Methods for the synthesis of phosphonate esters

Phsophonate esters can be synthesized in high yields by condensations of alcohols with methyl phosphonates followed by selective demethylation. The reaction is general, relativly insensitive to steric constraints of hindered phosphonic acids, and can also be carried out on a solid support to synthesize large collections of compounds to screen for pharmacological activity.

BACKGROUND OF THE INVENTION 
This invention relates generally to methods for the synthesis of 
phosphonate esters. More particularly, the invention provides methods for 
producing monoesters and diesters of phosphonic acids from condensations 
of alcohols with alkylphosphonates, such as methyl 
.alpha.-aminoalkyl-phosphonates. The methods can be carried out in 
solution phase, but in an important embodiment, the method is carried out 
on a solid support, such as beads or a glass slide, and used to create 
large arrays of diverse compounds, such as peptidylphosphonates and 
polyphosphonates, which can then be conveniently screened for the presence 
of compounds with desired pharmacological or other properties. The 
invention therefore relates to the fields of chemistry, enzymology, 
pharmacology, and medicinal chemistry. 
Aminophosphonic acids and aminophosphonates are derivatives of amino acids 
in which the amino acid carboxyl group has been replaced with a phosphonic 
acid or phosphonate moiety. In .alpha.-aminophosphonic acids and 
phosphonates, the .alpha.-carbon is often a chiral center, bearing a 
phosphonate moiety, an amine moiety, and one or more amino acid side 
chains. The structure can be represented as follows: 
##STR1## 
wherein R represents any amino acid side chain and R' and R" represent 
hydrogen (in the case of a phosphonic acid) or a group such as alkyl or 
aryl (in the case of a phosphonate ester). 
.alpha.-Aminophosphonates are used in the synthesis of peptide analogues 
(peptidylphosphonates) possessing a phosphonate linkage in the place of at 
least one amide link in the peptide main chain. The phosphonate linkage 
can impart various useful properties to peptides. Because the phosphonate 
linkage in a peptidylphosphonate exists as a charged moiety in the peptide 
backbone, it increases the water solubility of the peptide. Further, the 
phosphonate linkage can impart protease resistance and thereby increase 
the serum half-life of many therapeutic peptide. Still further, 
substitution of the phosphonate linkage for the amide linkage allows 
additional functionalities to be introduced in otherwise inaccessible 
regions of naturally occurring peptides. This is because the phosphonate 
linkages are tetrahedral, while amide linkages are planar. The tetrahedral 
geometry allows substituents to be placed above and below the plane of the 
amide linkage of a peptide. Moreover, the tetrahedral configuration of the 
phosphonate linkage can be exploited to optimize ligand-receptor binding. 
See Bartlett and Marlowe (1983) Biochemistry 22:4618-24. 
Literature examples of specific uses of peptidylphosphonates are numerous. 
For example, these compounds are recognized as effective transition-state 
analogue inhibitors for a variety of enzymes, including a number of other 
proteases and esterases (see, e.g., Morgan et al. (1991) J. Am. Chem. Soc. 
113:297 and Bartlett et al. (1990) J. Org. Chem. 55:6268). 
Peptidylphosphonate esters have been used as nonhydrolyzable analogues of 
phosphates to inhibit dinucleoside triphosphate hydrolase (see, e.g., 
Blackburn et al. (1987) Nucl. Acids Res. 15:6991), phosphatidyltransferase 
(see, e.g., Vargas (1984) Biochim. Biophys. Acta 796:123), and squalene 
synthetase (see, e.g., Biller et d. (1988) J. Med. Chem. 31:1869). In 
fact, the most potent non-covalent enzyme inhibitor known is a 
phosphonyltripeptide inhibitor of carboxypeptidase A, which binds with 11 
femptomolar (fM) K.sub.D. See Kaplan et al. (1991) Biochem. 30:8165-8170. 
In addition, phosphonate esters have been used as haptens for the 
production of catalytic antibodies possessing esterase activity ( see, 
e.g., Jacobs et al. (1987) J. Am. Chem. Soc. 109:2174; Tramontano et al. 
(1986) Science 234:1566; and Pollack et al. (1986) Science 234:1570; see 
also, U.S. patent application Ser. No. 858,298, filed Mar. 26, 1992). Some 
peptidylphosphonates analogues are commercially available therapeutics. 
For instance, the drugs Monopril and Fosinopril are available from Bristol 
Myers, Squibb (Evansville, Ind.). 
.alpha.-Aminophosphonates are a member of a broader class of compounds, 
phosphonic acid monoesters. These monoesters have typically been 
synthesized by one of two methods. In the first, a monomethyl 
alkylphosphoryl chloride is treated with an alcohol. Selective 
deesterification of the methyl ester yields the desired product. The 
monomethyl alkylphosphoryl chloride is produced by the direct action of 
PCl.sub.5 on a phosphonate diester (see Balthazor et al. (1980) J. Org. 
Chem. 45:530) or by base hydrolysis of the phosphonate diester followed by 
reaction with thionyl chloride (see Bartlett et al. (1990) J. Org. Chem. 
55:6268). 
Alternatively, the direct monoesterification of a phosphonic acid can be 
accomplished with the appropriate alcohol and condensing reagents (see 
Gilmore et al. (1974,) J. Pharm. Sci. 63:965), such as 
1,3-dicyclohexylcarbodiimide (DCC) or trichloroacetonitrile (see, e.g., 
Wasielewski et al. (1976) J. Rocz. Chem. 50:1613). A large excess of both 
the condensing agent and alcohol is required, and yields vary depending on 
the components being coupled. In addition, forcing conditions are 
required, such as heating to reflux temperature in a solution of THF with 
triethylamine and DCC, and to 50.degree. C.-80.degree. C. in pyridine with 
trichloroacetonitrile, conditions that can lead to decomposition of 
starting material. In addition, product yields are extremely sensitive to 
steric encumbrance from the reacting components, and in some cases, no 
product formation is observed. 
The Mitsunobu reaction is a mild and effective method utilizing the redox 
chemistry of triphenylphosphine and a dialkylazodicarboxylate to condense 
an acidic reagent with primary and secondary alcohols (see Mitsunobu, 
(1981) Synthesis 1-28). Mitsunobu does not describe the reaction of 
phosphate esters with secondary or tertiary alcohols or the reaction of 
phosphonates with alcohols. The Mitsunobu reaction has been used with 
carboxylic acids, phenols, and phosphates as the acidic component, but 
only one example using a phosphonic acid has been described. 
In this example (see Norbeck et al. (1987) J. Org. Chem. 52:2174), a 
monomethyl phosphonate and a primary alcohol were reacted under modified 
Mitsunobu conditions (triphenylphosphine and diisopropylazodicarboxylate) 
to produce a phosphonate diester. Demethylation using triethylamine and 
thiophenol produced the phosphonate monoester. Yields, however, were low 
for the condensation of a phosphate monoester with even a primary alcohol. 
Only moderate yields were obtained with elevated temperatures or with 
hexamethylphosphorous triamide (HMPT) as the solvent. 
Recently, innovative combinatorial strategies for synthesizing large 
numbers of polymeric compounds on solid supports have been developed. One 
such method, referred to as VLSIPS.TM. ("Very Large Scale Immobilized 
Polymer Synthesis"), is described in U.S. patent application Ser. No. 
07/805,727, filed Dec. 6, 1991, which is a continuation-in-part of Ser. 
No. 07/624,120, filed Dec. 6, 1990, which is a continuation-in-part of 
U.S. Pat. No. 5,143,854, which is a continuation-in-part of Ser. No. 
07/362,901, filed Jun. 7, 1989, and now abandoned. Such techniques are 
also described in PCT publication No. 92/10092. Related combinatorial 
techniques for synthesizing polymers on solid supports are discussed in 
U.S. patent application Ser. No. 07/946,239 filed Sep. 16, 1992which is a 
continuation-in-part of Ser. No. 07/762,522 filed Sep. 18, 1991 and U.S. 
patent application Ser. No. 07/980,523 filed Nov. 20, 1992 which is a 
continuation-in-part of Ser. No. 07/796,243 filed Nov. 22, 1991. Briefly, 
a combinatorial synthesis strategy is an ordered strategy for parallel 
synthesis of diverse polymer sequences by sequential addition of reagents 
to a solid support. 
To synthesize phosphonates, and particularly peptidylphosphonates, on a 
solid support, one needs a general method for introducing the phosphonic 
acid monoester functionality. However, the phosphonate synthesis 
procedures described above generally have not proved easily modified for 
solid phase applications, which presents a significant problem for the 
medicinal chemist. Thus, there remains a need for chemical synthesis 
methods for use in the VLSIPS.TM. method to generate and screen large 
numbers of peptidylphosphonates and other compounds containing phosphonate 
ester building blocks. This method should be compatible with a variety of 
functional groups and consistently produces high yields of the desired 
compounds and that can be applied to solution as well as solid phase 
chemistry. The present invention meets these needs. 
SUMMARY OF THE INVENTION 
The present invention provides methods for producing phosphonate esters. In 
one embodiment, the present invention relates to a method for synthesizing 
a phosphonic acid monoester from a phosphonate with a structure: 
##STR2## 
wherein R.sup.1 is selected from the group consisting of alkyl, aryl, 
substituted alkyl, substituted aryl, heteroaryl, alkylaryl, and 
aminoalkyl, and R.sup.2 is selected from the group consisting of methyl, 
ethyl, benzyl, and substituted benzyl, 
by treating said phosphonate with a dialkylazodicarboxylate, a 
triarylphosphine, and a primary or secondary alcohol with a structure 
R.sup.3 OH, wherein R.sup.3 is selected from the group consisting of 
alkyl, aryl, substituted alkyl, substituted aryl, and aminoalkyl and 
wherein said alcohol is present in excess, said excess being greater than 
a ratio of 1.1 equivalents of alcohol to one equivalent of phosphonate, in 
an aprotic solvent to yield a phosphonate diester with a structure: 
##STR3## 
wherein R.sup.2 and R.sup.3 are not identical, and R.sup.2 can be 
selectively hydrolyzed without hydrolyzing R.sup.3, and 
treating said phosphonate diester to hydrolyze selectively said phosphonate 
diester to yield said phosphonic acid monoester with a structure: 
##STR4## 
In a preferred embodiment, R.sup.2 is methyl, said dialkylazodicarboxylate 
is diisopropylazodicarboxylate (DIAD), said triarylphosphine is 
triphenylphosphine (TPP), said solvent is tetrahydrofuran (THF), and 
trimethylsilyl bromide (TMSBr) is added directly to the reaction mixture 
after completion of esterification to effect selective hydrolysis of the 
methyl ester to yield the desired phosphonic acid monoester. 
In another preferred embodiment, the present invention relates to a method 
for synthesizing a phosphonic acid monoester from a phosphonate with a 
structure: 
##STR5## 
wherein R.sup.1 is selected from the group consisting of alkyl, aryl, 
substituted alkyl, substituted aryl, heteroaryl, alkylaryl, and 
aminoalkyl, and R.sup.2 is selected from the group consisting of methyl, 
ethyl, benzyl, and substituted benzyl, by: 
treating said phosphonate with a dialkylazodicarboxylate, and a 
triarylphosphine, wherein at least one of the aryl groups of the 
triarylphosphine bears an electron withdrawing substituent; 
treating this mixture with a primary or secondary alcohol with a structure 
R.sup.3 OH, wherein R.sup.3 is selected from the group consisting of 
alkyl, aryl, substituted alkyl, substituted aryl, and aminoalkyl and 
wherein said phosphonate is present in excess, said excess being greater 
than a ratio of 1.1 equivalents of phosphonate to one equivalent of 
alcohol, and an exogenous base, in an aprotic solvent to yield a 
phosphonate diester with a structure: 
##STR6## 
wherein R.sup.2 and R.sup.3 are not identical, and R.sup.2 can be 
selectively hydrolyzed without hydrolyzing R.sup.3, and then 
treating said phosphonate diester to hydrolyze selectively said phosphonate 
diester to yield said phosphonic acid monoester with a structure: 
##STR7## 
In a preferred embodiment, R.sup.2 is methyl, said dialkylazodicarboxylate 
is diisopropylazodicarboxylate (DIAD), said triarylphosphine is 
tris(4-chlorotriphenyl)phosphine (CTPP), said base is 
diisopropylethylamine (DIEA), said solvent is tetrahydrofuran (THF), and 
trimethylsilyl bromide (TMSBr) is added directly to the reaction mixture 
after completion of esterification to effect selective hydrolysis of the 
methyl ester to yield the desired phosphonic acid monoester. 
In yet another preferred embodiment, the phosphonate is an 
.alpha.-aminoalkylphosphonate, because the resulting compound is an amino 
acid analogue particularly useful in the production of compounds that can 
be screened for pharmacological activity or used in the generation of 
catalytic antibodies. In addition, this preferred embodiment of the 
reaction can be carried out on a solid support to which is covalently 
linked the hydroxyl group of the alcohol moiety. The solid support can be 
a glass slide or other suitable substrate for light directed and other 
combinatorial synthesis strategies described below. In this embodiment, 
the invention also provides novel hydroxyacid and phosphonate ester 
building blocks protected with photoremovable protecting groups and 
methods for their production. The solid support can also be beads or 
particles, which are preferred for use in other combinatorial synthesis 
strategies. 
Thus, in an especially preferred embodiment of this aspect of the 
invention, the solid support comprises an amine group of an amino acid or 
peptide, and the support is treated so as to couple a hydroxy acid to said 
amine group to yield a hydroxyl moiety covalently linked to said support. 
The support is then treated with: 
an .alpha.-aminoalkylphosphonate with the following structure: 
##STR8## 
wherein R.sup.4 is an .alpha.-aminoalkyl group, and R.sup.2 is selected 
from the group consisting of methyl, ethyl, benzyl, and substituted 
benzyl; 
a dialkylazodicarboxylate; 
an exogenous base, such as DIEA; and 
a triarylphosphine, such as CTPP or TPP, 
to couple said .alpha.-aminoalkylphosphonate to said hydroxyl groups on 
said surface. In a preferred embodiment, a triarylphosphine wherein at 
least one of the aryl groups of the triarylphosphine bears an electron 
withdrawing substituent. This process can be continued by selective 
hydrolysis of the R.sup.2 group (e.g., by treatment with TMSBr or 
triethylammonium phenoxide) and/or by further couplings to the amine group 
of said .alpha.-aminoalkylphosphonate. 
The invention further provides a method of forming an array of phosphonate 
esters or phosphonic acids. These defined arrays or libraries of 
oligonucleotide will find a variety of uses, for example, to screen the 
substrate-bound compounds for biological activity. 
In another embodiment, the invention provides a method for producing 
symmetric phosphonate diesters. In this embodiment, a phosphonic acid with 
a structure: 
##STR9## 
wherein R.sup.1 is selected from the group consisting of alkyl, aryl, 
substituted alkyl, substituted aryl, heteroaryl, alkylaryl, and 
aminoalkyl, is treated with a dialkylazodicarboxylate, a triarylphosphine, 
and a primary or secondary alcohol with a structure R.sup.3 OH, wherein 
R.sup.3 is selected from the group consisting of alkyl, aryl, substituted 
alkyl, substituted aryl, and aminoalkyl, dissolved in solvent to yield a 
phosphonate diester with a structure: 
##STR10## 
In a preferred embodiment, said dialkylazodicarboxylate is DIAD, said 
triarylphosphine is TPP, and said solvent is THF. In a preferred 
embodiment for use with secondary alcohols, the alcohol is either present 
in excess, or the alcohol, dialkylazodicarboxylate, and triphenylphosphine 
are allowed to react to form an alkoxyphosphorane before the addition of 
the phosphonic acid to the reaction mixture. 
A further understanding of the nature and advantages of the inventions 
herein may be realized by reference to the remaining portions of the 
specification and the attached drawings.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS 
I. Terminology 
Unless otherwise stated, the following terms used in the specification and 
claims have the meanings given below: 
"Alkyl" refers to a cyclic, branched, or straight chain chemical group 
containing only carbon and hydrogen, such as methyl, heptyl, 
--(CH.sub.2).sub.2 --, and adamantyl. Alkyl groups can either be 
unsubstituted or substituted with one or more substituents, e.g., halogen, 
alkoxy, acyloxy, amino, hydroxyl, mercapto, carboxy, benzyloxy, phenyl, 
benzyl, or other functionality which may be suitably blocked, if necessary 
for purposes of the invention, with a protecting group. Typically, alkyl 
groups will comprise 1 to 12 carbon atoms, preferably 1 to 10, and more 
preferably 1 to 8 carbon atoms. 
An ".alpha.-amino acid" consists of a carbon atom, called the 
.alpha.-carbon, to which is bonded an amino group and a carboxyl group. 
Typically, this .alpha.-carbon atom is also bonded to a hydrogen atom and 
a distinctive group referred to as a "side chain." The hydrogen atom may 
also be replaced with a group such as alkyl, substituted alkyl, aryl, 
substituted aryl, heteroaryl, and other groups. The side chain of the 
amino acid is designated as group R.sup.1 in FIG. 3. The side chains of 
naturally occurring amino acids are well known in the art and include, for 
example, hydrogen (as in glycine), alkyl (as in alanine (methyl), valine 
(isopropyl), leucine (sec-butyl), isoleucine (iso-butyl), and proline 
(--(CH.sub.2).sub.3 --)), substituted alkyl (as in serine (hydroxymethyl), 
cysteine (thiomethyl), aspartic acid (carboxymethyl), asparagine, 
arginine, glutamine, glutamic acid, and lysine), arylalkyl (as in 
phenylalanine, histidine, and tryptophan), substituted arylalkyl (as in 
tyrosine and thyroxine), and heteroaryl (as in histidine). See, e.g., 
Harper et al. (1977) Review of Physiological Chemistry, 16th Ed., Lange 
Medical Publications, pp. 21-24. 
In addition to naturally occurring side chains, the amino acids used in the 
present invention may possess synthetic side chains. A "synthetic side 
chain" is any side chain not found in a naturally occurring amino acid. 
For example, a synthetic side chain can be an isostere of the side chain 
of a naturally occurring amino acid. Naturally occurring and synthetic 
side chains may contain reactive functionalities, such as hydroxyl, 
mercapto, and carboxy groups. One skilled in the art will appreciate that 
these groups may have to be protected to carry out the desired reaction 
scheme. As stated above, the hydrogen at the .alpha.-carbon can also be 
replaced with other groups; those of skill in the art recognize the 
medicinal importance of .alpha.-methyl amino acids and other .alpha.-, 
.alpha.-disubstituted amino acids. 
"Aminoalkyl" refer to the group --C(NR'R")R.sup.1 R.sup.2,where R.sup.1, 
R.sup.2, R' and R" are independently selected from the group consisting of 
hydrogen, alkyl, and aryl. Preferably, the amino alkyl group will be 
derived from an alpha-amino acid HOOC--C(NR'R")R.sup.1 R.sup.2 where 
R.sup.1 and R.sup.2 are the side chains of the amino acid. 
"Aryl" or "Ar" refers to a monovalent unsaturated aromatic carbocyclic 
group having a single-ring (e.g., phenyl) or multiple condensed rings 
(e.g., naphthyl or anthryl), which can optionally be unsubstituted or 
substituted with amino, hydroxyl, lower alkyl, alkoxy, chloro, halo, 
mercapto, and other substituents. Preferred aryl groups include phenyl, 
1-naphthyl, and 2-naphthyl. 
"Arylalkyl" refers to the groups R-Ar and R-HetAr, where Ar is an aryl 
group, HetAr is a heteroaryl group, and R is straight-chain or 
branched-chain aliphatic group. Examples of arylalkyl groups include 
benzyl and furfuryl. 
"Electron withdrawing group" refers to a substituent that draws electrons 
to itself more than a hydrogen atom would if it occupied the same position 
in a molecule. Examples of electron withdrawing groups include --NR.sub.3 
+, --COOH, --OR, --SR.sub.2.sup.+, --F, --COR, --Cl, --SH, --NO.sub.2, 
--Br, --SR, --SO.sub.2 R, --I, --OH, --CN, --C.tbd.CR, --COOR, --Ar, 
--CH.dbd.CR.sub.2, where R is alkyl, aryl, arylalkyl, or heteroaryl. 
"Exogenous base" refers to nonnucleophilic bases such as alkali metal 
acetates, alkali metal carbonates, alkaline metal carbonates, alkali metal 
bicarbonates, tri(lower alkyl) amines, and the like, for example, sodium 
acetate, potassium bicarbonate, calcium carbonate, diisopropylethylamine, 
triethylamine, and the like. 
"Heteroaryl" or "HetAr" refers to a monovalent unsaturated aromatic 
carbocyclic group having a single ring (e.g., pyridyl or furyl) or 
multiple condensed rings (e.g., indolizinyl or benzothienyl) and having at 
least one hetero atom, such as N, O, or S, within the ring, which can 
optionally be unsubstituted or substituted with amino, hydroxyl, alkyl, 
alkoxy, halo, mercapto, and other substituents. 
"Limiting reagent" refers to that substance which limits the maximum amount 
of product formed in a chemical reaction, no matter how much of the other 
reactants remains. 
"Lower alkyl" refers to an alkyl group of one to six carbon atoms. Lower 
alkyl groups include those exemplified by methyl, ethyl, n-propyl, 
i-propyl, n-butyl, t-butyl, i-butyl (2-methylpropyl), cyclopropylmethyl, 
i-amyl, n-amyl, and hexyl. Preferred lower alkyls are methyl and ethyl. If 
more than one alkyl group is present in a given molecule, then each may be 
independently selected from "lower alkyl" unless otherwise stated. 
"Phosphonate ester," "alkylphosphonate ester," "alkylphosphonic acid 
ester," or "phosphonic acid ester" refers to the group (RPO)(OR')(OR") 
where R, R' and R" are independently selected from the group consisting of 
hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, and 
heteroaryl, provided both R' and R" are not hydrogen, in which case the 
group is a phosphonic acid. "Phosphonate monoester," "alkylphosphonate 
monoester," "alkylphosphonic acid monoester", or "phosphonic acid 
monoester" refers to a phosphonate ester where R' is hydrogen and R" is 
alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, or 
heteroaryl. With respect to nomenclature, note that compound 1 in FIG. 1, 
if R is N-benzyloxycarbonylaminomethyl, is named methyl 
N-(benzyloxycarbonyl)aminomethylphosphonate. 
"Phosphonic acid" or "alkylphosphonic acid" refers to the group 
(RPO)(OH).sub.2. 
"Protecting group" refers to a chemical group that exhibits the following 
characteristics: (1) reacts selectively with the desired functionality in 
good yield to give a protected substrate that is stable to the projected 
reactions for which protection is desired; 2) is selectively removable 
from the protected substrate to yield the desired functionality; and 3) is 
removable in good yield by reagents compatible with the other functional 
group(s) generated in such projected reactions. Examples of protecting 
groups can be found in Greene et aI. (1991) Protective Groups in Organic 
Synthesis, 2nd Ed. (John Wiley & Sons, Inc., New York). Preferred terminal 
amino protecting groups include benzyloxycarbonyl (CBz), 
9-fluorenylmethyloxycarbonyl (Fmoc), or suitable photolabile protecting 
groups such as 6-nitroveratryloxy carbonyl (Nvoc), nitropiperonyl, 
pyrenylmethoxycarbonyl, nitrobenzyl, dimethyl dimethoxybenzyl, 
5-bromo-7-nitroindolinyl, and the like. Preferred hydroxyl protecting 
groups include Fmoc, photolabile protecting groups (such as nitroveratryl 
oxymethyl ether (Nvom)), Mom (methoxy methyl ether), and Mem (methoxy 
ethoxy methyl ether). Particularly preferred protecting groups include 
NPEOC (4-nitrophenethyloxycarbonyl) and NPEOM 
(4-nitrophenethyloxymethyloxy). 
"Stereospecific reaction" refers to a reaction in which bonds are broken 
and made at a single asymmetric atom (usually, but not necessarily 
carbon), and which lead largely to a single stereoisomer. If the 
configuration of the asymmetric carbon is altered in the process, the 
reaction is said to involve an inversion of configuration. If the 
configuration of the asymmetric carbon remains the same, the 
transformation occurs with retention of configuration. Stereospecific 
reaction sequences can be characterized as having a high percent 
asymmetric synthesis or percent enantiomeric excess where these terms 
serve as a measure of the extent to which one enantiomer is produced in 
excess over the other. (For example, a reaction sequence that produces 59% 
of one isomer and 41% of another isomer would have a percent asymmetric 
synthesis or percent enantiomeric excess of 59-41%=18%). Typically, the 
reaction sequences described herein will exhibit at least about a 50% 
percent enantiomeric excess; preferably, at least about a 60% percent 
enantiomeric excess; more preferably, at least about a 70% percent 
enantiomeric excess; and even more preferably, at least about an 80 % 
percent enantiomeric excess, where a perfectly stereospecific reaction 
sequence would have a 100% percent enantiomeric excess. See, e.g., 
Morrison and Mosher, "Asymmetric Organic Reactions", 2nd Ed., American 
Chemical Society, Washington, D.C. (1976), which is incorporated herein by 
reference. 
"Weak acid" refers to a substance having a pK.sub.a between about 3 and 8, 
and preferably between about 3 and 5. Preferred weak acids include acetic 
acid, citric acid, ascorbic acid, lactic acid, and the like. 
II. Synthesis of Monoalkyl Phosphonates 
A. The Starting Materials 
1. The Phosphonate Monoester 
Monomethyl phosphonates (compound 1, FIG. 1) are preferred for purposes of 
the present invention and can be readily obtained by the general 
procedures schematically outlined in FIG. 2. The R group of such compounds 
can be alkyl, aryl, heteroaryl, or arylalkyl, each of which may be 
optionally substituted. Some phosphonic acids (compound 4, FIG. 2) are 
commercially available, and for base sensitive compounds, a phosphonic 
acid can be mono-esterified directly to produce the monomethyl 
phosphonates. This esterification can be accomplished by treating a 
phosphonic acid with methanol and a small excess of thionyl chloride, as 
described by Hoffman (1986) Synthesis, at page 557 et seq. In addition, 
saponification of dimethyl phosphonates (compound 5, FIG. 2) can be used 
to produce the mono-methyl phosphonates in high yields. Dimethyl 
phosphonates can be produced either by the bis-esterification of 
phosphonic acids or obtained directly by the methodology of Arbuzov (see 
Arbuzov (1964) Pure Appl. Chem. 9:307) or Seebach (see Seebach et al. 
(1989) Helv. Chim. Acta 72:401). 
Again with reference to FIG. 1, the methyl group of compound 1 may 
optionally be replaced with an ethyl, benzyl, or substituted benzyl group, 
or any group that can be selectively hydrolyzed from compound 2 (with the 
methyl group replaced by the group that substitutes for the methyl group) 
to yield compound 3. 
2. The Alcohol 
A wide variety of alcohols may be used to produce the phosphonate 
monoesters by the methods of the present invention. The alcohol may be 
primary or secondary. The R' group in the alcohol and in compounds 2 and 3 
(FIG. 1 ) originates from the alcohol and may be alkyl, substituted alkyl, 
aryl, substituted aryl, arylalkyl, or heteroaryl, each of which may be 
optionally substituted. 
With sterically undemanding alcohols, such as ethanol and isopropanol, the 
reaction occurs instantly, regardless of the substitutents on the 
phosphonate. With more sterically hindered alcohols, such as methyl 
mandelate, the reaction may require up to two hours to go to completion. 
B. The Phosphonate as the Limiting Reagent 
1. Overview 
The present invention provides methods for producing phosphonate esters. In 
one embodiment, the present invention relates to a method for synthesizing 
a phosphonic acid monoester from a monoalkyl or monoaryl phosphonate, 
wherein the phosphonate is the limiting reagent, by treatment with a 
dialkylazodicarboxylate, preferably diisopropylazodicarboxylate (DIAD), a 
triarylphosphine, preferably triphenylphosphine (TPP), and an alcohol 
dissolved in a solvent, such as tetrahydrofuran (THF). In a preferred 
embodiment, approximately equimolar amounts of the alcohol, 
triarylphosphine, and dialkylazodicarboxylate are used. Typically, the 
ratio of phosphonate to alcohol will range from about 1:1.1 to about 1:5, 
preferably from about 1:1.1 to about 1:3, and more preferably about 1:1.5. 
The coupling reaction proceeds stereospecifically with inversion of the 
stereochemistry at the carbon bonded to the hydroxyl group of the alcohol 
reactant. Accordingly, if the alcohol reactant has an R configuration at 
the carbon bonded to the hydroxyl group, the resulting phosphonate has an 
S configuration at the corresponding carbon. 
When the coupling reaction is complete, the selective cleavage of the ester 
is carried out; preferably, trimethylsilyl bromide (TMSBr) is added to the 
reaction mixture to effect selective hydrolysis of the methyl ester and 
yield the desired compound (compound 3, FIG. 1). Other methods for 
cleaving the phosphonic acid ester can also be used; for instance, the 
protocols for cleaving esters of carboxylic acids described in Greene, 
supra, at page 156 et seq., can be used for this purpose, provided the 
desired selectivity of hydrolysis can be achieved. 
Although FIG. 1 exemplifies the reaction as an intermolecular reaction, one 
of skill in the art will appreciate that the reaction may also be 
performed intramolecularly (i.e., the alcohol and the monomethyl 
phosphonate are parts of the same molecule). This embodiment allows for 
the synthesis of cyclic and sterically restricted phosphonates. 
2. The General Procedure 
The general procedure for the reaction wherein the phosphonate is the 
limiting reagent is as follows. To a solution of a phosphonate (compound 
1, FIG. 1, 0.5 mmol), alcohol (0.75 mmol), and a triarylphosphine (0.75 
mmol) in anhydrous THF (5 mL, refluxed over potassium and distilled prior 
to use), is added a dialkylazodicarboxylate, preferably 
diisopropylazodicarboxylate (0.75 mmol). Upon completion of the 
condensation reaction (generally, 0.5-5 hours), TMSBr (1.5 mmol) is added. 
The reaction is stirred for about an additional hour. The reaction mixture 
is then diluted with ether (10 mL) and extracted with 5% NaHCO.sub.3 
(twice, 10 mL each extraction). The aqueous phase is washed with ether 
(three times, 10 mL each wash), acidified to pH=2 with concentrated HCl, 
and then extracted with ethyl acetate (EtOAc, three times, 10 mL each 
extraction). The EtOAC)Ac phase is dried over MgSO.sub.4, filtered, and 
concentrated to yield the desired compound. See Example 1 below. 
3. The Mechanism 
For a more complete understanding of the invention, one may find helpful 
the theoretical mechanism of the reaction. This theoretical reaction 
mechanism is illustrated in FIG. 3 (see also, for a discussion of the 
reaction mechanism for carboxylic acids, Hughes et al., (1988) J. Am. 
Chem. Soc. 21:6487; Grochowski et aI., (1982) J. Am. Chem. Soc. 104:6876; 
Crich et at., (1989) J. Org. Chem. 54:257; and Varasi et al., (1987) J. 
Org. Chem. 52:4235). 
In Path A, the betaine (compound 10) formed between triphenylphosphine and 
DIAD is protonated by an acidic component with pKa&lt;11 (see Kim and 
Rapoport (1990) J. Org. Chem. 55:3699), yielding the protonated betaine 
(compound 11, FIG. 3), which in turn reacts with an alcohol to form the 
alkoxyphosphonium salt (compound 13, FIG. 3). Subsequent S.sub.N 2 attack 
by the conjugate base of compound 13 yields the esterified product with 
inversion of configuration at the carbinol carbon. 
Path B only occurs when the acidic component is not present to protonate 
the betaine (compound 10, FIG. 3). Two equivalents of alcohol react with 
compound 10 to form a dialkoxyphosphorane (compound 12, FIG. 3; see Camp 
and Jenkins (1989) J. Org. Chem. 54:3045; Von Itzstein and Jenkins (1983) 
Aust. J. Chem. 36:557; and Guthrie and Jenkins (1982) Aust. J. Chem. 
35:767). Upon addition of the acidic component, compound 12 is converted 
to the phosphonium salt (compound 13, FIG. 3), followed by product 
formation as in Path A. Some investigators have suggested that compound 12 
(FIG. 3) is in equilibrium with an (acyloxy)alkoxyphosphorane, but whether 
such a compound plays a significant role in product formation is unknown 
(see Camp and Jenkins (1989) J. Org. Chem. 54:3049). 
Pyrophosphonate esters presumably arise from condensation of one molecule 
of an alkylphosphonic acid with an electrophilic phosphorus species formed 
via the protonated betaine (compound 11, FIG. 3), followed by further 
esterification with excess alcohol. The electrophilic species derived from 
benzylphosphonic acid was trapped with t-butanol, yielding t-butyl 
benzylphosphonate. Because Mitsunobu condensations are not normally 
observed with tertiary alcohols (see Camp and Jenkins, supra), and no 
reaction with t-butanol was observed with benzylpyrophosphonate, or when 
methyl benzylphosphonate (this compound, as well as methyl 
t-butylphosphonate were obtained by treatment of the respective 
alkylphosphonic acids (Aldrich Chemical Co.) with diazomethane, followed 
by saponification) was substituted for benzylphosphonic acid, any t-butyl 
ester that is formed must result from nucleophilic attack by the alcohol 
on the electrophilic phosphorus species. The availability of this 
additional pathway with phosphonic acids may result in a different 
stereochemical configuration of the alcohol component than the normally 
observed inverted carbinol carbon. 
To circumvent formation of the electrophilic phosphorus species, one can 
access Path B (FIG. 3), using reaction conditions similar to those 
described by Loibner and Zbiral (1976) Helv. Chim. Acta. 59:2100. More 
specifically, the alcohol, triarylphosphine, and DIAD are initially 
combined to produce the alkoxyphosphorane (compound 12, FIG. 3) before 
adding the acidic component. Formation of compound 12 prevents the acidic 
component from reacting with the betaine (compound 10, FIG. 3) to form the 
protonated betaine (compound 11, FIG. 3) and eliminates pyrophosphonate 
formation. Formation of compound 12 (FIG. 3) was inferred by the 
significant upfield shifts in the .sup.1 H-NMR spectrum of the alcohol 
protons upon addition of DIAD and triphenylphosphine. 
In the slower reactions, the protonated betaine (compound 11, FIG. 3) was 
formed rapidly, as evidenced by an upfield shift of the phosphonic acid 
monoester's methylene and methoxide .sup.1 H-resonances, and the 
appearance of a proton attributable to the protonated carbamate amine in 
compound 11 (FIG. 3). The protonated betaine is believed then to react 
slowly with the alcohol to yield the desired product and 
diisopropylhydrazodiformate (DIHD). 
4. Results 
The present method has broad applicability, as shown by Table 1, below. 
Table 1 shows that the present method has been successfully applied to 
t-butyl and benzylphosphonic esters and to the synthesis of phosphorus 
containing peptide analogues. 
TABLE 1 
______________________________________ 
Alkylphosphonic acid monoester synthesis via condensations 
of methyl alkylphosphonates with alcohols followed 
by TMSBr treatment 
Time 
Phosphonate Alcohol (hr) Yield 
______________________________________ 
Methyl benzyl- 
Ethanol 0.5 94% 
phosphonate Isopropanol 0.5 91% 
Methyl 3-L-phenyl- 
1 80% 
lactate 
Methyl S-mandelate 
2 81% 
Methyl t-butyl- 
Ethanol 0.5 81% 
phosphonate Isopropanol 0.5 88% 
Methyl 3-L-phenyl- 
2 82% 
lactate 
Methyl N-CBz-amino- 
Methyl glycolate 
0.5 86% 
methylphosphonate 
Methyl 3-L-phenyl- 
1 78% 
lactate 
Methyl 2-L-hydroxy- 
3.5 76% 
isovalerate 
Methyl N-CBz- 
Methyl glycolate 
0.5 83% 
valinyl- Methyl 3-L-phenyl- 
1 64% 
phosphonate lactate 
Methyl 2-L-hydroxy- 
4 69% 
isovalerate 
______________________________________ 
The phosphorus analogues of glycine and valine were used to observe the 
substituent effects of the phosphorus component on coupling rates and 
yields. These compounds were synthesized from the CBZ-protected amino 
acids by the methods of Seebach et al., supra, and Corcoran and Green 
(1990) Tetr. Lett. 31:6827. Likewise, the hydroxy analogues of glycine, 
phenylalanine, and valine were used to examine the scope of the method. 
.alpha.-L-Hydroxyisovaleric acid was purchased from Bachem Bioscience, 
Inc. Based on the results of previous workers comparing coupling rates for 
N.sup..alpha. -benzyloxycarbonylamino acid p-nitrophenyl ester derivatives 
with amino acid esters (see Kovacs, The Peptides: Analysis, Synthesis, 
Biology, vol. 2 (Academic Press, N.Y. 1979), pp. 485-539), one might 
expect that the rate of phosphonate ester formation in the reaction would 
be sensitive to steric effects at both the alcohol and phosphorus centers. 
Upon comparing the reaction times required to achieve greater than 99% 
coupling yields (estimated by .sup.1 H-NMR for the phosphonic acid 
couplings), one surprisingly finds, however, that the reaction rate is 
relatively insensitive to the steric bulk of-the phosphoryl residue side 
chain. The phosphorus Gly and Val analogues had similar reaction times, 
unlike the active ester condensations, which exhibited 16-22 fold 
differences. This tendency has been mentioned previously with regard to 
the coupling of amino-carboxylic acids and azido compounds via 
triarylphosphines. See Wilt et al., (1985) J. Org. Chem. 50:2601. The 
influence of steric effects from the hydroxy component on the reaction 
rate was comparable to that seen in the active ester condensations. 
The following compounds have been prepared using this general method, 
wherein the phosphonate was the limiting reagent: ethyl benzylphosphonate, 
isopropyl benzylphosphonate, methyl 
(D)-2-O-benzylphosphoryl-3-phenylpropionate, methyl 
(D)-2-O-benzylphosphoryl-2-phenylacetate, ethyl t-butylphosphonate, 
isopropyl t-butylphosphonate, methyl 
(D)-2-O-t-butylphosphoryl-3-phenylpropionate, methyl 
2-O-(1-N-(benzyloxycarbonyl)aminomethylphosphoryl)acetate, methyl 
(D)-2-O-(1-(N-(benzyloxycarbonyl)amino)methylphosphoryl)-3-phenylpropionat 
e, methyl (D)-2-O-(1-(N-(benzyloxycarbonyl)amino)methylphosphoryl)-3-m 
ethylbutyrate, methyl 
2-O-((R,S)-1-(N-(benzyloxycarbonyl)amino)-2-methylpropylphos 
phory)acetate, methyl 
(D)-2-O-((R,S)-1-(N-(benzyloxycarbonyl)amino)-2-methylpropylphosphoryl)-3- 
phenylpropionate, and methyl 
(D)-2-O-((R,S)-1-(N-(benzyloxycarbonyl)amino)-2-methylpropyl 
phosphoryl)-3-methylbutyrate. 
B. The Alcohol as the Limiting Reagent 
1. Overview 
Thus far, discussion has focused on a general methodology for the synthesis 
of phosphonate esters wherein the phosphonate formed the limiting reagent. 
Initial attempts at modifying this reaction such that the alcohol was the 
limiting reagent resulted in increased reaction time and decreased yield. 
Surprisingly, the addition of an exogenous base and a triarylphosphine 
that was more electronegative at phosphorus than triphenylphosphine 
overcame these deficiencies. 
According to this embodiment, the methyl phosphonate is treated with a 
dialkylazodicarboxylate, preferably DEAD or DIAD, and a triarylphosphine, 
wherein at least one of the aryl groups bears an electron withdrawing 
substituent. Typically, the electron withdrawing substituent will have a 
Hammett constant (s) from between about 0.05 to about 0.8, and preferably 
between about 0.25 to about 0.5. See Lowry and Richardson, Mechanism and 
Theory of Organic Chemistry, 2nd Ed., p. 131 (1981), and Hughes et al. 
(1988) J. Am. Chem. Soc. 110:6487, which are hereby incorporated by 
reference. Preferably, the phosphorus of the triarylphosphine will not be 
as electron deficient as tris(pentafluorophenyl)phosphine. The reaction 
mixture is then treated with an exogenous base and the alcohol. In a 
preferred embodiment, the exogenous base will be soluble in the reaction 
solvent. Particularly preferred exogenous bases include tri(lower 
alkyl)amines, such as diisopropylethylamine (DIEA) or triethylamine (TEA). 
In a preferred embodiment, approximately equimolar amounts of the 
triarylphosphine, dialkylazodicarboxylate, and phosphonate are used. The 
ratio of alcohol to phosphonate will typically range from about 1:1.1 to 
about 1:5, preferably from about 1:1.1 to about 1:3, and more preferably 
about 1:1.5. An excess of either the dialkylazodicarboxylate or the 
triarylphosphine (e.g., up to about 5 equivalents) can be used; however, 
to avoid elimination and other side reactions, excess amounts of both 
reagents should not be used. The exogenous base is used in excess, 
typically, between about 2 and 10 equivalents, preferably, between about 2 
and 8 equivalents, and more preferably, about 5 equivalents, based on the 
amount of alcohol. 
The coupling reaction proceeds stereospecifically with inversion of the 
stereochemistry at the carbon bonded to the hydroxyl group of the alcohol 
reactant. Accordingly, if the alcohol reactant has an R configuration at 
the carbon bonded to the hydroxyl group, the resulting phosphonate has an 
S configuration at the corresponding carbon. 
When the coupling reaction is complete, the selective cleavage of the ester 
is carried out; preferably, trimethylsilyl bromide (TMSBr) is added to the 
reaction mixture to effect selective hydrolysis of the methyl ester and 
yield the desired compound (compound 3, FIG. 1). Other methods for 
cleaving the phosphonic acid ester can also be used; for instance, the 
protocols for cleaving esters of carboxylic acids described in Greene, 
supra, at page 156 et seq., can be used for this purpose, provided the 
desired selectivity of hydrolysis can be achieved. 
Although FIG. 1 exemplifies the reaction as an intermolecular reaction, one 
of skill in the art will appreciate that the reaction may also be 
performed intramolecularly (i.e., the alcohol and the monomethyl 
phosphonate are parts of the same molecule). This embodiment allows for 
the synthesis of cyclic and sterically restricted phosphonates. 
2. The General Reaction 
To a solution of methyl alkylphosphonate (1.5 mmol), alcohol (1 mmol), and 
a triarylphoshine, preferably, tris(4-chlorophenyl)phosphine, (1.5 mmol) 
dissolved in anhydrous methylene chloride (5 ml) was added 
dialkylazodicarboxylate, preferably DIAD or DEAD, (1.5 mmol) and an 
exogenous base, such as TEA or DIEA (5 mmol). Upon completion of the 
reaction the reaction mixture was concentrated under vacuum and purified 
by flash chromatography (ethyl acetate/hexanes). 
3. The Mechanism 
Although not wanting to be limited to the following mechanism, it is 
currently believed that when an equimolar excess of phosphonic acid, 
triphenylphosphine, and DIAD relative to the alcohol are used (entry 2, 
Table 2) the protonated betaine 11 is formed and reacts sluggishly with 
the alcohol to form the alkoxyphosphonium salt 13. See FIG. 3. When a 
slight excess of unprotonated betaine 11 is present (entries 1 & 3, Table 
2) it assists formation of the alkoxyphosphonium salt 13 via general base 
catalysis. However, with a larger excess of 11 (entry 4, Table 2) path B 
is accessed resulting in formation of the dialkoxyphosphorane 12 which can 
undergo an E2 elimination instead of cascading to product. See, also, Von 
Itzstein and Jenkins (1983) Aust. J. Chem. 36:557 and Loibner and Zbiral 
(1977) Helv. Chim. Acta 60:417. 
This pathway is circumvented, at least in part, by the addition of an 
exogenous base as a general base catalyst which serves to increase the 
rate of reaction between the alcohol and 11 to form 13. Moreover, 
increasing the electrophilicity of phosphorus in 11 increases the rate of 
formation of 13. Additionally, to prevent access to path B and potential 
elimination reactions, an equimolar excess of phosphonic acid, phosphine, 
and DIAD should be used. 
4. The Results 
The results are summarized in Table 2 (entries 5-7). All couplings were 
performed with an equimolar excess of phosphonic acid, DIAD and phosphine 
relative to the alcohol. 
TABLE 2 
______________________________________ 
Reaction Times for Phosphonic Acid Couplings 
Phosphonic Coupling 
Acid Alcohol TPP CTPP.sup.1 
DIAD TEA Time.sup.2 
______________________________________ 
1.0 1.5 1.5 -- 1.5 -- 1 
2.0 1.0 2.0 -- 2.0 -- &gt;72 
1.5 1.0 2.0 -- 2.0 -- 12 
1.5 1.0 3.0 -- 3.0 -- --.sup.3 
2.0 1.0 2.0 -- 2.0 5.0 8 
2.0 1.0 -- 2.0 2.0 -- 6 
2.0 1.0 -- 2.0 2.0 5.0 0.5 
______________________________________ 
FNT .sup.1 Tris(4-chlorophenyl)phosphine .sup.2 Hours .sup.3 Negligible product 
formation 
Upon addition of TEA the reaction time was reduced to 8-hours (entry 5, 
Table 2). A number of commercially available phosphines with electron 
withdrawing substituents on the phenyl rings were investigated. The most 
electron deficient phosphine studied was tris(pentafluorophenyl)-phosphine 
(s=0), however it was unable to form the betaine 10 as evidenced by .sup.1 
H-NMR. Relative to triphenylphosphine only a modest improvement in 
reaction time was observed with tris(4-fluorophenyl)phosphine (s=0.06) 
while tris(4-chlorophenyl)phosphine (s=0.23) reduced the reaction time to 
6-hours (entry 6, Table 2). 
Combining the addition of an exogenous base with 
tris(4-chlorophenyl)phosphine substituted for TPP (entry 7, Table 2) 
reduced the reaction time to 0.5-hours which is comparable to the standard 
Mitsunobu coupling results (entry 1, Table 2). Additional improvements in 
rates and yields can be achievable with more electron deficient phosphines 
providing this doesn't result in an E2 elimination reaction of the 
alkoxyphosphonium salt. 
The effect of additional steric encumbrance on the yields and reaction 
times was also studied and the results summarized in Table 3. Satisfactory 
results were obtained even with the most sterically demanding couplings. 
The coupling times, and yields are comparable to the results we previously 
reported for the standard Mitsunobu procedure under phosphonic acid 
limiting conditions. Supra. If desired, selective demethylation of the 
resulting unsymmetrical phosphonate diesters can be accomplished with 
either TMSBr (see Campbell (1992) J. Org. Chem. 57:6331) or 
triethylammonium phenoxide (see Norbeck et al. (1987) J. Org. Chem. 
52:2174). 
TABLE 3 
______________________________________ 
Alkylphosphonic acid monoester synthesis via condensations 
of methyl alkylphosphonates with alcohols followed 
by TMSBr treatment 
Time 
Phosphonate Alcohol (hr) Yield 
______________________________________ 
Methyl benzyl- 
Isopropanol 0.5 91% 
phosphonate Methyl glycolate 
0.3 81% 
Methyl D-lactate 
0.3 88% 
Methyl 3-D-phenyl- 
0.5 79% 
lactate 
Methyl 2-D-hydroxy- 
3.0 61% 
isovalerate 
Methyl N-CBz-amino- 
Methyl D-lactate 
0.5 81% 
methylphosphonate 
Methyl 3-D-phenyl- 
0.5 72% 
lactate 
Methyl 2-D-hydroxy- 
4.0 50% 
isovalerate 
Methyl (R)-[N-CBz- 
Methyl D-lactate 
0.5 81% 
alanylphosphonate 
Methyl 3-D-phenyl- 
0.5 84% 
lactate 
Methyl 2-D-hydroxy- 
3.5 43% 
isovalerate 
Methyl (R,S)-[N- 
Methyl D-lactate 
0.5 88% 
CBz-valinyl- Methyl 3-D-hydroxy- 
0.5 80% 
phosphonate phenyl lactate 
Methyl 2-D-hydroxy- 
7.0 53% 
isovalerate 
______________________________________ 
C. .alpha.-Aminoalkylphosphonates 
In another important embodiment, an aminoalkylphosphonate (compound 9, FIG. 
4) serves as the phosphonate. In this embodiment, the R.sup.1 and R.sup.2 
groups of compound 9 (FIG. 4) typically are derived from an amino acid and 
are independently selected from hydrogen, alkyl, heteroalkyl, aryl, 
arylalkyl, and heteroaryl, each of which may be optionally substituted. In 
addition, R.sup.1, R.sup.2, and the nitrogen atom to which R.sup.2 is 
attached may form a heterocyclic ring. According to some embodiments, 
R.sup.2 will be a protecting group. 
When the phosphonate is an .alpha.-aminoalkylphosphonate in the method of 
the present invention, the resulting compound, e.g., a methyl 
.alpha.aminoalkylphosphonate diester, is particularly useful in the 
production of compounds that have pharmacological activity or can be used 
in the generation of catalytic antibodies. In one embodiment, this aspect 
of the invention is directed to the synthesis of peptide analogues that 
possess a phosphonate linkage in the place of an amide or amidomimetic 
linkage in the peptide or peptidomimetic. These peptide analogues can be 
prepared from the corresponding aminoalkylphosphonates, as discussed 
below. 
To prepare peptidylphosphonates according to the methods of the present 
invention, a monomethyl aminoalkylphosphonate (compound 9, FIG. 4) is 
first prepared. Monomethyl aminoalkylphosphonates can be produced from 
amino acids, as shown in FIG. 4. A variety of N-protected amino acids 
(compound 6, FIG. 4) can be used to prepare aminoalkylphosphonates. 
Although .alpha.-aminoalkylphosphonates are preferred for many purposes of 
the invention, the invention is equally applicable to other 
aminoalkylphosphonates, such as beta-amino acid phosphonate derivatives 
and other derivatives where the number of carbon atoms between the amino 
group and the phosphonate group is more than one. 
To prepare compounds such as compound 7 (FIG. 4), the amino group of the 
amino acid (compound 6, FIG. 4) must be protected. One skilled in the art 
will appreciate that any one of a variety of terminal amino protecting 
groups may be used. The terminal amino protecting group corresponds to the 
group R.sup.2 in compounds 6 through 9 (FIG. 4). Examples of terminal 
amino protecting groups may be found in Greene et al., supra, and include 
carbamates, amides, N-alkyl groups, N-aryl groups, etc. Preferred amino 
protecting groups include the carbobenzyloxy group (CBz), the 
9-fluorenylmethyl carbamate group (Fmoc), and the NPEOC group. 
Aminophosphonates can be produced from amino acids by decarboxylation 
according to the following reaction. Compound 7 (FIG. 4), where Z is 
acetoxy, can be prepared by the procedure of Corcoran et al. (1990) Tetr. 
Lett. 31:6827-6830. The Corcoran et aI. reference describes the oxidative 
decarboxylation of N-protected amino acids with lead tetraacetate. 
Compound 7, where Z is either iodo, bromo, or chloro, can be prepared 
through the treatment of an N-protected amino acid (compound 6, FIG. 4) 
with lead tetraacetate and halide ions, preferably iodine, bromide, and 
chloride. This general procedure is reported in Kochi (1965) J. Am. Chem. 
Soc. 87:2500. A review of this reaction can be found in Sheldon and Kochi 
(1972) Org. React. 19:279-421. Alternatively, compound 7, where Z is 
either iodo, bromo, or chloro, can be prepared from an N-protected amino 
acid using the procedures outlined in March, 1985, Advanced Organic 
Chemistry 3rd Ed. (John Wiley & Sons, New York), pp. 654-655. 
Compound 8 (FIG. 4) can be prepared through a variety of synthetic 
sequences. According to a preferred embodiment, these compounds can be 
produced stereospecifically using the procedure described in U.S. Pat. No. 
5,362,899, which is incorporated herein by reference, and illustrated in 
FIG. 5. This procedure provides for the conversion of a protected amino 
acid to the corresponding acyl aroyl or diacyl peroxide which then 
spontaneously rearranges to form an alpha amino ester. The ester 
subsequently can be converted to an appropriate leaving group which is 
displaced with a phosphite to yield Compound 8. 
Compound 8 also can be produced via the reaction of compound 7, where Z is 
acetoxy or halo, with trimethylphosphite. See Corcoran, supra; see also, 
Seebach, supra. A review of this general reaction can be found in Arbuzov, 
supra. In a preferred embodiment, compound 7 (FIG. 4), where Z is acetoxy, 
is treated with trimethylphosphite in the presence of titanium 
tetrachloride to produce compound 8. 
To illustrate this aspect of the invention, one can prepare dimethyl 
[1-[N-(benzyloxycarbonyl)-amino]-2-methylpropyl]phosphonate (see compound 
8, FIG. 4) from 1-acetoxy-1 -N-(benzyloxycarbonyl)amino-2-methylpropane 
(see compound 7, FIG. 4), or one can prepare dimethyl 
[1-[N-(benzyloxycarbonyl)-amino]-ethyl]phosphonate from 
1-acetoxy-1-N-(benzyloxycarbonyl)aminoethane by dissolving the starting 
compound in a polar aprotic solvent, such as methylene chloride, under an 
inert atmosphere. Trimethylphosphite is then added, and the mixture is 
cooled, preferably to between 0.degree. C. and -78.degree. C. A dilute 
solution, preferably 1M, of titanium tetrachloride in a polar aprotic 
solvent, such as methylene chloride, is slowly added to the solution of 
the compound, and the reaction is stirred at room temperature until the 
reaction is complete. See Example 3 below. 
The dimethyl phosphonates characterized by the structure of compound 8 
(FIG. 4) may be saponified to yield monomethyl phosphonates (compound 9, 
FIG. 4) using a variety of techniques. In a preferred embodiment, the 
saponification is accomplished under basic conditions. To illustrate this 
aspect of the invention, one can prepare methyl 
[1-[N-(benzyloxycarbinyl)-amino]-ethyl]phosphonate (see compound 9, FIG. 
4) from dimethyl [1-[N-(benzyloxycarbonyl)-amino]-ethyl]phosphonate (see 
compound 8, FIG. 4), or one can prepare methyl 
[1-[N-(benzyloxycarbonyl)-amino]-2-methylpropyl]phosphonate from dimethyl 
[1-[N-(benzyloxycarbonyl)-amino]-2-methylpropyl]phosphonate by dissolving 
the starting compound in a polar protic solvent and a strong aqueous base, 
preferably concentrated aqueous sodium hydroxide or concentrated aqueous 
lithium hydroxide, and heating the resulting mixture for a time sufficient 
to hydrolyze the ester group. See Example 3 below. 
D. Solid State Synthesis 
1. Overview 
One can synthesize many pharmaceutically important compounds by the method 
of the present invention. As already noted, the present methods are also 
amenable to synthesis reactions carried out on a solid support or 
substrate, an important aspect of the present invention. Synthesis in this 
format is desirable primarily because of the ease with which one can 
synthesize and screen large numbers of molecules for desired properties. 
In addition, this synthesis format may be preferred, because byproducts, 
such as unreacted starting materials, solvents, deprotection agents, etc., 
may simply be washed away from the desired solid support-bound product. 
2. Substrates 
In accordance with the present invention, solid phase substrates include 
beads and particles, such as peptide synthesis resins (see, e.g., 
Merrifield (1963) J. Am. Chem. Soc. 85:2149-54; U.S. patent application 
Ser. No. 762,522, filed Sep. 18, 1991; U.S. patent application Ser. No. 
876,792, filed Apr. 29, 1992; and Houghten, U.S. Pat. No. 4,631,211), rods 
(see, e.g., Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81:3998-4002, 
and Geysen et al., 1986, PCT patent publication No. WO 86/00991), glass 
slides (see Fodor et al. PCT patent publication No. WO 92/10092), and any 
additional support upon which peptides or other organic compounds can be 
synthesized. Thus, the solid substrate may be composed of any of a wide 
variety of materials, for example, polymers, plastics, resins, 
polysaccharides, silicon or silica-based materials, carbon, metals, 
inorganic glasses, and membranes. 
The surface of the substrate is preferably provided with a layer of linker 
molecules, although one of skill in the art understands that the linker 
molecules are optional and not strictly required. The linker molecules are 
of sufficient length to permit compounds attached to the substrate to 
interact freely with molecules exposed to the substrate. The linker 
molecules can be 6-50 atoms long or longer to provide optimum exposure. 
The linker molecules, may be, for example, aryl acetylene, or ethylene 
glycol oligomers, or diamines, diacids, amino acids, or combinations 
thereof in oligomer form, each oligomer containing 2 to 10 or more monomer 
units. Other linker molecules may also be used. 
The linker molecules can be attached to the substrate via carbon-carbon 
bonds using, for example, (poly)trifluorochloroethylene surfaces, or 
preferably, by siloxane bonds (using, for example, glass or silicon oxide 
surfaces). Siloxane bonds with the surface of the substrate may be formed 
via reactions of linker molecules bearing trichlorosilyl groups. The 
linker molecules may optionally be attached in an ordered array, i.e., as 
parts of the head groups in a polymerized Langmuir Blodgett film. In 
alternative embodiments, the linker molecules are adsorbed to the surface 
of the substrate. 
On the distal or terminal end of the linker molecule opposite the 
substrate, the linker molecule is provided with a functional group. This 
functional group is used to bind a protective group, a substrate, or a 
reactant, depending on the format desired and the stage of synthesis. 
Typically, the functional group will comprise a hydroxyl group, a mercapto 
group, an amino group, a carboxylic acid group, a thiol group, or other 
groups capable of bonding to the reactant. 
3. Coupling Chemistry 
For a better understanding of the wide variety of solid supports, linkers, 
and screening methods compatible with the present invention, the following 
specific method for incorporating the phosphonate transition-state analog 
pharmacaphore into a growing peptide chain via solid phase synthesis is 
provided. 
The solid phase synthesis of a peptidylphosphonate begins with the 
construction of a peptide of desired length and sequence using standard 
FMOC peptide synthesis methodology. Incorporation of a phosphonate linkage 
requires two unnatural (non-peptidyl) building blocks: (1) an 
appropriately protected aminophosphonic acid; and (2) an appropriately 
protected alpha-hydroxy acid (for example, an analog of a genetically 
coded .alpha.-amino acid). 
Secondary alcohols, such as the side chain hydroxyl of threonine, have been 
shown to be unreactive toward modern amino acid coupling agents, such as 
BOP and HBTu. Surprisingly, however, most of the secondary hydroxyls of 
the alpha-hydroxy acids are reactive toward these agents, at least in the 
present methods, and therefore must be suitably protected during coupling 
to an amino acid. The reaction is schematically outlined in FIG. 6. 
Chemical peptide synthesis typically proceeds in the carboxy-to-amino (C to 
N) direction. Incorporation of the phosphonate proceeds in two steps. 
First, a suitable protected hydroxy acid, typically, an 
alpha-O-FMOC-hydroxy acid or an alpha-O-NPEOM-hydroxy acid, is attached to 
the peptide's amino-terminus using standard peptide coupling conditions, 
in the example shown, HBTu 
(2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) 
chemistry. The hydroxyl protecting group is removed. Treatment with 30% 
piperidine or 5% DBU in NMP removes the FMOC group and the yields for the 
coupling of the hydroxy acid to the substrate can be assessed by UV 
analysis using the fluorene chromophore of the dibenzofulvene-piperidine 
adduct (301 nm, .epsilon.=7800 M-.sup.-1 cm-.sup.-1, see Bernatowicz et 
al. (1989) Tetrahedron Lett. 30:4645). 
Synthesis of the peptidylphosphonate proceeds by coupling the resin bound 
alcohol with a methyl .alpha.-N-NPEOC-aminoalkylphosphonate using the 
methods described herein, i.e., using the activating agents 
triarylphosphine, dialkylazodicarboxylate (such as DIAD or DEAD), and a 
base. In a preferred embodiment, a triarylphosphine wherein at least of 
the aryl groups bears an electronegative substituent, and more preferably 
tris(4-chlorophenyl)phosphine, is used. More preferably, the 
electronegative substituent(s) will have a Hammett constant(s) from 
between about 0.05 to about 0.8, and preferably between about 0.25 to 
about 0.5. These reagents activate the hydroxyl by forming a 
triphenylphosphonium derivative, which in turn undergoes an S.sub.N 2 
displacement by the phosphonic acid monoester to afford the phosphonate 
diester (compound 22, FIG. 6). 
Due to the lability of the FMOC group in the presence of the coupling 
reagents, according to this embodiment, preferred protecting groups 
include the NPEOC group and NPEOM group. These groups can be removed with 
5-10% DBU (1,8-diazabicyclo[5.4.0]undec-7-ene) in NMP (N-methyl 
pyrrolidone). UV analysis of the 4-nitrostyrene chromophore (308 nm, 
.epsilon.=13,200 M.sup.-1 cm.sup.-1) can be performed to determine the 
coupling efficiency. The CBz-protecting group also can be employed. 
Initial experiments were performed on Alltech Adsorbosphere Amino beads (5 
.mu.m glass beads derivatized with an amino functionality and a 50-80 
.mu.m pore size. Very slow and incomplete coupling was observed, possibly 
attributable to the small pore size and variations in pore geometries 
which may have hindered the accessibility of the a-hydroxy acid residues 
to the bulky protonated betaine. High coupling yields, however, could be 
obtained using either controlled pore glass (CPG) or a polyethyleneglycol 
crosslinked polystyrene resin (Rapp Polymere) as the solid substrate. 
Examples of these yields are shown below in Table 4. 
##STR11## 
TABLE 4 
__________________________________________________________________________ 
Time Required for &gt;90% Coupling.sup.4 
R.sup.2 R.sup.1 = H 
CH.sub.3 
CH.sub.2 (C.sub.6 H.sub.5) 
CH.sub.2 CH(CH.sub.3).sub.2 
__________________________________________________________________________ 
H &lt;0.5 &lt;1.5 
12 6.4 
(R)--CH.sub.3 
&lt;0.5 &lt;1.5 
12 8.4 
(R,S)--CH.sub.2 (C.sub.6 H.sub.5) 
&lt;0.5 &lt;1.5 
13 10 
(R,S)--CH.sub.2 CH(CH.sub.3).sub.2 
&lt;0.5 &lt;1.5 
14 10 
(R,S)--CH(CH.sub.3).sub.2 
&lt;0.5 &lt;1.5 
10 20 
__________________________________________________________________________ 
.sup.4 As determined by 4nitrostyrene absorbance at 308 nm. 
Upon completion of the coupling reaction, one has a variety of options: the 
N-terminal phosphonate can be deprotected to allow further couplings, for 
example, using standard peptide coupling chemistry, or synthesis can stop 
here, and the compound can be cleaved from the support, as shown in FIG. 
6. One can also selectively hydrolyze the ester by, e.g., treatment with 
TMSBr or triethylammonium phenoxide, either on the support or after the 
compound is cleaved from the support. 
One skilled in the art will appreciate that the cleavage conditions will 
vary with the type of substrate and the nature of the phosphonate ester. 
Typically, the substrate-bound phosphonate ester will first be washed with 
a polar aprotic solvent and dried under reduced pressure. If a or 
resin is used as the substrate, then cleavage of the phosphonate ester 
from the resin can be accomplished through the action of trifluoroacetic 
acid. The phosphonate ester can be isolated by filtration and concentrated 
under reduced pressure. The solid phase synthesis of phosphonate esters by 
the method of the present invention will generally produce phosphonate 
esters of greater than 75% purity. 
The methods described herein are superior to others in which a 
peptidylphosphonate is synthesized as a dimer and then incorporated as a 
cassette into a growing peptide. In the prior art methods, one must first 
synthesize the cassette in solution phase and purify the cassette to 
homogeneity prior to incorporation. The prior art method allows one to 
synthesize only one combination of phosphonate-hydroxy acid dimer. One 
would have to synthesize in solution phase an entirely new cassette to 
make a different phosphonate. 
5. The Building Blocks 
a. The Hydroxy Acid 
By the present method, a single phosphonic acid building block can be 
coupled to virtually any hydroxy acid to form a variety of different 
dimers. A number of the hydroxy acids are commercially available. In 
addition, alpha-hydroxy acid analogs of the amino acids can be prepared in 
from commercially available amino acids (or any hydroxy acid) using the 
protocol shown in FIG. 7. For carboxylic acids having additional side 
chain functional groups, such as serine, threonine, and tyrosine, 
typically the side chain functional group will be protected (with a 
protecting group PG) prior to diazotization. Examples of suitable side 
chain protecting groups include, but are not limited to, t-butyl esters 
(for Glu and Asp); t-butyl ethers (for Ser, Thr, and Tyr); BOC (for Lys); 
and trityl amides (for Gln and Asn). 
This synthetic route involves (1) diazotization with sodium nitrite in mild 
acid, such as acetic acid or citric acid; and (2) carboxy protection, 
preferably as the triisopropylsilyl group, and protection of the hydroxy 
group, typically with FMOC-Cl; and carboxy deprotection. This reaction 
sequence is stereospecific and results in retention of the chirality of 
the carbon bearing the amino group (and thus, the carbon bearing the 
hydroxyl group). This latter multi-step sequence can be performed without 
intermediate product isolation and purification. This reaction sequence 
will generally produce the desired hydroxy acid in 35-70% isolated overall 
yield.] 
b. The Phosphonate 
Like the hydroxy acids, phosphonic acid building blocks can be prepared 
from commercially available amino acids. Again, one should not limit the 
present invention to only the .alpha.-aminoalkylphosphonic acids or 
phosphonic acids that are similar to the naturally occurring amino acids 
or the 20 amino acids encoded genetically. The synthesis of a protected 
aminophosphonic acid starting with a commercially available amino acid is 
outlined in FIG. 8, and the synthesis of a protected aminophosphonic acid 
starting with a commercially available aminophosphonic acid is outlined in 
FIG. 9. As discussed above, .alpha.-aminoalkylphosphonic acids can also be 
produced by the procedures set forth in copending application U.S. Ser. 
No. (Attorney Docket No. 11509-84), which is incorporated herein by 
reference. 
6. Libraries 
The present methods for the solid phase synthesis of phosphonate esters are 
especially preferred for the production of libraries of peptidylphosphonic 
acid derivatives. These libraries can be assembled using, for example, the 
combinatorial synthesis strategies described in PCT patent publication No. 
92/10092; U.S. patent application Ser. No. 762,522, filed Sep. 18, 1991; 
and U.S. patent application Ser. No. 876,792, filed Apr. 29, 1992. 
Briefly, a combinatorial synthesis strategy is an ordered strategy for 
parallel synthesis of diverse polymer sequences by sequential addition of 
reagents to a solid support. In one embodiment, the solid support is a 
collection of beads, and each cycle of monomer addition is carried out in 
parallel in a series of reactions, each reaction using a different 
monomer. After each cycle, the beads from the different, parallel series 
of reactions are pooled and redistributed prior to the next cycle of 
monomer addition. These methods apply, for example, to the synthesis of 
protease inhibitors where one knows the position of the P.sub.1 -P.sub.1 ' 
site and wants to fix the phosphonate linkage while exploring amino acid 
preferences at the flanking regions. 
In another preferred embodiment, the combinatorial synthesis is performed 
using the VLSIPS.TM. technique described in Fodor et al. (1991) Science 
251:767-773, and in PCT patent publication No. 92/10092 (and corresponding 
U.S. counterparts, including Ser. No. 805,727, filed Dec. 6, 1991). 
Briefly, the VLSIPS.TM. technology is a method for synthesizing large 
numbers of different compounds simultaneously at specific locations on a 
silica chip. Supra. Using this technology, one can produce an array of 
phosphonic acid and phosphonate derivatives and screen that array for 
compounds with biological activity. 
To screen for biological activity, the array is exposed to one or more 
receptors such as antibodies, whole cells, receptors on vesicles, lipids, 
or any one of a variety of other receptors. The receptors are preferably 
labeled with, for example, a fluorescent marker, radioactive marker, or a 
labeled antibody reactive with the receptor. After the array has been 
exposed to the receptor, the location of the marker can be detected with, 
for example, photon detection or autoradiographic techniques. Through 
knowledge of the sequence of the material at the location where binding is 
detected, one can quickly determine which compounds bind to the receptor. 
This combinatorial synthesis approach can be utilized to produce a library 
of peptidylphosphonates using the reaction scheme illustrated in FIG. 6 
and the synthetic protocol shown in FIG. 10. Typically, the protecting 
groups will be present on the amine and hydroxyl functional groups on the 
surface of the support where coupling occurs and will be photolabile, 
although other protecting groups can be used to advantage in the protocol. 
Using lithographic methods, one exposes the photoremovable protecting 
group to light so as to remove the group from the reactant in the first 
selected region to free an amine or hydroxyl group on the compounds where 
the desired attachment is to occur. 
The substrate is then washed or otherwise contacted with a first hydroxy 
acid, amino acid, or phosphonate, depending on the step in the process. 
Once the hydroxy acid is coupled to the activated amine groups on the 
support and then irradiated to remove the protecting group from the 
hydroxyl group of the coupled hydroxy acid, an 
.alpha.-aminoalkylphosphonate, preferably a monomethyl 
.alpha.-aminoalkylphosphonate, is coupled to the hydroxyl group with the 
activating reagents dialkylazodicarboxylate and triphenylphosphine. 
Portions of the substrate which have not been deprotected do not react 
with the phosphonate (or other monomer, depending on the step and the 
compounds being synthesized). If the phosphonate (or other monomer) 
possesses other functionalities, these functionalities may need to be 
blocked with an appropriate protecting group, which may be photolabile but 
could also be removable with acid or base treatment. 
A second set of selected regions can then be exposed to light to remove the 
photoremovable protecting group from compounds in the second set of 
regions. The substrate is then contacted with a second monomer and 
coupling reagents, e.g., a monomethyl phosphonate, triphenylphosphine, and 
dialkylazodicarboxylate. Of course, one can also introduce and couple 
other monomers, such as amino acids, to the growing polymer chains on the 
surface, so long as appropriate chemistry and protecting groups are 
employed. This process is repeated until the desired array of compounds 
has been synthesized. 
Thus, libraries of potential ligands for binding with receptors can be 
prepared using the methods of the present invention. Typically, a peptide 
would first be identified as a potential ligand for binding with a given 
receptor. An array of peptide analogues would then be produced. These 
analogues are peptidylphosphonates in which amide linkages of the peptide 
have been replaced with a phosphonate linkage. Of course, one can also 
synthesize other polymers, such as polyphosphonates (using .alpha.-hydroxy 
alkylphosphonates), by the methods of the invention. 
The methods described herein have been employed to produce libraries of a 
combinatorial libray of .alpha.-N-CBz-aminoalkylphosphonic acid capped 
trimers on both cleavable and nonocleavable resin. Thermolysin, a well 
characterized metalloprotease, has been used as a model system to 
investigate a number of assay formats, both with soluble and resin 
supported libraries. 
III. Synthesis of Phosphonate Diesters 
A. Overview 
Thus, the present invention has diverse applications. For instance, and as 
shown in FIG. 11, the present invention also provides methods to 
synthesize symmetrical phosphonate diesters (compound 15). 
Bisesterification of commercially available benzylphosphonic acid 
(compound 14, FIG. 11, commercially available from Lancaster Synthesis) 
was initially used to exemplify this aspect of the invention, and the 
reaction was carried out by adding a dialkylazodicarboxylate, preferably 
DIAD, to a solution of compound 14, an alcohol, and a triarylphosphine, 
preferably triphenylphosphine, in THF. When primary alcohols were used, 
the reaction was instantaneous, and the symmetrical esters were isolated 
in high yields. 
According to a preferred embodiment, approximately equimolar amounts of the 
triarylphosphine, the dialkylazodicarboxylate, and the alcohol are used. 
Typically, the ratio of phosphonic acid to alcohol will range from about 
1:2.5 to about 1:50, and preferably from about 1:2.5 to about 1:25. 
B. The General Reaction 
The general procedure for the solution phase synthesis of benzylphosphonate 
diesters is as follows. To a solution of benzylphosphonic acid (0.5 mmol), 
alcohol (1.25 mmol), and triphenylphosphine (1.25 mmol) dissolved in 
anhydrous THF (5 mL), is added diisopropylazodicarboxylate (1.25 mmol). 
After about 30 minutes, the reaction mixture is concentrated under vacuum, 
and then the triphenylphosphineoxide is crystallized with acetone/pentane, 
and removed by filtration. The filtrate is optionally concentrated under 
vacuum and purified by chromatography (HOAc/EtOAc can be used). 
Illustrative compounds prepared by this protocol include dimethyl 
benzylphosphonate, diisopropyl benzylphosphonate, and dibenzyl 
benzylphosphonate. See Example 2. 
C. The Mechanism 
To understand the method for making phosphonate diesters, one may benefit 
from considering the reaction schemes shown in FIGS. 3 (supra) and 12. 
When methanol and benzyl alcohol were used, the alkoxyphosphorane formed 
reacted cleanly and rapidly with benzylphosphonic acid to form the 
phosphonate diesters. With isopropanol, the reaction was complicated by 
the ready decomposition of compound 12 (FIG. 3), presumably via an E2 
elimination as has been observed by others (see Von Itzstein and Jenkins, 
supra, and Loibner and Zbiral (1977) Helv. Chim. Acta. 60:417). 
One explanation for the observed reactivity of benzylphosphonic acid is 
that intramolecular dehydration of compound 11 (FIGS. 3 and 12) yields 
benzylmetaphosphonate (compound 18, FIG. 12), triphenylphosphine oxide, 
and diisopropylhydrazodiformate (see FIG. 12). Formation of 
phenylmetaphosphonate (see Eckes and Regitz (1975) Tetr. Lett., p.447 et 
seq.), mesitylenemetaphosphonate (see Sigal and Lowe (1978) J. Am. Chem. 
Soc. 100:6394), and monomeric metaphosphate (see Bodalski et al. (1991) J. 
Org. Chem. 56:2666; Freeman et al. (1987) J. Am. Chem. Soc. 109:3166; and 
Westheimer (1981) Chem. Rev. 81:313) have all been observed. The 
benzylmetaphosphonate (compound 18, FIG. 12) would be highly electrophilic 
and could readily undergo nucleophilic attack by t-butanol to produce the 
t-butyl ester, or by another molecule of benzylphosphonic acid producing 
benzylpyrophosphonate (compound 19, FIG. 12). Mitsunobu esterification of 
compound 19 (FIG. 12) with isopropanol would then yield diisopropyl 
benzylpyrophosphonate (compound 16, FIG. 11). 
D. The Results 
The results of these experiments are shown in Table 5 below. 
TABLE 5 
______________________________________ 
Symmetrical alkylphosphonate diester syntheses via 
condensations of benzylphosphonic acid and alcohols 
Reaction Alcohol 
Scheme* Alcohol Equivalents 
Yield 
______________________________________ 
A Methanol 2.5 85% 
A Methanol 25 91% 
A Isopropanol 2.5 11% 
A Isopropanol 25 76% 
A Benzyl alcohol 
2.5 87% 
B Methanol 5 81% 
B Isopropanol 5 37% 
B Benzyl alcohol 
5 90% 
______________________________________ 
*See discussion below. 
When isopropanol was used, diisopropyl benzylpyrophosphonate (compound 16, 
FIG. 11, where R' is isopropanol) was the major product. Similar products 
have been observed during the study of radical-based dephosphorylation in 
the production of monomeric metaphosphate. See Avila and Frost (1988) J. 
Am. Chem. Soc. 110:7904. Perturbations of the reaction conditions yielded 
mixed results: temperatures of -78.degree. C. to 25.degree. C. had no 
effect on the product ratios, and changing the concentration of the 
reactants likewise had no effect on the product ratios. However, as shown 
in Table 2, increasing the equivalents of the alcohol to the phosphonic 
acid improves the yield markedly. In similar fashion, reaction scheme B, 
described more fully below, could also be accessed to improve yields; 
however, this method may be problematic if the reaction intermediates are 
susceptible to elimination or degradation reactions (as is isopropanol; 
see Table 2). 
Thus, the present invention provides a variety of methods for making, 
using, and screening phosphonate esters and compounds comprising 
phosphonate esters. The above description and the following examples are 
intended to be illustrative and not restrictive. Many embodiments of the 
invention not explicitly set forth herein will be apparent to those of 
skill in the art upon reviewing this description of the invention. The 
disclosures of all articles and references, including patents and patent 
applications, in their entirety are incorporated herein by reference. The 
scope of the invention should be determined not with reference to the 
above description, but instead with reference to the appended claims, 
along with the full scope of equivalents to which such claims are 
entitled. 
The following examples are provided to aid one of skill in the art in 
understanding the invention. Isolation and purification of the compounds 
and intermediates described in the examples can be effected, if desired, 
by any suitable separation or purification procedure such as, for example, 
filtration, extraction, crystallization, column chromatography, thin-layer 
chromatography, thick-layer (preparative) chromatography, distillation, or 
a combination of these procedures. Specific illustrations of suitable 
separation and isolation procedures are in the examples below. Other 
equivalent separation or isolation procedures can, of course, also be 
used. These examples are for illustrative purposes only and are not to be 
construed as limiting this invention in any manner. 
EXPERIMENTAL 
.sup.1 H-NMR spectra were recorded at 300 MHz. Mass spectral measurements 
were performed by the Mass Spectrometry Laboratory at the University of 
California, Berkeley. 
Benzylphosphonic acid, tris(pentafluorophenyl), 
tris(4-fluorophenyl)-phosphine, and tris(4-chlorophenyl)phosphine were 
purchased from Lancaster Synthesis Inc., glycolic acid, D-lactic acid, 
3-D-phenyllactic acid, and a-D-hydroxyisovaleric acid from Fluka, and all 
remaining chemicals were purchased from Aldrich Chemical Co. THF was 
refluxed over potassium and distilled prior to use. 
EXAMPLE 1 
Preparation of Phosphonic Acid Monoesters 
1.1 The Phosphonate as the Limiting Reagent 
To a solution of methyl alkylphosphonate (0.5 mmol), an alcohol (0.75 
mmol), and triphenylphosphine (0.75 mmol) in anhydrous THF, was added 
diisopropylazodicarboxylate (0.75 mmol). Upon completion of the 
condensation reaction, TMSBr was added. Stirring was continued an 
additional hour. The reaction mixture was diluted with ether (10 mL), and 
extracted with 5% aqueous sodium bicarbonate (10 mL.times.2). The aqueous 
phase was washed with ether (10 mL.times.2), acidified to pH=2 with 
concentrated hydrochloric acid, and extracted with ethyl acetate (10 
mL.times.3). The ethyl acetate phase was dried over magnesium sulfate, 
filtered, and concentrated under reduced pressure to yield a phosphonic 
acid monoester. 
1.2 The Alcohol as the Limiting Reagent 
To the methyl alkylphosphonate (1.5 mmol), dissolved in THF (10 ml) was 
added tris(4-chlorophenyl)phosphine (1.5 mmol), DIAD (1.5 mmol) and DIEA 
(5 mmol) followed by the alcohol (1 mmol). Upon completion of the reaction 
the mixture was concentrated under vacuum then purified by flash 
chromatography (EtOAc/hexanes). 
Following this procedure the following compounds were prepared: 
methyl isopropyl benzylphosphonate: .sup.13 C-NMR (CDCl.sub.3) 
.differential.: 131.40 (d, J=9 Hz), 129.72 (d, J=7 Hz), 128.42 (d, J=3 
Hz), 126.76 (d, J=4 Hz), 70.89 (d, J=7 Hz), 52.31 (d, J=7 Hz), 33.75 (d, 
J=139 Hz), 24.00 (d, J=4 Hz), 23.66 (d, J=6 Hz). .sup.31 P-NMR 
.differential.: 27.15. MS (FAB.sup.+, m/z): 228. Anal. Calcd. for C.sub.11 
H.sub.17 0.sub.3 P.0.6H.sub.2 O: C, 55.26; H, 7.69; P, 12.95. Found: C, 
55.05; H, 7.29; 
methyl L-2-[(methyl benzylphosphoryl)oxy]acetate: .sup.13 C-NMR 
(CDCl.sub.3) .differential.: 168.86 (d, J=4 Hz), 130.84 (d, J=10 Hz), 
129.75 (d, J=7 Hz), 128.53 (d, J=3 Hz), 126.98 (d, J=4 Hz), 62.39 (d, J=4 
Hz), 52.54 (d, J=7 Hz), 52.16, 33.51 (d, J=139 Hz). .sup.31 P-NMR 
.differential.: 29.67. MS (FAB.sup.+, m/z): 258; 
methyl L-2-[(methyl benzylphosphoryl)oxy]propionate: .sup.13 C-NMR 
(CDCl.sub.3) .differential.: 171.52, 131.14 (d, J=10 Hz), 130.87 (d, J=9 
Hz), 129.78 (d, J=7 Hz), 128.49, 128.40 (d, J=3 Hz), 127.00 (d, J=4 Hz), 
126.82 (d, J=4 Hz), 70.64 (t, J=7 Hz), 52.69 (d, J=7 Hz), 52.31, 52.25, 
52.06 (d, J=7 Hz), 33.69 (d, J=140 Hz), 33.62 (d, J=139 Hz), 19.34 (d, J=4 
Hz), 18.81 (d, J=6 Hz). .sup.31 P-NMR .differential.: 28.99, 28.44. MS 
(FAB.sup.+,m/z): 272. Anal. Calcd. for C.sub.12 H.sub.17 0.sub.5 
P.0.5H.sub.2 O: C, 51.24; H, 6.46; P, 11.01. Found: C, 51.17; H, 6.46; 
methyl L-2-[(methyl benzylphosphoryl)oxy]-3-phenylpropionate: .sup.13 C-NMR 
(CDCl.sub.3) .differential.: 170.59, 135.72 (d, J=7 Hz), 129.86 (d, J=7 
Hz), 129.69 (d, J=7 Hz), 129.50, 128.50, 128.43, 128.34, 127.13, 127.01, 
126.86 (d, J=3 Hz), 126.74 (d, J=4 Hz), 75.80 (d, J=7 Hz), 74.94 (d, J=7 
Hz), 52.73 (d, J=8 Hz), 52.39, 52.26, 51.53 (d, J=7 Hz), 39.23, 39.15, 
39.09, 33.71 (d, J=140 Hz), 33.33 (d, J=140 Hz). .sup.31 p-NMR 
.differential.: 28.89. MS (FAB.sup.+, m/z): 348. Anal. Calcd. for C.sub.11 
H.sub.17 0.sub.3 P.0.5H.sub.2 O: C, 60.49; H, 6.22; P, 8.67. Found: C, 
60.49; H, 6.22; 
methyl L-2-[(methyl benzylphosphoryl)oxy]-3-methylbutyrate: .sup.13 C-NMR 
(CDCl.sub.3) .differential.: 170.72 (d, J=6 Hz), 131.54 (d, J=10 Hz), 
131.04 (d, J=9 Hz), 129.92 (d, J=6 Hz), 129.80, 128.53 (d, J=3 Hz), 128.36 
(d, J=3 Hz), 126.98 (d, J=4 Hz), 126.75 (d, J=4 Hz), 79.01 (d, J=7 Hz), 
78.63 (d, J=7 Hz), 53.09 (d, J=7 Hz), 52.29 (d, J=7 Hz), 52.44, 52.02, 
33.77 (d, J=139 Hz), 33.50 (d, J=141 Hz), 31.57, 31.49, 18.64, 18.27, 
16.76, 16.49. .sup.31 P-NMR .differential.: 29.17, 28.47. MS (FAB.sup.+, 
m/z): 300; 
methyl 
L-2-[[methyl[1-[N-(benzyloxycarbonyl)amino]-methyl]-phosphoryl]oxy]propion 
ate: .sup.13 C-NMR (CDCl.sub.3) .differential.: 171.76, 171.23, 156.18, 
156.10, 136.26, 136.14, 128.44, 128.41, 128.15, 128.03, 71.00, 70.91, 
70.82, 67.18, 67.10, 53.71 (d, J=7 Hz), 52.71, 52.46, 52.27 (d, J=6 Hz), 
36.86 (d, J=160 Hz), 36.75 (d, J=157 Hz), 19.12, 19.05. .sup.31 P-NMR 
.differential.: 25.51, 24.01. MS (FAB.sup.+, m/z): 346. Anal. Calcd. for 
C.sub.11 H.sub.17 0.sub.3 P: C, 48.70; H, 5.84; N, 4.06; P, 8.97. Found: 
C, 48.88; H, 6.08; N, 3.94; P, 8.87; 
methyl 
L-2-[[methyl[1-[N-(benzyloxycarbonyl)-amino]methyl]-phosphoryl]oxy]-3-phen 
ylpropionate: .sup.13 C-NMR (CDCl.sub.3) .differential.: 170.99, 170.25, 
156.14 (d, J=7 Hz), 155.87 (d, J=8 Hz), 136.25 (d, J=13 Hz), 135.67 (d, 
J=6 Hz), 129.41, 129.29, 128.80, 128.61, 128.17, 128.19, 128.08, 127.48, 
127.21, 75.33 (d, J=7 Hz), 75.14 (d, J=6 Hz), 67.18, 67.13, 53.04 (d, J=7 
Hz), 52.83, 52.58, 51.69 (d, J=7 Hz), 39.03 (d, J=6 Hz), 38.92 (d, J=6 Hz) 
37.10 (d, J=160 Hz), 36.54 (d, J=160 Hz). .sup.31 P-NMR .differential.: 
25.24, 24.45. MS (FAB.sup.+, m/z): 422. Anal. Calcd. for C.sub.11 H.sub.17 
0.sub.3 P: C, 57.00; H, 5.75; N, 3.32; P, 7.35. Found: C, 56.65; H, 6.07 ; 
N, 3.41; 
methyl 
L-2-[[methyl[1-[N-(benzyloxycarbonyl)-amino]methyl]phosphoryl]oxy]-3-methy 
lbutyrate: .sup.13 C-NMR (CDCl.sub.3) .differential.: 171.25, 170.49, 
156.25, 156.17, 156.05, 136.35, 136.16, 133.35, 133.20, 128.48, 128.45, 
128.18, 128.07, 79.20 (d, J=8 Hz), 79.05 (d, J=6 Hz), 67.23, 67.11, 53.38 
(d, J=7 Hz), 52.56, 52.47, 52.27, 36.93 (d, J=158 Hz), 36.64 (d, J=160 
Hz), 31.40, 31.30, 18.81, 18.51, 16.47, 16.21. .sup.31 P-NMR 
.differential.: 25.88, 24.31. MS (FAB.sup.+, m/z): 374; 
methyl 
L-2-[[methyl[(R)-1-[N-(benzyloxycarbonyl)-amino]ethyl]-phosphoryl]oxy]prop 
ionate: .sup.13 C-NMR (CDCl.sub.3) .differential.: 171.35, 171.31, 155.65, 
155.55, 155.52, 136.34, 136.17, 128.43, 128.37, 128.13, 128.01, 127.95, 
70.97 (d, J=8 Hz), 70.57 (d, J=7 Hz), 67.04, 66.93, 53.20 (d, J=7 Hz), 
52.64, 52.42, 52.29, 43.66 (d, J=160 Hz), 43.38 (d, J=169 Hz), 19.13 (d, 
J=4 Hz), 18.94 (d, J=6 Hz), 16.27, 15.88. .sup.31 P-NMR .differential.: 
27.76, 26.96. MS (FAB.sup.+, m/z): 360. Anal. Calcd. for C.sub.11 H.sub.17 
0.sub.3 P: C, 50.13; H, 6.18; N, 3.90; P, 8.62. Found: C, 49.79; H, 6.23; 
N, 4.14; 
methyl 
L-2-[[methyl[(R)-1-[N-(benzyloxycarbonyl)-amino]ethyl]-phosphoryl]oxy]-3-p 
henylpropionate: .sup.13 C-NMR (CDCl.sup.13) .differential.: 170.92, 
170.89, 170.32, 155.47, 155.36, 136.42, 136.20, 135.68, 135.39, 129.45, 
129.37, 128.64, 128.51, 128.43, 128.37, 128.10, 127.96, 127.93, 127.30, 
127.12, 75.34 (d, J=7 Hz), 74.87 (d, J=7 Hz), 66.99, 66.86, 53.25 (d, J=7 
Hz), 52.71 52.40, 51.69 (d, J=7 Hz), 43.81 (d, J=160 Hz), 43.37 (d, J=160 
Hz), 39.12, 39.03, 38.96, 16.36, 15.58. .sup.31 P-NMR .differential.: 
27.51, 26.77. MS (FAB.sup.+, m/z): 436. Anal. Calcd. for C.sub.11 H.sub.17 
0.sub.3 P.0.2H.sub.2 O: C, 57.44; H, 6.07; N, 3.19; P, 7.05. Found: C, 
57.44; H, 6.30; N, 3.19; 
methyl 
L-2-[[methyl[(R)-1-[N-(benzyloxycarbonyl)-amino]ethyl]-phosphoryl]oxy]-3-m 
ethylbutyrate: .sup.13 C-NMR (CDCl.sub.3) .differential.: 171.14, 171.10, 
170.67, 155.73, 136.48, 136.25, 128.17, 128.41, 128.14, 127.98, 79.18 (d, 
J=9 Hz), 78.75 (d, J=7 Hz), 67.04, 66.92, 53.56 (d, J=7 Hz), 52.71 (d, J=7 
Hz), 52.50, 52.24, 43.98 (d, J=159 Hz), 43.38 (d, J=160 Hz), 31.53, 31.45, 
31.39, 18.78, 18.48, 16.56, 16.51, 16.31, 16.21.31. .sup.31 P-NMR 
.differential.: 28.07, 27.10. MS (FAB+,m/z): 388. Anal. Calcd. for 
C.sub.11 H.sub.17 0.sub.3 P: C, 52.71; H, 6.77; N, 3.62; P, 8.00. Found: 
C, 52.33; H, 6.84; N, 3.71; 
methyl 
L-2-[[methyl[(R,S)-1-[N-(benzyloxycarbonyl)-amino]-2-methylpropyl]phosphor 
yl]oxy]propionate: .sup.13 C-NMR (CDCl.sub.3) .differential.: 171.39, 
171.36, 171.31, 156.34, 156.25, 156.17, 136.33, 136.28, 136.22, 136.15, 
128.37, 128.05, 127.91, 127.84, 70.80, 70.69, 70.57, 70.49, 70.41, 70.35, 
67.07, 67.01, 66.95, 54.06, 54.00, 53.81, 53.07, 52.98, 52.77, 52.67, 
52.44, 52.41, 52.29, 52.13, 52.03, 51.97, 51.76, 29.17, 29.11, 29.06, 
28.95, 28.89, 28.78, 28.72, 20.35, 20.30, 20.22, 20.13, 19.48, 19.44, 
19.24, 19.19, 19.02, 18.93, 18.84, 17.81, 17.71, 17.65, 17.56. .sup.31 
P-NMR .differential.: 27.08, 26.13, 26.03; 
methyl 
L-2-[[methyl[(R,S)-1-[N-(benzyloxycarbonyl)-amino]-2-methylpropyl]phosphor 
yl]oxy]-3-phenylpropionate: .sup.13 C-NMR (CDCl.sub.3) .differential.: 
170.35, 156.22 (m), 136.29 (m), 135.38 (m), 129.47, 129.41, 128.51, 
128.44, 128.17, 128.10, 128.05, 127.99, 127.90, 127.18, 127.10, 74.94 (m), 
67.09 (m), 54.04 (m), 53.19 (m), 52.30 (m), 39.19 (m), 29.18 (m), 20.35 
(m), 17.78 (m). .sup.31 P-NMR .differential.: 27.09, 26.85, 26.15, 26.00; 
methyl 
L-2-[[methyl[(R,S)-1-[N-(benzyloxycarbonyl)-amino]-2-methylpropyl]phosphor 
yl]oxy]-3-methylbutyrate: .sup.13 C-NMR (CDCl.sub.3) .differential.: 
170.78, 156.52, 156.42, 136.50, 128.46, 128.40, 128.16, 128.01, 127.96, 
127.89, 78.77 (m), 67.03 (m), 54.40 (m), 52.35 (m), 31.48 (m), 29.28 (m), 
20.52 (m), 18.70 (m), 17.79 (m), 16.57 (m). .sup.31 P-NMR .differential.: 
27.64, 27.24, 26.48, 26.08. Anal. Calcd. for C.sub.11 H.sub.17 0.sub.3 P: 
C, 54.93; H, 7.28; N, 3.37; P, 7.46. Found: C, 55.19; H, 7.24; N, 3.36. 
EXAMPLE 2 
Preparation of Phosphonate Diesters 
2.1 Method A 
To a solution of benzylphosphonic acid (0.5 mmol), an alcohol, and 
triphenylphosphine (1.25 mmol) in anhydrous THF (5 mL), was added 
diisopropylazodicarboxylate (1.25 mmol). After 30 minutes, the reaction 
mixture was concentrated under reduced pressure. The 
triphenylphosphineoxide was crystallized with acetone/pentane and removed 
by filtration. The filtrate was concentrated under reduced pressure and 
purified by chromatography (elution with acetic acid/ethyl acetate) to 
yield a phosphonate diester. 
2.2 Method B 
To a solution of an alcohol and triphenylphosphine (1.25 mmol) in anhydrous 
THF (5 mL), was added diisopropylazodicarboxylate (1.25 mmol). To the 
reaction mixture was then added a solution of benzylphosphonic acid (0.5 
mmol) in anhydrous THF. After 30 minutes, the reaction mixture was 
concentrated under reduced pressure. The triphenylphosphineoxide was 
crystallized with acetone/pentane and removed by filtration. The filtrate 
was concentrated under reduced pressure and purified by chromatography 
(elution with acetic acid/ethyl acetate) to yield a phosphonate diester. 
EXAMPLE 3 
Synthesis of Phosphonic Acid Building Blocks from Amino Acids 
3.1 Preparation of Fmoc-protected Methyl Hydrogen 
[(.+-.)-N-(fluorenylmethoxycarbonyl)-1-aminoethyl]phosphonate 
To an ice-cold solution of lead tetraacetate (34.1 g, 105 mmol) in 100 mL 
of dimethylformamide (DMF), was added dropwise a solution of CBz-alanine 
(20.0 g, 89.6 mmol) in 50 mL of DMF. The dark brown reaction mixture was 
allowed to warm to room temperature and then stirred for 8 hours. The now 
clear reaction mixture was diluted with saturated sodium bicarbonate (150 
mL), extracted with ethyl acetate (4.times.100 mL), and the combined 
organic layers washed with saturated sodium bicarbonate (1.times.100 mL) 
and then brine (1.times.100 mL). The organic layer was dried over 
anhydrous magnesium sulfate and then concentrated to afford 18.52 g of a 
white solid (93%). 
To the acetate (18.52 g, 83.3 mmol) in 333 mL of dichloromethane was added 
trimethylphosphite, the solution was cooled to -78.degree. C., and 1.0M 
titanium tetrachloride in dichloromethane solution (100 mL, 100 mmol) was 
added dropwise over 3 minutes. The reaction was allowed to warm to room 
temperature and then stirred overnight. The reaction was quenched by the 
addition of solid sodium carbonate (41.7 g, 146 mmol), stirred for 2 
hours, and then filtered through a glass wool plug. The filtrate was 
washed with water (4.times.100 ml), brine (1.times.100 mL), dried over 
magnesium sulfate, filtered, concentrated to a light yellow oil, and then 
purified by silica gel chromatography (8:2 ethyl acetate/hexane) to afford 
14.06 g of a light yellow oil (59%). 
To a solution (10.0 g, 35 mmol) of the light yellow oil (dimethyl 
[1-[N-(benzyloxycarbonyl)amino]-ethyl]phosphonate) in ethyl alcohol (218 
mL), was added 10M aqueous sodium hydroxide (8.75 mL, 87.5 mmol), and the 
reaction heated to reflux for 90 minutes. The reaction was cooled, diluted 
with water (50 mL), acidified to pH=2 with concentrated HCl, and then 
extracted with ethyl acetate (3.times.50 mL). The combined organic layers 
were dried over magnesium sulfate, filtered, and then concentrated to 
afford 6.73 g of an oily semi-solid (70%). 
To a solution (1.0 g, 3.60 mmol) of the oily semi-solid (methyl 
[1-[N-(benzyloxycarbonyl)amino]-ethyl]phosphonate) in methanol (18 mL) 
cooled to 0.degree. C., was added 10% Pd/carbon (100 mg) and a balloon of 
hydrogen gas. The reaction was warmed to room temperature and stirred 
overnight. The solution was then filtered through a sintered glass funnel 
and then concentrated to a solid. To some of this solid (100 mg, 0.92 
mmol), was added dioxane (1.9 mL), 10% aqueous sodium bicarbonate (1.9 
mL), and a solution of 9-fluorenylmethyloxycarbonyl (Fmoc) chloroformate 
(310 mg, 1.2 mmol) in 1 mL of dioxane, and the slightly turbid reaction 
mixture was stirred overnight. The now clear reaction mixture was diluted 
with water and then acidified to pHI with concentrated HC1. Upon standing, 
a white precipitate formed. This precipitate was collected by filtration 
and dried in vacuo to afford 100 mg of an off-white solid (30%). 
3.2 Preparation of Methyl Hydrogen 
[(.+-.)-N-(phenylmethoxycarbonyl)-1-amino-2-methylpropyl]phosphonate 
To a suspension of lead tetraacetate (6.537 g, 14.7 mmol) in dry 
N,N-dimethylformamide (20 mL) under argon at 0.degree. C., was added 
N-(carbobenzyloxy)-L-valine (3.082 g, 12.3 mmol). After 1 hour, cooling 
was stopped and stirring continued an additional 3 hours at room 
temperature. The reaction was quenched with saturated NaHCO.sub.3 (100 mL) 
and extracted with ethyl acetate (4.times.30 mL). The organic layers were 
combined, washed with saturated NaHCO.sub.3 (25 mL), then with H.sub.2 O 
(25 mL), and then brine (25 ml), and then dried over MgSO.sub.4, filtered, 
and concentrated under vacuum. 
The resulting oil was dissolved in methylene chloride (20 mL), then 
trimethylphosphite (2.2 mL, 18.7 mmol) was added, and the mixture was 
cooled to -78.degree. C. Then, TiC14 (15 mL, 15 mmol), as a 1M solution in 
methylene chloride, was added to the mixture. The reaction was warmed to 
room temperature, and after 12 hours, the reaction mixture was cooled to 
0.degree. C., quenched with Na.sub.2 CO.sub.3 (35 g, 120 mmol), and then 
stirred at room temperature for 30 minutes. The mixture was diluted with 
H.sub.2 O (100 mL), extracted with CH.sub.2 Cl.sub.2 (4.times.25 mL), and 
the combined organic layers were then washed with H.sub.2 O (25 ml) and 
brine (2.times.25 mL), dried over MgSO.sub.4, filtered, then concentrated 
to afford 3.855 g (99%) of a colorless oil. 
EXAMPLE 4 
Synthesis of Phosphonic Acid Building Blocks from Aminophosphonic Acids 
4.1 Preparation of N-phenylmethoxycarbonyl-amino Methylphosphonic Acid 
To aminomethylphosphonic acid (500 mg, 4.50 mmol) in 1M aqueous sodium 
hydroxide (11.5 mL, 11.5 mmol) cooled to 0.degree. C., was added 
benzyoxycarbonyl chloroformate (845 mg, 5.00 mmol, 0.71 mL) dropwise over 
one minute and the reaction stirred overnight. The reaction mixture was 
then extracted with ethyl ether (3.times.25 mL), the aqueous layer 
strongly acidified with concentrated HCl, and then extracted with ethyl 
acetate (3.times.25 mL). The organic layers were combined, dried 
(MgSO.sub.4), filtered, and then concentrated to afford 700 mg of a white 
solid (63%). 
4.2 Preparation of N-nitroveratryloxycarbonyl-aminomethylphosphonic Acid 
To aminomethylphosphonic acid (500 mg, 4.50 mmol) in 5% aqueous sodium 
bicarbonate (22.5 mL, 13.5 mmol), was added dioxane (20 mL) and then 
nitroveratryloxycarbonyl (NVOC) chloroformate (5.00 mmol, 1.36 g) in 20 mL 
of dioxane. The reaction was stirred overnight, extracted with ethyl ether 
(3.times.40 mL), and then the aqueous layer was strongly acidified with 
concentrated HCl. Upon standing, fine yellow needles formed and were 
filtered and dried in vacuo to afford 1.35 g of product (86%). 
EXAMPLE 5 
Preparation of a-Hydroxy Acids from .alpha.-Amino Acids 
5.1 Method A 
To D-phenylalanine (2.00 g, 12.4 mmol) in water (20 mL), was added 3M HCl 
(20 mL, 60 mmol) and acetic acid (10 mL). The solution was cooled to 
0.degree. C., and an aqueous solution of sodium nitrite (3.92 g, 56.8 
mmol, in 7.5 mL of water) was added dropwise over 10 minutes. The reaction 
was stirred for eight hours, then more sodium nitrite was added (3.92 g, 
56.8 mmol, in 7.5 mL of water). After six hours, the solution was washed 
with ether (3.times.25 mL), and the combined organic layers were extracted 
with saturated sodium bicarbonate (3.times.30 mL). The bicarbonate washes 
were combined, acidified to pH 1 with concentrated HCl, and then extracted 
with ether (3.times.30 mL). The organic layers were combined, dried 
(magnesium sulfate), filtered, and then concentrated to afford 1.65 g of a 
white waxy solid (80%). 
5.2 Preparation of (D)-O-nitroveratryloxy-methoxy-2-oxy-4-methylpentanoic 
Acid 
The hydroxy acid prepared above was esterified using diazomethane, and to 
the resulting compound, methyl (D)-2-hydroxy-4-methyl valerate (1.0 g, 
6.85 mmol) in 10 mL of methylene chloride was added Nvom chloride (2.69 g, 
10.5 mmol) and diisopropylethyleneamine (DIE& 1.8 mL). After 12 hours, the 
reaction mixture was diluted with methylene chloride (20 mL), washed with 
water (2.times.15 mL), and the organic layer dried (over MgSO.sub.4). The 
product was purified by silica gel chromatography (4:1 petroleum 
ether:ethyl acetate) to afford 1.2 g of material (47%). The free acid was 
obtained in quantitative yield by hydrolysis with lithium hydroxide, as 
previously described. One could also prepare this compound, with possibly 
higher yield and shorter reaction time, using sodium hydride/THF instead 
of DIEA/dichloromethane. 
5.3 Preparation of Side Chain Functionalized .alpha.-O-FMOC-Hydroxy Acids 
To a solution of the hydroxy acid prepared above (with the side chain 
functionality protected, 1 mmol) in dry pyridine at 0.degree. C. is added 
N-methylmorpholine (2.5 mmol) and triisopropylsilyl chloride (1.1 mmol). 
The solution was stirred for 15 minutes. To the reaction mixture is then 
added FMOC-Cl (1.1 mmol) and additional Nomethylmorpholine. The reaction 
is stirred for 4-8 hours until complete. To the mixture is then added 
tetrabutylammonium fluoride (2.5 mmol). The reaction mixture is stirred 
for 15 minutes and quenched. 
EXAMPLE 6 
Synthesis of a Peptidylphosphonate: Preparation of Z-Phe.sup.p (O)-Leu-Ala 
6.1 Coupling (D)-Nvom-2-oxy-4-methylpentanoic Acid to Ala Resin 
To alanine-derivatized resin (1.00 g, 534 gmol) swollen in DMF (5 mL) 
was added Nvom-2-oxy-4-methylvaleric acid (1.5 mmol, 537 mg), HBTu (1.5 
mmol, 720 mg), HOBt (1.5 mmol, 229 mg), and DIEA (3.75 mmol, 650 .mu.L), 
and the suspension was shaken for one hour. The resin was washed with DMF 
(4.times.10 mL), suspended in 30 mL of DMF, and photolyzed at 360 nm (10 
mW) for 24 hours. The resin was washed with DMF (4.times.10 mL) and then 
with methanol (4.times.10 mL) and then dried in vacuo. 
6.2 Coupling Methyl Hydrogen 
[(.+-.)-N-(phenylmethoxy-carbonyl)-1-aminoethyl]phosphonate to 
2-hydroxy-4-methylvaleryl Alanine Resin Preparation of (.+-.)CBz-Phe.sup.P 
-(O)-Leu-Ala 
To 2-hydroxy-4-methylvalerylalanine resin (50 mg, 26.5 .mu.gmol) in THF was 
added methyl hydrogen 
[(.+-.)-N-(phenylmethoxycarbonyl)-1-aminoethyl]phosphonate (70 mg, 260 
.mu.mol), TPP (65 mg, 260 .mu.mol), and DIAD (50 mg, 260 .mu.mol). 
Additional equivalents of TPP and DIAD were added at two 15 minute 
intervals, and the reaction mixture shaken for two hours. The resin was 
then washed with DMF (3.times.10 mL) and then with methanol (3.times.10 
mL) and then dried in vacuo. The peptidylphosphonate was cleaved from the 
resin by treatment with 0.5 mL of TFA for 30 minutes. 
EXAMPLE 7 
Solid Phase Synthesis of Peptidylphosphonates 
7.1 General Procedure 
The methyl N-NPEOC-aminoalkylphosphonate (100 .mu.mole), tris 
(4-chlorophenyl)phosphine (100 .mu.mole) and diethylazodicarboxylate (100 
.mu.mole) were pre-activated in anhydrous THF (900 .mu.L) for 5 minutes. 
The mixture was then added to a resin (previously dried under vacuum in 
the presence of phosphorous pentoxide) containing a terminal hydroxyl 
group (.ltoreq.20 .mu.mole) that had been suspending in anhydrous THF (100 
.mu.L) and DIEA (200 .mu.mole). The mixture was sonicated at 25.degree. C. 
until the reaction was complete. The resin was then washed with NMP and 
ether and dried. 
7.2 NPEOC-Deprotection and Coupling Efficiency Determination 
The resin was treated with 5% DBU in NMP (1 mL) for 2 hours. The released 
4-nitrostyrene was spectroscopically measured at 308 nm 
(.epsilon.=1.32.times.10.sup.4) to determine the coupling efficiency. The 
resin was washed with NMP and ether. 
7.3 Capping 
The resin was treated with commercially available capping reagents until 
negative ninhydrin tests were obtained. 
7.4 Peptide Elongation 
Uncapped peptidylphosphonate was further elongated using standard HBTU/HOBT 
amino acid coupling chemistry. 
7.5 Deprotection and Cleavage of the Peptidylphosphonate 
The resin bound peptidylphosphonate was treated with a 1:2:2 mixture of 
thiophenol:triethylamine:dioxane (1 mL) for 2 hours. However, if the 
phosphoryl methoxide group was to be retained, this treatment was not 
performed. Following washes with NMP, methanol, and ether, the 
peptidylphosphonate was treated with trifluoroacetic acid containing the 
appropriate scavengers (1 mL) for 1 hours. 
7.6 Half-life Coupling Time Determination 
Equal amounts of resin containing a terminal .alpha.-hydroxylalkyl acid 
were placed in separate containers then treated with the appropriate 
methyl N-NPEOC-aminoalkylphosphonate using the general procedure described 
above. After appropriate time intervals the resin in one container is 
washed as above then treated with 5% DBU/NMP and the coupling yield was 
determined. The coupling yields form each container at different time 
intervals was then plotted and the pseudo-first order rate constant 
calculated and used to determine the coupling time half-life. 
7.7 Results 
The following peptidylphosphonates, including both the phosphoryl methoxide 
and the phosphonic acid forms, have been synthesized and characterized: 
Z-(R,S)-Phe.sup.P (O)-Leu-Ala-OH; Z-Gly.sup.P (O)-Leu-Ala-OH; 
Z-(R)-Ala.sup.P (O)-Leu-Ala-OH; Z-(R,S)-Leu.sup.P (O)-Leu-Ala-OH; 
Z-(R,S)-Val.sup.P (O) -Leu-Ala-OH, where Z represents CBz. The following 
phosphonic acids were also synthesized and characterized: 
Z-(R,S)-Phe.sup.P (O)-Leu-Trp-OH; and Z-(R,S)-Phe.sup.P (O)-Leu-Leu. 
The K.sub.i values for thermolysin for the following peptidylphosphonates 
have also been determined: Z-Gly.sup.P (O)-Leu-Ala-OH, 13 .mu.M; 
Z-(R)-Ala.sup.P (O)-Leu-Ala-OH, 1.8 .mu.M; Z-(R,S)-Leu.sup.P 
(O)-Leu-Ala-OH, 680 nm/42 .mu.M (L/D isomers); and Z-(R,S)-Phe.sup.P 
(O)-Leu-Ala-OH, 45 nM/30 .mu.M (L/D isomers). 
7.8 Peptidylphosphonate Combinatorial Library Preparations 
The protocol shown in FIG. 10 using the monomer basis set shown in FIG. 13 
was used to prepare a library of N-CBz capped peptidylphosphonates bound 
to Tentagel resin. This protocol is based on the "divide and pool" 
technology described by Furka et al. (1991) Int. J. Pept. Protein Res. 
37:487, which is incorporated herein by reference. 
EXAMPLE 8 
Assay Protocols 
8.Depletion Assays 
Resin bound inhibitor was pre-incubated with the enzyme thermolysin (100 
nM). The support with resin bound inhibitor/enzyme complex was removed by 
filtration. An aliquot of the filtrate was treated with 
N-(3-[2-furyl]acryloyl)-Glu-Leu amide (1 mM). The turnover rate was 
analyzed in an attempt to rank order the binding affinities of the 
peptidylphosphonates. These results for a partial characterization of 
thermolysin's interactions with the peptidylphosponate libraries prepared 
in Example 7.8 above are shown in FIG. 14. 
8.2FITC-Thermolysin Binding Assay 
The binding assay described above can be conducted using the FACS 
instrument to quantify the relative fluorescence activity. This format 
does require the use of small diameters with narrow size distribution.