Chemical and biomedical motifs of the reactions of hydroxymethylphosphines with amines, amino acids, and model peptides

Article abstract of DOI:10.1021/ja9827604

The reactions of tris(hydroxymethyl)phosphine (THP, 1), 1,2- bis(bis(hydroxymethyl)phosphino)benzene (HMPB, 2), and 1,2- bis(bis(hydroxymethyl)phosphino)ethane (HMPE, 3) with various amines including amino acids and model peptides have been explored. The

Full text of DOI:10.1021/ja9827604

1658
J. Am. Chem. Soc. 1999, 121, 1658-1664
Chemical and Biomedical Motifs of the Reactions of
Hydroxymethylphosphines with Amines, Amino Acids, and Model
Peptides
Douglas E. Berning,†,‡,∇ Kattesh V. Katti,*,†,§ Charles L. Barnes,‡ and Wynn A. Volkert†,|
Contribution from the Departments of Radiology, Chemistry, and Missouri UniVersity Research Reactor,
UniVersity of Missouri-Columbia, Columbia, Missouri 65211, and H. S. Truman Memorial V. A. Hospital,
Columbia, Missouri 65203
ReceiVed July 30, 1998
Abstract: The reactions of tris(hydroxymethyl)phosphine (THP, 1), 1,2-bis(bis(hydroxymethyl)phosphino)-
benzene (HMPB, 2), and 1,2-bis(bis(hydroxymethyl)phosphino)ethane (HMPE, 3) with various amines including
amino acids and model peptides have been explored. The reactions of these multifunctional phosphines with
excess amino acids unexpectedly produced monomeric products. The reaction of THP with excess glycine
produced THP(glycine)3 (4) in high yield. The reactions of HMPB with the secondary amines N-methylaniline
and diethylamine produced the compounds HMPB(N-methylaniline)4 (5) and HMPB(diethylamine)4 (6),
respectively. However, the reactions of HMPB and HMPE with excess glycine produced trans annular-bonded
bicyclic compounds HMPB(glycine)2 (7) and HMPE(glycine)2 (10). The reactions of HMPB with excess alanine
and glycylglycylglycine were also explored to determine the generality of the reactions and correspondingly
yielded the novel heterocyclic compounds HMPB(alanine)2 (8) and HMPB(gly-gly-gly)2 (9), respectively.
The products are oxidatively stable in air and under a wide pH range. All of the new compounds have been
characterized by a combination of analytical and spectroscopic techniques, and the molecular structures of
compounds 4, 5, 7, and 10 have been confirmed by single-crystal X-ray diffraction studies.
Introduction
tides, or other biomolecules (e.g., sugars) are currently being
used for the formulation of larger biomolecular vectors.1-7
In this context, the reaction of tris(hydroxymethyl)phosphine
(P(CH2OH)3 (THP)) (Scheme 1) with primary amines, first
reported by Daigle and co-workers, provides an excellent
example of linking amine-containing compounds (e.g., amino
acids, peptides, or other biomolecules) in a single but yet
effective methodology.8 Although the utility of the reaction
outlined in Scheme 1 has been studied for cross-linking and
immobilization of specific enzymes,9,10 the vast scope that this
reaction offers to potentially link various amino acids and
peptides for applications in biomedical sciences is still untapped.
The Mannich-type condensation of CH2OH groups (of THP)
with NH2 groups of amines (Scheme 1) may be used in the in-
troduction of phosphine centers on peptide backbones. Phos-
phine-containing peptides present applications in a number of
areas. Immobilization of phosphines on peptides would yield
interesting peptide (or protein)-metal conjugates.11 In fact,
tailoring metal-binding sites in peptides and proteins have been
Chemical molecules that link two or more biologically useful
amines, peptides, or proteins, under mild (and biologically
benign) reaction conditions, are vital in a number of biomo-
lecular structural motifs,1,2 which include formulation of
synthetic â turns and â sheets3 and construction of three, four,
or six helix bundles.4-6 A number of chemical approaches to
linking basic subunits of simple amines, amino acids, pep-
* Author for Correspondence.
† Department of Radiology, University of Missouri-Columbia.
‡ Department of Chemistry, Universiyt of Missouri-Columbia.
§ Missouri University Research Reactor, University of Missouri-
Columbia.
| H. S. Truman Memorial V.A. Hospital.
∇ Current address: National Renewable Energy Laboratory, Golden, CO
80401.
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Metal Complexes of Phosphurus, Arsenic, and Antimony Ligands; McAu-
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10.1021/ja9827604 CCC: $18.00 © 1999 American Chemical Society
Published on Web 01/28/1999

Reactions of Hydroxymethyl Phosphines
J. Am. Chem. Soc., Vol. 121, No. 8, 1999 1659
and gly-gly-gly were obtained from Sigma Chemical Co. Diethylamine
and N-methylaniline were obtained from Aldrich Chemical Co. All
commercial reagents were used as received. The synthesis of THP (1),20
HMPB (2),21 and HMPE (3)22 has been previously described. Waters
Sep-Pak Vac C18 columns (10 g, 35 mL) were obtained from Fisher
Scientific Co.
Nuclear magnetic resonance spectra were recorded on a Bruker ARX-
300 spectrometer. The 1H and 13C chemical shifts are reported relative
to an external standard of TMS, and 31P NMR chemical shifts are
reported to an external standard of 85% H3PO4.
Scheme 1. Reaction of THP (1) with Amines
shown to stabilize biologically active conformations, enhance
structural integrity, and thereby promote new enzymatic activi-
ties.12
Since phosphines display versatile coordination chemistry
with transition metals and radiometals, phosphine-containing
peptides (or peptide-avid biomolecules) have gained importan-
tance in the design and development of tumor-specific radio-
pharmaceuticals.13-17 Despite the significant utility offered by
phosphine-containing peptides (and proteins), synthetic strategies
of producing such bioconjugates are still in infancy. The elegant
work by Gilbertson and co-workers on the incorporation of aryl
and cyclohexyl phosphines on specific peptides has provided
impetus to this burgeoning field of chemical and biomedical
sciences.18
From the aforementioned discussions, it is clear that the model
reaction, outlined in Scheme 1, can be used in multiple appli-
cations in chemistry, biochemistry, and biomedical sciences.
Therefore, a detailed understanding of the fundamental organic
chemistry of the reactions of hydroxymethylphosphines with
amines and amino acids will further aid the applications of these
reactions in chemical and biomedical sciences. As part of our
studies involving the development of new bioconjugates,19 we
have undertaken a systematic investigation of the reactions of
(a) tris(hydroxymethyl)phosphine (THP, 1), (b) 1,2-bis[bis-
(hydroxymethyl)phosphino]benzene (HMPB, 2), and (c) 1,2-
bis[bis(hydroxymethyl)phosphino]ethane (HMPE, 3) with primary/
secondary amines, amino acids, and model peptides. We, herein,
report isolation and full characterization of phosphine-amine
(or amino acid) and peptide conjugates and also X-ray structures
of several of the amino acid-phosphine conjugates: (i) mono-
phosphine (THP)-glycine conjugate, (ii) bisphosphine (HMPB)-
N-methylaniline conjugate, (iii) bisphosphine (HMPB)-glycine
conjugate, and (iv) bisphosphine (HMPE)-glycine conjugate.
These are the first examples of structurally characterized hy-
droxymethyphosphine-amino acid linkages, and they provide,
for the first time, definite evidence for the Mannich-type of
addition of -CH2OH groups with -NH2 groups as discussed
in the following sections
High-pressure liquid chromatography (HPLC) analyses and separa-
tions were performed using a Waters 600 dual-pump system equipped
with a 486 tunable absorbance detector and a 746 data module. Standard
reverse phase HPLC separations were performed on a C18 column
(Whatman Partisil 10 ODS-3 (9.5 × 500 mm)) using a gradient mobile
phase with solvent A composed of 0.1% trifluoroacetic acid in 3:1
acetonitrile/water mixture and solvent B composed of 0.1% trifluoro-
acetic acid in water. The gradient used is as follows: 0-3 min 2% A,
98% B; 3-18 min linear gradient to 100% A; 18-20 min 100% A;
20-30 min linear gradient to 2% A, 98% B. The flow rate and
wavelength were set to 4 mL/min and 254 nm; respectively. Mass
spectral analyses were performed by the Washington University
Resource for Biomedical and Bio-organic Mass Spectrometry, St. Louis,
Missouri. Elemental analyses were performed by Oneida Research
Services, Inc. Whitesboro, New York. Melting points were determined
on Mel-Temp II apparatus and are uncorrected.
2-Di(carboxymethylaminomethyl)phosphanylmethylamino-
acetic Acid (4). Tris(hydroxymethyl)phosphine (0.933 g, 7.52 mmol)
in 10 mL of distilled water was added dropwise to glycine (0.282 g,
3.76 mmol) in water (10 mL) at 25 °C. The reaction was stirred
under dry nitrogen for 3 h. The product was filtered off and dried
in vacuo to give the analytically pure compound in 82% yield as a
white solid. Anal. Calcd for C9H18N3O6P‚H2O: C, 34.49; H, 6.44;
N,13.42. Found: C, 32.75; H, 6.34; N, 13.14. HRFAB calcd for
[M + H]+ 295.0933, found 296.1011. Mp 202-204 °C dec. 1H NMR
(D2O, NaOD): δ 2.70 (s, 6H, NCH2COOH), 3.04 (s, 6H, PCH2N).
13C NMR (D2O, NaOD): δ 44.55 (d, NCH2COOH, J ) 5.21 Hz), 53.50
(d, PCH2N, J ) 9.96 Hz), 179.14 (s). 31P NMR (D2O, NaOD): δ
-38.1 (s).
1,2-Di[di(methylanilinomethyl)phosphanyl]benzene (5). N-meth-
ylaniline (0.453 g, 4.23 mmol) was added dropwise to 1,2-bis[bis(hy-
droxymethyl)phosphino]benzene (0.222 g, 0.850 mmol) in ethanol (5
mL) at 25 °C. The reaction was stirred under dry nitrogen for 1 h. The
product was filtered off and dried in vacuo to give the analytically
pure compound in 90% yield as a white solid. Anal. Calcd for
C38H44N4P2: C, 73.76; H, 7.17; N, 9.05. Found: C, 73.60; H, 7.05; N,
9.02. HRFAB calcd for C38H44N4P2 [M + H]+ 618.3041, found
1
619.3095. Mp 88-90 °C. H NMR (CDCl3): δ 2.76 (s, 12H, NCH3),
Experimental Section
3.83 (m, 8H, PCH2N), 6.63-6.68 (m, 12H, MeNC6H5-o,p), 7.07-7.19
(m, 8H, MeNC6H5-m), 7.39-7.44 (m, 2H, PC6H4-m), 7.59-7.64 (m,
2H, PC6H4-o). 13C (CDCl3) NMR: δ 39.2 (virtual triplet, NCH3, J )
6.7 Hz), 53.8 (virtual triplet, PCH2N, J ) 9.1 Hz), 113.4 (t, J ) 1.2
Hz), 116.9 (s), 128.9 (s), 129.4 (s), 131.3 (t, J ) 3.8 Hz), 143.9 (t, J
) 5.3 Hz), 149.4 (s). 31P (CDCl3) NMR: δ -43.6 (s).
All reactions were carried out under purified nitrogen by standard
Schlenk techniques. Solvents were purified and dried by standard
methods and distilled under nitrogen prior to use. Glycine, alanine,
(12) (a) Iverson, B. L.; Iverson, S. A.; Roberts, V. A.; Getzoff, E. D.;
Tainer, J. A.; Benkovic, S. J.; Lerner, R. A. Science 1990, 249, 659-662.
(b) Regan, L. Annu. ReV. Biophys. Biomol. Struct. 1993, 22, 257-281. (c)-
Kellis, J. T., Jr.; Todd, R. J.; Arnold, F. H. Biotechnology 1991, 9, 994-
995.
(13) DeRosch, M. A.; Brodack, J. W.; Grummon, G. D.; Marmion, M.
E.; Nosco, D. L.; Deutsch, K. F.; Deutsch, E. J. Nucl. Med. 1992, 33, 850.
(14) Dilworth, J. R; Parrott, S. J. Chem. Soc. ReV. 1998, 27, 43-55.
(15) Berning D. E.; Katti K. V.; Volkert W. A. unpublished results.
(16) Berning D. E.; Katti K. V.; Volkert W. A.; Higginbotham, C. J.;
Ketring, A. R. Nucl. Med. Biol. 1998, 25, 577-583.
1,2-Di[di(ethylaminomethyl)phosphanyl]benzene (6). Diethyl-
amine (3.00 g, 41.0 mmol) was added dropwise to 1,2-bis[bis(hy-
droxymethyl)phosphino]benzene (2.56 g, 9.77 mmol) in ethanol (10
mL) at 25 °C. The reaction was stirred under dry nitrogen for 1 h. The
solvent and excess diethylamine were removed in vacuo to obtain a
viscous oil. The residue was suspended in water (10 mL) and extracted
with CH2Cl2 (3 × 10 mL). The combined organic fractions were
concentrated to approximately 10 mL and purified on a silica gel column
to give the analytically pure product in 62% yield. Anal. Calcd for
(17) For recent references on the design and development of tumor-
specific radiopharmaceuticals, see: (a) McAffe, J. G.; Neumann, R. D. Nucl.
Med. Biol. 1996, 23, 673-676. (b) Lister-James, J.; Moyer, B. R.; Dean,
R. T. Q. J. Nucl. Med. 1996, 40, 221-233. (c) Lui, S.; Edwards, D. S.;
Barrett, J. A.; Bioconjugate Chem. 1997, 8, 621-636. (d) Fischmann, A.
J.; Babich, J. W.; Strauss, D. J. Nucl. Med. 1993, 34, 2253-2263.
(18) (a) Gilbertson S. R.; Chen G.; McLoughlin, M. J. Am. Chem Soc.
1994, 116, 4481-4482. (b) Gilbertson S. R.; Wang X.; Hoge G. S.; Klug
C. A.; Schaefer J. Organometallics 1996, 15, 4678-4680.
(20) (a) Ellis, J. W.; Harrison, K. N.; Hoye, P. A. T.; Orpen, A. G.;
Pringle, P. G.; Smith, M. B. Inorg. Chem. 1992, 31, 3026-3033. (b) Hoye,
P. A. T.; Pringle, P. G.; Smith, M. B.; Worboys, K. J. Chem. Soc., Dalton
Trans. 1993, 269-274.
(21) Reddy, V. S.; Katti, K. V.; Barnes, C. L. J. Chem. Soc., Dalton
Trans. 1996, 1301-1304.
(22) Reddy, V. S.; Katti, K. V.; Barnes, C. L. Inorg. Chim. Acta 1995,
240, 367-370.
(19) Katti, K. V.; Hariprasad, G.; Smith, C. J.; Berning, D. E. Acc. Chem.
Res. 1999, 32, 9-17.

1660 J. Am. Chem. Soc., Vol. 121, No. 8, 1999
Berning et al.
Table 1. Crystal Data and Details of Data Collection for Compounds 4, 5, 7, 10
4
5
7
10
formula
space group
crystal system
fw
a, Å
b, Å
C9H21N3O6P)(Cl)3‚H2O
P1h
C38H44N4P2
P2/c, No. 13
monoclinic
618.73
21.199(4)
8.1123(7)
20.854(3)
90.0
(C14H19N2O4P2)(Cl)
C2/c
monoclinic
376.71
13.573(1)
13.279(1)
10.653(1)
90.0
108.816(1)
90.0
C10H18N2O4P2
C2/c
triclinic
422.63
monoclinic
292.21
14.6181(11)
8.3199(7)
11.8780(9)
90.0
115.8770(10)
90.0
295(2)
6.8627(8)
9.354(2)
14.486(4)
84.51(2)
77.44(2)
86.25(2)
295(2)
c, Å
R, deg
â, deg
γ, deg
T, K
105.510(10)
90.0
295(2)
295(2)
λ, X
Z
1.54056
2
1.54056
4
0.71073
4
0.71073
4
F(000)
443.89
902.5(3)
1.555
not measured
5.89
2.85
1320
3455.7(9)
1.189
not measured
1.37
2.43
784
1817.4
1.377
not measured
0.40
1.64
0.031, 0.055
616
1299.77(18)
1.493
not measured
0.33
2.12
V, Å3
F
F
calcd, g/cm3
obsd, g/cm3
µ, mm-1
gof
a
RF, Rw
0.039, 0.073
0.47, 0.073
0.042, 0.069
a RF ) ∑(||Fo| - |Fc||)/∑(|Fo|); Rw ) [∑(w(|Fo| - |Fc|)2)/∑ (w|Fo|)2]1/2
.
C26H52N4P2: C, 64.68; H, 10.86; N, 11.61. Found: C, 64.55; H, 10.78;
N, 11.72. HRFAB calcd for C26H52N4P2 [M + H]+ 482.3667, found
483.3745. 1H NMR (CDCl3): δ 0.92-1.01 (m, 24H, NCH2CH3), 2.49-
2.81 (m, 16H, NCH2CH3), 3.02-3.14 (m, 8H, PCH2N), 7.28-7.34 (m,
2H, PC6H4-m), 7.46-7.53 (m, 2H, PC6H4-o). 13C (CDCl3) NMR: δ
12.1 (s, NCH2CH3), 48.2 (s, NCH2CH3), 55.3 (s, PCH2N), 128.6 (s),
131.1 (s), 144.9 (t, J ) 7.1 Hz). 31P (CDCl3) NMR: δ -60.3 (s).
2-(13-Carboxymethyl-10,13-diaza-1,8-diphosphatricyclo[6.3.3.02.7]-
tetradeca-2,4,6-triene-10-yl)acetic Acid (7). 1,2-Bis[bis(hydroxy-
methyl)phosphino]benzene (0.5007 g, 1.910 mmol) in water (5 mL)
was added dropwise to glycine (0.7209 g, 9.603 mmol) also in water
(10 mL) at 25 °C. The reaction mixture was continuously stirred under
dry nitrogen for 1 h. The product was filtered off, washed with water,
and dried in vacuo to give the analytically pure compound in 85%
yield. Anal. Calcd for C14H18N2O4P2‚H2O: C, 46.92; H, 5.63; N, 7.82.
Found: C, 46.81; H, 5.48; N, 7.86. HRFAB calcd for C14H18N2O4P2
340.0742. Found [M + H]+ 341.0820. Mp 210-212 °C. 1H NMR (D2O,
NaOD): δ 2.82 (s, 4H, NCH2COOH), 2.84-2.94 (m, 4H, PCH2N),
3.21-3.27 (m, 4H, PCH2N), 7.20-7.24 (m, 2H, PC6H4-m), 7.53-7.81
(m, 2H, PC6H4-o). 13C NMR (D2O, NaOD): δ 50.2 (d, PCH2N, J )
15.1 Hz), 65.2 (s, NCH2COOH), 130.5 (s), 137.8 (d, J ) 52.8 Hz),
142.0 (s), 178.8 (s). 31P NMR (D2O, NaOD): δ -9.9 (s).
HRFAB calcd for C22H30N6O8P2 [M + Na]+ 568.1600, found 591.1495.
1H NMR (D2O, NaOD): δ 2.82 (m, 4H, PCH2N), 3.16 (m, 4H, PCH2N),
2.90-3.62 (m, 12H, NCH2CO), 7.16-7.17 (br m, 2H, PC6H4-m), 7.44-
7.52 (m, 2H, PC6H4-o). 13C (D2O, NaOD): δ 43.2 (br s), 44.6 (s), 50.9
(d, J ) 15.1 Hz), 63.9 (s), 130.7 (d, J ) 15.1 Hz), 138.4 (d, J ) 52.8
Hz), 142.0 (br s), 170.6 (s) 174.1 (s) 176.5 (s), 31P NMR (D2O,
NaOD): δ -8.6 (s).
2-(7-Carboxymethyl-3,7-diaza-1,5-diphosphabicyclo[3.3.2]dec-3-
yl)acetic Acid (10). 1,2-Bis[bis(hydroxymethyl)phosphino]ethane (0.2800
g, 1.308 mmol) in water (5 mL) was added dropwise to glycine (0.5019
g, 6.686 mmol) also in water (5 mL) at 25 °C. After stirring for 3 h,
the reaction mixture was concentrated and purified on a Water’s Sep-
Pak Vac column (10 g, 35 mL). The solvent was removed in vacuo
and dried overnight in vacuo to give the compound in 78% yield. Anal.
Calcd for C10H18N2O4P2‚H2O: C, 38.43; H, 12.27; N, 8.97. Found: C,
38.12; H, 12.18; N, 8.82. HRFAB calcd for 292.0820, [M + H]+ found
293.0820. Mp 185-190 °C dec. 1H NMR (D2O): δ 2.26 (m, 4H,
PCH2CH2P, 2JPC ) 6.00 Hz), 3.33-3.39 (m, 4H, PCH2N), 3.52 (s, 4H,
NCH2COOH), 3.78-3.87 (m, 4H, PCH2N). 13C NMR (D2O): δ 24.2-
24.4 (m), 54.5 (d, J ) 30.2 Hz), 62.7 (s), 173.2 (s). 31P NMR (D2O):
δ -36.3 (s).
X-ray Data Collection and Process. The crystal data and details
of data collection for complexes 4, 5, 7, and 10 are given in Table 1.
Clear, colorless crystals of 4 and 7 were obtained from methanol/
hydrochloric acid at -20 °C. Clear, colorless crystals of 5 were obtained
from ethyl acetate at -20 °C. Clear, colorless crystals of 10 were
obtained from water/methanol at 25 °C. Intensity data for compounds
4 and 5 were collected on an Enraf-Nonius CAD-4 diffractometer with
Cu KR radiation and a graphite monochromator at 25 °C. Intensity
data for compounds 7 and 10 were collected on a Siemens SMART
CDD system using the omega scan mode. Data were collected for
absorption using the program SADABS, which is based on the method
of Blessing.23 Crystal decay were negligible, and corrections were
deemed unnecessary. The structures were solved by the Patterson
method using SHELXS-8624 and refined by the full-matrix least-squares
method on F2 using SHELXL-93.25
2-[13-(1-Carboxyethyl)-10,13-diaza-1,8-diphosphatricyclo[6.3.3.02,7]-
tetradeca-2,4,6-trien-10-yl)propanoic Acid (8). 1,2-Bis[bis(hydroxy-
methyl)phosphino]benzene (0.8603 g, 3.282 mmol) in water (15 mL)
was added dropwise to alanine (1.498 g, 16.81 mmol) also in water
(15 mL) at 25 °C. The reaction was continuously stirred under dry
nitrogen at room temperature for 1 h. The product was filtered off,
washed with water, and dried in vacuo to give the analytically pure
product in 78% yield. Anal. Calcd for C16H22N2O4P2‚H2O: C, 49.73;
H, 6.26; N, 7.25. Found: C, 50.02; H, 6.77; N, 7.55. HRFAB calcd
for C16H22N2O4P2 [M + H]+ 368.1055, found 369.1133. Mp 208-210
°C. 1H NMR (D2O, NaOD): δ 0.83 (d, 6H, NCHCH3COOH, J ) 6.91
Hz), 2.73-2.82 (m, 4H, NCHCH3COOH, PCH2N), 2.91-3.07 (m, 2H,
PCH2N), 3.14-3.21 (m, 4H, PCH2N), 7.12-7.15 (m, 2H, PC6H4-m),
7.44-7.52 (m, 2H, PC6H4-o). 13C NMR (D2O, NaOD): δ 15.1 (s,
NCHCH3COOH), 45.8 (br s), 48.8 (br s), 67.4 (s), 130.3 (d, J ) 18.1
Hz), 137.9 (d, J ) 51.3 Hz), 142.3 (s), 182.0 (s). 31P NMR (D2O,
NaOD): δ -8.3 (s).
2-(13-Carboxymethylcarbamoylmethylcarbamoylmethyl-10,13-
diaza-1,8-diphosphatricyclo[6.3.3.02,7]tetradeca-2,4,6-trien-10-yl-
methylcarboxamidomethylcarboxamido)acetic Acid (9). 1,2-Bis[bis-
(hydroxymethyl)phosphino]benzene (0.4370 g, 1.667 mmol) in water
(5 mL) was added dropwise to gly-gly-gly (1.578 g, 8.340 mmol) also
in water (100 mL) at 25 °C. After stirring for 3 h, the reaction mixture
was concentrated and chromatographed by semipreparatory HPLC to
give the analytically pure compound as a viscous oil in 52% yield.
Results and Discussion
Conjugation of Glycine with Tris(hydroxymethyl)phos-
phine (1). The addition of THP (1) to 5-6-fold excess glycine
in water (Scheme 2) produces a white precipitate (compound
4) which has been found to be soluble in acidic or alkaline
(23) Blessing R. H. Acta Crystallogr. 1995, A51, 33-38.
(24) Sheldrick, G. M. Acta Crystallogr. 1990, A46, 467-473.
(25) Sheldrick, G. M. SHELXL-93; University of Gottingen: Germany,
1993.

Reactions of Hydroxymethyl Phosphines
J. Am. Chem. Soc., Vol. 121, No. 8, 1999 1661
Scheme 3. Synthesis of C6H4[P(CH2NMePh)2]2 (5) and
C6H4[P(CH2NCH2CH3)2]2 (6)
Additionally, the high oxidative stability and aqueous solubility
of 4 will present new opportunities in the development of water-
soluble transition metal/organometallic compounds with poten-
tial applications in biphasic catalysis.
Figure 1. ORTEP drawing of 4 showing 50% probability ellipsoids.
Scheme 2. Synthesis of P(CH2NHCH2COOH)3 (4)
Conjugation of Secondary Amines with 1,2-Bis[bis(hy-
droxymethyl)phosphino]benzene (2). The addition of HMPB
(2) to the secondary amines, N-methylaniline and diethylamine,
in ethanol produces compounds 5 and 6, respectively (Scheme
3). The high-resolution fast atom bombardment mass spectra
of compounds 5 and 6 show molecular ions [M + H]+ with
m/z ) 619.3095 and 483.3745, respectively. The 31P NMR
spectrum of compound 5 shows an upfield resonance at -43.6
ppm (∆δ ) -12.4), while the 31P NMR spectrum of compound
6 shows an upfield resonance at -60.3 ppm (∆δ ) -29.1).
The 1H NMR and 13C NMR spectra for compounds 5 and 6 are
consistent with the proposed structures.
Table 2. Selected Bond Distances (Å) and Angles (deg) for 4
P-C1
P-C7
1.863(2)
1.866(2)
P-C4
1.870(2)
C1-P-C4
C4-P-C7
102.07(11)
95.52
C1-P-C7
96.54(11)
Crystals of 5 suitable for X-ray diffraction analysis were
obtained from an ethyl acetate solution at -20 °C. The crystal
data and details of data collection are listed in Table 1. The
ORTEP diagram of compound 5 is shown in Figure 2. The
selected bond distances and bond angles of complex 5 are listed
in Table 3. Atomic coordinates and their equivalent isotropic
displacement coefficients for compound 5 are included in the
Supporting Information. The asymmetric unit of the complex
[((C6H5)CH3NCH2)2PC6H4P(CH2NCH3(C6H5))2] (5) contains
two crystallographically independent molecules. As revealed by
the structure, all four of the hydroxymethyl groups of HMPB
(2) reacted with N-methylaniline to give the fully substituted
product. The bond distances for Pa-C1a, Pa-C4a, and Pa-
C12a are 1.851(2), 1.877(3), and 1.874(2) Å, respectively, with
an average bond distance of 1.867 Å. The bond distances for
Pb-C1b, Pb-C4b, and Pb-C12b are 1.840(2), 1.866(3), and
1.879(3) Å, respectively, with an average of 1.862 Å. The bond
angles C1a-Pa-C4a, C1a-Pa-C12a, and C4a-Pa-C12a are
100.5(1), 97.5(1), and 99.5(1)°, respectively. The bond angles
C1b-Pb-C4b, C1b-Pb-C12b, and C4b-Pb-C12b are 101.7-
(1), 97.1(1), and 99.9(1)°, respectively.
solutions, presumably as a result of the protonation of the amines
or the deprotonation of the carboxylic acid, respectively. The
high-resolution fast atom bombardment mass spectrum of
compound 4 shows a molecular ion [M + H]+ with a m/z )
296.1011. The 31P NMR spectrum of compound 4 shows a
single resonance upfield from THP (1) at -38.1 ppm (∆δ )
1
-14.7). Both the H and 13C NMR spectra are consistent with
the proposed structure.
Crystals of 4 suitable for X-ray diffraction analysis were
obtained from a methanol/hydrochloric acid solution at -20 °C.
The crystal data and details of data collection are listed in Table
1. The ORTEP diagram of compound 4 is shown in Figure 1.
The selected bond distances and bond angles of complex 4 are
listed in Table 2. Atomic coordinates and their equivalent
isotropic displacement coefficients for 4 are included in the
Supporting Information. The asymmetric unit for complex 4
consists of the cation [P(CH2NH2COOH)3]3+, three noncoor-
dinating chloride counterions, and one water molecule. As
revealed by the structure, all three of the hydroxymethyl groups
of THP (1) reacted with glycine to give the fully substituted
product. Growing the crystals in methanol/hydrochloric acid
resulted in the protonation of the amines to give a cationic
product. The P-C1, P-C4, and P-C7 bond distances are 1.863-
(2), 1.870(2), and 1.866(2) Å, respectively with an average of
1.866. The C1-P-C4, C1-P-C7, and C4-P-C7 bond angles
are 102.1(1), 96.5(1), and 95.5(1)°, respectively.
Conjugation of Glycine, Alanine, and Glyglyglycine with
1,2-Bis[bis(hydroxymethyl)phosphino]benzene (2) or 1,2-Bis-
[bis(hydroxymethyl)phosphino]ethane (3). The addition of
HMPB (2) to the amino acids glycine and alanine in water
produces compounds 7 and 8, respectively (Scheme 4). The
high-resolution fast atom bombardment mass spectra of com-
pounds 7 and 8 show molecular ions [M + H]+ with a m/z )
341.0820 and 369.1133, respectively. The 31P NMR spectrum
of compound 7 shows a downfield resonance at -9.9 ppm (∆δ
) 21.3), while the 31P NMR spectrum of compound 8 shows a
Compound 4 represents a novel example of a tertiary
phosphine functionalized with an amino acid moiety. The
presence of three amino acids on 4 may aid in the development
of large biomolecular vectors via the reaction with acid groups.

1662 J. Am. Chem. Soc., Vol. 121, No. 8, 1999
Berning et al.
Figure 3. ORTEP drawing of compound 7 showing 50% probability
ellipsoids.
Scheme 4. Synthesis of HMPB(Glycine)2 (7),
HMPB(Alanine)2 (8), and HMPB(gly-gly-gly)2 (9)
Figure 2. ORTEP drawings of the two crystallographically independent
molecules 5A and 5B showing 50% probability ellipsoids.
Table 3. Selected Bond Distances (Å) and Angles (deg) for 5
Pa-C1a
Pa-C4a
Pa-C12a
1.851(2)
1.877(3)
1.874(2)
Pb-C1b
Pb-C4b
Pb-C12b
1.840(2)
1.866(3)
1.879(3)
Table 4. Selected Bond Distances (Å) and Angles (deg) for 7
C1a-Pa-C4a
C4a-Pa-C12a
C1b-Pb-C12b
100.52(11)
99.49(11)
97.11(11)
C1a-Pa-C12a
C1b-Pb-C4b
C4b-Pb-C12b
97.49(10)
101.67(11)
99.94(12)
P1-C1
P1-C5
1.822(2)
1.852(2)
P1-C4
1.857(2)
1
C1-P1-C4
C4-P1-C5
C1-P1-C5
P1-C5-Na
106.99(9)
102.32(9)
103.63(10)
117.60
C4-N-C5a
C4-N-C6
C5a-N-C6
P1-C4-N
113.6(2)
113.65(14)
110.7(2)
downfield resonance at -8.6 ppm (∆δ ) 22.6). The H NMR
and 13C NMR spectra of 7 and 8 are consistent with the proposed
structures.
118.24(13)
Crystals of 7 suitable for X-ray diffraction analysis were
obtained from a methanol/hydrochloric acid solution at -20 °C.
The crystal data and details of data collection are listed in Table
1. The ORTEP diagram of compound 7 is shown in Figure 3.
The selected bond distances and bond angles of complex 7 are
listed in Table 4. Atomic coordinates and their equivalent
isotropic displacement coefficients for compound 7 are included
in the Supporting Information. The asymmetric unit for complex
7 consists of the cation [C14H19N2O4P2]+ and a noncoordinating
chloride counterion. As revealed by the structure, all four of
the hydroxymethyl groups of HMPB (2) reacted with 2 equiv
1
a Atom at 1 - x, y, /2 - z.
of glycine to give a tricyclic structure composed of a phenyl
ring attached to two, heterocyclic seven-membered rings as
shown above. An intriguing feature of the structure is that even
though the crystals were obtained from relatively concentrated
hydrochloric acid, the nitrogens are only hemiprotonated. The
hydrogen atom has been refined to be equidistant between the
two nitrogens. The bond distances P1-C1, P1-C4, and P1-
C5 are 1.822(2), 1.857(2), and 1.852(2) Å, respectively with
an average bond distance of 1.844 Å. The bond angles C1-

Reactions of Hydroxymethyl Phosphines
J. Am. Chem. Soc., Vol. 121, No. 8, 1999 1663
As revealed by the structure, all four of the hydroxymethyl
groups of HMPE (3) react with 2 equiv of glycine to give the
bicyclic compound. The bond distances P1-C1, P1-C2, and
P1-C3 are 1.846(2), 1.853(1), and 1.858(2) Å, respectively,
with an average bond distance of 1.852 Å. The bond angles
C1-P1-C2, C1-P1-C3, and C2-P1-C3 are 103.9(1), 104.5-
(1), and 102.6(1)°, respectively. The bond angles C2-N1-C3a
and C2-N1-C4 are 113.8(1), and 113.3(1)°, respectively. The
bond angle C3a-N1-C4 is 111.5(1)°. The bond angles P1-
C2-N1 and P1-C3-N1a are 116.4(1) and 118.6(1)°, respec-
tively.
Tris(hydroxymethyl)phosphine (1) has been shown to react
with ammonia, primary and secondary amines by the Mannich
reaction, as shown in Scheme 1.8,9,26,27 The reaction of THP
with ammonia and primary amines generally leads to polym-
erization which was of interest in the 1960s as a method of
producing flame-retardant textiles.26 In addition, the reaction
of THP with secondary amines to give monomeric products has
also been explored.8 Recently, there has been a renewed interest
in the use of THP for the immobilization of enzymes.9 However,
the reaction of THP (1) with amino acids, peptides, and
antibodies has not been explored. Thus, the ability of the
hydroxymethyl groups to react with amines and leave the
phosphines free for further coordination to transition metals may
provide a novel method of producing site-directed radiopharm-
aceuticals.13-17,19 Earlier work in our laboratory of conjugation
of THP to antibodies and subsequently labeling with Tc-99m
suggested that the idea was feasible.28 Due to the difficulty of
identifying the THP-antibody products, the reaction of THP
with simple peptides and amino acids, as described above, was
explored. The addition of THP to excess glycine leads primarily
to the formation of a monomer in which all three of the
hydroxymethyl groups react with a glycine molecule. Compound
4 is highly stable to oxidation in acidic and basic solution even
though the phosphine is nucleophilic (as suggested by the 31P
NMR chemical shift). This represents the first example of an
X-ray crystallographically characterized THP-amine conjugate.
When 5 or 6 equiv of peptides or amino acids were reacted
with HMPB or HMPE, only 2 equiv of the amines were found
to conjugate with the hydroxymethylphosphines. The degree of
substitution for compounds 7-10 has been determined by
elemental analyses, high-resolution mass spectroscopy, and
Figure 4. ORTEP drawing of compound 10 showing 50% probability
ellipsoids.
Scheme 5. Synthesis of HMPE(Gly)2 (10)
Table 5. Selected Bond Distances (Å) and Angles (deg) for 10
P1-C1
P1-C3
1.846(2)
1.858(2)
P1-C2
1.8531(14)
C1-P1-C2
C2-P1-C3
C1-P1-C3
P1-C2-N1
103.86(8)
102.56(7)
104.54(8)
116.36(9)
C2-N1-C3a
C2-N1-C4
C3a-N1-C4
P1-C3-N1a
113.75(11)
113.31(11)
111.47(12)
118.62(10)
a Atom at 1 - x, y, 1.5 - z.
P1-C4, C1-P1-C5, and C4-P1-C5 are 107.0(1), 103.6(1),
and 102.3(1)°, respectively. The bond angles C4-N-C5a, C4-
N-C6, and C5a-N-C6 are 113.6(2), 113.6(1), and 110.7(2)°,
respectively. The bond angles P1-C4-N and P1-C5-Na are
118.2(1) and 117.6(1)°, respectively.
The addition of HMPB (2) to excess glycylglyclyglycine in
water followed by removal of the solvent produces a viscous
oil as outlined in Scheme 4. The product (compound 9) was
purified by semipreparatory HPLC. The high-resolution fast
atom bombardment mass spectrum shows a molecular ion [M
+ Na]+ with a m/z ) 591.1495. The 31P NMR spectrum of 9
shows a single resonance downfield from HMPB at -8.6 ppm
1
integration of the protons in the H NMR spectra. For each
compound there are two possible isomers that fit the given data
as shown in Scheme 6. Although unlikely due to ring strain,
one possible isomer is where two hydroxymethyl groups from
each phosphine reacts with an amino acid or peptide to form
two four-membered rings (Scheme 6). The other possibility is
where two hydroxymethyl groups from adjacent phosphines
reacts with an amino acid to form two heterocyclic seven-
membered rings. The final molecular structure of the compounds
has been confirmed by X-ray diffraction analysis and proves to
be the latter of the two possible isomers described above. Similar
to compound 4, compounds 7-10 are highly stable to oxidation
in both acidic and alkaline media.
The reactions of HMPB (and HMPE) with primary amines,
as depicted in Schemes 4-6, provide important information on
the kinetic propensity of the reactivity of -P(CH2OH)2 groups
with primary amines. It must be recognized that HMPB (2) (and
HMPE (3)) possesses four -CH2OH groups (two on each
phosphine center) and they could conceivably produce cross-
1
(∆δ ) 22.6). In addition, the H NMR and 13C NMR spectra
for 9 are consistent with the proposed structure.
The addition of HMPE (3) to excess glycine produces com-
pound 10 in near quantitative yields as outlined in Scheme 5.
The high-resolution fast atom bombardment mass spectrum of
10 shows a molecular ion [M + H]+ with a m/z ) 293.0820.
The 31P NMR spectrum of compound 10 shows an upfield
1
resonance at -36.3 ppm (∆δ ) -11.2). Furthermore, the H
and 13C NMR spectra of 10 are consistent with the proposed
structure.
Crystals of 10 suitable for X-ray diffraction analysis were
obtained from a water/methanol mixture at 25 °C. The details
of data collection are listed in Table 1. The ORTEP diagram of
compound 10 is shown in Figure 4. The selected bond distances
and bond angles of complex 10 are listed in Table 5. Atomic
coordinates and their isotropic displacement coefficients for 10
are included in the Supporting Information. The asymmetric
unit for complex 10 consists only of the complex [C10H18N2O4P2].
(26) Daigle, D. J.; Frank, A. W. Tex. Res. J. 1982, 52, 751-755.
(27) Frank, A. W.; Drake, G. L., Jr. J. Org. Chem. 1972, 37, 2752-
2755.
(28) Berning, D. E.; Katti, K. V.; Volkert, W. A., unpublished results.

1664 J. Am. Chem. Soc., Vol. 121, No. 8, 1999
Berning et al.
Scheme 6. Possible Isomers of the Reaction of HMPB (2)
or HMPE (3) with Amino Acids or Peptides
amines can be conjugated to other materials that contain one
or more P(CH2OH)n moieties. It must be recognized that
although gluteraldehyde and other aldehydes are being routinely
used for conjugation of bioactive amines, the products from such
reactions lead to unstable imine (CHdNR) linkages and often
require further reduction, under harsh conditions, to produce
the more stable amines. In contrast, the reactions of hydroxy-
methylphosphines with amines result in stable amine (H2C-
NHR) linkages in a single reaction step in biologically benign
media, (i.e., water). Reactions of amino functionalities, present
on the backbone of biomolecules, with hydroxymethylphos-
phines functionalities may aid in altering the biochemical prop-
erties for potential use in protein/peptide structure modifications.
The conjugation of amines, amino acids, and peptides with hy-
droxymethylphosphines, as described in Schemes 1-6, and the
kinetic propensity of these reactions to produce well-defined
singular chemical/biochemical species are, therefore, unique.
Acknowledgment. This work was supported by funds pro-
vided by the Department of Energy (Grant No. DEFGO289-
ER60875), DuPont-Merck Pharmaceuticals, and the Depart-
ments of Chemistry, Radiology, and Research Reactor at the
University of Missouri-Columbia. Partial funding of the X-ray
diffractometer by the National Science Foundation, Grant No.
CHE[90-11804], is gratefully acknowledged.
linked polymers upon interactions with the N terminus of amino
acids and peptides. However, the observation that these reactions
strictly result in the formation of well-defined heterocyclic rings
suggests a strong kinetic propensity to monomeric product
formation. These are important results in the context of using
the water-soluble HMPB (2) (and HMPE (3)) phosphines as
synthons to modify the structures and properties of peptides
(and proteins) wherein specific primary amines would interact
to produce PCH2N linkages without cross-linking the biomol-
ecules.
Supporting Information Available: Tables of crystal data,
structure solution and refinement, atomic coordinates, bond
lengths and angles, and anisotropic thermal parameters for 4,
5, 7, and 10 (PDF). This material is available free of charge
via the Internet at http://pubs.acs.org.
Conclusions and Comments
The generality of reactions, outlined in Schemes 1-6, demon-
strate that virtually any molecule with primary or secondary
JA9827604