BACKGROUND OF THE INVENTION

The present invention is related to the area of chemical synthesis. More specifically, one embodiment of the present invention provides a method for the preparation of Fmoc-protected amines, and more particularly, for the preparation of Fmoc-protected amino acids.

Chemical protecting groups are used during synthesis reactions to temporarily protect certain functional groups on a compound against undesired reactions. When a reaction sequence is complete, and protection is no longer necessary, the protective group is removed to restore the protected functional group to its natural activity. Protective groups are removed by various procedures such as exposure to acidic or basic conditions or electromagnetic radiation (e.g., light of a prescribed wavelength).

A particularly preferred protecting group for amines and particularly, amino acids, is the 9-fluorenylmethoxycarbonyl (Fmoc) group. The Fmoc group was first introduced for the protection of an alpha-amino function of an amino acid and it was removable through the treatment of liquid ammonia in 10-12 hours at room temperature (see Carpino and Han (1970) T. Am. Chem. Sor. 92:5748); subsequently, other deprotection methods have been used. Greene et al (1991) Protective Groups in Organic Synthesis. 2nd Ed. (John Wiley & Sons, Inc. _, New York). The chemistry of its deprotection centers on the acidic nature of the proton on the beta-carbon atom, and hence, upon its abstraction by base.

A number of methods are known in the literature for preparing Fmoc-protected amino acids.

For example, Carpino supra; describes the use of Fmoc-Cl with a carbonate base in dioxane/water. Likewise, Sigler et al. (1983) Biopolymers 22:2157 describes the use of Fmoc-OSu with a carbonate base in dioxane/water. Ten Kortenaar et al. (1986) Intl. T. Pept. Prot. Res. 27:398 discusses the use of Fmoc-OSu with triethylamine in water /acetonitrile. Significantly, each of these methods rely upon the use of aqueous reaction conditions.

Bolin et al. (1989) Int. T. Pept. Prot. Res. 33:353 describes the use of trimethylsilyl chloride to convert amino acids into the corresponding 0,N- bis(trimethylsilyl)amino acids and subsequently, through treatment with Fmoc-Cl, into the desired Fmoc-protected amino acid. Although the reaction is conducted in methylene chloride with diisopropyl ethyl amine, this latter procedure also suffers the disadvantage that salt removal must be accomplished with an aqueous workup.

The use of a photolabile molecule as a linker to couple compounds to solid supports and to facilitate the subsequent cleavage reaction has received considerable attention during the last two decades. Photolysis offers a mild method of cleavage which complements traditional acidic or basic cleavage techniques. See, e.g., Lloyd-Williams et al. (1993) Tetrahedron 49:11065-111.33 The rapidly growing field of combinatorial organic synthesis (see, e.g., Gallop et al. (1994) I. Med. Chem. 37:1233-1251; and Gordon et al. (1994) T. Med. Chem. 37:1385- 1401) involving libraries of peptides and small molecules has markedly renewed interest in the use of photolabile linkers for the release of both ligands and tagging molecules.

A preferred photolinker is described in Holmes and Jones (1995) lournal of Organic Chemistry 60:2318: U.S. Serial No. 08/493,877, filed June 23, 1995, U.S. Serial No. 08/374,492, filed January 17, 1995, and PCT application US 95/07983, each of which is incorporated herein by reference, and has the formula:

wherein,

R ⁶ is hydrogen, Ci -Cs alkyl, aryl or arylalkyl; R^, R ⁴ and R^ are each independently hydrogen, Cj-Cβ alkyl, or Ci-Cs alkoxy; X ¹¹ and Y^ ¹ are each independently selected from the group consisting of halogen, — COOH, — SH, — SP, —OH, — OP, — NH2, — NHP, in which P is a suitable protecting or activating group, and — R^Rδ wherein R? and R * are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, aryl alkyl, substituted aryl alkyl, heteroaryl, and substituted heteroaryl; and q is an integer of from 1 to 10, and preferably, from 1 to 4.

In the scale up development work for the production of the photolinker 2 from its amino acid precursor 1,

MeO

practical difficulties were encountered when using traditional Fmoc-procedures The product 2 forms a gel in the presence of water which makes it extremely difficult to carry out the Fmoc-reaction with good control over the stirring and the pH-measurement Also the isolation of the product proved to be extremely time- consuming due to the fact that the filtration of the gel was very slow and centrifugation proved to be extremely inefficient

Thus, there is a need for a method of preparing Fmoc-protected amines under neutral, anhydrous conditions This invention fulfills this and other needs

SUMMARY OF THE INVENTION The present invention provides new methods for the preparation of Fmoc-protected amines. These methods involve the use of mild conditions and result in the production of high yields of Fmoc-protected amines, and particularly amino acids, having high chemical and optical purity. The methods can be conducted under anhydrous conditions when necessary.

More specifically, an amine component is treated with a silylating agent and then with an activated Fmoc-reagent to yield the Fmoc-protected amine. Preferably, the silylating agent is selected from the group consisting of N-methyl- N-(tπmethylsilyl)trifluoracetamide (MSTFA), N,0-bis(tnmethylsilyl)acetamide (BSA), N-methyl-N-(trimethylsilyl)acetamιde (MSA), N,0-bιs (tπmethylsilyl)trifluoroacetamide (BSTFA) and bistrimethyl silylurea (BSU) More preferably, N-methyl-N-(trimethylsilyl)trifluoracetamide (MSTFA) or N,0- B_s(tr_methylsilyl)acetamide (BSA) is used More preferably, at least two silylating equivalents of silylating agent are used.

Preferred activated Fmoc-reagents mclude the acid chloride derivative of Fmoc (Fmoc-Cl); Fmoc-N-pentafluorophenyl ester (Fmoc-OPfp) and N-(9-fluorenylmethoxycarbonyloxy)succιnιmide (Fmoc-OSu) A particularly preferred activated Fmoc-reagent is Fmoc-OSu

The amine component preferably is a primary or secondary amine having the formula -NR'R", where R' and R" are independently selected from

the group consisting of hydrogen alkyl, alkoxy, amino, aryl, aryloxy, heteroaryl, and arylalkyl or salts thereof, provided that either R' or R" is hydrogen. More preferably, the amine component will have the formula:

wherein Rl and R^ are independently selected from the groups consisting of hydrogen, alkyl, alkoxy, aryl, aryloxy, heteroaryl, carboxyl, carboxyalkyl, and arylalkyl. More preferably, the amine component will comprise an amino acid or peptide.

Most preferably, the amine component will have the formula:

wherein,

R6 is hydrogen, C^-Cg alkyl, aryl or arylalkyl; R^, R4 _anc j R5 _{are eacn} independently hydrogen, Cj-Cg alkyl, or C\-CQ alkoxy; X^l and γll are each independently selected from the group consisting of halogen, — COOH, — SH,

— SP, — OH, — OP, — H2, — NHP, in which P is a suitable protecting or activating group, and — NR^R^ wherein R? and R^> are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, aryl

alkyl, substituted aryl alkyl, heteroaryl, and substituted heteroaryl; and q is an integer of from 1 to 10, and preferably, from 1 to 4

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 PREFERRED EMBODIMENT I. Terminology Unless otherwise stated, the following terms used in the specification and claims have the meanings given below:

"Activated reagent" refers to those compounds which have been modified to render the compound more reactive toward covalent bond formation with another functional group or reactive site as compared with the unmodified compound. For example, activated Fmoc-reagents include those compounds which the carboxyl group has been modified to produce an anhydride, acid chloride, or other reactive species. Preferred activated Fmoc-reagents mclude the acid chloride derivative (Fmoc-Cl); Fmoc-N-pentafluorophenyl ester (Fmoc-OPfp) and N-(9-fluorenylmethoxycarbonyloxy)succinιmide (Fmoc-OSu). Other activating groups are known to those of skill in the art.

"Alkoxy" refers to the group alkyl-O-.

"Alkyl" refers to a cyclic, branched, or straight chain chemical group containing only carbon and hydrogen, such as methyl, heptyl, -(CH2)2", and adamantyl. Alkyl groups can either be unsubstituted or substituted with one or more substituents, e.g., halogen, alkoxy, acyloxy, ammo, aryl, 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 When "alkyl" or "alkylene" is used to refer to a linking group or a spacer, it is taken to be a group having two available valences for covalent attachment, for example, — CH CH ₂ — , — CH ₂ CH ₂ CH ₂ — — CH ₂ CH ₂ CH(CH3)CH2— and —

CH2(CH2CH2)2CH2 — Preferred alkyl groups as substituents are those containing

1 to 10 carbon atoms, with those containing 1 to 6 carbon atoms (i.e., lower alkyl groups) being particularly preferred Preferred alkyl or alkylene groups as linking groups are those containing 1 to 20 carbon atoms, with those containing 3 to 6 carbon atoms being particularly preferred "Ammo ^' or "amine group" refers to the group -NR'R", where R and

R" are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, aryl alkyl, substituted aryl alkyl, heteroaryl, and substituted heteroaryl In a primary amino group, both R' and R" are hydrogen, whereas m a secondary amino group, either, but not both, R' or R" is hydrogen

An "o -amino acid" consists of a carbon atom, called the α-carbon, to which is bonded an ammo group and a carboxyl group. Typically, this α-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 chains of naturally occurring amino acids are well known m the art and include, for example, hydrogen (as in glycine), alkyl [as in alanine (methyl), valine (isopropyl), leucme (sec-butyl), isoleuαne (iso-butyl), and proline (-(CH2)3-)], substituted alkyl [as in serine (hydroxymethyl), cysteine (thiomethyl), aspartic acid (carboxymethyl), asparagine, arginme, glutamme, glutamic acid, and lysine], aryl alkyl (as in phenylalarune, histidine, and tryptophan), substituted aryl alkyl (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 ammo 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 α-carbon can also be replaced with other groups; those of skill in the art recognize the medicinal importance of α-methyl amino acids and other α-, α-disubs tuted amino acids In addition, the term "amino acid" includes other molecules having both an amino group and a carboxyl group, such as beta-amino acids, gamma-amino acids, the photolinker described in detail below, and the like.

"Protected amino acid" refers to an ammo acid, typically an α-amino acid having either or both the amine functionality and the carboxylic acid functionality suitably protected by one of the groups described above Additionally, for those amino acids having reactive sites or functional groups on a side chain (i.e., seπne, tyrosme, glutamic acid), the term "protected ammo acid ^" is meant to refer to those compounds which optionally have the side chain functionality protected as well.

"Aryl" or "Ar" refers an aromatic substituent which may be a single ring or multiple rings which are fused together, linked covalently or linked to a common group such as an ethylene or methylene moiety. The aromatic rings may each contain heteroatoms, for example, phenyl, naphthyl, biphenyl, diphenylmethyl, 2,2-dipheny 1-1 -ethyl, thienyl, pyridyl and quinoxalyl. The aryl moieties may also be optionally substituted with halogen atoms, or other groups such as nitro, carboxyl, alkoxy, phenoxy and the like. Additionally, the aryl radicals may be attached to other moieties at any position on the aryl radical which would otherwise be occupied by a hydrogen atom (such as, for example, 2-pyridyl, 3- pyridyl and 4-pyridyl).

"Aryloxy" refers to the group aryl-O- or heteroaryl-O-. "Arylalkyl'Or "aralkyl" 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 (for example, benzyl, phenylethyl, 3-(4- nitrophenyl)propyl, and the like). Preferred aryl groups include phenyl, 1- naphthyl, 2-naphthyl, biphenyl, phenylcarboxylphenyl (i.e , derived from benzophenone), and the like.

"Carboxy" or "carboxyl" refers to the group -R'(COOH) where R' is alkyl, substituted alkyl, aryl, substituted aryl, aryl alkyl, substituted aryl alkyl, heterocyclic, heteroaryl, or substituted heteroaryl.

"Carboxyalkyl" refers to the group -(CO)-OR' where R' is alkyl or substituted alkyl.

"Carboxyaryl" refers to the group -(CO)-OR' where R' is aryl, heteroaryl, or substutited aryl or heteroaryl.

"Linker" refers to a molecule or group of molecules attached to a solid support and spacing a synthesized compound from the solid support, such as for exposure /binding to a receptor.

"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 derivative that is stable to the projected reactions for which protection is desired; 2) can be selectively removed from the derivatized solid support 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 al. (1991) Protective Groups in Organic Synthesis. 2nd Ed. Qohn Wiley & Sons, Inc., New York). Preferred protecting groups include photolabile protecting groups (such as methylnitropiperonyloxycarbonyl (Menpoc), methylnitropiperonyl (Menp), nitroveratryl (Nv), nitroveratryloxycarbonyl (Nvoc), or nitroveratryloxymethyl ether (Nvom)); acid-labile protecting group (such as Boc or DMT); base-labile protecting groups (such as Fmoc, F , phosphonioethoxycarbonyl (Peoc, see Kunz (1976) Chem. Ber. 1Q9_:2670); groups which may be removed under neutral conditions (e.g., metal ion-assisted hydrolysis ), such as DBMB (see Chattopadhyaya et al. (1979) T.C.S. Chem. Comm. 987-990), allyl or alloc (see, e.g., Greene and Wuts, "Protective Groups in Organic Synthesis", 2nd Ed., John Wiley & Sons, Inc., New York, NY (1991), 2-haloethyl (see Kunz and Buchholz (1981) Angew. Chem. Int F.H Engl. 20:894), and groups which may be removed using fluoride ion, such as 2- (trimethylsilyl)ethoxymethyl (SEM), 2-(trimethylsilyl)ethyloxycarbonyl (Teoc) or 2- (trimethylsilyl)ethyl (Te) (see, e.g., Lipshutz et al. (1980) Tetrahedron I .P. Γ. ?ι -:v . 3346)); and groups which may be removed under mild reducing conditions (e.g.,

with sodium borohydride or hydrazine), such as Lev Id. at 30-31, 97, and 112 Particularly preferred protecting groups include Fmoc, Fm, Menpoc, Nvoc, Nv, Boc, CBZ, allyl, alloc, Npeoc (4-nιtrophenethyloxycarbonyl) and Npeom (4- nitrophenethyloxy-methyloxy) "Solid support", "support", and "substrate" refer to a material or group of materials having a rigid or semi-rigid surface or surfaces. In many embodiments, at least one surface of the solid support will be substantially flat, although in some embodiments it may be desirable to physically separate synthesis regions for different compounds with, for example, wells, raised regions, pins, etched trenches, or the like. According to other embodiments, the solid support(s) will take the form of beads, resins, gels, microspheres, or other geometric configurations. A preferred solid support is a bead havmg a size is in the range of 1 nm to 100 μm, but a more massive solid support of up to 1 mm in size may sometimes be used. Particularly preferred resins include Sasrin resin (a polystyrene resin available from Bachem Bioscience, Switzerland); and TentaGel S AC, TentaGel PHB, or TentaGel S NH2 resm (polystyrene-polyethylene glycol copolymer resins available from Rappe Polymere, Tubingen, Germany). Other preferred supports are commercially available and described by Novabiochem, La Jolla, California. "Stereospecific reaction" refers to a reaction m 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 m 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.

Isolation and purification of the compounds and intermediates described herem 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 can be had by

references to the examples hereinbelow. However, other equivalent separation or isolation procedures can, of course, also be used

II General

A. The Amme Component

According to the present invention, an amine component is silylated and then treated with an activated Fmoc-reagent to yield a Fmoc protected amine. The amine component can be utilized in a soluble format or can be attached to a solid support. According to the latter embodiment, the amine component will include a functionality which can covalently bind the molecule to the solid support (e.g., an activated carbonyl, acyl halide, or activated hydroxyl) as well as the amino group or a protected derivative thereof.

Typically the amine component will comprise a primary or secondary amme having the formula: -NR'R", where R' and R" are independently selected from the group consisting of hydrogen alkyl, alkoxy, amino, aryl, aryloxy, heteroaryl, and arylalkyl or salts thereof, provided that either R' or R" is hydrogen. More preferably, the amine component will have the formula:

wherein Rl and R^ are independently selected from the groups consisting of hydrogen, alkyl, alkoxy, aryl, aryloxy, heteroaryl, carboxyl, carboxyalkyl, carboxyaryl, and arylalkyl. The amine component, if not commercially available, can be prepared by standard chemical procedures.

In a preferred embodiment, the amine component will comprise an amino acid, and more preferably, an amino acid bearing a substituent on the alpha carbon The amino acids finding utility in the present invention include the twenty naturally occurring α-ammo acids, in either their D- or L-enantiomeπc

forms. Unnatural amino acids such as α, α-disubstituted amino acids, N-al yl amino acids, lactic acid, and other unconventional amino acids are also suitable components. Examples of unconventional amino acids include, but are no: limited to: 4-hydroxyproline, O-phosphoseπne, 3-methylhistidine, 5- hydroxylysine, and other similar amino acids. Since peptides are composed of amino acid subunits, one of skill in the art will appreciate that peptides can also serve as amine components.

In a preferred embodiment, the amine component is a photochemically cleavable linking group which can be represented by the formula:

in which R > is hydrogen, C -Cg alkyl, aryl or arylalkyl; R3, R4 and R5 are each independently hydrogen, C -Cg alkyl, or Cj-Cg alkoxy; χll and Y l are each independently selected from the group consisting of halogen, — COOH, — SK,

— SP, — OH, — OP, — NH2, — NHP, in which P is a suitable protecting or activating group, and — NR^Rδ wherein R? and R^ are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, aryl alkyl, substituted aryl alkyl, heteroaryl, and substituted heteroaryl; and q is ar. integer of from 1 to 10, and preferably, from 1 to 4. These compounds can be prepared using methods known in the art.

In one embodiment, the photocleavable linking groups have the formula:

wherein,

R6 is hydrogen, Cj-Cg alkyl; R ⁴ and R^ are each independently hydrogen, C _} -Cg alkyl or -Cg alkoxy; R^ is Cg alkoxy; X ¹¹ and Y*l are each independently selected from the group consisting of — Br, — Cl, — OH, — OP, — NH2, — NHP, in which P is a suitable protecting or activating group, and —

NR ⁷ Rδ wherein R ⁷ and R^ are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, aryl alkyl, substituted aryl alkyl, heteroaryl, and substituted heteroaryl; and q is an integer of from 1 to 4. Preferably, R6 is hydrogen or methyl, R° and R^ are both hydrogen, R3 is methoxy, γll i _s —OH, and X ¹¹ is — NH2 or — NR ⁷ R8 wherein R ⁷ is hydrogen and R ⁸ is lower alkyl, more preferably, methyl.

In another embodiment, the photocleavable linking groups have the formula:

wherein,

R° is hydrogen, Cj-Cg alkyl; R^ and R are each independently hydrogen,

C^-Cg alkyl or Cj-Cg alkoxy; R^ is C^-Cg alkoxy; X^ and γll are each independently selected from the group consisting of — Br, — Cl, — OH, — NH2, —

OP, — HP, in which P is a suitable protecting or activating group, and — NR R8 wherein R ⁷ and R ⁸ are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, aryl alkyl, substituted aryl alkyl, heteroaryl, and substituted heteroaryl; and q is an integer of from 1 to 4. Preferably, R^ is hydrogen or methyl, R^ and R4 are both hydrogen, R3 is methoxy, γll i _s —OH, and X ¹¹ is — H2 or — NR ⁷ R ⁸ wherein R ⁷ is hydrogen and R ⁸ is lower alkyl, more preferably, methyl.

Particularly preferred photocleavable protecting groups are: wherein R6, R5 and R ⁴ are hydrogen, R3 is methoxy, Y ¹¹ is — OH, and X ¹¹ is — NH2, and q is 1; wherein R^, R5 and R4 are hydrogen, R^ is methoxy, YU is — OH, and χll is — NH2, and q is 3; wherein R6, R5 and R ⁴ are hydrogen, R^ is methoxy, Y*l is — OH, and X ¹¹ is — NHMe, and q is 1; wherein R6, R5 and R ⁴ are hydrogen, R^ is methoxy, Y l is — OH, and X ¹¹ is — NHMe, and q is 3; wherein is methyl, R^ and R4 are hydrogen, R^ is methoxy, YU is — OH, and X ¹¹ is — NH2, and q is 1; wherein R^ is methyl, R^ and R4 are hydrogen, R^ is methoxy, YU is — OH, and X ¹¹ is — NH2, and q is 3; wherein R6 is methyl, R^ and R4 are hydrogen, R^ is methoxy, YU is — OH, and X ¹¹ is —NHMe, and q is 1; and wherein R6 is methyl, R^ and R ⁴ are hydrogen, R^ is methoxy, YU is — OH, and X ¹¹ is — NHMe, and q is 3.

Although these compounds are shown with specific protecting groups, one of skill in the art will readily appreciate that any suitable amme, hydroxy, or carboxy protecting group can be used.

Methods for preparing the photolinkers can be found in Holmes and Jones (1995) Journal of Organic Chemistry 60:2318: U.S. Serial No. 08/493,877, filed June 23, 1995, U.S. Serial No. 08/374,492, filed January 17, 1995, and PCT application US 95/07983, each of which is incorporated herein by reference.

If the amine component possesses reactive sites other than the amino group, it may be necessary to protect them during the synthesis. Suitable protecting groups include acid-labile, base-labile, photoremovable, or removable under neutral conditions. See, e.g., Green, Protecting Groups in Organic Synthesis. Wiley 1985, pp. 218-288, which is incorporated herein by reference. The choice of a particular protecting group will be determined generally by the reaction conditions and by the types of protecting groups present on other components to be used in synthesis. In a most preferred embodiment, the protecting groups are photoremovable and their removal is accomplished by exposing the surface or selected regions thereof to light (e.g., from a light source through a mask) or removable under neutral conditions. Such protecting groups and techniques are described in U.S. Patent No. 5,148,854 and co-pending U.S. Patent Applications Serial No. 07/624,120, filed December 6, 1990, and 07/971,181, filed November 2, 1992.

B. The Silylation Reaction

The compound to be protected is first silylated through treatment with a mono- or disilyl acetamide or other agents capable of silylating an amine group without generating acid by-products. Preferably, N-methyl-N- (trimethylsilyl)trifluoracetamide (MSTFA), N,0-bis(trimethylsilyl)acetamide (BSA), N-methyl-N-(trimethylsilyl)acetamide (MSA), N,0-bis (trιmethylsilyl)trifluoroacetamide (BSTFA) or bistrimethyl silylurea (BSU) is used (each available from Aldrich Chemical Co., Milwaukee, WI; or Fluka, Buchs

Switzerland or Ronkonkoma, NY). Most preferably, MSTFA or BSA is used.

O

n I ³

SiM_ ₃ O

H-rT^ COOH MSTFA M ^β ₃ ^S _{N cooslM} 11

^l.-'^CF, H ³ H ³ or

.SiM- ₃

I SIMe.

BSA

Typically, an excess of the silylating agent , preferably, two or more silylating equivalents, and more preferably, between about two and five equivalents, is used. For compounds having a free hydroxyl group, one additional silylating equivalent of silylating agent is used.

The silylation reaction is conducted in an aprotic solvent, such as methylene chloride, and at a temperature between about room temperature and 100°C, preferably, between about room temperature and about 50°C, and most preferably, at about the temperature of the refluxing solvent.

C. Treatment with an activated Fmoc-reagent

To the resulting solution of the silylated compound is then added an activated Fmoc-reagent, preferably, Fmoc-Cl; Fmoc-OPfp or Fmoc-OSu, most preferably, Fmoc-OSu (each available from Aldrich Chemical Co., Milwaukee, WI; Fluka, Buchs Switzerland or Ronkonkoma, NY, or Chemlmpex).

M ^β -S . Fmoc-OSu Fmoov. _^s_

IT ^COOSiMe ₃

COOSiMe3

Preferably, between about 1.0 and 3.0 equivalents, more preferably, between about 1.0 and 2.0 equivalents of activated Fmoc-reagent is used. Most preferably, about 1.0 equivalents of Fmoc-OSu is used.

17

The reaction is typically conducted at a temperature between about 0°C and 100°C, preferably, between about 0°C and 50°C, and most preferably at about room temperature. The reaction generally will go to completion within about 24 hours.

D. The Work-up

The reaction can be worked up under anhydrous conditions, typically, by first adding a polar, protic solvent, such as a lower alkanol, such as methanol. This leads to the conversion of the trimethylsilyl ester into the free acid and methoxytrimethylsilane. The free acid may be a crystalline product that is insoluble in methanol and thus, can be further purified by washing it with methanol to obtain, after drying, a pure product. This is the method of choice for the preparation of the Fmoc-photolinker. If, however, the free acid is not insoluble in methanol, the reaction can be worked-up as shown below and described in detail in the examples:

5 Addition of Methanol

+ Me.Si-0-CH_

2) Solvent Evaporation I

SOLID OIL

J

Workup A Workup B

8

More specifically, if a solid has formed, it can be washed with a mixture of dilute aqueous acid:polar protic solvent, as described in more detail below. If an oil had formed after the solvent evaporation it can be worked up by an extractive procedure. The exact protocols involved in the work-up procedures are not critical.

E. Results

The present methods provide for a high yield of Fmoc-protected amines with a high degree of purity. Typically, the yield will be greater than 80%, preferably, greater than 90%, and more preferably, greater than 95%, as detailed in the experimental section below.

Moreover, as it has been reported in the literature that Fmoc dipeptides can occasionally be found as side-products in preparations of Fmoc amino acids, (see, Sigler supra.), the presence of possible Fmoc dipeptide impurities was investigated. Fmoc (Gly-Gly) and Fmoc (Phe-Phe) were prepared as reference compounds with the newly developed procedure. The results are shown in the next table:

The two compounds Fmoc (Gly-Gly) and Fmoc (Phe-Phe) were then used in an HPLC study to check the possible contamination with Fmoc dipeptides in the originally prepared Fmoc glycine and Fmoc phenylalanine. The HPLC

experiments clearly indicated that neither Fmoc glycine nor Fmoc phenylalanine contained any detectable Fmoc dipeptides ( detection limit: 0.1%).

In addition, an important aspect in the preparation of amino acid derivatives is the retention of configuration of the chiral center during the course of the reaction. The issue of racemization was investigated by using a chiral HPLC- method. A reversed-phase column derivatized with [L]-tert. leucine and 3,5- dinitro aniline proved very useful in the separation of the [D] and [LJ enantiomers. In none of the examined cases was there any detectable Fmoc-[D] amino acid present (detection limit: 0.5%). Thus, in the methods for preparing Fmoc-protected amines, the chirality of the original amine starting material was retained throughout the reaction sequence to produce a chiral product. The reaction sequence described herein therefore can be characterized as being highly stereoselective and may be stereospecific. (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 sequence described herein will exhibit at least about a 60% percent enantiomeric excess overall; preferably, at least about a 70% percent enantiomeric excess; more preferably, at least about a 80% percent enantiomeric excess; and even more preferably, at least about an 90% 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. Moreover, the reaction conditions are sufficiently mild that even complicated amines can be safely converted to the corresponding protected derivative.

EXAMPLES The following examples are included for the purpose of illustrating the invention and are not intended to limit the scope of the invention in any manner.

EXAMPLE 1

This example illustrates generically the use of MTSFA to produce

Fmoc-protected compounds.

The compound to be protected (20 mmol) was suspended in methylene chloride (20 ml). To the resulting suspension was added, N-Methyl-N- (trimethylsilyl)trifluoroacetamide (7.97 g, 40 mmol). For compounds having a free hydroxyl group, one additional equivalent N-Methyl-N-

(trimethylsilyl)trifluoroacetamide was added. The mixture was refluxed until a clear solution as obtained. This solution was then cooled to room temperature, and a solution of Fmoc-OSu (6.74 g, 20 mmol) in methylene chloride (30 ml) was added over 10-15 minutes.

When the Fmoc-OSu was completely consumed or when the amount of Fmoc-OSu did not decrease over a 2 hour period as judged by TLC or HPLC, the reaction was worked up by one of the following procedures.

Workup A

To the reaction mixture was added methanol (10 ml) and the mixture was stirred for 30 minutes. The reaction mixture was evaporated to dryness with a rotary evaporator. If a solid formed , water was added (30 ml) and the mixture was stirred for 30 min. The solid was filtered and then washed 3x with 1:1 10% aqueous citric acid:methanol (10 ml) followed by washes with water until the filtrate showed a neutral pH. The product was dried at 50°C under vacuum.

Workup B

To the reaction mixture was added methanol (10 ml) and the mixture was stirred for 30 minutes. The reaction mixture was evaporated to dryness with a rotary evaporator. If an oil formed, the product was isolated via an extractive workup by first adding ether and extracting the mixture with 5% aqueous potassium carbonate. The pH of the aqueous phase was then lowered to 2 with IN HCl. If a solid precipitated, it was collected by filtration, washed with water until the filtrate showed a neutral pH and then dried at 50°C under vacuum.

If an oil formed, the mixture was extracted three times with ethyl acetate. The organic phase was washed with water and brine and then dried over sodium sulfate. The solvent was removed by rotary evaporation and the product was dried at 50°C under vacuum.

Workup C

If the Fmoc-protected compound was insoluble in methanol, crystalline Fmoc products were obtained by adding more methanol, filtering the solid and washing it with methanol to obtain, after drying, a pure product. This is the method of choice for the preparation of the Fmoc-photolinker.

Using the MSTFA procedure above, the following results have been obtained:

By replacing MSTFA in the example above, with BSA, the following results have been obtained:

EXAMPLE 2

This example illustrates the use of MTSFA to produc Fmoc phenylalanine.

To methylene chloride (20 ml) was added L-phenylalanine (3.3 g, 20 mmol) and N-methyl-N-(trimethylsilyl)trifluoroacetamide (7.97 g, 40 mmol). The mixture was refluxed for 30 minutes after which a clear solution was obtained. It was cooled to room temperature and a solution of Fmoc-OSu (6.74 g, 20 mmol) in methylene chloride (30 ml) was added over 10-15 minutes.

After 23 hours, methanol (10 ml) was added and the mixture was stirred for 30 minutes. The reaction mixture was evaporated to dryness with a rotary evaporator and water (30 ml) was added. After stirring for 30 minutes, the white solid was filtered and washed 3x with 10 ml 10% aqueous citric acid /methanol 1:1 followed by washes with water until the filtrate showed a neutral pH. The white product was dried at 50°C under vacuum. This resulted in

7.13 g (92%) Fmoc phenylalanine with a melting point of 184-187°C.

EXAMPLE 3

This example illustrates the use of MTSFA to produce Fmoc-protected photolinker:

To a slurry of nitroamino acid 1 (59.6 g, 0.20 mol) in methylene chloride (180 ml) was added BSA (60.9 g, 0.30 mol), rinsed in with methylene chloride (20 ml). The mixture was refluxed for 75 minutes, whereupon the mixture became a solution. The heat was removed and at 30°C, a solution of Fmoc-OSu (70.8 g, 0.21 mmol) in methylene chloride (300 ml) was added over 15 minutes. Within 15 minutes, a heavy slurry developed, so additional methylene chloride (50 ml) was added. The slurry was stirred for 4 hours at room temperature. To it was then added methanol (500 ml) and stirring was continued for 3 hours. The mixture was filtered and the solid was twice reslurried with methanol (600 ml).

The product was dried at 70°C at 50 mm Hg to obtain 88.2 g photolinker, melting point 199-202°C. By HPLC, the product was 100% pure. The mother liquors and washes were combined and stripped to dryness. Trituration and thorough washing with methanol yielded, after drying, an additional 6.5 g product, melting point 197-200°C. The overall yield was 91.1%.

The above description is illustrative and not restrictive. Many variations of the invention will become apparent to those of skill in the art upon review of this disclosure. Merely by way of example a wide variety of process times, reaction temperatures, and other reaction conditions may be utilized, as well as a different ordering of certain processing steps. The scope of the invention should, therefore, be determined not with reference to the above description, but instead should be determined with reference to the appended claims along with their full scope of equivalents. Although certain embodiments and examples have been used to describe the invention, changes may be made to those embodiments

and examples without departing from the scope of the following claims or spirit of the invention.

The disclosures in this application of all articles and references, including patent documents, are incorporated herein by reference.

WHAT IS CLAIMED IS:

1. A method of making Fmoc-protected ammo acids comprising the steps of: (a) treating an amino acid with a silylating agent to form a silylated intermediate; and

(b) treating the silylated intermediate with an activated Fmoc-reagent to yield the Fmoc-protected amino acid.

2. The method of Claim 1, wherein said silylating agen: is selected from the group consisting of N-methyl-N-(trimethylsilyl)trifIuoracetamιde, N,0- bis(trimethylsilyl)acetamide, N-methyl-N-(trimethylsilvt)acetamice, N,0-bis (trimethylsilyl)trifluoroacetamide, and bistrimethylsilylurea.

3. The method of Claim 1, wherein at least two silylating equivalents of silylating agent are used.

4. The method of Claim 1, wherein said activated Fmoc-reagent is Fmoc- OSu.

5. The method of Claim 1, wherein said amino acid is an alpha-amino acid or a peptide.

6. The method of Claim 1, wherein said amino acid is