LABELING OF RAPAMYCIN USING RAPAMYCIN-SPECIFIC
METHYLASES
BACKGROUND OF THE INVENTION
Rapamycin is a macrocyclic triene antibiotic produced by Streptomyces
hygroscopicus, which was found to have antifungal activity, particularly against
Candida albicans, both in vitro and in vivo. [C. Vezina et al., J. Antibiot. 28, 721
(1975); S.N. Sehgal et al., J. Antibiot. 28, 727 (1975); H. A. Baker et al., J. Antibiot.
31, 539 (1978); US Patent No. 3,929, 992; and US Patent No. 3,993,749]. The
immunosuppressive effects of rapamycin have been described. FK-506, another
macrocyclic molecule, has also been shown to be an immunosuppressive agent.
These compounds have also been shown to be useful for a variety of other therapeutic
indications. Rapamycin is commercially available under the Rapamune® name.
A rapamycin ester, rapamycin 42-ester with 3-hydroxy-2-(hydroxymethyl)-2-
methylpropionic acid [described in US Patent No. 5,362,718], also known as CCI-
779, has been shown to have antitumor activity against a variety of tumor cell lines, in
in vivo animal tumor models, and in Phase I clinical trials. [Gibbons, J., Proc. Am.
Assoc Can. Res. 40: 301 (1999); Geoerger, B., Proc Am. Assoc. Can. Res. 40: 603
(1999); Alexandre, J., Proc. Am. Assoc. Can. Res. 40: 613 (1999); and Alexandre, J.,
Clin. Cancer. Res. 5 (November Supp.): Abstr. 7 (1999)].
The labeling of rapamycin with labeling precursor compounds, including
acetate, propionate or methionine [N. L. Paive and A.L. Demain, J Natl Products,
54(1): 167-177 (Jan-Feb 1991)], or shikimic acid [PAS. Lowden, et al, Angew.
Chem. Int. Ed. 40(4):777-779 (2001)] by adding these compounds to fermentation
cultures has been described. In these methods, as the bacteria synthesize rapamycin,
some of the labeled material is incorporated into the newly produced rapamycin. The
labeled rapamycin is purified from a mixture of other molecules, some of which might
also carry the label. However, these methods provide inconsistent results in that not
every rapamycin molecule isolated is labeled to the same extent, or in the same
position.

What is desired are methods of specifically labeling rapamycin to produce a
uniformly labeled molecule.
SUMMARY OF THE INVENTION
The procedure described in the invention uses a specific methylase to label
solely rapamycin in a uniform manner. The methylase, which is present in a crude
cell extract, adds a labeled methyl group to purified desmethyl-rapamycin in vitro. In
this system, rapamycin is the only molecule that is labeled. It may be tagged with
isotopic labels, e.g., radioactivity. Isolation of the labeled material is quite simple
using standard methods. Labeled rapamycin is readily identifiable based on its mass
and/or radioactive label.
Other aspects and advantages of the invention will be readily apparent from
the following detailed description of the invention.
DETAILED DESCRIPTION OF THE INVENTION
The gene cluster responsible for the biosynthesis of rapamycin has been
sequenced and analyzed [Schwecke et al., PNAS USA 92, 7839-43 (1995); Molnar et
al., Gene 169, 1-7 (1996); Aparicio et al., Gene 169, 9-16 (1996)]. Following the
synthesis and cyclization of the core polyketide, which is mediated by the protein
products rapA, rapB, rapC and rapP, further modifications are made to the
molecule. Among these modifications are oxidations and methylations.
Three genes have been identified as S-adenosyl-L-methionine (SAM)-
dependent methyltransferases, rapI, rapM and rapQ. Rapl methylates the C-41
hydroxyl, and RapM and RapQ methylate the C-7 and C-32 hydroxyl groups [Chung
et al., J. Antibiotics 54, 250-256 (2001)].
The method of the invention takes advantage of these rapamycim-specific
methyl transferases (methylases) to efficiently label a desmethyl rapamycin in vitro.
Three enzymes, encoded by the genes, rapl, rapM and rapQ, are used in the
method of the invention. These enzymes can be used individually, or mixtures
thereof can be used in the process of the invention.

As defined herein, the term "a rapamycin" defines a class of
immunosuppressive compounds which contain the following rapamycin nucleus:

The term "desmethylrapamycin" refers to the class of immunosuppressive
compounds which contain the basic rapamycin nucleus shown, but lacking one or
more methyl groups. In one embodiment, the rapamycin nucleus is missing a methyl
group from either positions 7, 32, or 41, or combinations thereof. The synthesis of
other desmemylrapamycins may be genetically engineered so that methyl groups are
missing from other positions in the rapamycin nucleus. Production of
desmethylrapamycins have been described. See, e.g., 3-desmethylrapamycin [US
PatentNo. 6,358,969], and 17-desmethylrapamycin [US PatentNo. 6,670,168].
The terms "desmethylrapamycin" and "-O-desmethylrapamycin" are used
interchangeably throughout the literature and the present specification, unless
otherwise specified.
The rapamycins used according to this invention include compounds which
may be chemically or biologically modified as derivatives of the rapamycin nucleus,
while still retaining immunosuppressive properties. Accordingly, the term "a
rapamycin" includes esters, ethers, oximes, hydrazones, and hydroxylamines of
rapamycin, as well as rapamycins in which functional groups on the nucleus have

been modified, for example through reduction or oxidation. The term "a rapamycin"
also includes pharmaceutically acceptable salts of rapamycins, which are capable of
forming such salts, either by virtue of containing an acidic or basic moiety.
As used herein, pharmaceutically acceptable salts include, but are not limited
to, hydrochloric, hydrobromic, hydroiodic, hydrofluoric, sulfuric, citric, maleic,
acetic, lactic, nicotinic, succinic, oxalic, phosphoric, malonic, salicylic, phenylacetic,
stearic, pyridine, ammonium, piperazine, diethylamine, nicotinamide, formic, urea,
sodium, potassium, calcium, magnesium, zinc, lithium, cinnamic, methylamino,
methanesulfonic, picric, tartaric, triethylamino, dimethylamino, and
tris(hydroxymethyl)aminomethane. Additional pharmaceutically acceptable salts are
known to those skilled in the art.
In one embodiment, the esters and ethers of rapamycin are of the hydroxyl
groups at the 42- and/or 31-positions of the rapamycin nucleus, esters and ethers of a
hydroxyl group at the 27-position (following chemical reduction of the 27-ketone),
and that the oximes, hydrazones, and hydroxylamines are of a ketone at the 42-
position (following oxidation of the 42-hydroxyl group) and of 27-ketone of the
rapamycin nucleus.
In another embodiment, 42- and/or 31-esters and ethers of rapamycin are
described in the following patents: alkyl esters (US Patent No. 4,316,885);
aminoalkyl esters (US Patent No. 4,650,803); fluorinated esters (US Patent No.
5,100,883); amide esters (US Patent No. 5,118,677); carbamate esters (US Patent No.
5,118,678); silyl ethers (US Patent No. 5,120,842); aminoesters (US Patent No.
5,130,307); acetals (US Patent No. 5,51,413); aminodiesters (US Patent No.
5,162,333); sulfonate and sulfate esters (US Patent No. 5,177,203); esters (US Patent
No. 5,221,670); alkoxyesters (US Patent No. 5,233,036); O-aryl, -alkyl, -alkenyl, and
-alkynyl emers (US Patent No. 5,258,389); carbonate esters (US Patent No.
5,260,300); arylcarbonyl and alkoxycarbonyl carbamates (US Patent No. 5,262,423);
carbamates (US Patent No. 5,302,584); hydroxyesters (US Patent No. 5,362,718);
hindered esters (US Patent No. 5,385,908); heterocyclic esters (US Patent No.
5,385,909); gem-disubstituted esters (US Patent No. 5,385,910); amino alkanoic
esters (US Patent No. 5,389,639); phosphorylcarbamate esters (US Patent No.

5,391,730); carbamate esters (US Patent No. 5,411,967); carbamate esters (US Patent
No. 5,434,260); amidino carbamate esters (US Patent No. 5,463,048); carbamate
esters (US Patent No. 5,480,988); carbamate esters (US Patent No. 5,480,989);
carbamate esters (US Patent No. 5,489,680); hindered N-oxide esters (US Patent No.
5,491,231); biotin esters (US Patent No. 5,504,091); O-alkyl ethers (US Patent No.
5,665,772); andPEG esters of rapamycin(US Patent No. 5,780,462). The preparation
of these esters and ethers is described in the patents listed above.
In yet another embodiment, 27-esters and ethers of rapamycin are described in
US Patent No. 5,256,790. The preparation of these esters and ethers is described in the
patent listed above.
In still another embodiment, oximes, hydrazines, and hydroxylamines of
rapamycin are described in US Patent Nos.: 5,373,014, 5,378,836, 5,023,264, and
5,563,145. The preparation of these oximes, hydrazones, and hydroxylamines is
described in the above-listed patents. The preparation of 42-oxorapamycin is
described in US Patent No. 5,023,263.
In another embodiment, rapamycins include rapamycin [US Patent No.
3,929,992], rapamycin. 42-ester with 3-hydroxy-2-(bydroxymethyl)-2-
methylpropionic acid [US Patent No. 5,362,718], and 42-O-(2-hydroxy)ethyl
rapamycin [US Patent No. 5,665,772]. The preparation and use of hydroxyesters of
rapamycin, including CCI-779, is described in US Patent Nos. 5,362,718 and
6,277,983.
Although the examples provided herein illustrate metbylation of 7-O-
desmethyl-rapamycin [US Patent No. 6,399,626] and 32-O-desmethylrapamycin,
these compounds are not a limitation of the invention.
I. The rapl, rapM. and rapQ enzymes
In one embodiment, the rapamycin methylating enzymes defined herein are
used in the form of crude enzyme extracts from Streptomyces hygroscopicus. In a
further embodiment, crude enzyme extracts are prepared from S. hygroscopicus cells
[available from the American Type Culture Collection, Manassas, Virginia, US,
accession number ATCC29253, or from other sources]. In one embodiment, these

cells are cultivated in shake flask fermentations using a method such as that described
in Kirn et al. (Kim, W-S. et al., 2000, Antimicrob. Agents Chemother. 44: 2908-
2910). In another embodiment, for preparing cell free extracts, cells are collected by
centrifugation, and about 1 gram of cell material is resuspended in about 20 mL of a
suitable buffer. In yet another embodiment, the buffer is 50 mM 2-(N-
morpholino)ethanesulfonic acid (MES) at a pH of about 6. In still another
embodiment, the buffer is 50 mM potassium phosphate at a pH of 7.5. Cells are then
disrupted and cell debris is removed by centrifugation. In one embodiment,
supernatants are adjusted to ~10% glycerol prior to freezing, e.g., at -70°C. In other
embodiments, alternative methods for preparing crude enzyme extracts from the cell
cultures will be readily apparent to one of skill in the art.
In yet another embodiment, these enzymes are further purified by classical
protein isolation methods such as ammonium sulfate precipitation, column
chromatography, etc.
In still another embodiment, the enzymes are synthesized by recombinant
techniques, using classical in vitro transcription and translation methodologies.
The nucleic acid sequences of the rapI, rapM and rapQ enzyme genes are
available from the PubMed NCBI on-line database, under accession No. X86780 for
S. hygroscopicus. The nucleic acid sequences of the rapQ methylases gene are
located at nt 90798-91433 of the CDS; protein ID# CAA60463.1 provides the amino
acid sequence. The nucleic acid sequences of the rapM methylases gene are located
on the complement of nt 92992-93945 of CDS; protein ED# CAA60466.1 provides the
amino acid sequence. The nucleic acid sequences of the rapl methylases gene are
located at nt 97622-98404 of the CDS, protein ID # CAA604701 provides the amino
acid sequence. See, also, T. Schwecke, et al, Proc. Natl Acad. Sci U.S.A. 92 (17),
7839-7843 (1995); 1. Molnar, et al, Gene 169 (1), 1-7 (1996), and J. F. Aparicio, et
al., Gene 169 (1), 9-16 (1996). The preceding nucleic acid and amino acid sequences
are hereby incorporated by reference.
In another embodiment, the genes encoding the rapamycin methylation
enzymes described herein are cloned into a suitable vector operably linked to
regulatory control sequences that control expression thereof. As used herein,

"operably linked" sequences include both expression control sequences that are
contiguous with the gene of interest and expression control sequences that act in trans or
at a distance to control the gene of interest. Expression control sequences include
appropriate transcription initiation (promoter) and termination; sequences that
enhance translation efficiency (e.g., Shine-Dalgarno site or ribosome binding site);
and when desired, sequences that enhance secretion of the encoded product. A great
number of expression control sequences, including promoters which are native,
constitutive, and/or inducible, are known in the art and may be utilized.
In one embodiment, the regulatory control sequences include a regulatable or
inducible promoter. Many such regulatable or inducible promoter systems have been
described and are available from a variety of sources. Inducible promoters allow
regulation of gene expression and can be regulated by exogenously supplied compounds,
or environmental factors such as temperature. Inducible promoters and inducible
systems are available from a variety of commercial sources, including, for example and
without limitation, Invitrogen, Clontech and Ariad. Many other systems have been
described and can be readily selected by one of skill in the art. For example, inducible
promoters include the T7 polymerase promoter system [International Patent Publication
No. WO 98/10088]. In one embodiment, the systems are selected for use in bacterial
systems.
In another embodiment, as illustrated below, one or more of the genes
encoding the enzyme are cloned into a commercial vector which expresses the
enzyme(s) under an inducible promoter, i.e., pET24 inducible plasmid expression
vector [Novagen]. However, one of skill in the art can readily select another vector
and/or another suitable promoter for expression of the enzymes.
The vector may be any vector known in the art or described above, including
naked DNA, a plasmid, phage, transposon, cosmids, episomes, viruses, etc.
Introduction into the host cell of the vector may be achieved by any means known in
the art or as described above, including transformation, transduction, and
electroporation. Introduction of the molecules (as plasmids or viruses) into the host
cell may also be accomplished using techniques known to the skilled artisan and as
discussed throughout the specification. In one embodiment, standard transformation
techniques are used, e.g., CaCl2-mediated transformation or electroporation.

Once cloned into a suitable expression vector, the nucleic acid sequences
encoding the enzyme are introduced into a suitable host cell for expression. In one
embodiment, a suitable host cell is selected from prokaryotic (i.e., bacterial) cells. In
the examples below, the host cells are Escherichia coli cells. However, one of skill in
the art can readily select another appropriate host cell for expression of the selected
enzymes.
In one embodiment, as illustrated below, crude enzyme extracts are prepared
using recombinant techniques. [See, generally, Sambrook et al., Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY] For
example, a cell transduced with the rapamycin methylase genes is cultured under
conditions that permit expression of the methylases(s). Where an inducible or
regulatable protein, these conditions include supplying the inducing agent. Following
culture, the cells are pelleted by centrifugation and resuspended in a suitable buffer,
including a reducing agent, and phosphate buffer, adjusted to a neutral pH. In one
embodiment, the buffer contains 50 to 100 mM potassium phosphate buffer, pH 7 to
7.5, containing 1 mM to 2 mM β-mercaptoethanol. In another embodiment, lysozyme
is added at a final concentration of 100 µg/ml. In yet another embodiment, a suitable
nuclease [e.g., Benzonase™ nuclease] is added at 0.5-2.0 µL/mL cells. In still another
embodiment, cell suspensions are incubated, e.g., for 15 minutes at 30°C. In yet
another embodiment, a protease inhibitor (e.g., phenyl methyl sulfonyl fluoride
(PMSF)) is added to the cells at a final concentration of 0.5-1.5 mM.
In a further embodiment, cells are fragmented by suitable means. In one
embodiment, fragmentation is by mechanical means, e.g., by sonication on ice. Cell
debris is removed and the resulting supernatants are adjusted to 5-15% glycerol (v/v)
before freezing. The resulting crude enzyme extracts are now available for use in the
rapamycin-specific methylation reaction of the invention.
In other embodiments, alternative methods for production and isolation of the
enzymes will be readily apparent to those of skill in the art [Sambrook J et al. 2000.
Molecular Cloning: A Laboratory Manual (Third Edition), Cold Spring Harbor Press,
Cold Spring Harbor, NY].

The methods for production, purification, and isolation are not limitations of
the present invention.
II. The Methylation Reaction
Using a rapamycin methylase as described herein, either as a crude extract or
another suitable form, the methylation reaction is performed as follows.
Approximately 45 to 65 % v/v crude methylase extract is added to a reaction
containing about 8-130 µM desmethyl rapamycin solution, about 0.2-0.4 mM
methylating reagent, about 4-10 mM magnesium (Mg, e.g., MgSO4), and a suitable
buffer at about 50-100 mM concentration, adjusted to a pH of 6.5 to 7.5. In another
embodiment, a more purified form of the methylases is utilized in lower volume, e.g.,
about 10 to about 45% v/v methylase.
In one embodiment, the methyl donor is S-adenosyl-L-methionine (SAM).
When selected for use in the invention, SAM is generally present at a final
concentration of about 0.2-0.4 mM.
In another embodiment, a rapamycin solution is about 0.5 mg/mL to about 5
mg/mL, about 1 mg/mL to 3 mg/mL, or about 1 mg/mL rapamycin in a suitable
solvent Suitable solvents for the selected rapamycin include methanol, ethanol and
dimethylformamide, tetrahydrofuran, or mixtures thereof.
Suitable buffers can be readily selected from among physiologically
compatible buffers, including, e.g., phosphate buffered saline, a 2-(N-Morpholino)-
ethanesulfonic acid (MES) buffer, Tris-(hydroxymethyl)arninomethane (Tris) buffer,
or potassium phosphate buffer.
Following mixture of these components, the reaction is allowed to proceed.
The reaction temperature can vary from 20°C to about 37°C for about 0.5 to 3 hours,
or about 1 to 2 hours. In one embodiment, the reaction mixture is incubated at about
34oC for approximately 1 hour.
At the end of incubation, 1 to 2 volumes of a quenching reaction is added to
terminate the reaction, e.g., ethanol, methanol or ethyl acetate.
Precipitated material is removed by conventional methods. In one
embodiment, the precipitated material is removed by centrifugation. In a further

embodiment, centrifugation is conducted at 14,000 rpm for 10 minutes. However,
other removal methods and/or centrifugation conditions are known in the art.
Purification can be accomplished by any suitable method known to those of
skill in the art. Suitable methods include recrystallization, silica gel column
chromatography, thin layer chromatography (TLC) and high performance liquid
chromatography (HPLC). In one embodiment, HPLC analysis is performed using a
C18 column (3.9 x 150mm) at 45°C with a mobile phase comprised of 60% dioxane,
0.05% acetic acid and.0.03% triethylamine. In another embodiment, HPLC analysis
is performed with a C18 column (4.6 x 250 mm) using a mobile phase gradient of
40%A:60%B going to 15%A:85%B over 75 minutes, where solvent A is 10 mM
ammonium acetate in water and solvent B is methanol.
III. COMPOSITIONS AND USES
Labeled rapamycin is needed to study and/or monitor the metabolic fate of
rapamycin in the body. In one embodiment, labeled rapamycin is used to identify
cells/structures that have bound to rapamycin. Rapamycin may be uniformly tagged
with either density or radioactive labels. Rapamycin labeled in the manner described
will have the conformation and properties of unlabeled, native rapamycin, but is
easily detectable because of the consistently incorporated density or radioactive label.
In one embodiment, the invention provides kits for specific labeling of
rapamycin, comprising one or more of the enzymes described herein. The kits may
further contain additional components, such as, e.g., a positive control (e.g., a
methylated rapamycin), a negative control, reagents (e.g., buffer, lysozyme, nuclease),
vials, tubes, and instructions for performing the method of the invention.
In certain circumstances, it is desirable to deliver the labeled rapamycin
produced according to the present invention in a composition comprising a
physiologically compatible carrier. These compositions are advantageous in that the
labeled rapamycin compounds produced according to the invention can be readily
tracked (i.e., monitored) using techniques known to those of skill in the art, e.g., mass
spectrometry or scintillation counting, among others.

The following examples are illustrative of the methods of the invention for
rapamycin specific methylatidn. It will be readily understood from a reading of the
detailed description of the invention these examples do not limit the invention to the
reaction conditions and reagents illustrated.
EXAMPLES
A. Amplification of Methylase Genes
The genes were amplified from genomic S. hygroscopicus
ATCC29253 DNA with oligonucleotide primers designed using the published
rapamycin gene cluster sequence (Schwecke, T. et a!., 1995, Proc. Natl. Acad. Sci.
USA 92: 7839-7843). The RapI, RapM and RapQ proteins were then expressed in E.
coli strain BL21(DE3) cells using the Novagen pET24 inducible plasmid expression
vector. In this vector, cloned genes are expressed from a T7 promoter by T7 RNA
polymerase, and expression is activated by IPTG addition.
B. Preparing Enzyme Extracts
To establish optimal conditions for an in vitro methylation reaction,
crude enzyme extracts were prepared from S. hygroscopicus [ATCC29253] cells
cultivated in shake flask, fermentations using a method like that described in Kim et
al. (Kim, W-S. et al., 2000, Antiraicrob. Agents Chemother. 44: 2908-2910). Cells
were collected by centrifugation, washed in 0.2 M MES buffer, pH 6.0, and cell
pellets were frozen prior to extraction. Approximately 8 g to 10 g of thawed cell
material was resuspended in 20 mL of 50 mM MES buffer, pH 6.0. For crude
extracts of the cloned methylase proteins, 25 mL cultures of induced cells were
collected by centrifugation and the pellets frozen. The pellets were resuspended in 10
mL 50 mM potassium phosphate buffer, pH 7.5, containing 1 mM β-mercaptoethanol.
Thereafter, lysozyme was added to a final concentration of 100 µg/ml and
Benzonase™ nuclease was added (1 µL/mL cells). Cells were sonicated for 1 to 2
min on ice and cell debris was removed by centrifugation at ~3 0,000 x g, 4°C for 15
min. Supematants were adjusted to ~10% glycerol prior to freezing at -70°C.

C. Methylation of 7-Desmethyl-rapamycin
Approximately 65 µL of crude methylase extract was added to a
reaction containing 3 µL of 1 mg/mL 7-desmethyl- rapamycin (7-dmr) solution, 5 µL
of 4 mM SAM, 4 µL of 0.1 M MgSO4, and 23 µL of a 0.5 M phosphate buffer,
adjusted to pH 7.5. Methylation reactions using the recombinant cell extracts were
carried out as described above, except that 50 µL of extract and 38 µL of buffer were
used. HPLC chromatograms from reactions containing the RapM methylase extract
and two desmethyl-rapamycin substrates, 7-O-desmethyl -rapamycin (7-dmr and 32-
O-desmethyl-rapamycin show that the RapM methylase generated rapamycin (rapa)
only when 7-dmr was the substrate. The enzyme did not modify 32-dmr, indicating
that the cloned enzyme retained its substrate specificity in vitro. In addition, samples
with no SAM added showed no conversion of 7-dmr to rapamycin.
D. Labeling Rapamycin with Methyl-tritiated SAM
For labeling of rapamycin, the same type of in vitro reaction was used.
For example, reaction mixtures for RapM methylation contained the following: 10
µL 0.5 M KPO4 buffer, pH 7.5, 4 µL of 0.1 M MgSO4, 33 µL of 100µM S-adenosyl-
L-methionine-(methyl-3H), 3 µL of 1 mg/mL 7-desmethyl-rapamycin (in ethanol),
and 50 µL crude extract. The following scheme shows an example of the labeled
rapamycin molecule that would be generated by the action of the RapM methylase on
the 7-dmr substrate.


The tritiated material was indistinguishable from the rapamycin standard by
HPLC analysis. Mass spectral data indicated that the labeled material was consistent
with tritiated rapamycin.
The present invention is not to be limited in scope by the specific
embodiments described herein. Indeed, various modifications of the invention in
addition to those described herein will become apparent to those skilled in the art
from the foregoing description and the accompanying figures. Such modifications are
intended to fall within the scope of the appended claims.
It is further to be understood that values are approximate, and are provided for
description.
Patents, patent applications, publications, procedures, and the like are listed
throughout this application, the disclosures of which are incorporated herein by
reference in their entireties. To the extent that a conflict may exist between the
specification and another document, the language of the disclosure made herein
controls.

We claim
1. A method for specifically labeling a rapamycin comprising the step of reacting a desmethylrapamycin with a rapamycin-specific methylase in the presence of a methylating reagent.
2. The method according to claim 1, wherein the methylating reagent is
S-adenosyl-L-methionine.
3. The method according to claim 1 or claim 2, wherein the methylase is
selected from the group consisting of rapI methylase, rapM methylase, and rapQ
methylase.
4. The method according to any one of claims 1 to 3, wherein the
methylase is in the form of a crude enzyme extract.
5. The method according to claim 4, wherein the crude enzyme extract is
prepared by the steps comprising:
(a) expressing the rapamycin-specific enzyme from a cell culture
transduced with a nucleic acid sequence encoding an enzyme operably linked to
regulatory control sequences, said enzyme selected from the group consisting of rapI
methylase, rapM methylase and rapQ methylase;
(b) concentrating the cells and resuspending them in a buffer;
(c) incubating the mixture with lysozyme and nuclease;

(d) fragmenting the cells; and
(e) centrifuging and collecting the supernatant.
6. The method according to claim 5, wherein the incubation in step (c)
further comprises a protease inhibitor.

7. The method according to any one of claims 1 to 6, wherein the reaction
mixture is incubated at about 34 °C for about 1 hour.
8. The method according to any one of claims 1 to 7, wherein methanol is
added to the reaction at the end of incubation.
9. The method of any one of claims 1 to 8, wherein precipitated material
is removed by centrifugation prior to HPLC analysis.

10. The method of claim 9, wherein HPLC analysis is performed in a C18
column at 45°C with a mobile phase comprising dioxane, acetic acid and
triethylamine.
11. A specifically labeled rapamycin produced according to the method of
any one of claims 1 to 10.
12. A composition comprising a specifically labeled rapamycin produced
according to the method of any one of claims 1 to 10 and a physiologically
compatible carrier.
13. A kit for producing a specifically labeled rapamycin comprising a
methylated rapamycin produced according to the method of any one of claims 1 to 10,
and one or more components selected from the group consisting of a negative control,
a methylation reagent, a vial, a tube, and instructions.
14. A kit comprising the labeled rapamycin according to claim 11.

A method for rapamycin-specific labeling using rapI, rapM and/or rapQ enzymes is described. Also are methods
for generating crude enzyme extracts useful in the method of the invention. Uses of the specifically labeled rapamycin as diagnostic
tools are provided.