CURRENT RESEARCH PROJECTS AND PROBLEMS OF THE INSTITUTE OF CHEMISTRY OF NATURAL PRODUCTS, MOSCOW/INTENSE SOVBLOC INTEREST IN STREPTOTHRYCIN, A TOXIC SUBSTANCE WITH POSSIBLE BW IMPLICATIONS

• Document Type: FOIA
• Collection: FOIA Collection
• Document Number (FOIA) /ESDN (CREST): 06265251
• Release Decision: RIPPUB
• Original Classification: U
• Document Page Count: 22
• Document Creation Date: March 9, 2023
• Document Release Date: November 19, 2021
• Case Number: F-2020-01174
• Publication Date: December 23, 1965

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ATION REPORT INFORMATION REPORT
CENTRAL INTELLIGENCE AGENCY
This material contains information affecting the National Defense of the United States within the meaning of the Espionage laws, Title
18, U.S.C. Secs. 793 and 794, the transmission or revelation of which in any manner to an unauthorized person is prohibited by law.
CONTROLLED DISSEM
COUNTRY USSR
2 9 DEC 1965
CONTTIAL
REPORT NO.
SUBJECT Current Research Projects and Problems DATE DISTR.
of the Institute of Chemistry of Natural
Products, Moscow/Intense Savbloc Interest NO. PAGES
in Streptothrycin, a Toxic Substance with
Possible BW Implications REFERENCES
DATE OF
INFO.
PLACE &
DATE ACQ.
to Nov 65
��� 410
to Nov 65
(b)(3)
NO DISSEM ABROAD
(b)(3)
23 December 1965
2
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Tills Is UNEVALUATED INFORMATION
SOURCE:
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1. During his visit to US microbiological facilities in October and November
1965 Dr MikhailMShemyakin, an active member of the Soviet Academy of
Sciences and director of the Institute of Chemistry of Natural Compounds,
Moscow, indicated his interest in the development of US mass spectrometry
of natural products. He expressed a genuine surprise when he learned
that even major US microbiological_research laboratories lacked mass
spectrometers, and boasted that his institute had four normal spectro-
meters and one high resolution mass spectrometer. (The latter costs
about US$200,000.)
2. According to Shemyakin, hiss institute is engaged among others, in the
following research projects:
a. Application of mass spectrometry to amino acid sequence
AnAlysis in poly-peptides.
b. Classification of actinomytes using chemistry of the cell
wall.- --------
c. Biological effects of DNA and its breakdown products.
d. Research on streptothricin.
_
3. To underline his vivid interest in streptothricin, Shemyakin brought
with him a hitherto unpublished paper written by Professor A S Khokhlov
deputy director, and his co-worker, Dr P D Reshetov, Institute of the
Chemistry of Natural Products. Shemyakin stated that Khokhlov and
his associates have been working on streptothricin, a highly toxin
'substance,, for several years and that they needed US streptothricin "for
comparison."
4. As is known, streptothricin was first reported by US investigators in
U-NO
(b)(1)
(b)(3)
GROUP I
Excluckd from (Moos*
downproding end
decleisifwetko
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1940. After two years of research, streptothricin was declared nephro-
toxic and it was agreed that further study would be useless. However,
the researchers also found that many peptide-like antibiotics related
to streptothricin have varied biological activities against both
Gram-positive and Gram-negative bacteria, fungi and viruses. Since
some US microbiologists felt that various members of the streptothricin
family of antibiotics may found non-toxic and may prove to be
highly important antibiotics, research has not entirely been abandoned.
5. Judging from private requests made by Soviet, Czechoslovak and Hungarian
microbiologists visiting in the US during the past few years, there
appears to be a concerted effort on the part of Sovbloc researchers
to obtain samples of streptothricin from their US colleagues. US
microbiologists recall that a Sovbloc visitor indicated his interest
in streptothricin in connection with his research in the field of
equine encephalitis. Several US researchers are convinced that the
Soviet sId satellite work on streptothricin has LW implications.
6. Shemyakin's institute is not doing any work on ergot alkaloids. However,
a new research organization, the Institute of the Biochemistry and
Physiology of Microorganisms, Soviet Academy of Sciences, now located
at Ul Vavilova 18, Moscow, but scheduled to move into its own building
in 1966, is conducting some work on ergot alkaloid production. The
director of the new institute is Dr Nikolay Yerusalimskiy.,) a former
deputy director of the Institute of Microbiology, MOSCOW. Ierusalemskiy's
deputy is Dr Georgiy K Skryabin.
7. The work of the Institute of Chemistry of Natural Products is determined
by-the following criteria:
a. The research problem has to be interesting from a biological
point of view.
� b. The problem has to be of chemical interest from the point
of view of chemistry of new products.
c. The problem has to have practical biological implications
since funds allocated for fundamental research are limited.
8. The institute's major efforts now center around natural product research
for which more funds are available than for, work on synthetic drugs.
9. Shemyakin's institute has only a small, insignificant project concerning
Soviet "popular" medicinals, and as a rule, Soviet microbiologists have
a low opinion of Chinese Communist research efforts in this field.
10. One of the top priorities of Shemyakin's institute is research on interferon.
Soviet researchers are of the opinion that interferon which has been
investigated by several US scientists could be important in virology as
an antiviral and, possibly an antitumor agent. Some Soviet scientists
believe that interferon maybe involved in the prevention of tumor.
However, they admit that they have been hindered by assay problems.
,Shemyakin requested several US researchers doing work on interferon to
help his institute with the solution of the assay problem.
end.
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�k,
TM PRESENT STA.T21 OP INV2STIGATION OP ,
pTR2PPO2IMICIN ANTIBIOTICS
Prof.A.S.Khokhlov and P.D�ReshetoV
Institute for Chemistry of Natural Products,.
� USSR Academy of Sciences, Moscow, USSR.
��
raioroorganisms producing stroptothricin and. related
� antibiotics are very: widespread. in nature. Subabances belong.� .
in to this clasS are frequently encountered. by all engaged in
.� screening tests' for new antibiotics from actinortycetes.
Tho high antibiotio activity of the streptothricins and.
,their delayed. toxicity which becomes apparent only on prolonged
advtrri stration have time and again attracted the attention of
� ��thd4 research workers to those compounds. Streptothricin.
o�
, the parent compound of this group aI4 its best known. member was'
discovered by S.Waksman and R.B.Woodruff in 1942. Le.ter a
number of ,iscaa344.1--9111.y purified antibiotic preparations with pro...-
,
pertios similar Ito the streptothcin was described. However
� in tho majority of oases the tiailecuonfiziod themselves only
to general description of nonhomopneous preparations to
which they save .new names orVcit4eacee-Zusually on inisu.ffioient
13 CtA,U>
�
grounds),Ift-vtartue of this the presi-l-nriim streptothriOin
\III .1.0,,;,...17c �
atabl, mat about 70 6,, which thowevev, as our
studios have shown are without, rational basis.
The structure of streptothricin was established in
a/
1961-1963 as lam result of extensive work by Amorican and.
� 1
�
English gEoups 01, soienti -4 4..
r7
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w. ma
�t
CIOCOn'hfe, "rat., I
H07,1 0 t's(H�c I .
1\1
�
QH
�H 014
00 020H CH2011.2CH2NE2
It is noteworthy that staptomyeetes produce strepto..
thricia free of other antibiotics of the same group; this
greatly facilitates its isolation and purification.
s.
Almost e=t4 .propo a were madith-.57-----v
,structures of streptolin(II.1.0arter� B'Esvan Tamelen),
raCemomycin 0 (S.Takemura), and partially or roseothricinT4
((oto et al ) and goomycin (Harookmann). However, our in.
Vestigations have shownAdme structures.Zii either inaq.'
curate, or (as in the case oT racemomycia 0) lacking _or-sound,.
� basis, This is probably duo to the tact that the preparation400
studied were mixtures rather then individual compounds.
It must be emphasized, that the isolation of individual.
ek.
streptothricins from crude preparations was fo1r long(a-very
difficult task. All spreptothrioins are insoluble in. nonpolar
solvents (practicallythey are soluble Only in water), they
are unstable in acid and alkaline solutions, and. at elevated
temperatures also(htiii-ral medium; they display no characteristic
. bands in the UV-spectra and so on. We have found, that different
streptothricins,lwhen Obtained intro-individual state, are .
almost indistinguishable from each other in many of their 'pro.
parties "16 for a:maples with the exception of only the most \
t/
simple otreptothricin P, i.e. streptahricin itselfothey have
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the same IR spectrum. All this great4 limits the posstbilitrn
-k 1
ies ofA investigatorinrseleottng methods for fractionatirij the .
streptothrioins. Preparative partition chromatography on
lulose in butanol-pyridine-acetio acid-water (15100112) sol.
vent system used, for instance,in the separation of the compost,
nonts of racemomytin and antibiotic A-8265 proved to be very.
laboroup and of little officieniy and did not receive wide applies
cation. Hence the first stage in our Investigations was the -
elaboration of methods for the isolation and purification of ,
individual streptothricins, on a preparative scale..
The different basicities of streptothrioin and otreptolin
led us to the assumption that, perhapsIthe other streptothricins j
differ from each other in the number of free amino groups.
�
On these grounds we believed ion-exchange chromatography might'
prove to be a suitable method for tractionatfng of the strepto.1, "
thmicin preparations,. For rational selection of the fraction**
, I
tion conditions we first determined the distribution coefti- .
j
tionts CO of one of our crude preparations-grisia (grisemin),-
between the carboxylcontaining ionr.exchange resins Amberlite
IRC-50 and carboxymethylcellulose (the sodium form) and soli*. 1
tions or sodium chloride and sodium acetate of various concentraft
The optimal .K values were found for adsorption of the
antibiotic on carboxymethylaelIulose from 0.2-.0.4 molar soluf,
'tions of sodium chloride.
The ana*tical experiments (with a column 0.9cm diameter
and 40 cm long) showed that under such conditions this method,
was highly efficient*
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By these means all six antibiotios which were found to.
be simultaneousll present in the mixture, could be reliably
separated.
Thus, ionrioxchango chromatography on carboxymethylcel*
lulose in a sodium chloride concentration gradient formed the
basis for a general whew we developed for the isolation and
purification of streptothricinsi apd-,in this wavy we were able to
overcome obstacles presented,by the similarity of the properties
� of these antibiotics.
The operations used in each stage of tho purification are
given in the following scheme, but some of them re4uire explaaa,*
tion. �
�
� Stage in the Isolaticin and purification of individeal,
streptothricins.
Principal stages' in the
' purification process
1.Preliminary purification .
of crude' preparations
\?.Fractionation of crude
preparations
3.Isolation or individual
Corresponding operations.
lareatment with active charcoal
2.Preparation of picrate and its �
transformation into hydro/chic)...
ride .
�
Lion exchange chromatography on
carpoxymethylcelluloae
10Adsorption on Atberlite 3110.50
streptothrioins from eluatoe 2�Desalting of ion exchange resin\
3�Desorption and drying of anti-
, biotics �
4.Furthor purification of ' l.Precipition as the picrate and
obtained compounds transformation into the hydro
chloride
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2.Fractiona3. precipitation
3.Proparation of pure sulphates
and oxalates.
After isolation, from culturea broth streptothricin prepare...
tions usually contain pigmented impurities and certain amounts
of inorganic salts so that they must be subjected to a IR pro MIMI.,
3.7 purification.
The results. of the chromatography of four preparations .at
optimal charge of the column (grisomin 5 mgiral,phytobacterio-
mycin 2 mg/mi, polvinycin and antibiotio N 4714-12495 me(m1)
are given on the next slide.
Because the qualitative and quantitative composition of
the crude streptothricin mixtures depends on the fermentation '
conditions, the fractionation was sometimes repeated., as a rule �
using for the first fractionation maximal charges of columns,
for exnmple for polyrnycin uo 6 mg/m1.
The fractions were analysed by measuring the optical den-
sity at 215 mi,i,which is a linear function of the concentration
of the compounds.. After fractionation the separated. Streptothri..
cins wore dissolved. in 3-65 liters of 0.2-0.3 molar solutions of �
sodium chloride,' i.e. the ratio by weight of antibiotic to so-
-;.
dium chloride thus being about 1 to 50. Concentrating and desalt."'
ins were achieved by adsorption on the sodium forme of Amberlite
IRC.50. However the preparations obtained at this stage still
contained small amounts of sodium chloride and. traces of inaa-
tivation products. The neutral sulphates and. oxalates of the
streptothrioins were prepared by means of thoroughly-washed
"I
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finoIy ground (400-600 mosh),,oe anion-exchange resin Arnberlite
IRA-400 in the appropriate (sulphate or oxalate) form,
As a result 18 dhromatographically pure streptothricias
belonging to six: different typos of compounds were obtained
from five crude preparations - paymycia, phytdbacteriovoin,
Japanese antibiotic racemomycia, griserain and antibiotic
M 4714-12
AU compounds, belonging to the series A. to B have the
- same characteristic /a-spectrum with strongly marked amide
� bmies 3250,1658,1563 and 1310 cm'. The IR, .4spoctraa of
� stroptothricins belonging to P-type differs only, by the absence
of absorption band in the 1563 eel region. Table 1 gives the ,
optical activity of the majority of the compounds obtained,
Table 3.,
Optical activity of streptothricin antibiotics
Name s . f streptothricin hydrochlorides
of initial pro-s (o 0.9 in methanol)
paration
BO D E
Polyqycin
l'hytobacteriomy-
cm
Grinemin
Antibiotic
M 4714-12
, Streptothricin
.8treptolin
�-9,2 -9,6
x) -.21,6
4
.21,0 x)
.42,2 -22,0 -36,6 -34,4
-42,0
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---x) These compounds wore tabtained in small amounts ezfd their fail
were determined, only approxima hay.
xx)Data from the literature.
A: very important characteristic of,streptothricin compounds.
is the number of free amino groups in their molecules, which
.!)
doterminoa their basicity. Ve determinEi.iaa 54=t1e number of
free amino groups we made use of a method described irA the 2i-.
toraturo based. on the electrophoresis of N-3,5-dinitrosu3,pho-
phenyl derivatives (DUSE) of different degrees of substitution.. �
The use of DN1Z6derivatives for this purpose has the advantage
over that of =4P-derivatives in high solubility of the DNSP-de-
e
rivativea in aqueous electrolytes and in jump--of 2 unitO of
charge for each substitution. Ebwever, preliminary experiments
revealed. that the interaction of potassium, 4-chloro-315-dinitro-,
benzenesulphonato with amino groups in, the presence of triethylft
amine was accompanied by partial inactivation of the strepto-
thricins and led to extra spots on theelectrophoregrams. In
analysing the electrophoregraas these extra spots could. be
excluded, by comparing the mobility DNSB.derivatives of .ihe
types of streptothrioins with that of their inactivation pro-.
duets 0b2?aine4 by treating antibiotics with dilute mineral
acids.
It was therefore possible to interpret the electrophoreg..
rams of DNSP-dertvativosnof streptothrioins as follows. The
streptothrioin, with two free amino groups (streptothricin 0)
can, however, take up three equivalents of acid. Therefore its
DNSP-derivatives on eleotrophoresis in acid electrolytes must
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+2 .
+5 , +1
0 +4 +2
have charges of +1 and .l rather then 0 and. .2. And. in fact :I '
these derivatives are readily noticeable as two intensely s
coloured spots, simmotrically aituated on both sides of the
zero-lino,
These conclusions were o-X essential importance in analyzing
4-the electrophoregrams or WW.derivatives of the other anti-
biotics. In this way it became possible to determine the number
of free amino groups Lind the basicity or all six types of stropto
thricins ( see Table 2)
Table 2.
Blectrophoresis of MU-derivatives of Streptothricins
�
�
�
Type'of
stropto.�
thricia
DNSP.derivatives of the streptothri.:NUMber- :Basicitj
cm' charges and location of the lat.:or amino:or strev
ter on the electrophoregrams ;groups � stothri.
mins
.;+1'
6. �-2
.1. -.3
0 s�Z. �
11 +5 .+3 I-C+1 '/(71. i-;73
4 +6' *�--.(+4 -,-(4.2 0. 4