After separation
of the ion exchanger by filtration, the solvent of the combined
aqueous layers was removed in vacuo (0.1 mbar, 20 °C) and
the residue dried at 20 °C/0.01 mbar for 1 h and then dissolved
in ethanol (5 mL). After addition of propylene oxide (2 mL),
the mixture was heated under reflux for 10 min. The precipi-
tated product was isolated by centrifugation, washed with
ethanol (2 × 2 mL) and diethyl ether (2 × 2 mL), and then
dried in vacuo (0.01 mbar, 40 °C, 36 h) to give (R)-4 in 53%
yield as a colorless crystalline solid (126 mg, 564 µmol). IR
P r ep a r a tion of (R)-2-Am in o-3-(tr im eth ylsilyl)p r op i-
on ic Acid [(R)-â-(Tr im eth ylsilyl)a la n in e; (R)-2]. A solution
of (R)-15 (1.20 g, 6.34 mmol) in 6 M hydrochloric acid (30 mL)
was heated under reflux for 1 h. After cooling to room
temperature, the solvent was removed in vacuo (0.1 mbar, 20
°C) and the residue dried at 40 °C/0.01 mbar for 1 h and then
dissolved in ethanol (5 mL). After addition of propylene oxide
(2 mL), the mixture was heated under reflux for 10 min. The
precipitated product was isolated by centrifugation, washed
with ethanol (2 × 2 mL) and diethyl ether (2 × 2 mL), and
then dried in vacuo (0.01 mbar, 40 °C, 36 h) to give (R)-2 in
79% yield as a colorless crystalline solid (808 mg, 5.01 mmol).
1
IR (KBr, cm-1): ν 1671 (CdO). H NMR (400.1 MHz, D2O): δ
0.15 (s, 9 H, SiCH3), 1.16 (δA), 1.22 (δB), and 3.81 (δX) (2J AB
)
3
3
14.6 Hz, J AX ) 5.7 Hz, J BX ) 10.3 Hz, 3 H, Si-CHAHB-CHX);
OH and NH2 not detected (H/D exchange). 13C NMR (100.6
MHz, D2O): δ -1.6 (SiCH3), 20.5 (SiCH2CH), 54.3 (SiCH2CH),
176.5 (CdO). 29Si NMR (79.5 MHz, D2O): δ -0.3. EI MS: 146
[7%, M+ - Me], 116 [100%, M+ - CO2H], 73 [90%, SiMe3+].
Anal. Calcd for C6H15NO2Si: C, 44.68; H, 9.37; N, 8.68.
Found: C, 44.6; H, 9.5; N, 8.6.
1
(KBr, cm-1): ν 1669 cm-1 (CdO). H NMR (300.1 MHz, D2O):
δ 0.40 (s, 3 H, SiCH3), 0.43 (s, 3 H, SiCH3), 1.35 (δB), 1.43 (δA),
3
3
and 3.92 (δX) (2J AB ) 14.5 Hz, J AX ) 10.1 Hz, J BX ) 5.9 Hz,
3 H, Si-CHAHB-CHX), 7.46-7.48 and 7.51-7.54 (m, 5 H,
SiC6H5); OH and NH2 not detected (H/D exchange). 13C NMR
(75.5 MHz, D2O): δ -3.7 (SiCH3), -3.2 (SiCH3), 19.3 (SiCH2-
CH), 53.6 (SiCH2CH), 128.6 (C-3/C-5, SiC6H5), 130.0 (C-4,
SiC6H5), 134.1 (C-2/C-6, SiC6H5), 137.7 (C-1, SiC6H5), 165.2
(CdO). 29Si NMR (59.6 MHz, D2O): δ -1.2. CI MS (negative
ions): 222 [100%, (M - H)+]. Anal. Calcd for C11H17NO2Si: C,
59.16; H, 7.67; N, 6.27. Found: C, 59.2; H, 7.7; N, 6.1.
P r ep a r a tion of (S)-2-Am in o-3-(tr im eth ylsilyl)p r op i-
on ic Acid [(S)-â-(Tr im eth ylsilyl)a la n in e; (S)-2]. This com-
pound was prepared analogously to the synthesis of (R)-2 by
heating a mixture of (S)-15 (252 mg, 1.33 mmol) in 6 M
hydrochloric acid (10 mL). After treatment of the solution of
the residue [(S)-2‚HCl] in ethanol (3 mL) with propylene oxide
(2 mL), (S)-2 was isolated in 60% yield as a colorless crystalline
solid (128 mg, 794 µmol). The IR, NMR, and MS data of the
product were identical with those obtained for (R)-2. Anal.
Calcd for C6H15NO2Si: C, 44.68; H, 9.37; N, 8.68. Found: C,
44.7; H, 9.5; N, 8.6.
P r ep a r a tion of (R)-2-Am in o-3-[d im eth yl(p h en yl)ger -
m yl]p r op ion ic Acid [(R)-â-[Dim et h yl(p h en yl)ger m yl]-
a la n in e; (R)-5]. This compound was prepared analogously to
the synthesis of (R)-4 starting from (R)-24 (280 mg, 946 µmol).
The product was isolated in 45% yield as a colorless crystalline
P r ep a r a tion of (R)-2-Am in o-3-(tr im eth ylger m yl)p r o-
p ion ic Acid [(R)-â-(Tr im eth ylger m yl)a la n in e; (R)-3]. This
compound was prepared analogously to the synthesis of (R)-2
by heating a mixture of (R)-16 (580 mg, 2.48 mmol) in 6 M
hydrochloric acid (30 mL). After treatment of the solution of
the residue [(R)-3‚HCl] in ethanol (10 mL) with propylene
oxide (3 mL), (R)-3 was isolated in 64% yield as a colorless
crystalline solid (329 mg, 1.60 mmol). IR (KBr, cm-1): ν 1672
(CdO). 1H NMR (400.1 MHz, D2O): δ 0.28 (s, 9 H, GeCH3),
1
solid (114 mg, 426 µmol). IR (KBr, cm-1): ν 1674 (CdO). H
NMR (300.1 MHz, D2O): δ 0.52 (s, 3 H, GeCH3), 0.53 (s, 3 H,
GeCH3), 1.50 (δB), 1.56 (δA), and 3.87 (δX) (2J AB ) 13.5 Hz, 3J AX
3
) 10.7 Hz, J BX ) 5.0 Hz, 3 H, Ge-CHAHB-CHX), 7.43-7.52
and 7.62-7.67 (m, 5 H, GeC6H5); OH and NH2 not detected
(H/D exchange). 13C NMR (75.5 MHz, D2O): δ -1.8 (GeCH3),
-1.6 (GeCH3), 19.7 (GeCH2CH), 53.3 (GeCH2CH), 127.9 (C-3/
C-5, GeC6H5), 128.3 (C-4, GeC6H5), 133.2 (C-2/C-6, GeC6H5),
141.0 (C-1, GeC6H5), 177.5 (CdO). CI MS (negative ions): 268
[5%, (M - H)+], 226 [100%], 224 [80%, (M - CO2H)+]. Anal.
Calcd for C11H17GeNO2: C, 49.32; H, 6.40; N, 5.23. Found: C,
49.2; H, 6.3; N, 5.3.
3
1.29 (δA), 1.33 (δB), and 3.85 (δX) (2J AB ) 13.5 Hz, J AX ) 5.5
3
Hz, J BX ) 10.2 Hz, 3 H, Ge-CHAHB-CHX); OH and NH2 not
detected (H/D exchange). 13C NMR (100.6 MHz, D2O): δ -0.2
(GeCH3), 21.6 (GeCH2CH), 56.7 (GeCH2CH), 178.1 (CdO). EI
MS: 192 [12%, M+ - Me], 162 [58%, M+ - CO2H], 119 [100%,
GeMe3+]. Anal. Calcd for C6H15GeNO2: C, 35.02; H, 7.35; N,
6.81. Found: C, 35.2; H, 7.3; N, 6.7.
P r ep a r a tion of (R)-2-Am in o-3-[d im eth yl(vin yl)silyl]-
p r op ion ic Acid [(R)-â-[Dim eth yl(vin yl)silyl]a la n in e; (R)-
6]. This compound was prepared analogously to the synthesis
of (R)-4 starting from (R)-25 (264 mg, 1.31 mmol). The product
was isolated in 42% yield as a colorless crystalline solid (94.4
mg, 545 µmol). IR (KBr, cm-1): ν 1670 (CdO). 1H NMR (300.1
MHz, D2O): δ 0.228 (s, 3 H, SiCH3), 0.234 (s, 3 H, SiCH3),
P r ep a r a tion of (S)-2-Am in o-3-(tr im eth ylger m yl)p r o-
p ion ic Acid [(S)-â-(Tr im eth ylger m yl)a la n in e; (S)-3]. This
compound was prepared analogously to the synthesis of (R)-2
by heating a mixture of (S)-16 (46.1 mg, 197 µmol) in 6 M
hydrochloric acid (5 mL). After treatment of the solution of
the residue [(S)-3‚HCl] in ethanol (2 mL) with propylene oxide
(2 mL), (S)-3 was isolated in 39% yield as a colorless crystalline
solid (15.9 mg, 77.3 µmol). The IR, NMR, and MS data of the
product were identical with those obtained for (R)-3. Anal.
Calcd for C6H15GeNO2: C, 35.02; H, 7.35; N, 6.81. Found: C,
35.1; H, 7.2; N, 6.7.
P r ep a r a tion of (R)-2-Am in o-3-[d im eth yl(p h en yl)silyl]-
p r op ion ic Acid [(R)-â-[Dim et h yl(p h en yl)silyl]a la n in e;
(R)-4]. Water (2.5 mL) and a 1 M aqueous lithium hydroxide
solution (1.07 mL, 1.07 mmol LiOH) were added to a solution
of (R)-23 (269 mg, 1.07 mmol) in dioxane (7 mL). After stirring
the mixture at room temperature for 16 h, the solvent was
removed in vacuo. The residue was washed with n-pentane
(10 mL) and then dried in vacuo (0.01 mbar, 20 °C, 2 h) to
give the lithium salt of (R)-4 in quantitative yield as a colorless
3
1.24 (δB), 1.30 (δA), and 3.84 (δX) (2J AB ) 14.7 Hz, J AX ) 9.6
3
Hz, J BX ) 6.1 Hz, 3 H, Si-CHAHB-CHX), 5.88 (δA), 6.12 (δM),
3
3
and 6.31 (δX) (2J AM ) 3.6 Hz, J AX ) 20.7 Hz, J MX ) 14.8 Hz,
3 H, Si-CHXdCHAHM); OH and NH2 not detected (H/D
exchange). 13C NMR (75.5 MHz, D2O): δ -3.8 (SiCH3), -3.6
(SiCH3), 19.0 (SiCH2CH), 53.5 (SiCH2CH), 131.4 (SiCHdCH2),
136.1 (SiCHdCH2), 175.7 (CdO). 29Si NMR (59.6 MHz, D2O):
δ -7.7. CI MS (negative ions): 172 [100%, (M - H)+]. Anal.
Calcd for C7H15NO2Si: C, 48.52; H, 8.72; N, 8.08. Found: C,
48.2; H, 8.5; N, 8.0.
P r ep a r a tion of th e Deca p ep tid es Acetyl-D-2-n a p h th yl-
a la n yl-D-4-ch lor op h en yla la n yl-D-3-p yr id yla la n yl-ser yl-
ter t-b u t yla la n yl-D-cit r u llyl-leu cyl-a r gin yl-p r olyl-D-a la -
n in a m id e (Ac-D-Na l1-4-Cl-D-P h e2-D-P a l3-Ser 4-Me3C-Ala 5-
D-Cit6-Leu 7- Ar g8-P r o9-D-Ala10-NH2; 7), Acetyl-D-2-n aph th yl-
a la n yl-D-4-ch lor op h en yla la n yl-D-3-p yr id yla la n yl-ser yl-

Si- and Ge-Containing R-Amino Acids and Peptides
Organometallics, Vol. 19, No. 18, 2000 3493
(tr im eth ylsilyl)a la n yl-D-citr u llyl-leu cyl-a r gin yl-p r olyl-D-
a la n in a m id e (Ac-D-Na l1-4-Cl-D-P h e2-D-P a l3-Ser 4-Me3Si-
Ala 5-D-Cit6-Leu 7-Ar g8-P r o9-D-Ala 10-NH2; 8), a n d Acetyl-D-
2 -n a p h t h y l a l a n y l -D -4 -c h l o r o p h e n y l a l a n y l -D -3 -
p yr id yla la n yl-ser yl-(tr im eth ylger m yl)a la n yl-D-citr u llyl-
leu cyl-a r gin yl-p r olyl-D-a la n in a m id e (Ac-D-Na l1-4-Cl-D-
(Ch lor om eth yl)tr im eth ylsila n e (11). This compound was
commercially available (Aldrich).
P r ep a r a tion of (Ch lor om eth yl)tr im eth ylger m a n e (12).
This compound was synthesized from trichloro(chloromethyl)-
germane21 according to ref 22.
P r ep a r a tion of (2R,5R)- a n d (2S,5R)-3,6-Dieth oxy-5-
isop r op yl-2-[(t r im et h ylsilyl)m et h yl]-2,5-d ih yd r op yr a -
zin e [(2R,5R)-13 a n d (2S,5R)-13]. A 1.6 M solution of
n-butyllithium in n-hexane (5.81 mL, 9.30 mmol n-BuLi) was
added dropwise at -70 °C within 30 min to a stirred solution
of (R)-10 (1.97 g, 9.28 mmol) in THF (40 mL). After the mixture
was stirred at -70 °C for 15 min, a solution of 11 (1.14 g, 9.29
mmol) in THF (10 mL) was added dropwise at -70 °C within
30 min, and the reaction mixture was stirred at this temper-
ature for 2 h. The mixture was allowed to warm to room
temperature within 12 h, followed by addition of diethyl ether
(50 mL) and water (50 mL). The organic phase was separated
and the aqueous layer extracted with diethyl ether (3 × 25
mL), and the combined organic extracts were dried over
anhydrous Na2SO4. The solvent was removed under reduced
pressure and the oily residue distilled in a Kugelrohr ap-
paratus (oven temperature 150 °C, 0.01 mbar) to give a 75:25
mixture of (2R,5R)-13/(2S,5R)-13 (molar ratio 85:15, GC
analysis) and (R)-10. The diastereomerically pure compounds
(2R,5R)-13 and (2S,5R)-13 were obtained by liquid-chromato-
graphic separation (for the general procedure, see below).
Da ta for (2R,5R)-13. Yield: 52% (1.43 g, 4.79 mmol),
relative to (R)-10; diastereomeric purity g99% de. IR (film,
cm-1): ν 1688 (CdN). 1H NMR (600.1 MHz, CDCl3): δ 0.02
(s, 9 H, SiCH3), 0.69 (δQ), 0.84 (δA), 1.01 (δT), 1.19 (δB), 2.24
P h e2-D-P al3-Ser 4-Me3Ge-Ala5-D-Cit6-Leu 7-Ar g8-P r o9-D-Ala10
-
NH2; 9). Compounds 7-9 were prepared by solid-phase
synthesis using a Labortec SP650 semiautomatic peptide
synthesizer. [(Fluoren-9-yl)methoxycarbonyl]-4-methoxy-4′-(γ-
carboxypropyloxy)benzhydrylamine resin (Fmoc-MBHA resin;
Bachem AG, D 1600) was used as polymeric support. The
sequential synthesis was performed as follows: N-Fmoc-
protected D-alanine (C-terminal) was covalently linked to the
unprotected MBHA resin by using a 3-fold molar excess of each
diisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazole
(HOBt) in a mixture of dichloromethane and DMF [1:1 (v/v)].
Removal of the Fmoc group was performed with a 20% solution
of piperidine in DMF as standard routine. Chain elongation
according to the respective amino acid sequence was ac-
complished with a 3-molar excess of each N-Fmoc-amino acid,
DIC, and HOBt within 90 min of coupling time. Completion
of acylation was checked via the chloroanil test. Cleavage of
the decapeptides from the polymeric support was accomplished
by treatment with a mixture of acetic acid/2,2,2-trifluoroet-
hanol/dichloromethane [1:1:8 (v/v/v)] at 60 °C for 2.5 h. The
solvents were removed under reduced pressure, and the
residue was washed with diethyl ether. The precipitate was
filtered off and dried at room temperature for 12 h. The
decapeptides were purified by high-performance liquid chro-
matography using a Nucleoprep RP-C-18 silica gel column. The
experimental conditions were as follows: HPLC pump, Shi-
madzu LC 8A; detector, Shimadzu SPD 68; column (50 mm
i.d. × 250 mm); eluent, water/acetonitrile/2,2,2-trifluoroacetic
acid [A: 97:3:1 (v/v/v); B: 30:70:1 (v/v/v); 45% B f 90% B
within 50 min]; injection volume, 20 mL; flow rate, 60.0 mL/
min. After removal of the solvents under reduced pressure,
the isolated decapeptides were lyophilized. The yields of the
products (white fluffy residues) were as follows: 7, 1.64 g; 8,
1.33 g; 9, 1.22 g.
3
(δN), 3.87 (δK), and 4.00 (δG) [2J AB ) 14.5 Hz, J AG ) 9.8 Hz,
5
3
3
3J BG ) 4.9 Hz, J GK ) 3.6 Hz, J KN ) 3.3 Hz, J NQ ) 6.8 Hz,
3J NT ) 6.9 Hz, 11 H, Si-CHAHB-CHG-NdC-CHK-CHN(CHQ3)-
(CHT3)], 1.24 (δX), 4.03 (δA), and 4.11 (δB) (2J AB ) 10.7 Hz, 3J AX
3
) 7.0 Hz, J BX ) 7.2 Hz, 5 H, O-CHAHB-CHX3), 1.26 (δX), 4.03
(δA), and 4.16 (δB) (2J AB ) 10.7 Hz, J AX ) 7.0 Hz, J BX ) 7.1
Hz, 5 H, O-CHAHB-CHX3). 13C NMR (100.6 MHz, CDCl3): δ
-0.5 (SiCH3), 14.40 (OCH2CH3), 14.43 (OCH2CH3), 16.8
(CHCH3), 19.1 (CHCH3), 23.2 (CHCH2Si), 31.9 [CHCH(CH3)2],
53.3 (CHCH2Si), 60.46 (OCH2CH3), 60.50 (OCH2CH3), 60.6
[CHCH(CH3)2], 162.2 (CdN), 165.1 (CdN). 29Si NMR (79.5
3
3
Da ta for 7. ESI MS: 1416 [2%, (M + Na)+], 1394 [10%, (M
MHz, CDCl3): δ 1.2. EI MS: 298 [87%, M+], 283 [43%, M+
-
+ H)+], 698 [100%, (M + 2 H)2+]. ESI MS/MS [698; (C68H96
-
Me], 269 [13%, M+ - Et], 255 [44%, M+ - CHMe2], 211 [100%,
M+ - CH2SiMe3], 73 [50%, SiMe3+]. Anal. Calcd for C15H30N2O2-
Si: C, 60.36; H, 10.13; N, 9.38. Found: C, 60.3; H, 10.2; N,
9.3.
ClN17O13 + 2 H)2+]: 1209 [20%, B8+], 1053 [14%, B7+], 1010
[7%, (B7 - HNCO)+], 974 [2%, Y8′′+], 940 [7%, B6+], 897 [9%,
(B6 - HNCO)+], 826 [3%, Y7′′+], 783 [5%, B5+], 739 [3%, Y6′′+],
676 [100%, (M + 2 H - HNCO)2+], 612 [5%, Y5′′+], 605 [12%,
B8′2+], 569 [2%, B3+], 455 [10%, Y4′′+], 342 [12%, Y3′′+].
Da ta for (2S,5R)-13. Yield: 8% (208 mg, 697 µmol), relative
to (R)-10; diastereomeric purity g99% de. IR (film, cm-1): ν
1686 (CdN). 1H NMR (600.1 MHz, CDCl3): δ 0.06 (s, 9 H,
SiCH3), 0.67 (δA), 0.75 (δQ), 1.02 (δT), 1.21 (δB), 2.14 (δN), 3.84
Da ta for 8. ESI MS: 1432 [10%, (M + Na)+], 1410 [20%,
(M + H)+], 706 [100%, (M + 2 H)2+]. ESI MS/MS [706; (C67H96
-
ClN17O13Si + 2 H)2+]: 1225 [19%, B8+], 1069 [13%, B7+], 1026
[13%, (B7 - HNCO)+], 990 [2%, Y8′′+], 956 [8%, B6+], 913 [11%,
(B6 - HNCO)+], 842 [3%, Y7′′+], 799 [5%, B5+], 755 [4%, Y6′′+],
684 [100%, (M + 2 H - HNCO)2+], 656 [6%, B4+], 613 [13%,
B8′2+], 612 [5%, Y5′′+], 569 [2%, B3+], 455 [12%, Y4′′+], 342 [11%,
Y3′′+].
3
3
(δK), and 3.97 (δG) [2J AB ) 14.5 Hz, J AG ) 11.5 Hz, J BG ) 4.8
5
3
3
3
Hz, J GK ) 4.2 Hz, J KN ) 4.1 Hz, J NQ ) 6.8 Hz, J NT ) 6.9
Hz, 11 H, Si-CHAHB-CHG-NdC-CHK-CHN(CHQ3)(CHT3)], 1.23
(δX), 4.03 (δA), and 4.12 (δB) (2J AB ) 10.7 Hz, J AX ) 6.9 Hz,
3
3J BX ) 7.2 Hz, 5 H, O-CHAHB-CHX3), 1.25 (δX), 4.05 (δA), and
3
3
4.08 (δB) (2J AB ) 10.7 Hz, J AX ) 7.2 Hz, J BX ) 6.9 Hz, 5 H,
O-CHAHB-CHX3). 13C NMR (100.6 MHz, CDCl3): δ -0.5
(SiCH3), 14.1 (OCH2CH3), 14.4 (OCH2CH3), 17.9 (CHCH3), 19.6
(CHCH3), 24.4 (CHCH2Si), 31.6 [CHCH(CH3)2], 53.4 (CHCH2-
Si), 60.3 (OCH2CH3), 60.4 (OCH2CH3), 61.0 [CHCH(CH3)2],
162.1 (CdN), 165.1 (CdN). 29Si NMR (79.5 MHz, CDCl3): δ
1.4. EI MS: 298 [76%, M+], 283 [46%, M+ - Me], 269 [16%,
M+ - Et], 255 [62%, M+ - CHMe2], 211 [100%, M+ - CH2-
SiMe3], 73 [52%, SiMe3+]. Anal. Calcd for C15H30N2O2Si: C,
60.36; H, 10.13; N, 9.38. Found: C, 60.4; H, 10.2; N, 9.3.
P r ep a r a tion of (2R,5R)- a n d (2S,5R)-3,6-Dieth oxy-5-
isop r op yl-2-[(tr im eth ylger m yl)m eth yl]-2,5-d ih yd r op yr a -
Da ta for 9. ESI MS: 1478 [10%, (M + Na)+], 1456 [14%,
(M + H)+], 729 [100%, (M + 2 H)2+]. ESI MS/MS [727; (C67H96
-
Cl70GeN17O13 + 2 H)2+]: 1267 [30%, B8+], 1111 [90%, B7+], 1068
[35%, (B7 - HNCO)+], 1032 [2%, Y8′′+], 998 [40%, B6+], 955
[42%, (B6 - HNCO)+], 884 [14%, Y7′′+], 841 [20%, B5+], 797
[14%, Y6′′+], 705 [100%, (M + 2 H - HNCO)2+], 656 [15%, B4+],
634 [48%, B8′2+], 670 [23%, Y5′′+], 569 [8%, B3+], 455 [48%,
Y4′′+], 342 [52%, Y3′′+].
P r ep a r a tion of (R)-3,6-Dieth oxy-2-isop r op yl-2,5-d ih y-
d r op yr a zin e [(R)-10]. This compound was synthesized in
analogy to ref 13. In this context, see also refs 19 and 20.
(19) Scho¨llkopf, U.; Groth, U.; Deng, C. Angew. Chem. 1981, 93,
793-795; Angew. Chem., Int. Ed. Engl. 1981, 20, 798-799.
(20) Groth, U.; Schmeck, C.; Scho¨llkopf, U. Liebigs Ann. Chem. 1993,
321-323.
(21) Tacke, R.; Becker, B. J . Organomet. Chem. 1988, 354, 147-
153, and references therein.
(22) Seyferth, D.; Rochow, E. G. J . Am. Chem. Soc. 1955, 77, 907-
910.

3494 Organometallics, Vol. 19, No. 18, 2000
Tacke et al.
zin e [(2R,5R)-14 a n d (2S,5R)-14]. These compounds were
prepared analogously to the synthesis of (2R,5R)-13/(2S,5R)-
13 by addition of a 1.6 M solution of n-butyllithium in n-hexane
(5.01 mL, 8.02 mmol n-BuLi) to a solution of (R)-10 (1.70 g,
8.01 mmol) in THF (40 mL), followed by treatment with a
solution of 12 (1.35 g, 8.07 mmol) in THF (10 mL). The crude
product was purified by Kugelrohr distillation (oven temper-
ature 160 °C, 0.01 mbar) to give a 85:15 mixture of (2R,5R)-
14/(2S,5R)-14 (molar ratio 86:14) and (R)-10. The diastereo-
merically pure compounds (2R,5R)-14 and (2S,5R)-14 were
obtained by liquid-chromatographic separation (for the general
procedure, see below).
M+ - SiMe3], 73 [79%, SiMe3+]. Anal. Calcd for C8H19NO2Si:
C, 50.75; H, 10.12; N, 7.40. Found: C, 50.7; H, 10.1; N, 7.5.
P r ep a r a tion of (S)-2-Am in o-3-(tr im eth ylsilyl)p r op i-
on ic Acid Eth yl Ester [(S)-15]. This compound was prepared
analogously to the synthesis of (R)-15 by treatment of a
solution of (2S,5R)-13 (150 mg, 503 µmol) in ethanol (10 mL)
with 3 M hydrochloric acid (5 mL). After liquid-chromato-
graphic separation on silica gel [see preparation of (R)-15; Rf-
[(S)-15] > Rf[(R)-valine]], (S)-15 was isolated in 84% yield as
a colorless liquid (80.3 mg, 424 µmol). The IR, NMR, and MS
data of the product were identical with those obtained for (R)-
15. Anal. Calcd for C8H19NO2Si: C, 50.75; H, 10.12; N, 7.40.
Found: C, 50.6; H, 10.4; N, 7.3.
Da ta for (2R,5R)-14. Yield: 54% (1.47 g, 4.29 mmol),
relative to (R)-10; diastereomeric purity g99% de. IR (film,
cm-1): ν 1688 (CdN). 1H NMR (400.1 MHz, CDCl3): δ 0.15
(s, 9 H, GeCH3), 0.69 (δQ), 1.00 (δT), 1.03 (δA), 1.34 (δB), 2.24
P r ep a r a tion of (R)-2-Am in o-3-(tr im eth ylger m yl)p r o-
p ion ic Acid Eth yl Ester [(R)-16]. This compound was
prepared analogously to the synthesis of (R)-15 by treatment
of a solution of (2R,5R)-14 (1.01 g, 2.94 mmol) in ethanol (20
3
(δN), 3.88 (δK), and 4.03 (δG) [2J AB ) 13.5 Hz, J AG ) 9.4 Hz,
5
3
3
3J BG ) 5.2 Hz, J GK ) 3.4 Hz, J KN ) 3.5 Hz, J NQ ) 6.8 Hz,
3J NT ) 6.9 Hz, 11 H, Ge-CHAHB-CHG-NdC-CHK-CHN(CHQ3)-
(CHT3)], 1.24 (δX), 4.03 (δA), and 4.10 (δB) (2J AB ) 10.7 Hz, 3J AX
mL) with
3 M hydrochloric acid (5 mL). After liquid-
chromatographic separation on silica gel [see preparation of
(R)-15; Rf[(R)-16] > Rf[(R)-valine]], (R)-16 was isolated in 73%
yield as a colorless liquid (500 mg, 2.14 mmol). IR (film, cm-1):
3
) 7.1 Hz, J BX ) 7.1 Hz, 5 H, O-CHAHB-CHX3), 1.26 (δX), 4.04
(δA), and 4.16 (δB) (2J AB ) 10.7 Hz, J AX ) 7.1 Hz, J BX ) 7.1
Hz, 5 H, O-CHAHB-CHX3). 13C NMR (100.6 MHz, CDCl3): δ
-1.0 (GeCH3), 14.40 (OCH2CH3), 14.42 (OCH2CH3), 16.8
(CHCH3), 19.1 (CHCH3), 23.6 (CHCH2Ge), 31.9 [CHCH(CH3)2],
53.8 (CHCH2Ge), 60.45 (OCH2CH3), 60.54 (OCH2CH3), 60.8
[CHCH(CH3)2], 162.4 (CdN), 164.9 (CdN). EI MS: 344 [8%,
M+], 329 [40%, M+ - Me], 301 [15%, M+ - CHMe2], 119 [100%,
GeMe3+]. Anal. Calcd for C15H30GeN2O2: C, 52.52; H, 8.82; N,
8.17. Found: C, 52.4; H, 9.0; N, 8.3.
3
3
1
ν 1734 (CdO). H NMR (400.1 MHz, CDCl3): δ 0.12 (s, 9 H,
GeCH3), 0.94 (δA), 1.13 (δB), and 3.42 (δX) (2J AB ) 13.6 Hz, 3J AX
3
) 9.0 Hz, J BX ) 6.1 Hz, 3 H, Ge-CHAHB-CHX), 1.20 (δX), 4.06
3
3
(δA), and 4.09 (δB) (2J AB ) 10.8 Hz, J AX ) 7.2 Hz, J BX ) 7.0
Hz, 5 H, O-CHAHB-CHX3), 1.40 (s, 2 H, NH2). 13C NMR (100.6
MHz, CDCl3): δ -1.5 (GeCH3), 14.1 (OCH2CH3), 23.4 (GeCH2-
CH), 52.6 (GeCH2CH), 60.6 (OCH2CH3), 177.2 (CdO). EI MS:
220 [18%, M+ - Me], 162 [90%, M+ - CO2Et], 119 [100%,
GeMe3+]. Anal. Calcd for C8H19GeNO2: C, 41.09; H, 8.19; N,
5.99. Found: C, 41.2; H, 8.3; N, 6.1.
Da ta for (2S,5R)-14. Yield: 6% (175 mg, 510 µmol), relative
to (R)-10; diastereomeric purity g99% de. IR (film, cm-1): ν
1691 (CdN). 1H NMR (400.1 MHz, CDCl3): δ 0.18 (s, 9 H,
GeCH3), 0.76 (δQ), 0.85 (δA), 1.03 (δT), 1.35 (δB), 2.16 (δN), 3.85
P r ep a r a tion of (S)-2-Am in o-3-(tr im eth ylger m yl)p r o-
p ion ic Acid Eth yl Ester [(S)-16]. This compound was
prepared analogously to the synthesis of (R)-15 by treatment
of a solution of (2S,5R)-14 (114 mg, 332 µmol) in ethanol (10
3
3
(δK), and 3.98 (δG) [2J AB ) 13.3 Hz, J AG ) 11.4 Hz, J BG ) 5.2
5
3
3
3
mL) with
3 M hydrochloric acid (5 mL). After liquid-
Hz, J GK ) 4.1 Hz, J KN ) 4.2 Hz, J NQ ) 7.0 Hz, J NT ) 6.8
Hz, 11 H, Ge-CHAHB-CHG-NdC-CHK-CHN(CHQ3)(CHT3)], 1.23
chromatographic separation on silica gel [see preparation of
(R)-15; Rf[(S)-16] > Rf[(R)-valine]], (S)-16 was isolated in 80%
yield as a colorless liquid (62.4 mg, 267 µmol). The IR, NMR,
and MS data of the product were identical with those obtained
for (R)-16. Anal. Calcd for C8H19GeNO2: C, 41.09; H, 8.19; N,
5.99. Found: C, 40.7; H, 8.1; N, 5.6.
(δX), 4.02 (δA), and 4.09 (δB) (2J AB ) 10.7 Hz, J AX ) 7.0 Hz,
3
3J BX ) 7.0 Hz, 5 H, O-CHAHB-CHX3), 1.24 (δX), 4.04 (δA), and
4.15 (δB) (2J AB ) 10.7 Hz, J AX ) 7.0 Hz, J BX ) 7.0 Hz, 5 H,
O-CHAHB-CHX3). 13C NMR (100.6 MHz, CDCl3): δ -1.1
(GeCH3), 14.4 (OCH2CH3), 14.5 (OCH2CH3), 17.9 (CHCH3),
19.6 (CHCH3), 24.6 (CHCH2Ge), 31.7 [CHCH(CH3)2], 53.9
(CHCH2Ge), 60.9 (OCH2CH3), 61.1 (OCH2CH3), 61.0 [CHCH-
(CH3)2], 162.2 (CdN), 165.3 (CdN). EI MS: 344 [1%, M+], 329
[29%, M+ - Me], 301 [18%, M+ - CHMe2], 119 [100%, GeMe3+].
Anal. Calcd for C15H30GeN2O2: C, 52.52; H, 8.82; N, 8.17.
Found: C, 52.8; H, 8.8; N, 8.4.
3
3
(Ch lor om eth yl)d im eth yl(p h en yl)sila n e (17). This com-
pound was commercially available (Aldrich).
P r ep a r a tion of (Ch lor om eth yl)d im eth yl(p h en yl)ger -
m a n e (18). A Grignard reagent was prepared from bromoben-
zene (27.2 g, 173 mmol) and magnesium turnings (4.20 g, 173
mmol) in diethyl ether (250 mL) and then added dropwise at
room temperature within 2 h to a stirred solution of trichloro-
(chloromethyl)germane21 (39.5 g, 173 mmol) in diethyl ether
(200 mL). After stirring at room temperature for 14 h and
heating under reflux for 1 h, n-pentane (100 mL) was added
and the precipitate was filtered off. The solvent of the filtrate
was removed under reduced pressure and the residue distilled
in vacuo (Vigreux column) to give 53.2 g of a mixture consisting
of dichloro(chloromethyl)phenylgermane, bromochloro(chlo-
romethyl)phenylgermane, and dibromo(chloromethyl)phe-
P r ep a r a tion of (R)-2-Am in o-3-(tr im eth ylsilyl)p r op i-
on ic Acid Eth yl Ester [(R)-15]. Hydrochloric acid (3 M, 10
mL) was added dropwise at 0 °C within 10 min to a stirred
solution of (2R,5R)-13 (1.02 g, 3.42 mmol) in ethanol (25 mL).
After stirring the mixture at 0 °C for 2 h, the solvent was
removed in vacuo (0.1 mbar, 20 °C), the residue dissolved in
dichloromethane, and the resulting solution extracted with a
2 M aqueous Na2CO3 solution. After drying of the organic layer
over anhydrous Na2SO4, the solvent was removed under
reduced pressure (rotary evaporator) and the product isolated
and purified by liquid chromatography on silica gel [eluent
diethyl ether/n-hexane (2:1, v/v; before using the eluent, it was
washed with concentrated aqueous ammonia solution); Rf[(R)-
15] > Rf[(R)-valine]] to give (R)-15 in a 86% yield as a colorless
liquid (555 mg, 2.93 mmol). IR (film, cm-1): ν 1734 (CdO). 1H
NMR (300.1 MHz, CDCl3): δ 0.00 (s, 9 H, SiCH3), 0.79 (δA),
1
nylgermane (molar ratio 17:47:36, H NMR analysis; 130 °C/
17 mbar to 85 °C/0.1 mbar). A stirred solution of this mixture
in diethyl ether (500 mL) was treated dropwise at -70 °C
within 2 h with a 1.6 M solution of methyllithium in diethyl
ether (136 mL, 218 mmol MeLi). After stirring for 4 h at this
temperature, the mixture was allowed to warm to room
temperature within 6 h, followed by addition of water (300
mL). The organic phase was separated and the aqueous layer
extracted with diethyl ether (4 × 150 mL), and the combined
organic extracts were dried over anhydrous Na2SO4. The
solvent was removed under reduced pressure and the residue
distilled in vacuo (Vigreux column) to give 18 in 51% yield as
3
3
1.00 (δB), and 3.42 (δX) (2J AB ) 14.6 Hz, J AX ) 8.9 Hz, J BX
)
6.1 Hz, 3 H, Si-CHAHB-CHX), 1.22 (δX), 4.07 (δA), and 4.10 (δB)
(2J AB ) 10.9 Hz, J AX ) 7.1 Hz, J BX ) 7.1 Hz, 5 H, O-CHAHB-
CHX3), 1.48 (s, 2 H, NH2). 13C NMR (100.6 MHz, CDCl3): δ
-1.0 (SiCH3), 14.1 (OCH2CH3), 23.4 (SiCH2CH), 52.0
(SiCH2CH), 60.7 (OCH2CH3), 177.4 (CdO). 29Si NMR (79.5
MHz, CDCl3): δ 0.9. EI MS: 174 [6%, M+ - Me], 116 [100%,
3
3
1
a colorless liquid (20.4 g, 89.0 mmol); bp 124 °C/29 mbar. H
NMR (300.1 MHz, CDCl3): δ 0.63 (s, 6 H, GeCH3), 3.19 (s, 2

Si- and Ge-Containing R-Amino Acids and Peptides
H, GeCH2Cl), 7.44-7.49 and 7.52-7.57 (m, 5 H, GeC6H5). 13
Organometallics, Vol. 19, No. 18, 2000 3495
C
16.9 (CHCH3), 19.1 (CHCH3), 22.8 (GeCH2CH), 32.0 [CHCH-
(CH3)2], 53.6 (GeCH2CH), 60.5 (OCH2CH3), 60.6 (OCH2CH3),
60.9 [CHCH(CH3)2], 127.8 (C-3/C-5, GeC6H5), 128.1 (C-4,
GeC6H5), 133.3 (C-2/C-6, GeC6H5), 142.4 (C-1, GeC6H5), 162.5
NMR (75.5 MHz, CDCl3): δ -4.8 (GeCH3), 30.6 (GeCH2Cl),
128.2 (C-3/C-5, GeC6H5), 129.0 (C-4, GeC6H5), 133.3 (C-2/C-6,
GeC6H5), 138.7 (C-1, GeC6H5). EI MS: 230 [1%, M+], 215 [4%,
M+ - Me], 181 [100%, M+ - CH2Cl]. Anal. Calcd for C9H13
-
(CdN), 164.6 (CdN). EI MS: 406 [4%, M+], 391 [14%, M+
-
ClGe: C, 47.15; H, 5.72. Found: C, 47.3; H, 5.7.
Me], 363 [8%, M+ - CHMe2], 329 [14%, M+ - Ph], 211 [5%,
M+ - CH2GeMe2Ph], 181 [100%, GeMe2Ph+]. Anal. Calcd for
P r ep a r a tion of (Ch lor om eth yl)d im eth yl(vin yl)sila n e
(19). This compound was synthesized according to ref 23.
P r ep a r a tion of (2R,5R)-3,6-Dieth oxy-5-isop r op yl-2-
{[dimethyl(phenyl)silyl]methyl}-2,5-dihydropyrazine[(2R,5R)-
20]. This compound was prepared analogously to the synthesis
of (2R,5R)-13/(2S,5R)-13 by addition of a 1.6 M solution of
n-butyllithium in n-hexane (7.00 mL, 11.2 mmol n-BuLi) to a
solution of (R)-10 (2.36 g, 11.1 mmol) in THF (40 mL), followed
by treatment with a solution of 17 (2.07 g, 11.2 mmol) in THF
(10 mL). The crude product was purified by Kugelrohr distil-
lation (oven temperature 170 °C, 0.01 mbar) to give a 65:35
mixture of (2R,5R)-20/(2S,5R)-20 (molar ratio 80:20) and (R)-
10. The diastereomerically pure compound (2R,5R)-20 was
isolated by liquid-chromatographic separation (for the general
procedure, see below) as a colorless liquid in 41% yield [relative
to (R)-10; 1.65 g, 4.58 mmol; g99% de]. IR (film, cm-1): ν 1691
C
20H32GeN2O2: C, 59.30; H, 7.96; N, 6.92. Found: C, 59.3; H,
7.7; N, 6.9.
P r ep a r a tion of (2R,5R)-Dieth oxy-5-isop r op yl-2-{[d i-
m eth yl(vin yl)silyl]m eth yl}-2,5-dih ydr opyr azin e [(2R,5R)-
22]. This compound was prepared analogously to the synthesis
of (2R,5R)-13/(2S,5R)-13 by addition of a 1.6 M solution of
n-butyllithium in n-hexane (5.11 mL, 8.18 mmol n-BuLi) to a
solution of (R)-10 (1.72 g, 8.10 mmol) in THF (40 mL), followed
by treatment with a solution of 19 (1.10 g, 8.17 mmol) in THF
(10 mL). The crude product was purified by Kugelrohr distil-
lation (oven temperature 150 °C, 0.01 mbar) to give a 80:20
mixture of (2R,5R)-22/(2S,5R)-22 (molar ratio 87:13) and (R)-
10. The diastereomerically pure compound (2R,5R)-22 was
isolated by liquid-chromatographic separation (for the general
procedure, see below) as a colorless liquid in 58% yield [relative
to (R)-10; 1.47 g, 4.73 mmol; g99% de]. IR (film, cm-1): ν 1698
1
(CdN). H NMR (600.1 MHz, CDCl3): δ 0.33 (s, 3 H, SiCH3),
1
(CdN). H NMR (400.1 MHz, CDCl3): δ 0.10 (s, 3 H, SiCH3),
0.34 (s, 3 H, SiCH3), 0.67 (δQ), 1.00 (δT), 1.10 (δA), 1.48 (δB),
3
2.22 (δN), 3.85 (δK), and 4.03 (δG) [2J AB ) 14.6 Hz, J AG ) 10.0
0.11 (s, 3 H, SiCH3), 0.68 (δQ), 0.90 (δA), 1.00 (δT), 1.27 (δB),
3
2.23 (δN), 3.86 (δK), and 4.01 (δG) [2J AB ) 14.5 Hz, J AG ) 9.9
3
5
3
3
Hz, J BG ) 4.7 Hz, J GK ) 3.7 Hz, J KN ) 3.2 Hz, J NQ ) 6.8
3
5
3
3
3
Hz, J BG ) 4.8 Hz, J GK ) 3.3 Hz, J KN ) 3.5 Hz, J NQ ) 6.8
Hz, J NT ) 6.9 Hz, 11 H, Si-CHAHB-CHG-NdC-CHK-CHN-
3
(CHQ3)(CHT3)], 1.19 (δX), 3.97 (δA), and 4.13 (δB) (2J AB ) 10.7
Hz, J NT ) 6.9 Hz, 11 H, Si-CHAHB-CHG-NdC-CHK-CHN-
(CHQ3)(CHT3)], 1.23 (δX), 4.02 (δA), and 4.11 (δB) (2J AB ) 10.5
3
3
Hz, J AX ) 6.9 Hz, J BX ) 7.1 Hz, 5 H, O-CHAHB-CHX3), 1.24
3
3
(δX), 3.81 (δA), and 4.00 (δB) (2J AB ) 10.7 Hz, J AX ) 7.1 Hz,
3
Hz, J AX ) 7.1 Hz, J BX ) 7.1 Hz, 5 H, O-CHAHB-CHX3), 1.25
(δX), 4.03 (δA), and 4.15 (δB) (2J AB ) 10.6 Hz, J AX ) 7.1 Hz,
3
3J BX ) 6.9 Hz, 5 H, O-CHAHB-CHX3), 7.31-7.42 and 7.53-7.62
(m, 5 H, SiC6H5). 13C NMR (100.6 MHz, CDCl3): δ -2.0
(SiCH3), -1.7 (SiCH3), 14.37 (OCH2CH3), 14.38 (OCH2CH3),
16.8 (CHCH3), 19.1 (CHCH3), 22.3 (SiCH2CH), 31.9 [CHCH-
(CH3)2], 53.1 (SiCH2CH), 60.46 (OCH2CH3), 60.51 (OCH2CH3),
60.7 [CHCH(CH3)2], 127.6 (C-3/C-5, SiC6H5), 128.6 (C-4,
SiC6H5), 133.7 (C-2/C-6, SiC6H5), 140.1 (C-1, SiC6H5), 162.3
(CdN), 164.8 (CdN). 29Si NMR (59.6 MHz, CDCl3): δ 3.3. EI
3J BX ) 7.1 Hz, 5 H, O-CHAHB-CHX3), 5.64 (δA), 5.89 (δM), and
6.17 (δX) (2J AM ) 3.8 Hz, J AX ) 20.3 Hz, J MX ) 14.7 Hz, 3 H,
Si-CHXdCHAHM). 13C NMR (75.5 MHz, CDCl3): δ -2.4 (SiCH3),
-2.2 (SiCH3), 14.40 (OCH2CH3), 14.43 (OCH2CH3), 16.8
(CHCH3), 19.1 (CHCH3), 22.2 (SiCH2CH), 31.9 [CHCH(CH3)2],
53.1 (SiCH2CH), 60.5 (OCH2CH3), 60.71 (OCH2CH3), 60.76
[CHCH(CH3)2], 130.9 (SiCHdCH2), 139.9 (SiCHdCH2), 162.3
(CdN), 164.9 (CdN). 29Si NMR (59.6 MHz, CDCl3): δ -6.1.
3
3
MS: 360 [42%, M+], 345 [45%, M+ - Me], 331 [11%, M+
-
EI MS: 310 [1%, M+], 295 [5%, M+ - Me], 283 [5%, M+
-
Et], 317 [38%, M+ - CHMe2], 283 [36%, M+ - Ph], 211 [88%,
CHdCH2], 267 [24%, M+ - CHMe2], 211 [49%, M+ - CH2-
SiMe2CHdCH2], 85 [100%, SiMe2CHdCH2+]. Anal. Calcd for
M+ - CH2SiMe2Ph], 135 [100%, SiMe2Ph+]. Anal. Calcd for
C
20H32N2O2Si: C, 66.62; H, 8.95; N, 7.77. Found: C, 66.6; H,
C
16H30N2O2Si: C, 61.89; H, 9.74; N, 9.02. Found: C, 61.7; H,
8.8; N, 7.9.
9.5; N, 9.2.
P r ep a r a tion of (2R,5R)-Dieth oxy-5-isop r op yl-2-{[d i-
methyl(phenyl)germyl]methyl}-2,5-dihydropyrazine [(2R,5R)-
21]. This compound was prepared analogously to the synthesis
of (2R,5R)-13/(2S,5R)-13 by addition of a 1.6 M solution of
n-butyllithium in n-hexane (5.11 mL, 8.18 mmol n-BuLi) to a
solution of (R)-10 (1.72 g, 8.10 mmol) in THF (40 mL), followed
by treatment with a solution of 18 (1.87 g, 8.16 mmol) in THF
(10 mL). The crude product was purified by Kugelrohr distil-
lation (oven temperature 200 °C, 0.01 mbar) to give a 82:18
mixture of (2R,5R)-21/(2S,5R)-21 (molar ratio 83:17) and (R)-
10. The diastereomerically pure compound (2R,5R)-21 was
isolated by liquid-chromatographic separation (for the general
procedure, see below) as a colorless liquid in 41% yield [relative
to (R)-10; 1.35 g, 3.33 mmol; g99% de]. IR (film, cm-1): ν 1691
(CdN). 1H NMR (600.1 MHz, CDCl3): δ 0.410 (s, 3 H, GeCH3),
0.414 (s, 3 H, GeCH3), 0.67 (δQ), 0.99 (δT), 1.28 (δA), 1.62 (δB),
P r ep a r a tion of (R)-2-Am in o-3-[d im eth yl(p h en yl)silyl]-
p r op ion ic Acid Eth yl Ester [(R)-23]. This compound was
prepared analogously to the synthesis of (R)-15 by treatment
of a solution of (2R,5R)-20 (1.13 g, 3.13 mmol) in ethanol (20
mL) with 3 M hydrochloric acid (10 mL). After liquid-
chromatographic separation on silica gel [see preparation of
(R)-15; Rf[(R)-23] > Rf[(R)-valine]], (R)-23 was isolated in 83%
yield as a colorless liquid (654 mg, 2.60 mmol). IR (film, cm-1):
1
ν 1733 (CdO). H NMR (300.1 MHz, CDCl3): δ 0.33 (s, 3 H,
SiCH3), 0.36 (s, 3 H, SiCH3), 1.08 (δA), 1.30 (δB), and 3.45 (δX)
(2J AB ) 14.7 Hz, 3J AX ) 9.1 Hz, 3J BX ) 5.9 Hz, 3 H, Si-CHAHB-
3
CHX), 1.19 (δX), 3.97 (δA), and 4.05 (δB) (2J AB ) 11.0 Hz, J AX
3
) 7.1 Hz, J BX ) 7.1 Hz, 5 H, O-CHAHB-CHX3), 1.45 (s, 2 H,
NH2), 7.35-7.43 and 7.53-7.62 (m, 5 H, SiC6H5). 13C NMR
(75.5 MHz, CDCl3): δ -2.6 (SiCH3), -2.1 (SiCH3), 14.0
(OCH2CH3), 22.5 (SiCH2CH), 51.9 (SiCH2CH), 60.6 (OCH2-
CH3), 127.7 (C-3/C-5, SiC6H5), 128.9 (C-4, SiC6H5), 133.5 (C-
2/C-6, SiC6H5), 138.7 (C-1, SiC6H5), 177.1 (CdO). 29Si NMR
(59.6 MHz, CDCl3): δ -3.7. EI MS: 236 [1%, M+ - Me], 135
[100%, SiMe2Ph+]. Anal. Calcd for C13H21NO2Si: C, 62.11; H,
8.42; N, 5.57. Found: C, 61.9; H, 8.6; N, 5.7.
3
2.21 (δN), 3.84 (δK), and 4.06 (δG) [2J AB ) 13.6 Hz, J AG ) 9.5
3
5
3
3
Hz, J BG ) 4.8 Hz, J GK ) 3.4 Hz, J KN ) 3.4 Hz, J NQ ) 6.8
3
Hz, J NT ) 6.9 Hz, 11 H, Ge-CHAHB-CHG-NdC-CHK-CHN-
(CHQ3)(CHT3)], 1.19 (δX), 3.83 (δA), and 4.01 (δB) (2J AB ) 10.7
3
3
Hz, J AX ) 7.1 Hz, J BX ) 7.1 Hz, 5 H, O-CHAHB-CHX3), 1.23
(δX), 3.96 (δA), and 4.13 (δB) (2J AB ) 10.7 Hz, J AX ) 7.1 Hz,
3
3J BX ) 7.1 Hz, 5 H, O-CHAHB-CHX3), 7.25-7.32 and 7.45-7.48
(m, 5 H, GeC6H5). 13C NMR (75.5 MHz, CDCl3): δ -2.5
(GeCH3), -2.3 (GeCH3), 14.36 (OCH2CH3), 14.39 (OCH2CH3),
P r ep a r a tion of (R)-2-Am in o-3-[d im eth yl(p h en yl)ger -
m yl]p r op ion ic Acid Eth yl Ester [(R)-24]. This compound
was prepared analogously to the synthesis of (R)-15 by
treatment of a solution of (2R,5R)-21 (608 mg, 1.50 mmol) in
ethanol (20 mL) with 3 M hydrochloric acid (10 mL). After
liquid-chromatographic separation on silica gel [see prepara-
(23) Altamura, M.; Giammaruco, M.; Taddei, M.; Ulivi, P. J . Org.
Chem. 1995, 60, 8403-8406.

3496 Organometallics, Vol. 19, No. 18, 2000
Tacke et al.
3
3
tion of (R)-15; Rf[(R)-24] > Rf[(R)-valine]], (R)-24 was isolated
in 74% yield as a colorless liquid (328 mg, 1.11 mmol). IR (film,
cm-1): ν 1735 cm-1 (CdO). 1H NMR (300.1 MHz, CDCl3): δ
0.45 (s, 3 H, GeCH3), 0.46 (s, 3 H, GeCH3), 1.20 (δX), 3.49 (δA),
4.20 (δX) (2J AB ) 14.5 Hz, J AX ) 5.8 Hz, J BX ) 10.2 Hz, 3 H,
Si-CHAHB-CHX), 4.35-4.41 (m, 3 H, CHCH2O), 7.27-7.73 (m,
8 H, aromat. H), OH and NH not localized. 13C NMR (CDCl3,
75.5 MHz): δ -1.0 (SiCH3), 20.2 (SiCH2CH), 46.9 (CHCH2O),
51.3 (SiCH2CH), 65.9 (CHCH2O), 120.3, 125.4, 125.5, 127.6,
127.9, 141.8, 144.9, and 143.8 (fluorenyl), 155.6 (CdO), 175.2
(CdO). 29Si NMR (CDCl3, 59.6 MHz): δ 1.8. CI MS (negative
ions): 765 [82%, (2 M - H)+], 382 [100%, (M - H)+], 180 [80%].
Anal. Calcd for C21H25NO4Si: C, 65.77; H, 6.57; N, 3.65.
Found: C, 65.5; H, 6.6; N, 3.6.
3
3
and 3.52 (δB) (2J AB ) 11.0 Hz, J AX ) 7.1 Hz, J BX ) 7.1 Hz, 5
H, O-CHAHB-CHX3), 1.23 (δA), 1.44 (δB), and 3.46 (δX) (2J AB
)
3
3
14.6 Hz, J AX ) 9.1 Hz, J BX ) 5.7 Hz, 3 H, Ge-CHAHB-CHX),
1.84 (s, 2 H, NH2), 7.31-7.38 and 7.42-7.50 (m, 5 H, GeC6H5).
13C NMR (75.5 MHz, CDCl3): δ -3.0 (GeCH3), -2.6 (GeCH3),
14.1 (OCH2CH3), 22.7 (GeCH2CH), 52.5 (GeCH2CH), 60.8
(OCH2CH3), 128.0 (C-3/C-5, GeC6H5), 128.4 (C-4, GeC6H5),
133.2 (C-2/C-6, GeC6H5), 144.1 (C-1, GeC6H5), 177.0 (CdO).
EI MS: 282 [17%, M+ - Me], 224 [18%, M+ - CO2Et], 181
[100%, GeMe2Ph+]. Anal. Calcd for C13H21GeNO2: C, 52.76;
H, 7.15; N, 4.73. Found: C, 52.7; H, 7.0; N, 4.6.
P r ep a r a t ion of (R)-N-[(F lu or en -9-yl)m et h oxyca r b -
on yl]-2-a m in o-3-(tr im eth ylger m yl)p r op ion ic Acid [(R)-
28]. This compound was prepared analogously to the synthesis
of (S)-26 starting from (R)-3 (1.16 g, 5.64 mmol). The product
(R)-28 was isolated in 47% yield as a colorless solid (1.13 g,
2.64 mmol). IR (KBr, cm-1): ν 1717 (CdO). H NMR (CDCl3,
1
P r ep a r a tion of (R)-2-Am in o-3-[d im eth yl(vin yl)silyl]-
p r op ion ic Acid Eth yl Ester [(R)-25]. This compound was
prepared analogously to the synthesis of (R)-15 by treatment
of a solution of (2R,5R)-22 (991 mg, 3.19 mmol) in ethanol (20
mL) with 3 M hydrochloric acid (10 mL). After liquid-
chromatographic separation on silica gel [see preparation of
(R)-15; Rf[(R)-25] > Rf[(R)-valine]], (R)-25 was isolated in 70%
yield as a colorless liquid (449 mg, 2.23 mmol). IR (film, cm-1):
300.1 MHz): δ 0.18 (s, 9 H, GeCH3), 1.26 (δA), 1.30 (δB), and
3
3
4.19 (δX) (2J AB ) 13.5 Hz, J AX ) 5.4 Hz, J BX ) 10.1 Hz, 3 H,
Ge-CHAHB-CHX), 4.33-4.43 (m, 3 H, CHCH2O), 7.25-7.74 (m,
8 H, aromat. H), OH and NH not localized. 13C NMR (CDCl3,
75.5 MHz): δ -1.6 (GeCH3), 21.1 (GeCH2CH), 47.1 (CHCH2O),
51.8 (GeCH2CH), 67.0 (CHCH2O), 120.0, 125.0, 125.1, 127.1,
127.7, 141.3, 143.7, and 143.8 (fluorenyl), 155.8 (CdO), 178.2
(CdO). CI MS (negative ions): 857 [94%, (2 M - H)+], 428
[100%, (M - H)+]. Anal. Calcd for C21H25GeNO4: C, 58.93; H,
5.89; N, 3.27. Found: C, 58.8; H, 5.8; N, 3.3.
1
ν 1732 (CdO). H NMR (300.1 MHz, CDCl3): δ 0.12 (s, 6 H,
SiCH3), 0.90 (δA), 1.11 (δB), and 3.47 (δX) (2J AB ) 14.7 Hz, 3J AX
3
) 9.1 Hz, J BX ) 5.9 Hz, 3 H, Si-CHAHB-CHX), 1.24 (δX), 4.10
3
3
(δA), and 4.13 (δB) (2J AB ) 10.8 Hz, J AX ) 7.1 Hz, J BX ) 7.2
Hz, 5 H, O-CHAHB-CHX3), 1.60 (s, 2 H, NH2), 5.68 (δA), 5.95
(δM), and 6.15 (δX) (2J AM ) 3.4 Hz, 3J AX ) 20.3 Hz, 3J MX ) 14.7
Hz, 3 H, Si-CHXdCHAHM). 13C NMR (75.5 MHz, CDCl3): δ
-2.8 (SiCH3), -2.7 (SiCH3), 14.2 (OCH2CH3), 22.3 (SiCH2CH),
51.9 (SiCH2CH), 60.8 (OCH2CH3), 132.0 (SiCHdCH2), 138.8
(SiCHdCH2), 177.0 (CdO). 29Si NMR (59.6 MHz, CDCl3): δ
-6.4. EI MS: 186 [5%, M+ - Me], 128 [83%, M+ - CO2Et], 85
[100%, SiMe2CHdCH2+]. Anal. Calcd for C9H19NO2Si: C,
53.69; H, 9.51; N, 6.96. Found: C, 53.0; H; 9.3; N, 7.1.
P r ep a r a tive Liqu id -Ch r om a togr a p h ic Sep a r a tion of
th e Mixtu r es (2R,5R)-13/(2S,5R)-13/(R)-10, (2R,5R)-14/
(2S,5R)-14/(R)-10, (2R,5R)-20/(2S,5R)-20/(R)-10, (2R,5R)-21/
(2S,5R)-21/(R)-10, a n d (2R,5R)-22/(2S,5R)-22/(R)-10. The
(2R,5R)- and (2S,5R)-isomers of 13, 14, 20, 21, and 22 were
separated by liquid chromatography (MPLC) on silica gel (30-
60 µm; Baker, 7024-01) starting from the respective mixtures
(2R,5R)-13/(2S,5R)-13/(R)-10, (2R,5R)-14/(2S,5R)-14/(R)-10,
(2R,5R)-20/(2S,5R)-20/(R)-10, (2R,5R)-21/(2S,5R)-21/(R)-10, and
(2R,5R)-22/(2S,5R)-22/(R)-10. The experimental conditions
were as follows: LC pump, Bu¨chi Pump 688; detector, dif-
ferential refractometer Knauer K-2300; column, 20 mm i.d. ×
250 mm; pressure, 5 bar; eluent, n-hexane/diethyl ether (60:
1, v/v); injection volume, 1.15 mL (150 mg of the sample
material dissolved in 1 mL of diethyl ether); flow rate, 8.0 mL/
min. The solvent of the respective fractions obtained [(2R,5R)-
isomers, first fraction; (2S,5R)-isomers, second fraction; (R)-
10, third fraction] was removed (rotary evaporator), and the
diastereomerically products (2R,5R)-13, (2S,5R)-13, (2R,5R)-
14, (2S,5R)-14, and (2R,5R)-20-(2R,5R)-22 [(2S,5R)-20-
(2S,5R)-22 not isolated] were purified by Kugelrohr distillation
[(2R,5R)-13 and (2S,5R)-13, oven temperature 150 °C, 0.01
mbar; (2R,5R)-14 and (2S,5R)-14, oven temperature 160 °C,
0.01 mbar; (2R,5R)-20, oven temperature 170 °C, 0.01 mbar;
(2R,5R)-21, oven temperature 200 °C, 0.01 mbar; (2R,5R)-22,
oven temperature 150 °C, 0.01 mbar].
P r epar ation of (S)-N-[(Flu or en -9-yl)m eth oxycar bon yl]-
2-a m in o-4,4-d im eth ylp en ta n oic Acid [(S)-26]. A solution
of (fluoren-9-yl)methyl chloroformate (3.93 g, 15.2 mmol) in
dioxane (40 mL) was added dropwise at 0 °C over 30 min to a
stirred solution of (S)-1 (2.00 g, 13.8 mmol) and Na2CO3 (2.93
g, 27.6 mmol) in water/dioxane (3:5, v/v) (80 mL). After stirring
at 10 °C for 1 h, the reaction mixture was allowed to warm to
room temperature within 12 h. The solvents were removed
under reduced pressure (0.1 mbar, 20 °C), and the residue was
acidified with 6 M hydrochloric acid (f pH 2). The aqueous
solution was extracted with ethyl acetate (3 × 5 mL), and the
combined organic extracts were dried over anhydrous Na2SO4.
After concentrating the solution under reduced pressure, the
resulting precipitate was isolated by filtration, washed with
petroleum ether (40-60 °C) (2 × 10 mL) and then dried in
vacuo (0.1 mbar, 20 °C, 2 h) to give (S)-26 in 80% yield as a
colorless solid (4.04 g, 11.0 mmol). IR (KBr, cm-1): ν 1719 (Cd
1
Sep a r a tion of th e (2R,5R)- a n d (2S,5R)-Dia ster eom er s
of th e 2,5-Dih yd r op yr a zin es 13, 14, a n d 20-22 by Ca p il-
la r y Ga s Ch r om a togr a p h y: Deter m in a tion of Dia ster -
eom er ic P u r ities. The (2R,5R)- and (2S,5R)-isomers of 13,
14, and 20-22 were separated by capillary gas chromatogra-
phy [gas chromatograph, Shimadzu GC-14A; SE-30 column
(0.32 i.d. × 10 m), Macherey-Nagel; carrier gas, nitrogen;
temperature program, 100 °C (15 min) to 280 °C (10 min) with
7 °C/min; injector temperature, 200 °C; split 1:45; detector,
FID; detector temperature, 320 °C]. The retention times of the
diastereomers of 13, 14, and 20-22 are listed in Table 4.
O). H NMR (CDCl3, 300.1 MHz): δ 0.89 (s, 9 H, CCH3), 1.19
3
(δA), 1.24 (δB), and 4.20 (δX) (2J AB ) 13.9 Hz, J AX ) 5.5 Hz,
3J BX ) 10.1 Hz, 3 H, C-CHAHB-CHX), 4.35-4.41 (m, 3 H,
CHCH2O), 7.27-7.74 (m, 8 H, aromat. H), OH and NH not
localized. 13C NMR (CDCl3, 75.5 MHz): δ 30.3 (CCH3), 31.5
(CCH3), 46.6 (CCH2CH), 47.9 (CHCH2O), 52.4 (CCH2CH), 67.0
(CHCH2O), 120.8, 125.7, 125.8, 127.8, 128.4, 142.0, 144.4, and
144.6 (fluorenyl), 156.7 (CdO), 178.4 (CdO). CI MS (negative
ions): 733 [100%, 2 M - H+], 366 [15%, M - H+]. Anal. Calcd
for C22H25NO4: C, 71.91; H, 6.86; N, 3.81. Found: C, 71.3; H,
6.7; N, 3.8.
Deter m in a tion of th e En a n tiom er ic P u r ities of th e
Am in o Acid Ester s (R)-15, (S)-15, (R)-16, (S)-16, a n d (R)-
23-(R)-25 by 1H NMR Sp ectr oscop y. The enantiomeric
purities of (R)-15, (S)-15, (R)-16, (S)-16, and (R)-23-(R)-25
were determined by 1H NMR experiments using the chiral
solvating agent (R)-2,2,2-trifluoro-1-(9-anthryl)ethanol [(R)-
TFAE; Aldrich]. The NMR spectra were recorded at 22 °C on
P r ep a r a t ion of (R)-N-[(F lu or en -9-yl)m et h oxyca r b -
on yl]-2-a m in o-3-(tr im eth ylsilyl)p r op ion ic Acid [(R)-27].
This compound was prepared analogously to the synthesis of
(S)-26 starting from (R)-2 (1.20 g, 7.44 mmol). The product
(R)-27 was isolated in 69% yield as a colorless solid (1.96 g,
1
5.11 mmol). IR (KBr, cm-1): ν 1720 (CdO). H NMR (CDCl3,
300.1 MHz): δ 0.01 (s, 9 H, SiCH3), 1.18 (δA), 1.24 (δB), and

Si- and Ge-Containing R-Amino Acids and Peptides
Organometallics, Vol. 19, No. 18, 2000 3497
a Bruker DRX-300 NMR spectrometer operating at 300.1 MHz.
The composition of the samples used for the H NMR experi-
ments was as follows: (R)-15, (S)-15, (R)-16, (S)-16, (R)-23-
(R)-25, 53 µmol; (R)-TFAE, 159 µmol; CDCl3, 1.0 mL.
oxidation occurs from luciferyl-CoA with favorable kinetics
(enhanced flash). Briefly, after removal of culture medium from
the microtiter plate, cells were lysed by adding 100 µL of lysis
buffer (25 mM tris-phosphate pH 7.8, 2 mM dithiothreitol, 2
mM 1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid (CDTA),
10% (v/v) glycerol, 1% (v/v) Triton X-100). After incubation for
15 min at room temperature, 10 µL of cell lysate was
transferred into a white microplate suitable for luminometric
detection (Dynatech). The enzymatic reaction was initiated by
adding 50 µL of assay buffer (20 mM Tricine pH 7.8, 1.07 mM
4MgCO3‚Mg(OH)2‚5H2O, 2.67 mM MgSO4, 0.1 mM EDTA, 33.3
mM dithiothreitol, 270 µM coenzyme A, 470 µM firefly lu-
ciferin, 530 µM rATPNa2). Luminescence with a signal half-
life of 5 min was quantified after 1 min for a total time of 1 s
by using the EG&G Berthold MicroLumat LB96P.
1
Recep tor Bin d in g Assa y for Deter m in a tion of An ta go-
n ist ic P ot en cy of t h e Deca p ep t id es 7-9.24 For receptor
binding studies, Cetrorelix was iodinated with 125I (Amersham;
specific activity 80.5 Bq/fmol) by using the Iodo-Gen reagent
(Pierce). The reaction mixture was purified by reversed-phase
high-performance liquid chromatography yielding mono-iodi-
nated Cetrorelix without unlabeled peptide. About 80% of [125I]-
Cetrorelix was capable of specific receptor association. The
receptor binding assay was performed with intact cells under
physiological conditions as follows. Subconfluent cultures of
stably transfected LTK- cells expressing the human GnRH
receptor were detached by incubation in NaCl/Pi (137 mM
NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 11.5 mM KH2PO4)/1
mM EDTA and collected by centrifugation. The cell pellet was
resuspended in binding buffer (Dulbecco’s modified Eagle’s
medium DMEM) without H2CO3, with 4.5 g/L glucose, 10 mM
Hepes pH 7.5, 0.5% (m/v) BSA, 1 g/L bacitracin, 0.1 g/L soy
bean trypsin inhibitor (SBTI), 0.1% (m/v) NaN3. For displace-
ment assays, 0.25 × 106 cells/100 µL were incubated with
approximately 225 pM [125I]Cetrorelix (specific activity (5-10)
× 105 dpm/pmol) and different concentrations of unlabeled
peptide as competitor. The cell suspension in 100 µL binding
medium was layered on top of 200 µL 84% (by vol) silicone oil
(Merck type 550)/16% (by vol) paraffin oil in 400 µL assay
tubes. After incubation for 1 h at 37 °C under slow continuous
agitation, the cells were separated from the incubation medium
by centrifugation for 2 min at 9000 rpm (rotor type HTA13.8;
Heraeus Sepatec). The tips of the tubes containing the cell
pellet were cut off. Cell pellet and supernatant were subse-
quently analyzed by γ-radiation counting. The amount of
unspecific binding was determined by including unlabeled
Cetrorelix at 1 µM final concentration and was typically e10%
of total binding. The analysis of binding data was accomplished
with the EBDA/Ligand analysis program (Biosoft V3.0).
F u n ction a l Assa y for Deter m in a tion of An ta gon istic
P oten cy of th e Deca p ep tid es 7-9. The assay was per-
formed as described in ref 25 with some modifications. Briefly,
10 000 cells/well expressing the human GnRH receptor and a
luciferase reporter gene were cultivated for 24 h in microtiter
plates using DMEM with supplements and 1% (v/v) FCSi. Cells
were subsequently stimulated for 6 h with 1 nM D-Trp6-GnRH.
Antagonistic GnRH analogues were added prior to the stimu-
lation, and cells were finally lysed for quantification of cellular
Luc (Photinus pyralis luciferase) activity. Calculation of the
IC50 values from dose-response curves was done by nonlinear
regression analysis using the Hill model (program EDX 2.0
by C. Grunwald, unpublished). Quantification of Luc activity
was carried out essentially as described (Promega Technical
Bulletins 101/161) by using the respective luciferase assay
system (Promega E4030). By inclusion of coenzyme A (CoA),
In Vivo Exp er im en ts for Deter m in a tion of An ta gon is-
tic P oten cy of th e Deca p ep tid es 7-9. Male Sprague-
Dawley rats weighing 250-290 g were caged under “specific
pathogen-free” conditions (SPF) and injected subcutaneously
into the right flank with single doses of 1.5 mg/kg of 7-9. The
treatment groups consisted of 5 rats each, and the injection
volume was 0.5 mL/kg. The test compounds were prepared in
5% mannitol just before used. The solution was then applied
to the animal according to the individual body weight. Blood
samples were taken under CO2 short narcosis from V. sublin-
gualis at 0, 1, 6, 24, 48, and 72 h after injection for the
measurement of testosterone. Analysis of testosterone was
performed by using an EIA kit provided by DRG Instruments
GmbH and runs on microtiter 96-well plates. Evaluation of
the microtiter plates was performed in a Mikrotrak EIA
System (Syva) and evaluated via a software based on Excel.
The specificity of the test is >98%. A significant suppression
is evident with testosterone values below 2 ng/mL since this
concentration represents the lower normal values that can be
seen sporadically in untreated controls.26
To evaluate the effects of the test compounds on luteinizing
hormone (LH) serum levels, male rats were castrated one week
before the start of treatment in order to ensure high LH levels
due to the positive feed back induced by ablation of the testes.
Treatment was then performed as described for testosterone,
but since the LH in castrated rats is very sensitive to
suppression, the dose applied was reduced to 0.05 mg/kg. Blood
samples were taken under CO2 short narcosis from V. sublin-
gualis at 0, 2, 4, 6, 24, 48, 72, and 96 h after injection for the
LH measurements. Determination of LH in rat serum was
performed in triplicate by using a specific rat-LH RIA provided
by the NIADDK program (Bethesda, MD).
Ack n ow led gm en t. Financial support of this work
by Degussa AG and the Fonds der Chemischen Indu-
strie is gratefully acknowledged. M.M. thanks the Fonds
der Chemischen Industrie for a postgraduate fellowship.
OM000169L
(24) Beckers, T.; Marheineke, K.; Reila¨nder, H.; Hilgard, P. Eur. J .
Biochem. 1995, 231, 535-543.
(25) Beckers, T.; Reila¨nder, H.; Hilgard, P. Anal. Biochem. 1997,
251, 17-23.
(26) Reissmann, T.; Klenner, T.; Deger, W.; Hilgard, P.; McGregor,
G. P.; Voigt, K.; Engel, J . Eur. J . Cancer 1996, 32A, 1574-1579.